Genetic Disorders and the Fetus: Diagnosis, Prevention and Treatment (Milunsky, Genetic Disorders and the Fetus) [6 ed.] 1405190876, 9781405190879

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Genetic Disorders and the Fetus: Diagnosis, Prevention and Treatment

Edited by Aubrey Milunsky Jeff M. Milunsky

WILEY-BLACKWELL

Genetic Disorders and the Fetus

Dedicated to

Laura and Kiran For their love, support and understanding and to our grandchildren and children,

Julie, Miranda, and Cody, who endow life with joy and meaning

“Make assurance double sure.” Shakespeare, MacBeth

Genetic Disorders and the Fetus Diagnosis, Prevention and Treatment EDITED BY

Aubrey Milunsky

MB BCh, DSc, FRCP,

FACMG, DCH Professor of Human Genetics, Pediatrics, Pathology, and Obstetrics and Gynecology, Co-Director, Center for Human Genetics Boston University School of Medicine and Boston Medical Center Boston, MA, USA

Jeff M. Milunsky

MD, FACMG

Professor of Pediatrics and Genetics and Genomics Co-Director, Center for Human Genetics Director, Clinical Genetics Boston University School of Medicine and Boston Medical Center Boston, MA, USA

SIXTH EDITION

A John Wiley & Sons, Ltd., Publication

This edition first published 2010, © 2010 by Aubrey Milunsky and Jeff Milunsky Previous editions: 1979, 1986, 1992, 1998, 2004 © Aubrey Milunsky Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical and Medical business to form Wiley-Blackwell. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by physicians for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. ISBN: 978-1-4051-9087-9 A catalogue record for this book is available from the British Library. Set in 9.5/12pt Minion by SNP Best-set Typesetter Ltd., Hong Kong Printed in Singapore 1

2010

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x 1 Genetic Counseling: Preconception, Prenatal and Perinatal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Aubrey Milunsky and Jeff M. Milunsky 2 Amniocentesis and Fetal Blood Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Sherman Elias 3 Amniotic Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Aubrey Milunsky 4 Amniotic Fluid Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 Daniel L. van Dyke 5 Prenatal Genetic Diagnosis through Chorionic Villus Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Giovanni Monni, Rosa Maria Ibba and Maria Angelica Zoppi 6 Prenatal Diagnosis of Chromosomal Abnormalities through Amniocentesis . . . . . . . . . . . . . . . . . . . . . 194 Peter A. Benn 7 Prenatal Diagnosis of Sex Chromosome Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Jeff M. Milunsky 8 Molecular Cytogenetics and Prenatal Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Stuart Schwartz 9 Prenatal Diagnosis and the Spectrum of Involvement from Fragile X Mutations . . . . . . . . . . . . . . . . . . 349 Randi Hagerman, Vivien Narcisa and Paul Hagerman 10 Prenatal Diagnosis by Microarray Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Joris Robert Vermeesch 11 Molecular Genetics and Prenatal Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 John A. Phillips III 12 Prenatal Diagnosis of Disorders of Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445 Bryan G. Winchester 13 Prenatal Diagnosis of the Peroxisomal and Mitochondrial Fatty Acid Oxidation Deficiencies . . . . . . . 489 Ronald J.A. Wanders 14 Prenatal Diagnosis of the Mucopolysaccharidoses and Postnatal Enzyme Replacement Therapy . . . . . 495 John J. Hopwood 15 Disorders of the Metabolism of Amino Acids and Related Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . 514 Vivian E. Shih and Roseann Mandell 16 Prenatal Diagnosis of Disorders of Carbohydrate Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554 Yuan-Tsong Chen and Deeksha S. Bali 17 Prenatal Diagnosis of Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 Gerald L. Feldman and Kristin G. Monaghan

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Contents

18 Prenatal Diagnosis and Treatment of Congenital Adrenal Hyperplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . 600 Phyllis W. Speiser 19 Prenatal Diagnosis of Miscellaneous Biochemical Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614 David S. Rosenblatt and David Watkins 20 Prenatal Diagnosis of Primary Immunodeficiency Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628 Jennifer M. Puck 21 Prenatal Diagnosis of the Hemoglobinopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646 John M. Old 22 Prenatal Diagnosis of Disorders of Bone and Connective Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 680 Andrea Superti-Furga and Sheila Unger 23 Maternal Serum Screening for Neural Tube and Other Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705 Aubrey Milunsky and Jacob A. Canick 24 Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities . . . . . . . . . . . . . . . . . . . . . . 771 Howard S. Cuckle and Peter A. Benn 25 Prenatal Diagnosis of Fetal Malformations by Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819 Yves G. Ville and Durata Nowakowska 26 Prenatal Diagnosis and Management of Abnormal Fetal Development with Emphasis on the Third Trimester of Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 882 Juriy W. Wladimiroff and Titia E. Cohen-Overbeek 27 Prenatal Diagnosis by Fetal Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911 Nadine Girard and Kathia Chaumoitre 28 Induced Abortion for Genetic Indications: Techniques and Complications . . . . . . . . . . . . . . . . . . . . . . . 929 Lee P. Shulman 29 Preimplantation Genetic Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950 Yury Verlinsky* and Anver Kuliev 30 Prenatal Diagnosis through Analysis of Intact Fetal Cells and Cell-Free Nucleic Acids in the Maternal Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 978 Diana W. Bianchi and Y.M. Dennis Lo 31 Fetal Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1001 Diana L. Farmer, Hammin Lee, Elizabeth Gress, Aubrey Milunsky and Michael R. Harrison 32 Prenatal Diagnosis of Fetal Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020 Yves Ville and Guillaume Benoist 33 Medicolegal Aspects of Prenatal Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053 Ellen Wright Clayton and Mary Z. Pelias 34 Prenatal and Preimplantation Diagnosis: International Policy Perspectives . . . . . . . . . . . . . . . . . . . . . . 1081 Bartha Maria Knoppers and Thu Minh Nguyen 35 Ethical Issues in the Diagnosis and Management of Genetic Disorders in the Fetus . . . . . . . . . . . . . . 1097 Frank A. Chervenak and Laurence B. McCullough Appendix, Prenatal Diagnosis of Additional Miscellaneous Genetic Disorders. . . . . . . . . . . . . . . . . . . . . . . 1122 Aubrey Milunsky Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1136 Color plate appears facing page 948

* Deceased.

Preface In the search for accurate and reliable information, a discerning reader would welcome a source that reliably dispenses evidence-based facts embellished by knowledge, experience and wisdom. This precious distillate, tinctured with recommendations and guidance born of long experience, is not reachable by the most avid electronic voyeur. Sifting through mountains of unfiltered, irrelevant, unreliable or misleading information, electronic searches simply spawn reams of paper, mostly lacking critical analysis of the subject in question. At best, authors will describe “limitations” in their studies, while awaiting guidance from their clinical Colleges or Societies, which often takes years. Fortunately this volume, a major repository of facts about prenatal diagnosis, provides a critical analysis and synthesis of established and new knowledge based on the long experience of authorities in their respective fields. The guidance provided and the insights and perspectives of these authors make this volume a valuable and indispensable resource for all whose focus is securing fetal health through prenatal diagnosis. A broad international perspective is presented in this volume with authoritative contributions from authors in nine countries. All chapters have been revised and updated, new guidelines emphasized, and three new important chapters added. The first addition is the use of chromosomal microarrays in prenatal diagnosis. Clinical trials, now underway, will help determine the frequency of detecting a microdeletion/duplication of clinical significance which would have otherwise been missed by routine cytogenetics. At the same time, a clear measure should emerge of how often copy number variations of uncertain significance are determined and how often they are deemed “probably benign”. Compounding the normal anxiety expectant mothers experience during prenatal diagnosis with significant degrees of uncertainty will not only be unhelpful, but may cause harm if unnecessary pregnancy termination is pursued. Major reservation is expressed in this chapter

about application of this new technology, which is constantly being refined, to preimplantation genetic diagnosis (PGD). The second addition is the chapter that is focused on the social, legal and public policy issues with special reference to international approaches to prenatal diagnosis. The third addition expands previous coverage of the important peroxisomal and related fatty acid oxidation disorders. The fundamental pillars of this sixth (and earlier) edition(s) are represented by the other 32 chapters which are replete with the factual basis of prenatal diagnosis, synthesis, critical analysis and guidelines. In the opening chapter, the principles and practice that underscore preconception, prenatal and perinatal genetic counseling is presented in detail with emphasis on lessons learned since the inception of prenatal genetic diagnosis. An enormous factual base is provided in a chapter on amniotic fluid function and constituents including pesticides, carcinogens and other environmental contaminants, while the essentials of cell culture for prenatal diagnosis are provided in another. Chapters on amniocentesis and fetal blood sampling, and chorion villus sampling are anchored by authors with a lifetime of experience. Prenatal chromosome diagnosis, the bedrock upon which this subject was built, is again captured in an authoritative extensive chapter that is pertinent to all engaged in prenatal diagnosis, and enhanced by a separate chapter on molecular cytogenetics and the use of fluorescent in situ hybridization. Clear guidance is provided for the genetic counseling and management following the frequently incidental prenatal diagnosis of a sex chromosome disorder, while the Fragile X syndrome is extensively addressed by pioneers. Recent and continuing advances in molecular genetics now command a central role in prenatal diagnosis reflected by the many serious monogenic disorders amenable to early detection. Comprehensive, authoritative and important chapters on biochemical genetics encompass much that is known about the prenatal

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Preface

diagnosis of the lipid storage disorders, the mucopolysaccharidoses, the organic and amino acid and related disorders of metabolism, the disorders of carbohydrate metabolism, the peroxisomal and related fatty acid oxidation disorders and the disorders of folate and cobalamin metabolism. Revised and updated chapters on cystic fibrosis, congenital adrenal hyperplasia and the primary immunodeficiency disorders are followed by a masterly discourse on the hemoglobinopathies. New advances combining molecular genetics and fetal imaging provide valuable information in the re-written chapter on connective tissue disorders. An extremely thorough exposition of maternal serum screening for neural tube defects, Down syndrome and chromosome abnormalities, occupies two chapters reflecting current practice. Biochemical screening for neural tube defects heralded previously as one of the most important advances in prenatal diagnosis, may well be largely replaced soon by ultrasonography once the technical skills have been universally mastered. Sophisticated ultrasonic imaging for fetal structural and functional abnormalities are superbly covered in two updated chapters, while the growing importance of fetal magnetic resonance imaging in resolving difficult diagnostic quandaries is expertly covered in a re-written chapter. Any attempt at prenatal diagnosis must be preceded by counseling at which time the provider must be fully informed about the details if induced abortion will need to be considered. An updated expert revision of a chapter on the subject provides the necessary facts and guidance to be shared with a patient. Avoidance of abortion is facilitated by preimplantation genetic diagnosis (PGD) which is fully updated in a revised chapter detailing a remarkable array of achieved diagnoses. Screening for aneuploidy prior to implantation seems common sense, but uniform supportive data are lacking, some concluding that randomized controlled trials are needed, while others maintain that sufficient data exists to indicate an unfavorable practice. Clearly, more definitive research is required. Among the most anticipated and exciting developments is non-invasive prenatal diagnosis by analysis of fetal DNA and RNA in the maternal circulation. The technological innovations, now developing rapidly and including shot-gun

sequencing of fetal DNA, are described and assessed in a re-written expert chapter. Advances in fetal therapy, either directly or via the maternal circulation, have continued and require attention given opportunities to intervene, especially where surgical or medical treatment can save the fetus, as described in two re-written and revised chapters. Cogent issues of law, ethics and public policy as they apply to prenatal diagnosis are explored in depth by acknowledged experts in the three last chapters. Greater public awareness of genetics has alerted many to the opportunities of preventing adverse outcomes in pregnancy. One consequence has been escalating litigation by those deprived of the chance to avoid harm, which is also discussed in the first chapter. This reference text, with contributions uniquely first authored by senior professors and directors, is a veritable repository of information on prenatal genetic diagnosis, is very heavily referenced, full of guidance and reflective of the lifetime experience and wisdom of the authors. This addition encompasses 162 tables, 129 figures, including 14 color plates, and nearly 9000 references. An extensive table of additional disorders amenable to prenatal diagnosis is added as an appendix. A valuable index will enrich the reader’s search for specific information. Exciting progress marks the 45th year since the introduction of prenatal cytogenetic diagnosis by amniocentesis and cell culture. Major recent advances include significant progress in the development of chromosomal microarrays, gene discovery and fast next-generation gene sequencing, fetal imaging, non-invasive prenatal diagnosis and preimplantation genetic diagnosis. We hope that this edition will once again provide evidence-based guidance, insight and perspective, combined with an enormous factual base. Recognition of many new and unresolved challenges should provide inspiration for novel research initiatives. Mostly however we hope that the progress mirrored in this volume and the anticipated progress will help reassure many parents at risk that they can avoid either conceiving offspring with serious/lethal genetic disorders or having affected offspring that could have been detected prenatally. Aubrey Milunsky and Jeff M. Milunsky Boston

Acknowledgments

Only rarely does one encounter a major reference text in which every chapter is written by an acknowledged authority or internationally recognized expert. Such is the nature of this sixth edition in which outstanding physicians, scientists and academicians have again considered it worthy to have taken the time to share their expertise, experience, and wisdom. Readers in many disciplines in

which fetal and maternal health and welfare are paramount will be the beneficiaries of the information and guidance proffered. We are extremely grateful to all our authors for their superb contributions. We are also most grateful to my senior executive secretary, Mrs. Marilyn McPhail, who yet again effectively demonstrated the art of multi-tasking.

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List of Contributors

Deeksha S. Bali, PhD, FACMG Assistant Professor and Laboratory Director Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, NC, USA

Ellen Wright Clayton, MD, JD Rosalind E. Franklin Professor of Genetics and Health Policy, Director, Center for Biomedical Ethics and Society; Professor of Pediatrics; Professor of Law; Vanderbilt University, Nashville, TN, USA

Peter A. Benn, MSc, PhD, FACMG, DSc Professor, Departments of Genetics and Developmental Biology, Pediatrics, and Laboratory Medicine; and Director, Human Genetics Laboratories, University of Connecticut Health Center, Farmington, CT, USA

Titia E. Cohen-Overbeek, MD, PhD Senior Physician Prenatal Medicine, Erasmus University Medical Center, Department of Obstetrics and Gynecology, Rotterdam, the Netherlands

Guillaume Benoist,

MD Department of Obstetrics and Gynecology, Centre Hospitalier Intercommunal de Poissy-St. Germain en Laye, Poissy, France

Howard S. Cuckle,

Diana W. Bianchi,

MD Natalie V. Zucker Professor of Pediatrics, Obstetrics and Gynecology and Vice-Chair for Research and Academic Affairs, Department of Pediatrics, Tufts University School of Medicine; Tufts Medical Center, Boston, MA, USA

Sherman Elias, MD, FACOG, FACMG, FACS John J. Sciarra Professor and Chair, Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, and Prentice Women’s Hospital, Chicago, IL, USA

Jacob A. Canick, PhD, FACB Professor, Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School of Brown University; and Director, Division of Prenatal and Special Testing, Women and Infants Hospital, Providence, RI, USA

Diana L. Farmer, MD Professor of Surgery, Pediatrics, and Obstetrics, Gynecology and Reproductive Sciences; Chief, Division of Pediatric Surgery, and Vice Chair, Department of Surgery, University of California, San Francisco and Surgeon-in-Chief, UCSF Children’s Hospital, San Francisco, CA, USA

Yuan-Tsong Chen, MD, PhD Professor of Pediatrics and Genetics, Duke University Medical Center, Durham, NC, USA and Institute of Biomedical Sciences, Academia Sinica, Taiwan Frank A. Chervenak,

MD Given Foundation Professor and Chair, Department of Obstetrics and Gynecology and Gynecologist-in-Chief, New York-Presbyterian Hospital and Weill Medical College of Cornell University, New York, NY, USA

Kathia Chaumoitre,

MD, PhD Laboratorire d’anthropologie, Faculte de Medicine Marseille, Universite de la Mediterranee, France and Department of Radiology, Hopital Nord, Marseille, France

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MSc, DPhil Adjunct Professor, Obstetrics and Gynecology, Colombia University, New York, NY, USA and Emeritus Professor, Reproductive Epidemiology, University of Leeds, Leeds, UK

Gerald L. Feldman,

MD, PhD Professor, Center for Molecular Medicine and Genetics, and Departments of Pediatrics and Pathology, Wayne State University School of Medicine; Director, Clinical Genetics Services, and Director, Molecular Genetics Diagnostic Laboratory, Detroit Medical Center-University Laboratories, Detroit, MI, USA

Nadine Girard, MD, PhD Professor of Neuroradiology, Centre de Resonance Magnetique Biologique et Medicale,, Centre National de la Recherche Scientifique, Faculte de Medicine la Timone, Marseille, France, Universite de la Mediterranee, and Head of Neuroradiology, Timone Hospital, Marseille, France

List of Contributors

Elizabeth Gress, BA Contracts and Grants Analyst, Department of Surgery, University of California, San Francisco, CA, USA Paul J. Hagerman,

MD, PhD Professor, Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA, USA

Randi J. Hagerman,

MD Medical Director of MIND Institute and Endowed Chair in Fragile X Research, University of California Davis Health System, Sacramento, CA, USA

Michael R. Harrison, MD Professor Emeritus of Surgery, Pediatrics, and Obstetrics, Gynecology and Reproductive Sciences and Founding Director, UCSF Fetal Treatment Center, University of California, San Francisco, CA, USA John J. Hopwood,

PhD Professor and Head, Lysosomal Diseases Research Unit, Women’s and Children’s Hospital, North Adelaide, Australia

Rosa Maria Ibba,

MD Department of Obstetrics and Gynecology, Prenatal and Preimplantation Genetic Diagnosis, Fetal Therapy, Ospedale Regionale per le Microcitemie, Cagliari, Sardinia, Italy

Bartha M. Knoppers, PhD, OC Centre of Genomics and Policy, McGill University Montreal, Quebec, Canada Anver Kuliev, MD, PhD Director of Research, Reproductive Genetics Institute, Chicago, IL, USA Hammin Lee,

MD Associate Professor of Surgery, Pediatrics, and Obstetrics, Gynecology and Reproductive Sciences and Director, UCSF Fetal Treatment Center, University of California, San Francisco, CA, USA

Y.M. Dennis Lo,

MD Professor of Pathology and Li Ka Shing Professor of Clinical Medicine, The Chinese University of Hong Kong, Hong Kong, China

Roseann Mandell,

BA Neurology Department, Massachusetts General Hospital, Boston, MA, USA

Laurence B. McCullough, PhD Dalton Tomlin Chair in Medical Ethics and Health Policy, Center for Medical Ethics and Health Policy, Baylor College of Medicine, Houston, TX, USA

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Aubrey Milunsky, MB BCh, DSc, FRCP, FACMG, DCH Professor of Human Genetics, Pediatrics, Pathology, and Obstetrics & Gynecology, and Co-Director, Center for Human Genetics, Boston University School of Medicine and Boston Medical Center, Boston, MA, USA Jeff M. Milunsky, MD, FACMG Professor of Pediatrics, Genetics and Genomics; CoDirector, Center for Human Genetics, Director, Clinical Genetics, Boston University School of Medicine, and Director, Clinical Genetics, Boston Medical Center, Boston, MA, USA Kristin G. Monaghan, PhD Director, DNA Diagnostics Laboratory, Department of Medical Genetics, Henry Ford Health System; and Assistant Professor, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA Giovanni Monni,

MD Professor, Post-Graduate Pediatric School, University of Cagliari, and Obstetrician-in-Chief, Department of Obstetrics and Gynecology, Prenatal and Preimplantation Genetic Diagnosis, Fetal Therapy, Ospedale Regionale per le Microcitemie, Cagliari, Sardinia, Italy

Vivien Narcisa, BS MIND Institute, Fragile X Research and Treatment Center, School of Medicine, University of California, Davis, CA, USA Thu Minh Nguyen, LLB Research Associate, Centre of Genomics and Policy, McGill University, Montreal, Quebec, Canada Durata Nowakowska,

MD, PhD Research Institute Polish Mother’s Memorial Hospital Department of Fetal Maternal Medicine, Łodz´ , Poland

John M. Old, PhD, FRCPath Consultant Clinical Scientist and Reader in Haematology, National Haemoglobinopathy Reference Laboratory, Oxford Haemophilia Centre, Churchill Hospital, Oxford, UK Mary Z. Pelias,

PhD, JD Professor Emerita, Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA, USA

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List of Contributors

John A. Phillips III,

MD David T. Karzon Professor Department of Pediatrics, Vanderbilt University School of Medicine and the Monroe Carell Jr. Children’s Hospital at Vanderbilt, Professor of Biochemistry and Medicine, Harvie Branscomb Distinguished Professor, Director, Division of Medical Genetics and Genomic Medicine, Department of Pediatrics, Vanderbilt University School of Medicine and Adjunct Professor of Microbiology, Meharry Medical College, Nashville, TN, USA

Sheila Unger,

MD Institute for Human Genetics, and Center for Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg, Germany

Daniel L. van Dyke, PhD Professor of Laboratory Medicine and Pathology, Mayo Medical School and Mayo Clinic Cytogenetics Laboratory, Rochester, MN, USA

Jennifer M. Puck,

Yury Verlinsky, PhD (Deceased) Director, Reproductive Genetics Institute, Chicago, IL, USA

David S. Rosenblatt,

Joris Robert Vermeesch, PhD Professor of Molecular Cytogenetics and Genome Research, Head of Constitutional Cytogenetics, Coordinator of Genomics Core, Center for Human Genetics, Katholicke Universitett, Leuven, Belguim

MD Professor of Pediatrics, Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA MDCM Professor and Chair, Department of Human Genetics, and Professor, Departments of Medicine, Pediatrics, and Biology, McGill University, Montreal, Quebec, Canada

Stuart Schwartz,

PhD Strategic Director, Cytogenetics, Laboratory Corporation of America, North Carolina, NC, USA

Vivian E. Shih, MD Professor, Department of Neurology, Harvard Medical School and Unit Chief, Metabolic Disorders; Director, Massachusetts General Hospital Neurochemistry/Amino Acid Disorders Laboratory, Boston, MA, USA

Yves G. Ville,

MD Professor of Obstetrics and Gynecology, University of Paris, and Director, Department of Obstetrics and Gynecology, Centre Hospitalier Intercommunal de Poissy-St. Germain en Laye, Poissy, France

Ronald J.A. Wanders,

MD Laboratory of Genetic Metabolic Diseases, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands

David Watkins, PhD Research Associate, Department of Human Genetics, McGill University, Montreal, Quebec, Canada

Lee P. Shulman, MD The Anna Ross Lapham Professor in Obstetrics and Gynecology, Director, Division of Reproductive Genetics, Co-Director, Northwestern Ovarian Cancer Early Detection and Prevention Program, Feinberg School of Medicine of Northwestern University, Adjunct Professor of Medicinal Chemistry and Pharmacognosy University of Illinois at Chicago College of Pharmacy, Chicago, IL, USA

Bryan G. Winchester, MA, PhD Emeritus Professor of Biochemistry, University College, Institute of Child Health at Great Ormond Street Hospital, London, UK

Phyllis W. Speiser, MD Professor, Department of Pediatrics, New York University School of Medicine, New York; and Chief, Division of Pediatric Endocrinology, Schneider Children’s Hospital, North Shore–LIJ Health System, New Hyde Park, NY, USA

Juriy W. Wladimiroff, MD, PhD, FRCOG Emeritus Professor, Department of Obstetrics and Gynaecology, Erasmus University Medical Center, Rotterdam, the Netherlands

Andrea Superti-Furga, MD Professor and Chair, Department of Pediatrics, University of Freiburg and Director, Centre for Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg, Germany

Maria Angelica Zoppi,

MD Department of Obstetrics and Gynecology, Prenatal and Preimplantation Genetic Diagnosis, and Fetal Therapy, Ospedale Regionale per le Microcitemie, Cagliari, Sardinia, Italy

1

Genetic Counseling: Preconception, Prenatal and Perinatal Aubrey Milunsky and Jeff M. Milunsky Center for Human Genetics, Boston University School of Medicine, Boston, MA, USA

Advances in molecular genetics and fetal imaging have enriched our ability to secure early prenatal diagnosis of a rapidly enlarging spectrum of genetic and developmental disorders. Pari passu, a newly added layer of diagnostic uncertainty has dawned created by an extant lack of knowledge about polymorphisms and developmental structural and functional variations. Cognizance of “normal” has always been important and is especially critical in the evolution of fetal health. Analyses via chromosomal microarrays and whole-genome sequencing make mandatory the need to first delineate normal variation, if erroneous decision making is to be avoided. The widening scope of molecular diagnostics and fetal imaging has increased opportunities for predictive, preconception, preimplantation and prenatal diagnosis. Consequently, genetic counseling for prenatal diagnosis can be expected increasingly to involve newly recognized microdeletion and microduplication syndromes (see Chapter 10), early adult-onset malignancies, neurodegenerative, cardiovascular and other fatal genetic disorders, as well as those with significant morbidity. Against this background, physicians in all specialties are expected to be cognizant of new developments in genetics that facilitate the prevention

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

or avoidance of genetic or acquired defects. In context, women at risk for having progeny with defects expect to be informed about their odds and options, preferably during preconception counseling. Their concerns are serious, given the significant contribution of genetic disorders to morbidity and mortality in children and adults.

The incidence, prevalence and burden of genetic disorders and congenital malformations Various measures reflect the population burden of genetic disease and congenital anomalies. Common assessments include the incidence or prevalence of the disorder/defect, the associated morbidity and mortality, the degree of disability and suffering, life expectancy and economic burden. Indeed, many factors influence efforts to accurately determine the incidence or prevalence of congenital anomalies or genetic disorders. Box 1.1 encompasses the majority of known etiologic categories, discussed below, which help explain sometimes striking differences among major studies. It is almost impossible to account for all these potentially confounding factors in a study and rarely has any one study come close. Incidence and prevalence Estimates of aneuploidy in oocytes and sperm reach 18–19 percent and 3–4 percent, respectively.1 Not surprisingly, then, about one in 13 conceptions results in a chromosomally abnormal

1

2

Genetic Disorders and the Fetus

Box 1.1 Factors that influence estimates of the incidence or prevalence in the newborn of a congenital malformation (CM) or genetic disorder

Availability and use of expertise in prenatal diagnostic ultrasound Case selection, bias and ascertainment Consanguinity Definitions of major and minor congenital anomalies Economic level in developed or developing world Family history Frequency, inclusion and exclusion of stillbirths, fetal deaths and elective pregnancy termination Frequency of certain infectious diseases History of recurrent spontaneous abortion In vitro fertilization Incidence and severity of prematurity Intracytoplasmic sperm injection Later manifestation or onset of disorder Maternal age Maternal alcohol abuse Maternal diabetes and gestational diabetes Maternal diet Maternal epilepsy, lupus erythematosus and other illnesses

conceptus,2 while about 50 percent of first-trimester spontaneous abortions are associated with chromosomal anomalies.3 Clinically significant chromosomal defects occur in 0.65 percent of all births; an additional 0.2 percent of babies are born with balanced structural chromosome rearrangements (see Chapter 6) that have implications for reproduction later in life. Between 5.6 and 11.5 percent of stillbirths and neonatal deaths have chromosomal defects.4 Congenital malformations with obvious structural defects are found in about 2 percent of all births.5 This was the figure in Spain among 710,815 livebirths,6 with 2.25 percent in Liberia,7 2.03 percent in India,8 and 2.53 percent among newborn males in Norway.8a The Mainz Birth Defects Registry in Germany in the 1990–1998 period reported a 6.9 percent frequency of major malformations among 30,940 livebirths, stillbirths and abortions.9 Factors that had an impact on the incidence/prevalence of congenital malformations are discussed below.

Maternal fever or use of hot tub in the first 6 weeks of pregnancy Maternal grandmother’s age Maternal obesity Maternal use of medication Multiple pregnancy rate Paternal age Previous affected child Previous maternal immunization/vaccination Season of the year Training and expertise in examination of newborns Use of chromosomal microarray Use of death certificates Use of folic acid supplementation Use of maternal serum screening for Down syndrome Use of maternal serum screening for neural tube defects Use of prenatal necropsy Use of registry data

More than 12,000 monogenic disorders and traits have been catalogued.10 Estimates based on 1 million consecutive livebirths in Canada suggested a monogenic disease in 3.6 in 1,000, consisting of autosomal dominant (1.4 in 1,000), autosomal recessive (1.7 in 1,000) and X-linked-recessive disorders (0.5 in 1,000).11 Polygenic disorders occurred at a rate of 46.4 in 1,000 (Table 1.1). At least 3–4 percent of all births are associated with a major congenital defect, mental retardation or a genetic disorder, a rate that doubles by 7–8 years of age, given later-appearing and/or laterdiagnosed genetic disorders.12,13 If all congenital defects are considered, Baird et al.11 estimated that 7.9 percent of liveborn individuals have some type of genetic disorder by about 25 years of age. These estimates are likely to be very low given, for example, the frequency of undetected defects such as bicuspid aortic valves that occur in 1–2 percent of the population.14 The bicuspid aortic valve is the most common congenital cardiac malformation

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Genetic Counseling: Preconception, Prenatal and Perinatal

Table 1.1 The frequencies of genetic disorders in 1,169,873 births, 1952–198311 Category

Rate per million

Percentage of

livebirths

total births

A Dominant

1,395.4

0.14

Recessive

1,665.3

0.17

532.4

0.05

Chromosomal

1,845.4

0.18

Multifactorial

46,582.6

4.64

1,164.2

0.12

Total

53,175.3

5.32a

All congenital

52,808.2

5.28

26,584.2

2.66

79,399.3

7.94

X-linked

Genetic unknown

B anomalies 740–759b Congenital anomalies with genetic etiology (included in section A) C Disorders in section A plus those congenital anomalies not already included a

Sum is not exact owing to rounding.

b

International Classification of Disease numbers.

and in the final analysis may cause higher mortality and morbidity rates than all other congenital cardiac defects.15 A metropolitan Atlanta study (1998–2005) showed an overall prevalence of 81.4 per 10,000 for congenital heart disease among 398,140 livebirths.16 These numbers lead to a significant genetic disease burden and have accounted for 28–40 percent of hospital admissions in North America, Canada and England.17,18 Notwithstanding their frequency, the causes of over 60 percent of congenital malformations remain obscure.19,20 The availability of prenatal diagnosis and maternal serum screening for neural tube defects (NTDs) and Down syndrome (DS) has also affected the birth frequency of these two most common congenital defects. One French study of the impact of

3

prenatal diagnosis over a 21-year period (1979– 1999) in a well-defined population showed a drop of 80 percent in the birth prevalence of DS.21 A later report from the Paris Registry of Congenital Anomalies (2001–2005) noted a “fairly stable prevalence of DS (7.1 per 10,000 livebirths) over time.”22 A study from Newcastle, England, based on ascertainment of all cases of NTDs revealed a twofold reduction in the birth prevalence between 1984–1990 and 1991–1996.23 A Scottish study aimed at assessing the impact of prenatal diagnosis on the prevalence of DS from 1980 to 1996. Both births and pregnancy terminations were included. Pregnancy terminations for DS rose from 29 percent to about 60 percent.24 In contrast, the prevalence of DS noted by the Dutch Paediatric Surveillance Unit in 2003 was 16 per 10,000 livebirths, exceeding earlier reports and thought to reflect an older maternal age cohort.25 In the US, a DS prevalence rate of 13 per 10,000 was found in metropolitan Atlanta (1979–2003).26 The effect of folic acid supplementation, via tablet or food fortification, on the prevalence of NTDs, now well known to reduce the frequency of NTDs by up to 70 percent,27,28 (see Chapter 23) has only recently been assessed in this context. A Canadian study focused on the effect of supplementation on the prevalence of open NTDs among 336,963 women. The authors reported that the prevalence of open NTDs declined from 1.13 in 1,000 pregnancies before fortification to 0.58 in 1,000 pregnancies thereafter (see Chapter 23).29 In a population-based cohort study by the Metropolitan Atlanta Congenital Defects Program, the risk of congenital malformations was assessed among 264,392 infants with known gestational ages born between 1989 and 1995. Premature infants (20-fold excess

Obstructive sleep apnea

Frequency greater than

Epilepsy

13.6

Testicular cancer

Standardized incidence

Alzheimer disease and

>50

in general population

ratio 4.8 dementia a

Includes cataracts, strabismus, nystagmus, refractive

errors, keratoconus, glaucoma, and lens opacities. Data from references 56, 57, 57a–d, 58.

natal genetic studies are used in Western society virtually exclusively for the detection of defects generally characterized by irreparable mental retardation and/or irremediable, serious to fatal genetic disease. Sadly, at present, the ideal goal of prevention or treatment rather than abortion after prenatal detection of a fetal defect is achieved only rarely, with the exception of NTDs (see Chapter 23). Preimplantation genetic diagnosis (see Chapter 29) does, however, provide another option that avoids abortion. All couples or individuals concerned about the risks of genetic defects in their offspring should seek genetic counseling before conceiving. Such counseling is best provided in medical genetics departments of university medical centers with

9

multiple-specialty clinical, counseling and laboratory teams. For the more common indications for prenatal diagnosis (such as advanced maternal age), the well-informed obstetrician should be able to provide the necessary information. However, a salutary observation in one study revealed that 43.3 percent of patients referred for amniocentesis exclusively for advanced maternal age had additional genetic risks or significant concerns regarding one or more genetic or congenital disorders.64 Neither a questionnaire in the physician’s office nor limited consultation time is likely to reveal many of these disorders. This group required more extensive genetic counseling. In a Hungarian study 98 percent and 92 percent of counsellees expected detailed information and the possibility of control over decision making, respectively.65

Prerequisites for genetic counseling Genetic counseling is a communication process concerning the occurrence and the risk of recurrence of genetic disorders within a family. The aim of such counseling is to provide the counselee(s) with as complete an understanding of the disorder and/or problem as possible and of all the options and implications. The counseling process is also aimed at helping families cope with their problems and at assisting and supporting them in their decision making. The personal right to found a family is considered inviolable. Such reproductive autonomy is enhanced by genetic counseling, a process that both emphasizes freedom of choice and reviews the available options in order to enrich the decisionmaking process. All couples have a right to know whether they have an increased risk of having children with genetic disease and to know which options pertain to their particular situation. The physician and genetic counselor has a clear duty and obligation to communicate this information, to offer specific tests or to refer couples for a second or more expert opinion. In the United States, at least, the full force of law supports the prospective parents’ right to know (see Chapter 33). As Kessler66 stated so succinctly, “Because genetic counselors work with people filled with uncertainty, fear of the future, anguish and a sense

10

Genetic Disorders and the Fetus

of personal failure,” they have unusual challenges and opportunities “to understand clients, give them a sense of being understood and help them feel more hopeful, more valued and more capable of dealing with their life problems.” The physician and genetic counselor providing genetic counseling should have a clear perception of the necessary prerequisites, guiding principles and potential problems. Knowledge of disease The need for a counselor to have extensive factual knowledge about disease in general, as well as about the disease for which counseling is being provided, hardly needs emphasis. Such knowledge should include how the diagnosis is made and confirmed, the test accuracy and limitations, the important co-morbidities, the recurrence risks, the mode of inheritance, the tests available to detect a carrier (and their detection rates), the heterogeneity and pleiotropic nature of the disease, the quality of life associated with survival, prognosis and the causes of death. When relevant, it is necessary to know about treatment and its efficacy. The physician or genetic counselor who initiates genetic counseling for an apparently straightforward indication (e.g. advanced maternal age) may find one or more other familial conditions with which he or she has little or no familiarity. Such circumstances dictate referral for specialist consultation. A National Confidential Enquiry into counseling for genetic disorders by nongeneticists in the United Kingdom revealed that less than half of those with known high genetic risks were referred to medical geneticists.67 This study focused on a review of 12,093 “genetic events” involving potentially avoidable cases of DS, NTDs, cystic fibrosis, β-thalassemia and multiple endocrine neoplasia. Medical record reviews were frustrated by the poor quality of clinical notes, which lacked evidence of counseling. An urgent call was made for genetic management to be at least as well documented as surgical operations, drug records and informed consent. A Dutch study evaluated the levels of knowledge, practical skills and clinical genetic practices of 643 cardiologists. They noted low levels of self-reported knowledge and that only 38 percent had referred patients to clinical geneticists.68 Other physicians too have been found

lacking in the necessary knowledge and communication skills.69–72 After the prenatal diagnosis of a serious genetic disorder, the physician should be able to inform the family fully about the anticipated burden and to detail the effects of this burden on an affected child, the family, other siblings, the family economics and marital relations, along with any other pros and cons of continuing pregnancy. The reality of early Alzheimer disease in DS and the care requirements that may devolve on the siblings should not be omitted from the discussion. Exact details should also be known about the risks of elective abortion (see Chapter 28). Expertise in genetic counseling Genetic counseling is best provided by board-certified clinical geneticists and genetic counselors. In countries with this specialization, such service is provided by a team composed of clinical geneticists (physicians) and genetic counselors, working in concert with clinical cytogeneticists, biochemical and molecular geneticists. It is, however, impractical and not cost effective to provide such formal counseling for every woman before prenatal diagnosis for advanced maternal age. It is necessary for the obstetrician to be fully informed about the indications for amniocentesis and to explain the techniques and requirements for obtaining the fluid, the limitations of the studies, the risks of chromosomal abnormality in the offspring of the patient being counseled, the risks of the procedure and, when pertinent, all matters concerned with elective abortion of an abnormal fetus. Gordis et al.73 concluded that the way in which an obstetrician managed patients at risk regarding referral for genetic screening was closely related to that obstetrician’s attitudes and education. Physicians in practice should be aware of the nuances and needs in the genetic counseling process, including the key psychologic aspects.74 Perhaps most important is the requirement that they recognize limitations in their knowledge of uncommon or rare genetic disorders and be alert to situations requiring referral. Obstetricians or family practitioners are not expected to have an extensive knowledge of all diseases but they should be able to recognize that a condition could be genetic. Concern about litigation should not act as

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a constant reminder to physicians of the need to consult or refer75–77 (see Chapter 33). Ability to communicate Many physicians are not born communicators and most have not had formal teaching and training to hone their communication skills. Recognizing these deficiencies, the American Academy of Pediatrics has provided valuable guidance and made specific recommendations for the development and teaching of communication skills,78 as have others.79a,79b Simple language, an adequate allocation of time, and care and sensitivity are keys to successful genetic counseling. Technical jargon, used with distressing frequency,80 is avoided only through conscious effort. How an issue requiring a decision is framed,81 and the nature of the language used,82 may influence the patient’s choice.83 Counseling is facilitated when three key questions are asked: “Why did you come?” “What exactly do you hope to learn?” and “Have I answered all your questions and concerns?” Although the explanation of exact statistical risks is important, patients often pay more attention to the actual burden or severity of the disease in question. How risks are explained and expressed is a skill to be mastered. Key to the exposition is the patients’ educational level, cultural background and the requirement of an interpreter (who may even bedevil a superb counselor). The use of numeric probabililties, relative risk, risk reduction or simple numbers of chance (1 in 100) or words (almost never, sometimes, more often than not)84 are choices a counselor must make. Clearly, the simpler, the better and the more likely the information is understood. Patients’ perceptions of risk not infrequently differ markedly from those of the counselor, a realization that should elicit no comment. An essential ingredient of the counseling process is time. The busy practitioner can hardly expect to offer genetic counseling during a brief consultation. Distress and misunderstanding are invariable sequelae of such hastily delivered counseling. Knowledge of ancillary needs For the couple at high risk of having a child with a serious genetic disorder, prenatal diagnosis is not

11

the sole option. Even in situations in which a particular disease is diagnosable prenatally, it is important to be certain that other avenues are explored. Prospective parents who are known, for example, to be carriers of an autosomal recessive disorder may be unaware of the possibility of sperm or ovum donation or may be unwilling to raise the question. This option may be viewed more favorably than prenatal diagnosis and elective abortion. Physicians should be certain that their patients are familiar with all the aforementioned important options, as well as with adoption, vasectomy, tubal ligation, treatments of the mother and/or fetus during pregnancy and other methods of assisted reproduction (e.g. intracytoplasmic sperm injection,85 epididymal sperm aspiration,86 and preimplantation genetic diagnosis) (see Chapters 7 and 29). Empathy Empathy embodies the ability to not only understand the perspectives and emotions of others but to communicate that understanding.87 Much more than the communication of risk figures for a particular disorder is required in the genetic counseling process. Warmth, care, sympathy, understanding and insight into the human condition are necessary for effective communication. The difficulty of assimilating information and making rational decisions in the face of anxiety88 should be recognized and vocalized. Empathy and sensitivity enable the counselor to anticipate and respond to unspoken fears and questions and are qualities that make the counseling experience most beneficial and valuable to the counselees. For example, a couple may have been trying to conceive for 10 years and, having finally succeeded, may be confronted by a callous physician who is impatient about their concerns regarding amniocentesis and elective abortion. Another couple may have lost their only child to a metabolic genetic disease and may be seeking counseling to explore the possibilities for prenatal diagnosis in a subsequent pregnancy or even treatment following prenatal diagnosis, as in the case of galactosemia. They may have in mind past problems encountered in prenatal diagnosis or may be aware of the uncertain outcome of treatment. Sensitivity and awareness of the plight of prospective parents are critical prerequisites and

12

Genetic Disorders and the Fetus

include the need to recognize and address the usually unspoken fears and anxieties. They may have had a previous affected child with physical/ mental deficits and experienced stigmatizing encounters, including intrusive inquiries, staring and pointing, devaluing remarks and social withdrawal.89 Beyond the qualifications and factual knowledge of the counselor is the person, who is key to successful and effective counseling. Attitude, body language, warmth, manners, dress, tone of voice and personality are facets that seriously influence the credibility and acceptance of the counseling offered. Curiously, counselors rarely realize during their counseling session that they are simultaneously being assessed. Patients assess the apparent knowledge and credibility of the counselor, seek and are encouraged by evidence of experience and consider the information provided in light of the counselor’s attitude, body language and other nonverbal characteristics. Quintessential prerequisites for the empathetic genetic counselor include the following. 1. Acknowledge the burden and empathize about the sadness or loss (e.g. a previous child; recurrent miscarriage; a deceased affected parent; a patient who has experienced mastectomy and chemotherapy for breast cancer with daughters at risk). 2. Vocalize the realization of the psychologic pain and distress the person or couple has experienced (e.g. recurrent pregnancy loss followed by multiple IVF efforts and subsequently a successful pregnancy with a fetal defect). 3. Compliment the coping that has been necessary, including the stress a couple might have to endure, despite sometimes conflicting feelings. 4. Recognize (and explain) psychologic difficulties in decision making when faced with a prenatal diagnosis of the same disorder affecting one parent (discussion of self-extinction, self-image and issues of guilt and survival). 5. Fulfill the patient’s need for hope and support and actively avoid any thoughtless comments66 that may erode these fundamental prerequisites. Wellintentioned statements are not infrequently perceived in a very different way.78 It is self-evident that empathy would engender greater patient satisfaction and may well be correlated with clinical competence.90

Sensitivity to parental guilt Feelings of guilt invariably invade the genetic consultation; they should be anticipated, recognized and dealt with directly. Assurance frequently does not suffice; witness the implacable guilt of the obligate maternal carrier of a serious X-linked disease.90a Explanations that we all carry harmful genes often helps. Mostly, however, encouragement to move anguish into action is important. This might also help in assuaging any blame by the husband in such cases.91 Guilt is not only the preserve of the obligate carrier. Affected parents inevitably also experience guilt on transmitting their defective genes.92,93 Frequently, a parent expresses guilt about an occupation, medication or illegal drug that they feel has caused or contributed to their child’s problem. Kessler et al.93 advised that assuaging a parent’s guilt may diminish their power of effective prevention, in that guilt may serve as a defense from being powerless. Guilt is often felt by healthy siblings of an affected child, who feel relatively neglected by their parents and who also feel anger toward their parents and affected sibling. What is termed “survivor guilt” is increasingly recognized, as the new DNA technologies are exploited. Experience with Huntington disease and adult polycystic kidney disease94–100 confirm not only survivor guilt with a new reality (a future) but also problems in relationships with close family members. Huggins et al.97 found that about 10 percent of individuals receiving low-risk results experienced psychologic difficulties.

Principles in genetic counseling Eleven key principles are discussed that guide genetic counseling in the preconception, prenatal and perinatal periods. This section is in concert with consensus statements concerning ethical principles for genetics professionals101,102 and surveyed international guidelines.103 Accurate diagnosis Clinical geneticists, obstetricians or pediatricians are frequently confronted by patients seeking guidance because of certain genetic diseases in their families. A previous child or a deceased sibling or parent may have had the disease in question. The

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Genetic Counseling: Preconception, Prenatal and Perinatal

genetic counseling process cannot begin, however, without an accurate diagnosis. Information about the exact previous diagnosis is important not only for the communication of subsequent risks but also for precise future prenatal diagnosis. Hence, it is not sufficient to know that the previous child had a mucopolysaccharidosis; exactly which type and even subtype must be determined because each may have different enzymatic deficiencies (see Chapter 14). A history of limb girdle muscular dystrophy will also not facilitate prenatal diagnosis because there are two dominant types (1A and 1B) and at least six autosomal recessive types (2A– 2F).104 Similarly, a history of epilepsy gives no clear indication of which of over 45 genes and susceptibility loci are involved.105–107 Birth of a previous child with craniosynostosis requires precise determination of the cause (where possible) before risk counseling is provided. Mutations in seven genes (FGFR1, FGFR2, FGFR3, TWIST1, EFNB1, MSX2, RAB23) are clearly associated with monogenic syndromic forms of craniosynostosis.108 Moreover, a chromosomal abnormality may be the cause. Awareness of genetic heterogeneity and of intraand interfamily phenotypic variation of a specific disorder (e.g. tuberous sclerosis)109 is also necessary. The assumption of a particular predominant genotype as an explanation for a familial disorder is unwarranted. The common adult-dominant polycystic kidney disease due to mutations in the ADPKD1 gene has an early infancy presentation in 2–5 percent of cases.110 However, mutations in the ADPKD2 gene may result in polycystic kidney disease and perinatal death111 and, further, should not be confused with the autosomal recessive type due to mutations in the ARPKD gene. Instead of simply accepting the patient’s description of the disease – for example, muscular dystrophy or a mucopolysaccharidosis – the counselor must obtain confirmatory data. The unreliability of the maternal history, in this context, is remarkable, a positive predictive value of 47 percent having been documented.112 Photographs of the deceased, autopsy reports, hospital records, results of carrier detection or other tests performed elsewhere and other information may provide the crucial confirmation or negation of the diagnosis made previously. Important data after miscarriage may also influence counseling. In a study of 91

13

consecutive, spontaneously aborted fetuses, almost one-third had malformations, most associated with increased risks in subsequent pregnancies.113 Myotonic muscular dystrophy type 1 (DM), the most common adult muscular dystrophy, with an incidence of about 1 in 8,000,114 serves as the paradigm for preconception, prenatal and perinatal genetic counseling. Recognition of the pleiomorphism of this disorder will, for example, alert the physician hearing a family history of one individual with DM, another with sudden death (cardiac conduction defect) and yet another relative with cataracts. Awareness of the autosomal dominant nature of this disorder and its genetic basis due to a dynamic mutation reflected in the number of trinucleotide (CTG) repeat units raises issues beyond the 50 percent risk of recurrence in the offspring of an affected parent. As the first disorder characterized with expanding trinucleotide repeats, the observation linking the degree of disease severity to the number of triplet repeats was not long in coming.114 In addition, the differences in severity when the mutation was passed via a maternal rather than a paternal gene focused attention on the fact that congenital DM was almost always a sign of the greatest severity and originating through maternal transmission. However, at least one exception has been noted.115 There is about a 93–94 percent likelihood that the CTG repeat will expand on transmission. This process of genetic anticipation (increasing clinical severity over generations) is not inevitable. An estimated 6–7 percent of cases of DM are associated with a decrease in the number of triplet repeats or no change in number.116 Rare cases also exist in which complete reversal of the mutation occurs with spontaneous correction to a normal range of triplet repeats.117–120 There are also reports of patients born with a decreased number of triplet repeats who nevertheless show no decrease in the severity of their DM.121–123 It is unclear whether these cases in part reflect somatic or germline (either or both combined) mosaicism.116 Somatic mosaicism is certainly well documented in DM with, for example, larger expansions being observed in skeletal muscle than in peripheral blood.124 Another problem that complicates molecular diagnosis is that 1200

Huntington disease

4p16.3

CAG

6–36

–

35–121

Kennedy disease (spinal bulbar

Xq11-12

CAG

12–34

–

40–62

Machado–Joseph disease

14q32.1

CAG

13–36

–

68–79

Myotonic dystrophy type 1

19q13.3

CTG

5–37

–

50 to >2000

Myotonic dystrophy type 2c

3q21.3

CCTG

90

Spinocerebellar ataxia type 10d

22q13-qter

ATTCT

10–22

–

>19,000

Spinocerebellar ataxia type 12

5q31-33

CAG

7–28

–

66–78

Spinocerebellar ataxia type 17

6q27

CAG

27–44

–

>45

muscular atrophy)

a

Variable ranges reported and overlapping sizes may occur.

b

Mutation may not involve an expansion.

c

Expansion involves four nucleotides.

d

Expansion involves five nucleotides.

Box 1.3 Selected genetic disorders with anticipation

Disorders with anticipation See Table 1.6 of disorders with trinucleotide repeats (exception: Friedreich ataxia) Disorders with suspected anticipation Adult-onset idiopathic dystonia Autosomal dominant acute myelogenous leukemia Autosomal dominant familial spastic paraplegia Autosomal dominant polycystic kidney disease (PKD1) Autosomal dominant rolandic epilepsy Behçet syndrome Bipolar affective disorder Charcot–Marie–Tooth disease Crohn disease Dyskeratosis congenita Facioscapulohumeral muscular dystrophy Familial adenomatous polyposis Familial amyloid polyneuropathy Familial breast cancer

Familial chronic myeloproliferative disorders Familial intracranial aneurysms Familial pancreatic cancer Familial paraganglioma Familial Parkinson disease Familial primary pulmonary hypertension Familial rheumatoid arthritis Graves disease Hereditary nonpolyposis colorectal cancer Hodgkin and non-Hodgkin lymphoma Holt–Oram syndrome Lattice corneal dystrophy type I (LCDI) Li–Fraumeni syndrome Ménière disease Obsessive-compulsive spectrum disorders Oculodentodigital syndrome Paroxysmal kinesigenic dyskinesia (PKD) Restless legs syndrome Schizophrenia Total anomalous pulmonary venous return Unipolar affective disorder

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37

Table 1.7 Examples of imprinting and human disease Syndrome

Chromosomal location

Parental origin

Selected references

Angelman syndrome

15q11-q13

Maternal

383

Autism

15q11-q13

Maternal

384 385–387

Beckwith–Wiedemann syndrome

11p15.5

Paternal

Birk Barel mental retardation syndrome

8q24

Maternal

388

Congenital hyperinsulinism

11p15

Maternal

389

Congenital myotonic muscular dystrophy

19q13.3

Maternal

390

Early embryonic failure

21

Maternal

391

Familial paraganglioma

11q23

Paternal

392

Hereditary myoclonus–dystonia

7q21

Maternal

393

Intrauterine and postnatal growth restriction

7

Maternal

394

Intrauterine growth restriction or miscarriage

16

Maternal

395

Mental retardation and dysmorphism

14

Paternal

396

Prader–Willi syndrome

15q11-q13

Paternal

397

Progressive osseous heteroplasia

20q13.3

Paternal

399

Pseudohypoparathyroidism

20q13.3

Paternal

398

Rett syndrome

Xq28

Paternal

400, 401

Russell–Silver syndrome

7p11.2

Maternal

402

11p15

Maternal

402a

Short stature

14

Maternal

403

Transient neonatal diabetes

6q22-q23

Paternal

404–406

abnormalities. They hypothesized that somatic mosaicism with different amplification rates in various tissues may be one possible explanation for the variable phenotypes. This phenomenon of parent-of-origin difference in the expression of specific genes introduces genomic imprinting into the genetic counseling considerations. Some genes are genetically marked before fertilization so that they are transcriptionally silent at one of the parental loci in the offspring.381 A number of disorders have been recognized in which genomic imprinting is especially important382 (Table 1.7). In addition, parentof-origin affects anticipation in triplet repeat expansions such as in Huntington disease. Paternal transmission of the gene is associated with earlier and more severe manifestations than would be the case after maternal transmission. Families at risk may not realize that Huntington disease may manifest in childhood, not only in the teens but as early as 18 months of age.364,407 Genotype–phenotype associations DNA mutation analysis has clarified few genotype– phenotype associations but extensive databases will

help.408 Notwithstanding this limitation, mutation analysis does provide precise prenatal diagnosis opportunities and detection of affected fetuses with compound heterozygosity. Simple logic might have concluded that genotype at a single locus might predict phenotype. For monogenic disorders, this is frequently not the case. In the autosomal dominant Marfan syndrome (due to mutations in the chromosome 15 fibrillin gene), family members with the same mutation may have severe ocular, cardiovascular and skeletal abnormalities, while siblings or other close affected relatives with the same mutation may have mild effects in only one of these systems.409 In Gaucher disease with one of the common Ashkenazi Jewish mutations, only about one-third of homozygotes have significant clinical disease.410 At least two-thirds have mild or late-onset disease or remain asymptomatic. Compound heterozygotes for this disorder involving mutations L444P and N370S have included a patient with mild disease first diagnosed at 73 years of age, while another requiring enzyme replacement therapy was diagnosed at the age of 4 years.411 In CF, a strong correlation exists between genotype and pancreatic function but only a weak

38

Genetic Disorders and the Fetus

Table 1.8 Selected monogenic disorders with established germline mosaicism Disorder

Inheritance

Achondrogenesis type II

AD

Achondroplasia

AD

Adrenoleukodystrophy

X-L rec

Albright hereditary osteodystrophy

AD

α-Thalassemia mental retardation syndrome

X-L

Amyloid polyneuropathy

AD

Aniridia

AD

Apert syndrome

AD

Becker muscular dystrophy

X-L rec

Cantu syndrome

AD

Central hypoventilation syndrome

AD

Cerebellar ataxia with progressive macular dystrophy (SCA7)

AD

Charcot–Marie–Tooth disease type 1B

AD

Coffin–Lowry syndrome

X-L dom

Congenital contractural arachnodactyly

AD

Conradi–Hunnermann–Happle syndrome

X-L dom

Cowden disease

AD

Danon disease (lysosome-associated membrane protein-2 deficiency)

X-L rec

Dejerine–Sotas syndrome (HNSN III) with stomatocytosis

AD

Duchenne muscular dystrophy

X-L rec

Dyskeratosis congenita

X-L

EEC syndrome (ectrodactyly, ectodermal dysplasia, orofacial clefts)

AD

Epidermolysis bullosa simplex

AR

Fabry disease

AR

Facioscapulohumeral muscular dystrophy

AD

Factor X deficiency

AR

Familial focal segmental glomerulosclerosis

AD

Familial hypertrophic cardiomyopathy

AD

Fibrodysplasia ossificans progressiva

AD

Fragile X syndrome (deletion type)

X-L

Hemophilia B

X-L rec

Herlitz junctional epidermolysis bullosa

A rec

Holt–Oram syndrome

AD

Hunter syndrome

X-L rec

Incontinentia pigmenti

X-L dom

Karsch–Neugebauer syndrome

AD

Lesch–Nyhan syndrome

X-L rec

Lissencephaly (males); “subcortical band heterotopia” (almost all females)

X-L rec

Multiple endocrine neoplasia I

AD

Myotubular myopathy

X-L rec

Neurofibromatosis type 1

AD

Neurofibromatosis type 2

AD

Oculocerebrorenal syndrome of Lowe

X-L

Ornithine transcarbamylase deficiency

X-L rec

Osteocraniostenosis

AD

Osteogenesis imperfecta

AD

Otopalatodigital syndrome

X-L dom

Pseudoachondroplasia

AD

Severe combined immunodeficiency disease

X-L rec

Spondyloepimetaphyseal dysplasia

AD

CHAP T E R 1

Genetic Counseling: Preconception, Prenatal and Perinatal

39

Table 1.8 Continued Disorder

Inheritance

Renal-coloboma syndrome

AD

Retinoblastoma

AD

Rett syndrome

X-L dom

Tuberous sclerosis

AD

von Hippel–Lindau disease

AD

von Willebrand disease (type 2b)

X-L rec

Waardenburg syndrome

AD

Wiskott–Aldrich syndrome

X-L rec

AD, autosomal dominant; AR, autosomal recessive; X-L rec, X-linked recessive; X-L dom, X-linked dominant.

association has been noted with the respiratory phenotype412 (see Chapter 17). Although individuals who are homozygous for the common CF mutation (ΔF508) can be anticipated to have classic CF, those with the less common mutation (R117H) are likely to have milder disease.413 On occasion, an individual who is homozygous for the “severe” ΔF508 mutation might unexpectedly exhibit a mild pancreatic-sufficient phenotype. Illustrating the complexity of genotype–phenotype associations is the instance noted by Dork et al.414 of a mildly affected ΔF508 homozygote whose one chromosome 7 carried both the common ΔF508 mutations and a cryptic R553Q mutation. Apparently, a second mutation in the same region may modify the effect of the common mutation, permitting some function of the chloride channel415 and thereby ameliorating the severity of the disease. The extensive mutational heterogeneity in hemophilia A416 is related not only to variable clinical severity but also to the increased likelihood of anti-factor VIII antibodies (inhibitors) developing. Miller et al.417 found about a fivefold higher risk of inhibitors developing in hemophiliac males with gene deletions compared with those without deletions. Given the history of a previously affected offspring with a genetic disorder, the preconception visit serves as an ideal time to refocus on any putative diagnosis (or lack thereof) and to do newly available mutation analyses when applicable. Mosaicism Mosaicism is a common phenomenon (witness the normal process of X-inactivation and tissue dif-

ferentiation) that results in functional mosaicism in females. Mosaicism might occur in somatic or germline cells. Its recognition is important, because a disorder may not be due to a new dominant mutation, despite healthy parents. Erroneous counseling could follow, with the provision of risks very much lower than would be the case if germline mosaicism existed. After the birth to healthy parents of a child with achondroplastic dwarfism, random risks of one in 10,000 might be given for recurrence. However, germline mosaicism has been described after the birth of a second affected child.418 Similarly, the birth of a male with Duchenne muscular dystrophy (DMD), no family history and no detectable mutation on DNA analysis of maternal peripheral leukocytes might lead to counseling based on spontaneous mutation rates. Once again, germline mosaicism is now well recognized in mothers of apparently sporadic sons with DMD and the risk of recurrence in such cases approximates 7–14 percent if the at-risk X-haplotype is determined.419 Germline mosaicism has also been documented for other disorders (Table 1.8) and undoubtedly occurs in some others yet to be discovered. Somatic cell mosaicism with mutations has been recognized in a number of distinctly different disorders, such as hypomelanosis of Ito, other syndromes with patchy pigmentary abnormalities of skin associated with mental retardation and in some patients with asymmetric growth restriction.420,421 Germline mosaicism should be distinguished from somatic cell mosaicism in which there is also gonadal involvement. In such cases, the

40

Genetic Disorders and the Fetus

patient with somatic cell mosaicism is likely to have some signs, although possibly subtle, of the disorder in question, while those with germline mosaicism are not expected to show any signs of the disorder. Examples of somatic and gonadal mosaicism include autosomal dominant osteogenesis imperfecta,422,423 Huntington disease424 and spinocerebellar ataxia type 2.425 Lessons from these and the other examples quoted for germline mosaicism indicate a special need for caution in genetic counseling for disorders that appear to be sporadic. Very careful examination of both parents for subtle indicators of the disorder in question is necessary, particularly in autosomal dominant and sex-linked recessive conditions. The autosomal dominant disorders are associated with 50 percent risks of recurrence, while the sex-linked disorders have 50 percent risk for males and 25 percent risk for recurrence in families. Pure germline mosaicism would likely yield risks considerably lower than these figures, such as 7–14 percent for females with gonadal mosaicism and X-linked DMD. A second caution relating to counseling such patients with an apparent sporadic disorder is the offer of prenatal diagnosis (possibly limited) despite the inability to demonstrate the affected status of the parent. Chromosomal mosaicism is discussed in Chapter 6 but note can be taken here of a possibly rare (and mostly undetected) autosomal trisomy. A history of subfertility with mostly mild dysmorphic features and normal intelligence has been reported in at least 10 women with mosaic trisomy 18.426

Genetic counseling when the fetus is affected The fateful day when the anxious, waiting couple hears the grim news that their fetus has a malformation or genetic disorder will live on in their memories forever. Cognizance of this impact should inform the thoughts, actions and communications of the physician called on to exercise consummate skill at such a poignant time. Couples may have traveled the road of hope and faith for many years, battling infertility only to be confronted by the devastating reality of a fetal anomaly. With hopes and dreams so suddenly dashed, doubt, anger and denial surface rapidly. The com-

passionate physician will need to be fully armed with all the facts about the defect or be ready to obtain an immediate expert clinical genetics consultation for the couple. Care should be taken in selecting a quiet, comfortable, private location that is safe from interruption. Ptacek and Eberhardt,427 in reviewing the literature, noted consensus recommendations in breaking bad news that included the foregoing and sitting close enough for eye contact without physical barriers. Identifying a support person if the partner cannot/will not attend the consultation is important and knowledge of available resources is valuable. All of the above points are preferences that have been vocalized by parents receiving bad news about their infants.428 Almost all couples would have reached this juncture through maternal serum screening, an ultrasound study or amniocentesis/CVS for maternal age, for established known carriers, because of a previously affected child, being an affected parent or having a family history of a specified disorder. Not rarely, an anxious patient insists on a prenatal study. On one such occasion, the patient stated, “My neighbor had a child with Down syndrome,” only to discover from the requested amniocentesis study that her fetus also had a serious abnormality. Physicians are advised not to dissuade patients away from prenatal diagnosis but rather to inform them about the risks of fetal loss balanced against the risk of fetal defects, distinctly different from recommendations for accepted indications. Recognition of a fetal abnormality by imaging, molecular or cytogenetic study may reveal, for the first time, the genetic disorder in an asymptomatic parent. Robyr et al.429 described 20 such parents with disorders including spinal muscular atrophy, DiGeorge syndrome, osteogenesis imperfecta, arthrogryposis and Noonan-like syndrome. Not infrequently, second-trimester ultrasound studies reveal fetal abnormalities of uncertain etiology. For example, on one (legal) case, sequential observations noted prominent lateral cerebral ventricles, multiple thoracic hemivertebrae and intrauterine growth restriction. Amniocyte chromosome studies were normal. The parents were not counseled about the potential for mental retardation despite no definitive diagnosis. The child was born with holoprosencephaly with marked psychomo-

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Genetic Counseling: Preconception, Prenatal and Perinatal

tor delay. Diagnostic uncertainty must be shared with parents at risk. Decision making The presence of both parents for the consultation concerning possible elective abortion for a fetal defect is critical in this situation. All the principles governing the delivery of genetic counseling and discussed earlier apply when parents need to decide whether or not to continue their pregnancy. A brief explanation of some of the key issues follows, culled from over 45 years of experience in this very subject. Doubt and disbelief crowd the parental senses in the face of such overwhelming anxiety. Was there a sample mix-up? How accurate is this diagnosis? How competent is the laboratory? Have they made errors in the past? How can we be certain that there has been no communication failure? Is there another couple with the same name? There are endless questions and endless doubts. Each and every one needs to be addressed carefully, slowly and deliberately, with painstaking care to provide the necessary assurance and reassurance. Needless to say, the clinical geneticist must have thoroughly checked all the logistics and potential pitfalls before initiating this consultation. Errors have indeed occurred in the past. The central portion of the communication will focus on the nature of the defect and the physician or counselor providing the counseling should be fully informed about the disorder, its anticipated burden, the associated prognosis, life expectancy and the possible need for lifetime care. A clear understanding of the potential for pain and suffering is necessary and an exploration concerning the effect on both parents and their other children is second only to a discussion about the potential effects on the child who is born with the condition in question. Any uncertainties related to diagnosis, prognosis, pleiotropism or heterogeneity should emerge promptly. Questions related to possible future pregnancies should be discussed, together with recurrence risks and options for prenatal diagnosis. The question concerning a repeat prenatal study is invariable, at least if not stated then certainly in the mind of the parents. There are occasions when a repeat test might be appropriate, especially if

41

there is a failure to reconcile cytogenetic or molecular results with expected high-resolution ultrasound observations. Maternal cell contamination (see Chapter 6), while extremely unlikely in almost all circumstances, requires exclusion in some others. Some prenatal diagnoses may not easily be interpretable and a phenotype may not be predictable with certainty. A de novo supernumerary chromosome fragment in the prenatal cytogenetic analysis (see Chapter 6) is a key example that can mostly be settled by a chromosomal microarray study. The sensitive counselor should offer a second opinion to anxious parents facing an uncertain prenatal diagnosis. The “compleat physician” anticipates virtually all of the patient’s questions, answers them before they are asked and raises all the issues without waiting for either parent to vocalize them. Occasionally, it is apparent that there are powerful disparate attitudes to abortion between the spouses. Such differences would best be considered during the preconception period, rather than for the first time when faced with a serious fetal defect. Resolution of this conflict is not the province of the physician or counselor, nor should either become arbitrator in this highly charged and very personal dispute, in which religious belief and matters of conscience may collide. The physician’s or counselor’s duty is to ensure that all facts are known and understood and that the pros and cons of various possible scenarios are identified in an impartial manner. A return appointment within days should be arranged. Questions of paternity have also suddenly emerged in this crisis period and can now be settled, sometimes with painful certainty. Elective abortion: decision and sequel Among the greatest challenges clinical geneticists and genetic counselors face is the consultation in which the results of prenatal studies indicating a serious fetal defect are communicated to parents for the first time. The quintessential qualities a counselor will need include maturity, experience, warmth and empathy, sensitivity, knowledge, communication skill and insight into the psychology of human relationships, pregnancy and grieving. Ample time (with follow-up visits) is critical. The principles and prerequisites for counseling discussed earlier apply fully in these circumstances and the fact that this is a parental decision, not a

42

Genetic Disorders and the Fetus

Table 1.9 The frequency of emotions and somatic symptoms of 84 women and 68 men: overall and 24 months after terminating a pregnancy for fetal abnormality430 Women (%)

Men (%)

Women after 24 months (%)

Men after 24 months (%)

Sadness

95

85

60

47

Depression

79

47

12

6

Anger

78

33

27

7

Fear

77

37

46

17

Guilt

68

22

33

7

Failure

61

26

24

14

Shame

40

9

18

4

Vulnerability

35

0

18

0

Relief

30

32

16

16

Isolation

27

20

11

6

Numbness

23

0

0

0

Panic spells

20

0

5

0

Withdrawal

0

32

0

13

Left out

0

12

0

0

Crying

82

50

22

5

Irritable

67

38

19

3

No concentration

57

41

7

1

Listlessness

56

17

2

0

Sleeplessness

47

19

2

1

Tiredness

42

21

6

3

Loss of appetite

31

10

0

0

Nightmares

24

7

5

0

Palpitations

17

6

0

Headaches

9

2

0

Feeling

Somatic symptom

– 8

medical “recommendation,” should not need reiteration. Anticipatory counseling in these consultations has been characterized by in-depth discussions of two areas: first, all medical and scientific aspects of the prenatal diagnosis made (and discussed earlier) and second, recognition and vocalization of emotional responses and reference to experiences (preferably published) of other couples in like circumstances when it was helpful. These sessions have then included explorations concerning guilt, a possible feeling of stigma (because of abortion), anger, upset and how other couples have coped. All of this anticipatory counseling has been tinctured with support and hope when possible. Many couples have expressed their appreciation of this approach and indicated the benefits of having had these discussions before elective termination.

The importance of continuing follow-up visits with couples who have terminated pregnancy for fetal defects cannot be overemphasized. In an important study on the psychosocial sequelae in such cases, White-van Mourik et al.430 showed the long-range effects (Table 1.9). Displays of emotional and somatic symptoms 1–2 years after abortion were not rare and included partners. Although some couples grew closer in their relationships, separations, especially because of failed communication, increased irritability and intolerance, were noted in 12 percent of the 84 patients studied.430 Marital discord in these circumstances has been noted previously.431,432 At least 50 percent of couples admitted to having problems in their sexual relationship. In addition, many couples indicated changed behavior toward their existing children, including overprotectiveness, anxiety,

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irritability and consequent guilt and indifference.430 Women with secondary infertility and those younger than 21 years of age (or immature women) had the most prolonged emotional, physical and social difficulties.430 Grief counseling becomes part of the consultation after elective termination, in which full recognition of bereavement is necessary. The psychology of mourning has been thoroughly explored by both Parkes433 and Worden.434 Worden emphasized how important it is for a bereaved individual to complete each of four stages in the mourning process. 1. Acceptance of the loss. 2. Resolving the pain of grieving. 3. Adjusting to life without the expected child. 4. Placing the loss in perspective. The importance of allowing parents the option of holding tthe fetus (or later, the child), when appropriate, is well recognized.435,436 These authors have also called attention to the complex tasks of mourning for a woman who is faced with one defective twin when pregnancy reduction or birth might occur. Notwithstanding anticipated loss and grief, Seller et al.,436 reflecting our own experience, emphasized that many couples recover from the trauma of fetal loss “surprisingly quickly.” Insinuation of this reality is helpful to couples in consultations both before and after elective termination. Moreover, couples’ orientation toward the grieving process achieves an important balance when they gain sufficient insight into the long-term emotional, physical, economic and social consequences they might have needed to contemplate if prenatal diagnosis had not been available. Testing the other children Invariably, parents faced with the news of their affected fetus question the need to test their other children. Answers in the affirmative are appropriate when diagnosis of a disorder is possible. Carrier detection tests, however, need careful consideration and are most appropriately postponed until the late teens, when genetic counseling should be offered. Given the complex dilemmas and farreaching implications of testing asymptomatic children for disorders that may manifest many years later, parents would best be advised to delay consideration of such decisions while in the midst

43

of dealing with an existing fetal defect. In later consultations, the thorny territory of predictive genetic testing of children can be reviewed at length.437–440 Fanos437 emphasized that testing adolescents “may alter the achievement of developmental tasks, including seeking freedom from parental figures, establishment of personal identity, handling of sexual energies and remodeling of former idealizations of self and others.” Fanos also emphasized that parental bonding may be compromised by genetic testing when the child’s genetic health is questionable. Parents may react to the possible loss or impairment of a child by developing an emotional distance, recognized as the vulnerable child syndrome.441 Other aspects, including interference with the normal development of a child’s self-concept, introduce issues of survivor guilt or increase levels of anxiety already initiated by family illnesses or loss.441 Predictive testing of children for later-manifesting neurodegenerative or other disorders would rarely be recommended, except in circumstances in which early diagnosis could offer preventive or therapeutic benefit.

Perinatal genetic counseling A similar spectrum of issues and concerns is faced after the detection and delivery of a child with a genetic disorder or an anomaly. Pregnancy with a defective fetus may have been continued from the first or second trimester or a diagnosis may be made in the third trimester or at the delivery of a living or stillborn child. The principles and prerequisites for genetic counseling discussed earlier apply equally in all these circumstances.442 Special attention should be focused on assuaging aspects of guilt and shame. Difficult as it may be for some physicians,443,444 close rapport, patient visitation and sincerity are necessary at these times, even when faced with commonly experienced anger. A misstep by the physician in these circumstances in failing to continue (it is to be hoped) the rapport already established during pregnancy care provides the spark that fuels litigation in relevant cases. Despite anger, grief and the gamut of expected emotions, the attending physician (not an inexperienced healthcare provider) should take care to

44

Genetic Disorders and the Fetus

urge an autopsy when appropriate. Diagnosis of certain disorders (e.g. congenital nephrosis) can be made by promptly collected and appropriately prepared renal tissue for electron microscopy, if mutation analysis (see Chapters 10 and 23) is unavailable. In circumstances in which parents steadfastly withhold permission for autopsy (which is optimal), magnetic resonance imaging could provide some useful acceptable alternative when fetal anomalies are expected.445 The autopsy is the last opportunity parents will have to determine causation, which may ultimately be critical in their future childbearing plans and also for their previous children. A formal protocol for evaluating the cause of stillbirth or perinatal death is important (Box 1.4) to secure a definitive diagnosis, thereby laying the foundation for providing accurate recurrence risks and future precise prenatal diagnosis. In addition, in the face of known or suspected genetic disorders in which mutation analysis now or in the future may be critical, care should be taken to obtain tissue for DNA banking or for establishing a cell line. Later, parents may return and seriously question the failure of the physician to secure tissues or DNA that would have been so meaningful in future planning (e.g. X-linked mental retardation, spinal muscular atrophy). Psychologic support is important for couples who have lost an offspring from any cause, a situ-

ation compounded by fetal or congenital abnormality.446 The birth (or prenatal detection) of twins discordant for a chromosomal disorder is not rare, given the increased frequency of multiple pregnancy associated with advanced maternal age and the use of assisted reproductive techniques. Pregnancy reduction (see Chapter 28) or the death of one twin or delivery of both evokes severely conflicting emotions that may well affect the mother’s care for the surviving child.447 Considerable psychologic skill must be marshaled by physicians if meaningful care and support are to be provided.448 Supporting telephone calls from doctor and staff and encouragement to attend appointments every 6 weeks, or more frequently when appropriate, are often appreciated by patients. Review of the autopsy report and discussion with reiterative counseling should be expected of all physicians. Frequently, parents receive an autopsy report by mail without further opportunity for explanation and discussion. In one study, 27 percent failed to receive autopsy results.449 Providing contact with support groups whose focus is the disorder in question is also valuable. In the United States, the vast majority of these groups have combined to form the Alliance of Genetic Support Groups, which acts as a central clearinghouse and referral center.

Box 1.4 Protocol for evaluating the cause of stillbirth or perinatal death

1. Review genetic, medical and obstetric history. 2. Determine possible consanguinity. 3. Gently and persistently recommend that parents permit a complete autopsy. 4. Obtain photographs, including full face and profile, whole body and, when applicable, detailed pictures of any specific abnormality (e.g. of digits). 5. Obtain full-body skeletal radiographs. 6. Consider full-body magnetic resonance imaging,390 if autopsy is not permitted. 7. Carefully document any dysmorphic features.

8. Obtain heparinized cord or fetal blood sample for chromosomal or DNA analysis. 9. Obtain fetal serum for infectious disease studies (e.g. parvovirus, cytomegalovirus, toxoplasmosis). 10. Obtain fetal tissue sample (sterile fascia best) for cell culture aimed at chromosome analysis or biochemical or DNA studies. 11. Obtain parental blood samples for chromosome analysis, when indicated. 12. Communicate final autopsy results and conclusions of special analyses. 13. Provide follow-up counseling, including a summary letter.

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Genetic Counseling: Preconception, Prenatal and Perinatal

Family matters Beyond all the “medical” steps taken in the wake of stillbirth or perinatal death due to fetal defects are critical matters important to the family and its future. Active, mature and informed management is necessary in these difficult and frequently poignant situations. Regardless of the cause of the child’s defect(s), maternal guilt is almost invariable and sometimes profound. Recognition of a definitive cause unrelated to a maternal origin should be explained in early discussions and reiterated later. For autosomal recessive disorders or with even more problematic X-linked disorders, maternal “culpability” is real and not easily assuaged. The fact that we all carry harmful genes, some of which we may have directly inherited, while others may have undergone mutation, may need in-depth discussion. Mostly, it is possible and important to reassure mothers that the outcome was not due to something they did wrong. Where the converse is true, much effort will be needed for management of guilt450 and shame, and for planning actions that promise a better future with ways to avert another adverse outcome. Attention to details that have a very important role in the mourning process include ensuring that the child be given a name and, in the case of the death of a defective fetus in the third trimester, that the parents’ wishes for a marked grave be determined. As noted earlier, most caretakers feel that parents are helped by both seeing and holding the baby.435,436,451 Although some may experience initial revulsion when the subject is mentioned, gentle coaxing and explanations about the experiences of other couples may help grieving parents. Even with badly disfigured offspring, it is possible for parents to cradle a mostly covered baby whose normal parts, such as hands and feet, can be held. Important mementos that parents should be offered are photographs, a lock of hair, the baby’s name band or clothing.447,448 Ultimately, these concrete emblems of the baby’s existence assist parents in the mourning process, although the desperate emptiness that mothers especially feel is not easily remedied. Photos may also be helpful in providing comfort for other children and for grandparents. Parents will also vary in their choice of traditional or small, private funerals. Physicians should ensure

45

that parents have the time to make these various decisions and assist by keeping the child in the ward for some hours when necessary. Both parents should be encouraged to return for continuing consultations during the mourning period.452 Mourning may run its course for 6–24 months. These consultations will serve to explore aspects of depression, guilt, anger, denial, possible marital discord and physical symptoms such as frigidity or impotence. Impulsive decisions for sterilization should be discouraged in the face of overwhelming grief. Advice should be given about safe, reliable and relatively long-term contraception.453 Similarly, parents should be fully informed about the consequences of having a “replacement child” very soon after their loss.454,455 That child may well become a continuing vehicle of grief for the parents, who may then become overanxious and overprotective. Subsequently, they may bedevil the future of the replacement child with constant references to the lost baby, creating a fantasy image of perfection that the replacement child could never fulfill. Such a child may well have trouble establishing his or her own identity. The surviving children Distraught parents frequently seek advice about how to tell their other children. Responses should be tailored to the age of the child in question, to the child’s level of understanding and against a background of the religious and cultural beliefs of the family. A key principle to appreciate is that having reached the stage of cognizance regarding the loss, a child needs and seeks personal security. Hence, the parents’ attention should be focused on love, warmth and repetitive reassurance, especially about (possibly) unstated feelings of previous wrongdoing and personal culpability. Advice about grieving together instead of being and feeling overwhelmed in front of their children is also helpful advice. Focusing on the children’s thoughts and activities is beneficial rather than lapsing into a state of emotional paralysis, which can only serve to aggravate the family’s psychodynamics adversely.

The efficacy of genetic counseling The essential goal of the communication process

46

Genetic Disorders and the Fetus

in genetic counseling is to achieve as complete an understanding by the counselee(s) as possible, thereby enabling the most rational decision making. Parental decisions to have additional affected progeny should not be viewed as a failure of genetic counseling. Although the physician’s goal is the prevention of genetic disease, the orientation of the prospective parents may be quite different. A fully informed couple, both of whom had achondroplasia, requested prenatal diagnosis with the expressed goal of aborting a normal unaffected fetus so as to be able to raise a child like themselves. Would anyone construe this as a failure in genetic counseling? Clarke et al.456 considered three prime facets that could possibly evaluate the efficacy of genetic counseling: (1) recall of risk figures and other relevant information by the counselee(s); (2) the effect on reproductive planning; and (3) actual reproductive behavior. Their conclusions, reflecting a Western consensus, were that there are too many subjective and variable factors involved in the recall of risk figures and other genetic counseling information to provide any adequate measure of efficacy. Further, assessing reproductive intentions may prejudge the service the counselee wishes as well as the fact that there are too many confounding factors that have an impact on reproductive planning. Moreover, how many years after counseling would be required to assess the impact on reproductive planning? They regarded evaluation of reproductive plans as “a poor proxy for reproductive behavior.” In dispensing with assessments of actual reproductive behavior in the face of counseling about such risks, they pointed to the complex set of social and other factors that confound the use of this item as an outcome measure. They did, however, recommend that efficacy be assessed against the background goals of genetic counseling aimed at evaluation of the understanding of the counselee(s) of their own particular risks and options. Evaluation of the efficacy of genetic counseling12,169 should therefore concentrate on the degree of knowledge acquired (including the retention of the counselee(s) with regard to the indicated probabilities) and the rationality of decision making (especially concerning further reproduction). Frequent contraceptive failures in high-risk

families highlight the need for very explicit counseling. Important points made by Emery et al.457 in their prospective study of 200 counselors included the demonstrated need for follow-up after counseling, especially when it is suspected that the comprehension of the counselee(s) is not good. This seemed particularly important in chromosomal and X-linked recessive disorders. They noted that the proportion deterred from having children increased with time and that more than one-third of their patients opted for sterilization within 2 years of counseling. A number of studies457–459 document the failure of comprehension by the counselee(s). The reports do not reflect objective measures of the skill or adequacy of genetic counseling and the possible value of a summary letter to the patient of the information provided after the counseling visit. Sorenson et al.460 prospectively studied 2,220 counselees who were seen by 205 professionals in 47 clinics located in 25 states and the District of Columbia. They gathered information not only on the counselees but also on the counselors and the clinics in which genetic counseling was provided. They, too, documented that 53 percent of counselees did not comprehend their risks later, while 40 percent of the counselees given a specific diagnosis did not appear to know it after their counseling. They thoroughly explored the multiple and complex issues that potentially contributed to the obvious educational failure that they (and others) have observed. In another study of parents with a DS child, Swerts408 noted that of those who had genetic counseling, 45 percent recalled recurrence risks accurately, 21 percent were incorrect and 34 percent did not remember their risks. The expected post-counseling letter to the referring physician with a copy (or a separate letter) to the patient plays a vital role in securing comprehension of risks and issues. Printed materials, especially covering risks, test limitations, psychologic and social aspects, enrich the counseling benefits.78 Genetic counseling can be considered successful when counselees, shown to be well informed, make careful, rational decisions regardless of whether their physicians consider their position to be ill advised. Clearly, counselees and counselors may differ in their perception of the consultation and

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the degree of satisfaction.461 Notwithstanding the obvious benefits of counseling, reproductive uncertainty is often not eliminated because it is related to factors beyond the scope of counseling.462 In considering the effectiveness of genetic counseling, Sorenson et al.460 summarized the essence of their conclusion. In many respects, an overall assessment of the effectiveness of counseling, at least the counseling we assessed in this study, is confronted with the problem of whether the glass is half full or half empty. That is, about half of the clients who could have learned their risk did but about half did not. And, over half of the clients who could have learned their diagnosis did but the remainder did not. In a similar vein, clients report that just over half of their genetic medical questions and concerns were discussed but about half were not. The picture for sociomedical concerns and questions was markedly worse, however. And, reproductively, just over half of those coming to counseling to obtain information to use in making their reproductive plans reported counseling influenced these plans but about half did not. Any overall assessment must point to the fact that counseling has been effective for many clients but ineffective for an almost equal number.

A critical analysis of the literature by Kessler463 concluded that published studies on reproductive outcome after genetic counseling reveal no major impact of counseling. Moreover, decisions made before counseling largely determined reproduction after counseling. A more recent study of patients’ expectations of genetic counseling revealed that the majority had their expectations fulfilled, especially with perceived personal control.464 When patients’ expectations for reassurance and advice were met, they were subsequently less concerned and had less anxiety compared with when such expectations were not fulfilled. The limited efficacy of genetic counseling revealed in the study by Sorenson et al.460 reflects the consequences of multiple factors, not the least of which are poor lay understanding of science and a lack or inadequacy of formal training of counselors in clinical genetics,465 which is no longer the case for genetic counselors, at least in the USA and Canada. Efficacy, of course, is not solely related to

47

counselee satisfaction. Efforts to educate the public about the importance of genetics in their personal lives have been made by one of us in a series of books (translated into nine languages) over a quarter of a century.167,168,171–173,256 In addition to public education and its concomitant effect of educating physicians generally, formal specialist certification in the United States, Canada and the United Kingdom, acceptance of clinical genetics as a specialty approved by the American Medical Association and new degree programs for genetic counselors certified by the National Board of Genetic Counselors will undoubtedly improve the efficacy of genetic counseling.

References 1. Martin RH, Ko E, Rademaker A. Distribution of aneuploidy in human gametes: comparison between human sperm and oocytes. Am J Med Genet 1991;39: 321. 2. Plachot M. Chromosome analysis of oocytes and embryos. In: Verlinsky Y, Kuliev A, eds. Preimplantation genetics. New York: Plenum Press, 1991:103. 3. Boué J, Boué A, Lazar P. Retrospective and prospective epidemiological studies of 1500 karyotyped spontaneous human abortions. Teratology 1975;12:11. 4. Alberman ED, Creasy MR. Frequency of chromosomal abnormalities in miscarriages and perinatal deaths. J Med Genet 1977;14:313. 5. Holmes-Seidle M, Ryyvanen M, Lindenbaum RH. Parental decisions regarding termination of pregnancy following prenatal detection of sex chromosome abnormality. Prenat Diagn 1987;7:239. 6. Martinez-Frias ML, Bermejo E, Cereijo A, et al. Epidemiological aspects of Mendelian syndromes in a Spanish population sample. II. Autosomal recessive malformation syndromes. Am J Med Genet 1991;38:626. 7. Njoh J, Chellaram R, Ramas L. Congenital abnormalities in Liberian neonates. West Afr J Med 1991;10:439. 8. Verma IC. Burden of genetic disorders in India. Indian J Pediatr 2001;67:893. 8a. Lie RT, Wilcox AJ, Skjaerven R. Survival and reproduction among males with birth defects and risk of recurrence in their children. JAMA 2001;285:755. 9. Queisser-Luft A, Stolz G, Wiesel A, et al. Malformations in newborn: results based on 30,940 infants and fetuses from the Mainz congenital birth defect monitoring system (1990–1998). Arch Gynecol Obstet 2002;266:163. 10. McKusick VA. Mendelian inheritance in man, 12th ed. Baltimore: Johns Hopkins University Press, 1998.

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after predictive testing for Huntington disease. Am J Hum Genet 1999;64:1293. Markel DS, Young AB, Penney JB. At-risk persons’ attitudes toward presymptomatic and prenatal testing of Huntington’s disease in Michigan. Am J Med Genet 1987;26:295. Lamport AN. Presymptomatic testing for Huntington’s chorea: ethical and legal issues. Am J Med Genet 1987;26:307. Taylor CA, Myers RH. Long-term impact of Huntington disease linkage testing. Am J Med Genet 1997: 70:365. Hayden MR. Predictive testing for Huntington disease: are we ready for widespread community implementation? Am J Med Genet 1991;40:515. Nance MA, Leroy BS, Orr HT, et al. Protocol for genetic testing in Huntington disease: three years of experience in Minnesota. Am J Med Genet 1991;40:518. Decruyenaere M, Evers-Kiebooms G, Cloostermans T, et al. Psychological distress in the 5-year period after predictive testing for Huntington’s disease. Eur J Hum Genet 2003;11:30. Evans DGR, Maher EF, Macleod R, et al. Uptake of genetic testing for cancer predisposition. J Med Genet 1997;34:746. Deltas CC, Christodoulou K, Tjakouri C, et al. Presymptomatic molecular diagnosis of autosomal dominant polycystic kidney disease using PKD1- and PKD2-linked markers in Cypriot families. Clin Genet 1996;50:10. Pirson Y, Chaveau D. Intracranial aneurysms in autosomal dominant polycystic kidney disease. In: Watson ML, Torres VE, eds. Polycystic kidney disease. Oxford: Oxford University Press, 1996:530. Sujansky E, Kreutzer SB, Johnson AM, et al. Attitudes of at-risk and affected individuals regarding presymptomatic testing for autosomal dominant polycystic kidney disease. Am J Med Genet 1990;35:510. Hannig VL, Hopkins JR, Johnson HK, et al. Presymptomatic testing for adult onset polycystic kidney disease in at-risk kidney transplant donors. Am J Med Genet 1991;40:425. Giardiello FM, Brensinger JD, Petersen GM, et al. The use and interpretation of commercial APC gene testing for familial adenomatous polyposis. N Engl J Med 1997;336:823. Telander RL, Zimmerman D, Sizemore GW, et al. Medullary carcinoma in children: results of early detection and surgery. Arch Surg 1989;124:841. Ross LF. Predictive genetic testing for conditions that present in childhood. Kennedy Inst Ethics J 2002;12:225. Wertz DC, Fanos JH, Reilly PR. Genetic testing for children and adolescents: who decides? JAMA 1994; 272:875.

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364. Milunsky JM, Maher TA, Loose BA, et al. XL PCR for the detection of large trinucleotide expansions in juvenile Huntington’s disease. Clin Genet 2003;64:70. 365. Tassicker R, Savulescu J, Skene L, et al. Prenatal diagnosis requests for Huntington’s disease when the father is at risk and does not want to know his genetic status: clinical, legal and ethical viewpoints. BMJ 2003;326:331. 366. Gusella JF, McNeil S, Persichetti F, et al. Huntington’s disease. Cold Spring Harb Symp Quant Biol 1996; 61:615. 367. Kremer B, Goldberg P, Andrew SE, A worldwide study of the Huntington’s disease mutation: the sensitivity and specificity of measuring CAG repeats. N Engl J Med 1994;330:1401. 368. Alonso ME, Yescas P, Rasmussen A, et al. Homozygosity in Huntington’s disease: new ethical dilemma caused by molecular diagnosis. Clin Genet 2002;61:437. 369. Lancaster JM, Wiseman RW, Berchuck A. An inevitable dilemma: prenatal testing for mutations in the BRCA1 breast-ovarian cancer susceptibility gene. Obstet Gynecol 1996;87:306. 370. DudokdeWit AC, Tibben A, Frets PG, et al. BRCA1 in the family: a case description of the psychological implications. Am J Med Genet 1997;71:63. 371. Julian-Reynier C, Eisinger F, Vennin P, et al. Attitudes towards cancer predictive testing and transmission of information to the family. J Med Genet 1996;33:731. 372. Lancaster JM, Wiseman RW, Berchuck A. An inevitable dilemma: prenatal testing for mutations in the BRCA1 breast-ovarian cancer susceptibility gene. Obstet Gynecol 1996;87:306. 373. Antoniou A, Pharoah PD, Narod S, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet 2003;72:1117. 374. National Comprehensive Cancer Network. Practice guidelines in oncology. Genetic/familial high-risk assessment: breast and ovarian. Version 1. Fort Washington, PA: National Comprehensive Cancer Network, 2008. 375. Burke W, Daly M, Garber J, et al. Recommendations for follow-up care of individuals with an inherited predisposition to cancer. II. BRCA1 and BRCA2. Cancer Genetics Studies Consortium. JAMA 1997;277:997. 376. King M-C, Marks JH, Mandell JB. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 2003:302;643. 377. Laken SJ, Petersen GM, Gruber SB, et al. Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC. Nat Genet 1997;17:79. 378. Warburton D, Kline J, Stein Z, et al. Does the karyotype of a spontaneous abortion predict the karyotype of a

379.

380.

381. 382. 383.

384.

385. 386.

387.

388.

389.

390.

391.

392.

59

subsequent abortion? Evidence from 273 women with two karyotyped spontaneous abortions. Am J Hum Genet 1987;41:465. Campuzano V, Montermini L, Molot MD, et al. Friedreich’s ataxia: autosomal recessive disease caused by an intronic GA triplet repeat expansion. Science 1996; 271:1423. Jaspert A, Fahsold R, Grehl H, et al. Myotonic dystrophy: correlation of clinical symptoms with the size of the CTG trinucleotide repeat. J Neurol 1995;242:99. Kalousek DK, Barrett I. Genomic imprinting related to prenatal diagnosis. Prenat Diagn 1994;14:1191. Deal CL. Parental genomic imprinting. Curr Opin Pediatr 1995;7:445. Clayton-Smith J, Laan L. Angelman syndrome: a review of the clinical and genetic aspects. J Med Genet 2003;40:87. Dindot SV, Antaiffy BA, Bhattacharjee MB, et al. The Angelman syndrome ubiquitin ligase localizes to the synapse and nucleus and maternal deficiency results in abnormal dendritic spine morphology. Hum Mol Genet. 2008;17:111. Walter J, Paulsen M. Imprinting and disease. Semin Cell Dev Biol 2003;14:101. Rossignol S, Steunou V, Chalas C, et al. The epigenetic imprinting defect of patients with Beckwith–Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region. J Med Genet 2006;43:902. Wilkins-Haug L, Porter A, Hawley P, et al. Isolated fetal omphalocele, Beckwith–Wiedemann syndrome and assisted reproductive technologies. Birth Defects Res A Clin Mol Teratol 2009;85:58. Barel O, Shalev SA, Ofir R, et al. Maternally inherited Birk Barel mental retardation dysmorphism syndrome caused by a mutation in the genomically imprinted potassium channel KCNK9. Am J Hum Genet 2008; 83:193. Fournet JC, Mayaud C, de Lonlay P, et al. Loss of imprinted genes and paternal SUR1 mutations lead to focal form of congenital hyperinsulinism. Horm Res 2000;53:2. Passos-Bueno MR, Cerqueira A, Vainzof M, et al. Myotonic dystrophy: genetic, clinical and molecular analysis of patients from 41 Brazilian families. J Med Genet 1995;32:14. Judson H, Hayward BE, Sheridan E, et al. A global disorder of imprinting in the human female germ line. Nature 2002;416:539. van Schothorst EM, Jansen JC, Bardoel AF, et al. Confinement of PGL, an imprinted gene causing hereditary paragangliomas, to a 2-cM interval on 11q22–q23 and exclusion of DRD2 and NCAM as candidate genes. Eur J Hum Genet 1996;4:267.

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393. Muller B, Hedrich K, Kock N, et al. Evidence that paternal expression of the epsilon-sarcoglycan gene accounts for reduced penetrance in myoclonusdystonia. Am J Hum Genet 2002;71:1303. 394. Fokstuen S, Ginsburg C, Zachmann M, et al. Maternal uniparental disomy 14 as a cause of intrauterine growth retardation and early onset of puberty. J Pediatr 1999;134:689. 395. Eggermann T, Zerres K, Eggermann K, et al. Uniparental disomy: clinical indications for testing in growth retardation. Eur J Pediatr 2002;161:305. 396. Davies W, Isles AR, Wilkinson LS. Imprinted genes and mental dysfunction. Ann Med 2001;33:428. 397. Perk J, Makedonski K, Lande L, et al. The imprinting mechanism of the Prader–Willi/Angelman regional control center. EMBO J 2002;21:5807. 398. Bastepe M, Juppner H. Pseudohypoparathyroidism: new insights into an old disease. Endocrinol Metab Clin North Am 2000;29:569. 399. Shore EM, Ahn J, Jan de Beur S, et al. Paternally inherited inactivating mutations of the GNAS1 gene in progressive osseous heteroplasia. N Engl J Med 2002;346:99. 400. Balmer D, Arredondo J, Samaco RC, et al. MECP2 mutations in Rett syndrome adversely affect lymphocyte growth but do not affect imprinted gene expression in blood or brain. Hum Genet 2002;110:545. 401. Girard M, Couvert P, Carrie A, et al. Parental origin of de novo MECP2 mutations in Rett syndrome. Eur J Hum Genet 2001;9:231. 402. Hitchins MP, Stanier P, Preece MA, et al. Silver–Russell syndrome: a dissection of the genetic aetiology and candidate chromosomal regions. J Med Genet 2001;38:810. 402a. Netchine I, Rossignol S, Dufourg MN, et al. 11p15 imprinting center region 1 loss of methylation is a common and specific cause of typical Russell–Silver syndrome: clinical scoring system and epigeneticphenotypic correlations. J Clin Endocrinol Metab 2007;92:3148. 403. Hannula K, Lipsanen-Nyman M, Kristo P, et al. Genetic screening for maternal uniparental disomy of chromosome 7 in prenatal and postnatal growth retardation of unknown cause. Pediatrics 2002;109:441. 404. Temple IK, Shield JP. Transient neonatal diabetes, a disorder of imprinting. J Med Genet 2002;39:872. 405. Mackay DJ, Boonen SE, Clayton-Smith J, et al. A maternal hypomethylation syndrome present as transient neonatal diabetes mellitus. Hum Genet 2006; 120:262. 406. Amor DJ, Halliday J. A review of known imprinting syndromes and their associtation with assisted reproduction technologies. Hum Reprod 2008;23:2826. 407. Turpin JC. Huntington chorea in children. Arch Fr Pediatr 1993;50:119.

408. Thorisson GA, Muilu J, Brookes AJ. Genotypephenotype databases: challenges and solutions for the post-genomic era. Nat Rev Genet 2009;10:9. 409. Pyeritz RE, McKusick VA. The Marfan syndrome: diagnosis and management. N Engl J Med 1979;300:772. 410. Beutler E, Nguyen NJ, Henneberger MW, et al. Gaucher disease: gene frequencies in the Ashkenazi Jewish population. Am J Hum Genet 1993;53:85. 411. Lewis BD, Nelson PV, Robertson EF, et al. Mutation analysis of 28 Gaucher disease patients: the Australasian experience. Am J Med Genet 1994;49:218. 412. Kerem E, Corey M, Kerem B, et al. The relationship between genotype and phenotype in cystic fibrosis: analysis of the most common mutation. N Engl J Med 1990;323:1517. 413. Cystic Fibrosis Genotype-Phenotype Consortium. Correlation between genotype and phenotype in patients with cystic fibrosis. N Engl J Med 1993;329:1308. 414. Dork T, Wulbrand U, Richter T, et al. Cystic fibrosis with three mutations in the cystic fibrosis transmembrane regulator gene. Hum Genet 1991;87:441. 415. Le C, Ramjeesingh M, Reys E, et al. The cystic fibrosis mutation (F508) does not influence the chloride channel activity of CFTR. Nat Genet 1993;3:311. 416. Tuddenham EGD. Factor VIII and haemophilia A. Baillière’s Clin Haematol 1989;2:849. 417. Miller DS, Steinbrecher RA, Wieland K, et al. The molecular genetic analysis of haemophilia A: characterization of six partial deletions in the factor VIII gene. Hum Genet 1990;86:219. 418. Hoo JJ. Alternative explanations for recurrent achondroplasia in siblings with normal parents. Clin Genet 1984;25:553. 419. Bakker E, Veenema H, Den Dunnen JT, et al. Germinal mosaicism increases the recurrence risk for “new” Duchenne muscular dystrophy mutations. J Med Genet 1989;26:553. 420. Donnai D, Read AP, McKeown C, et al. Hypomelanosis of Ito-A manifestation of mosaicism or chimerism. J Med Genet 1988;25:809. 421. Thomas IT, Frias JL, Cantu ES, et al. Association of pigmentary anomalies with chromosomal and genetic mosaicism and chimerism. Am J Hum Genet 1989;45: 193. 422. Raghunath M, Mackay K, Dalgleish R, et al. Genetic counseling on brittle grounds: recurring osteogenesis imperfecta due to parental mosaicism for a dominant mutation. Eur J Pediatr 1995;154:123. 423. Lund AM, Nicholls AC, Schwartz M, et al. Parental mosaicism and autosomal dominant mutations causing structural abnormalities of collagen I are frequent in families with osteogenesis imperfecta type III/ IV. Acta Paediatr 1997;86:711.

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424. Telenius H, Kremer B, Goldberg YP, et al. Somatic and gonadal mosaicism of the Huntington disease gene CAG repeat in brain and sperm. Nat Genet 1994;6: 409. 425. Cancel G, Durr A, Didierjean O, et al. Molecular and clinical correlations in spinocerebellar ataxia 2: a study of 32 families. Hum Mol Genet 1997;6:709. 426. Satge D, Geneix A, Goburdhun J, et al. A history of miscarriages and mild prognathism as possible mode of presentation of mosaic trisomy 18 in women. Clin Genet 1996;50:470. 427. Ptacek JT, Eberhardt TL. Breaking bad news. JAMA 1996;276:496. 428. Bond CF, Anderson EL. The reluctance to transmit bad news: private discomfort or public display? J Eur Soc Psychol 1987;23:176. 429. Robyr R, Bernard JP, Roume J, et al. Familial diseases revealed by a fetal anomaly. Prenat Diagn 2006;26: 1224. 430. White-van Mourik MCA, Connor JM, Ferguson-Smith MA. The psychosocial sequelae of a second-trimester termination of pregnancy for fetal abnormality. Prenat Diagn 1992;12:189. 431. Blumberg BD, Golbus MC, Hanson K. The psychological sequelae of abortion performed for a genetic indication. Am J Obstet Gynecol 1975;122:799. 432. Blumberg BD. The emotional implications of prenatal diagnosis. In: Emery, AEH, Pullen IM, eds. Psychological aspects of genetic counselling. London: Academic Press, 1984:202. 433. Parkes CM. Bereavement. Studies of grief in adult life. London: Tavistock Publications, 1972. 434. Worden JW. Grief counseling and grief therapy, 2nd edn. New York: Springer, 1991. 435. Appleton R, Gibson B, Hey E. The loss of a baby at birth: the role of the bereavement officer. Br J Obstet Gynaecol 1993;100:51. 436. Seller M, Barnes C, Ross S, et al. Grief and midtrimester fetal loss. Prenat Diagn 1993;13:341. 437. Fanos JH. Developmental tasks of childhood and adolescence: implications for genetic testing. Am J Med Genet 1997;71:22. 438. Wertz DC, Fanos JH, Reilly PR. Genetic testing for children and adolescents: who decides? JAMA 1994; 272:875. 439. Clinical Genetics Society (UK). Report of a Working Party: the genetic testing of children. J Med Genet 1994;31:785. 440. American Society of Human Genetics and American College of Medical Genetics. Points to consider: ethical, legal and psychosocial implications of genetic testing in children and adolescents. Am J Hum Genet 1995; 57:1233.

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441. Green M, Solnit AJ. Reactions to the threatened loss of a child: a vulnerable child syndrome. Pediatrics 1964; 34:58. 442. McIntosh N, Eldrige C. Neonatal death: the neglected side of neonatal care? Arch Dis Child 1984;59:585. 443. Bourne S. The psychological effects of a stillbirth on women and their doctors. J R Coll Gen Pract 1968; 16:103. 444. Crowther ME. Communication following a stillbirth or neonatal death: room for improvement. Br J Obstet Gynaecol 1995;102:952. 445. Brookes JAS, Hall-Craggs MA, Sams VR, et al. Noninvasive perinatal necropsy by magnetic resonance imaging. Lancet 1996;348:1139. 446. Nicholas AM, Lewin TJ. Grief reactions of parental couples: congenital handicap and cot death. Med J Aust 1986;144:292. 447. Lewis E, Bryan E. Management of perinatal loss of a twin. BMJ 1988;297:1321. 448. Lewis E. Stillbirth: psychological consequences and strategies of management. In: Milunsky A, ed. Advances in perinatal medicine, vol. 3. New York: Plenum, 1983:205. 449. McPhee SJ, Bottles K, Lo B, et al. To redeem them from death: reactions of family members to autopsy. Am J Med 1986;80:665. 450. Irvin NA, Kennell JH, Klaus MH. Caring for the parents of an infant with a congenital malformation. In: Warkany J, ed. Congenital malformations: notes and comments. Chicago: Year Book Medical Publishers, 1971. 451. Klaus MH, Kennell JH. Caring for parents of an infant who dies: maternal–infant bonding. St Louis, MO: CV Mosby, 1976. 452. Furlong RM, Hobbins JC. Grief in the perinatal period. Obstet Gynecol 1983;61:497. 453. Shulman LP, Grevengood C, Phillips OP, et al. Family planning decisions after prenatal detection of fetal abnormalities. Am J Obstet Gynecol 1994;171:1373. 454. Rowe J, Clyman R, Green C, et al. Follow-up of families who experience a perinatal death. Pediatrics 1978; 62:166. 455. Forrest GC, Standish E, Baum JD. Support after perinatal death: a study of support and counseling after bereavement. BMJ 1982;285:1475. 456. Clarke A, Parsons E, Williams A. Outcomes and process in genetic counseling. Clin Genet 1996;50: 462. 457. Emery AEH, Raeburn JA, Skinner R. Prospective study of genetic counseling. BMJ 1979;1:253. 458. Sibinga MS, Friedman CG. Complexities of parental understanding for phenylketonuria. Pediatrics 1971; 48:216.

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459. Reynolds BD, Puck MH, Robinson A. Genetic counseling: an appraisal. Clin Genet 1974;5:177. 460. Sorenson JR, Swazey JP, Scotch NA. Effective genetic counseling: more informed clients. In: Reproductive pasts, reproductive futures: genetic counseling and its effectiveness. New York: Alan R. Liss, 1981:79. 461. Aalfs CM, Oort FJ, de Haes JC, et al. A comparison of counselee and counselor satisfaction in reproductive genetic counseling. Clin Genet 2007;72:74. 462. Wertz DC, Sorenson JR, Heeren TC. Clients’ interpretation of risks provided in genetic counseling. Am J Hum Genet 1986;39:253.

463. Kessler S. Psychological aspects of genetic counseling. VI. A critical review of the literature dealing with education and reproduction. Am J Med Genet 1989;34: 340. 464. Davey A, Rostant K, Harrop K, et al. Evaluating genetic counseling: client expectations, psychological adjustment and satisfaction with service. J Genet Couns 2005;14:197. 465. Swerts A. Impact of genetic counseling and prenatal diagnosis for Down syndrome and neural tube defects. Birth Defects Orig Artic Ser 1987;23(2): 61.

2

Amniocentesis and Fetal Blood Sampling Sherman Elias Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

Amniocentesis was first used in Germany in the early 1880s to treat hydramnios.1,2 In 1930, Menees et al.3 used amniocentesis to inject contrast media into the amniotic sac, to evaluate the fetus and to localize the placenta. The procedure of using amniocentesis to introduce hypertonic saline into the amniotic sac to terminate pregnancy was first used in 1937.4 In 1950 Alvarez of Uruguay performed amniocentesis to assess fetal well-being.5 The use of amniocentesis increased rapidly in the 1950s, when spectrophotometric analysis of bilirubin proved valuable in monitoring fetuses with Rh isoimmunization.5,6 Amniocentesis for exclusively genetic indications evolved in the mid-1950s. Several investigators demonstrated that fetal sex could be determined by X-chromatin analysis of amniotic fluid cells (AFCs).7–9 In 1966, Steele and Breg10 reported the feasibility of performing chromosomal analysis of AFCs, thereby formally introducing the prenatal diagnosis of genetic disorders. The next year, Jacobson and Barter11 reported the first prenatal diagnosis of a chromosomal abnormality (a balanced D/D translocation). In 1968, Valenti et al.12 and Nadler13 reported the in utero detection of Down syndrome, with confirmation after elective abortion. That same year, Nadler13 diagnosed galactosemia in fetal AFCs, the first detection of a mendelian disorder. Within the next few years,

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

numerous investigators reported successful prenatal diagnosis of a wide variety of chromosomal and metabolic disorders.14 In 1970, Nadler and Gerbie15 summarized their initial experience in performing genetic amniocentesis in 142 patients during 155 pregnancies. By demonstrating both diagnostic accuracy and a relatively low risk, their landmark report helped establish prenatal diagnosis through amniocentesis as an integral part of modern obstetric care.16 This chapter addresses current technique and the safety of genetic amniocentesis as well as fetal blood sampling, the second most commonly employed invasive prenatal diagnostic procedure in the second and third trimesters. Indications and methods of prenatal diagnosis are considered in detail throughout this text, as well as elsewhere by the author.17–22

Amniocentesis Prerequisites Consistently reliable and safe diagnosis can be achieved only by a team that provides the necessary expertise. Ideally, couples should have the opportunity to discuss their genetic risks and available antenatal studies before pregnancy.17,21,23 The counselor should elicit an accurate history, confirm the diagnosis of any abnormality in question, be aware of diagnostic capabilities, and be cognizant of psychologic defenses (e.g. denial, guilt reactions, and blame) engendered during genetic counseling. Couples must understand the risks of amniocentesis itself, the accuracy and limitations of antenatal

63

64

Genetic Disorders and the Fetus

diagnosis, the time required before results become available, technical problems potentially necessitating a second amniocentesis, and the rare possibility of an inability to make a diagnosis. Amniocentesis should be performed only by an obstetrician who (1) is experienced in this procedure, (2) has the availability of high-quality ultrasonography, and (3) has access to a laboratory with experience in performing prenatal diagnostic studies.20,24,25 Only obstetricians should perform the procedure, not because of technical difficulty but because the operator must always be prepared to deal with the potential complications of the procedure. According to the American College of Obstetricians and Gynecologists (ACOG), if an abnormality is detected and the couple elects to terminate the pregnancy, the obstetrician must either perform the abortion or refer the family to an obstetrician who will act on their request.26 Technique of amniocentesis

Timing Traditionally, amniocentesis has been performed at about the 15th and 16th weeks of gestation (menstrual weeks). At this time, the ratio of viable to nonviable cells is greatest compared with procedures performed later in gestation.27 In addition, the uterus is accessible by an abdominal approach and contains sufficient amniotic fluid (AF) (200– 250 mL) to permit 20–30 mL to be aspirated safely. Amniocentesis earlier in gestation (i.e. 46

129

2.33

0.77

–

–

–

–

–

–

1.55

1.55

0.77

–

–

–

–

6.98

–

–

6.98

≥35

52,965

1.16

0.23

0.07

0.06

0.04

0.04

0.18

0.05

0.12

0.16

0.03

0.04

0.04

0.02

0.02

2.01

0.25

0.03

2.26

Reproduced by permission from J. Wiley & Sons, Ltd. Table 2 in, Ferguson-Smith and Yates, 1984.40 Abn, all unbalanced abnormalities; Bal, balanced structural abnormalities (excluding pericentic inversion 9); Unbal, duplications, deficiencies arising from structural abnormalities.

CHAP T E R 6

Prenatal Diagnosis of Chromosomal Abnormalities through Amniocentesis

199

Table 6.5 The incidence of de novo balanced structural

Table 6.6 The frequency of chromosomal abnormalities in

rearrangements and supernumerary markers in 337,357

unselected spontaneous abortions

genetic amniocenteses Total analyzed De novo rearrangement

Number of cases

Percentage

Reciprocal translocation

176

0.047

42

0.011

33

0.009

162

0.04

Robertsonian translocation Inversion Supernumerary small marker chromosome

13,369

–

Normal

6,850

–

Abnormal

6,519

48.8%

Autosomal trisomy (inc mos)

3,610

55.4%

Monosomy X (inc mos)

1,033

15.8%

Triploid (inc hypo- & hyper-)

996

15.3%

Tetraploid (inc hypo- & hyper-)

328

5.0%

Abnormalities

Satellited marker

77

0.02

Double trisomy (inc mos)

170

2.6%

Nonsatellited marker

85

0.023

Structural

278

4.3%

413

0.109

Other (+X, +Y, monosomy 21,

105

1.6%

Total

Data from Warburton 1991.43

+mar, complex) Data from references 50–66. Inc mos, includes mosaicism.

translocations appeared to be underestimated, probably because of under-reporting. Data from chorionic villus sampling Published data are available for more than 16,000 chorionic villus specimens from women of advanced maternal age. These data show maternal age-specific prevalence for chromosomal abnormalities higher than that seen at amniocentesis (see Table 6.2). Data from spontaneous abortuses Major chromosomal abnormalities have been found in nearly one half of all first-trimester spontaneously aborted fetuses.50–66 Of 13,369 spontaneous abortuses studied (Table 6.6), 6,519 (48.8 percent) were found to have chromosomal abnormalities. Of these, 55 percent were autosomal trisomies, 16 percent were 45,X, 20 percent were polyploidies, and 8 percent had other anomalies, such as a structural aberration, mosaicism, double trisomies, monosomy 21 or other complex karyotypes. Among the autosomal trisomies, any chromosome can be involved but trisomy 16 accounts for 25 pecent of the cases (Table 6.7). The acrocentric chromosomes (13–15, 21–22) are also overrepresented. The frequency of autosomal trisomies in spontaneous abortions increases with maternal age.67 Monosomy X (45,X), however, was found to be associated with young maternal age;68 32 percent of 45,X abortuses came from women with ages between 20 and 24 years.67

Table 6.7 Autosomal trisomy in spontaneous abortions Chromosome

Cases

% All trisomies

16

131

24.7

22

116

13.9

21

121

12.3

15

67

8.3

13

35

6.8

18

46

4.8

14

25

4.4

7

17

3.4

2

14

3.2

8

15

3.0

9

25

2.9

4

20

2.8

20

20

2.7

10

6

1.5

12

9

1.2

6

6

1.0

3

3

0.9

17

14

0.9

11

4

0.5

5

3

0.4

19

1

0.2

1

1

0.0

total

699

100

Data from references 50–66, including extraction of numbers from Figure 1 in reference 66.

200

Genetic Disorders and the Fetus

Data from induced abortuses The largest series of cytogenetic studies of induced abortuses is to be found in the report of Kajii et al.69 More than 7,000 induced abortuses were karyotyped. Chromosomal abnormalities were found in 5 percent of the 3,237 specimens that included both complete and incomplete tissues and in 1.1 percent of 3,816 specimens with complete tissue specimens alone. It is likely that the incomplete specimens contained a significant number of “blighted ova,” either with no embryo or with a stunted embryo.

births, 5.2 percent for neonatal deaths in the first 7 days after birth and 3.4 percent for neonatal deaths between 8 and 28 days. The most common abnormalities reported were trisomies 18, 13, and 21, as well as sex chromosome aneuploidies and unbalanced translocations. These frequencies of chromosomal abnormality in stillbirths and neonatal deaths are approximately 10 times higher than those in newborns.

Data from stillbirths and neonatal deaths Stillbirth is defined as the birth of a dead fetus during the late second or the third trimester of pregnancy (gestational age >20 weeks), whereas neonatal death refers to death occurring within the first 4 weeks after birth. To provide adequate counseling for parents, all cases of stillbirth and neonatal death must be properly investigated. Thus, cytogenetic evaluation has become an important component of perinatal autopsy (see Chapter 1). In a combined group of stillbirths and neonatal deaths, 160 (6.8 percent) of 2,344 karyotyped cases were found to have a major chromosomal abnormality70–76 (Table 6.8). The average frequency of abnormal karyotypes was 9.5 percent for macerated stillbirths, 6.2 percent for non-macerated still-

Amniocentesis is considered an invasive procedure involving a risk of fetal loss (see Chapter 2) and is usually offered only to women with an increased risk of having an affected child. Many women will receive both first- and/or second-trimester screening, fetal ultrasound evaluation or may have other risk factors. Individual patients may therefore have multiple indications for prenatal cytogenetic diagnosis. These indications are discussed separately below.

Table 6.8 The frequency of chromosomal abnormalities in stillbirths and neonatal deaths Number karyotyped

Abnormal Number

Percentage

Stillbirths Macerated

369

35

Nonmacerated

693

43

6.2

85

16

18.8

1,147

94

8.2

Early (0–7 days)

1,018

53

5.2

Late (8–28 days)

147

5

3.4

32

8

25.0

1,197

66

5.5

Unspecified Total

9.5

Neonatal deaths

Unspecified Total

Data from references 70–76.

Indications for prenatal cytogenetic diagnosis

Advanced maternal age The data from livebirths, genetic amniocenteses, and spontaneous abortions all demonstrate the close association between advanced maternal age and risk for fetal autosomal trisomies and trisomies involving sex chromosomes (except XYY). Prior to the widespread availability of prenatal screening using maternal serum and ultrasound markers, it became standard practice to offer amniocentesis to all advanced maternal age women. Based on the increasing risks for fetal aneuploidy, the amniocentesis procedure-related risk for fetal loss, and the availability of cytogenetic laboratory resources, advanced maternal age was usually defined as 35 years or more (corresponding to a second-trimester risk for DS of approximately 1 : 270 or more). The most recent American College of Obstetrics and Gynecology (ACOG) and American College of Medical Genetics (ACMG) guidelines77,78 recommend the availability of prenatal screening and diagnosis for women of all ages. Serum and ultrasound screening tests do not identify all ageassociated aneuploidies. Moreover, the association between maternal age and fetal chromosomal abnormality can be a source of considerable

CHAP T E R 6

Prenatal Diagnosis of Chromosomal Abnormalities through Amniocentesis

anxiety, and a negative screening result is sometimes not sufficiently reassuring. Significant numbers of amniocenteses are therefore still performed on the basis of maternal age alone. First-trimester screening for aneuploidy The combination of maternal age, maternal serum concentration of pregnancy-associated plasma protein-A (PAPP-A) , human chorionic gonadotropin (hCG), ultrasound measurement of the fetal nuchal translucency (NT) and sometimes other ultrasound markers provide an effective screening for fetal Down syndrome (see Chapter 24). These same markers can also be used to screen for trisomy 18 and the algorithm can be extended to trisomy 13. Although the screening result is available in the first trimester, many women will reject CVS and opt for second-trimester amniocentesis. Different screening programs choose different criteria to identify their high-risk (screen-positive) groups and results may be quoted on the basis of firsttrimester, second-trimester or term risk. Individual patients should be presented with their risk figure, regardless of whether they are screen positive or negative, so they can make their own determination on whether they wish to pursue invasive testing.77 Table 6.9 summarizes the pattern of markers typically seen for various fetal chromosome abnormalities.79 Although not formally part of the screening algorithm, first-trimester screening will identify many cases of 45,X because these pregnancies frequently show increased NT, low PAPP-A but usually normal hCG.80 The screening also preferentially identifies triploid pregnancies with molar cases showing increased NT, elevated hCG, and mildly decreased PAPP-A while nonmolar triploid pregnancies generally have normal/low NT, very low hCG and PAPPA.81 Spencer et al.80 also suggested that first-trimester screening also helped identify 47,XXX, 47,XXY and 47,XYY but their study probably had incomplete ascertainment of screen-negative sex chromosome aneuploidies. Increased NT hs been reported to be present in fetuses with an extremely broad range of disorders and syndromes, including congenital cardiac defects82 (see Chapter 25). A significant number of congenital cardiac defects are associated with 22q11.2 microdeletions and therefore it is not sur-

201

prising that there are case reports of 22q11.2 microdeletions found in fetuses with increased NT.83,84 In euploid fetuses with NT ≥3.5 mm, the prevalence of major cardiac defect is expected to be 78.4/1,00085 and of those with cardiac defects, approximately 3 percent are likely attributable to 22q11.2 deletion.86 Therefore, only 2.4/1,000 cases with NT ≥3.5 mm will be expected have the deletion. Consistent with this, Hollis et al.87 failed to identify any such deletions by FISH in a series of 75 cases with NT ≥3.5 mm and they concluded that routine FISH analysis was of limited value. However, it has been suggested that it may be appropriate to perform FISH on stored cells if there is an abnormal fetal echocardiogram later in pregnancy.83,84,87 Currently, saving cells for possible follow-up FISH testing later in pregnancy is not a routine practice. Additional data are needed to determine what degree of NT enlargement or other clinical criteria would justify the additional cell culture and handling that would be required for routinely enabling this contingent FISH testing for 22q11.2 microdeletions. Second-trimester maternal serum screening for aneuploidy Second-trimester screening for DS and trisomy 18 is discussed in Chapter 24. Screening reports provide risks for DS and trisomy 18 with different patterns of markers characterizing these aneuplodies (see Table 6.9). Although some trisomy 13-affected pregnancies will be identified because of low uE3 and elevated MS-AFP (when neural tube defects (NTDs), omphalocele or urogenital defects are present), most cases of trisomy 13 are not detected.88 Many cases of 45,X in which fetal hydrops or cystic hygromas are present are picked up through second-trimester DS screening while some nonedematous cases will be screen positive for trisomy 18.89,90 Triploid pregnancies show bimodal serum marker patterns with most partial molar cases characterized by very high hCG and INH-A, high MS-AFP and low uE3 and nonmolar cases usually showing low or very low levels of all four serum markers.91 Microdeletions in Xp22, specifically of the steroid sulfatase (STS) gene, are identified because maternal serum uE3 levels are essentially undetectable in these cases.92 STS deficiency is present in approximately 1 in 1,500–3,000 males causing

202

Genetic Disorders and the Fetus

Table 6.9 Typical prenatal screening marker patterns for some specific chromosome abnormalities Trimester

Chromosome abnormality

Marker pattern

First

+21

↑ NT, ↓ PAPP-A, ↑ hCG

+18

↑ NT, ↓ PAPP-A, ↓ hCG

+13

↑ NT, ↓ PAPP-A, ↓ hCG

45,X

↑ NT, ↓ PAPP-A, ↔ hCG

3n

↑ NT, ↓ PAPP-A, ↑↑ hCG, or ↔ NT, ↓↓ PAPP-A, ↓↓ hCG

XXX, XXY, XYY

↑ NT (?), ↔ PAPP-A, ↔ hCG

Second

+21

↓ AFP, ↓ uE3, ↑ hCG, ↑ INH-A

+18

↓ AFP, ↓ uE3, ↓ hCG

+13

↔ AFP, ↓ uE3, ↔ hCG

45,X

↑ AFP, ↓ uE3, ↑ hCG or ↓ AFP, ↓ uE3, ↓ hCG

3n

↑ AFP, ↓ uE3, ↑↑ hCG, ↑↑ INH-A or ↓ AFP, ↓↓ uE3, ↓↓ hCG, ↓↓ INH-A

del(X)(p22)

uE3 = 0

+16 mosaicism

↑ AFP, ↓ uE3, ↑↑ hCG, ↑↑ INH-A

XXX, XXY, XYY

↔ AFP, ↔ uE3, ↔ hCG

↑ denotes elevation of the marker in an affected pregnancy, ↓ reduction, and ↔ levels similar to that in unaffected pregnancies. Two arrows denote very large departures from normal.

X-linked ichthyosis and perhaps learning disabilities.93,94 In a small proportion of cases the deletion can involve genes contiguous with STS and this can include genes associated with mental retardation and Kallman syndrome.95,96 For these cases, additional testing using molecular approaches or FISH with probes for STS and perhaps additional loci may be indicated. Most STS deletions are inherited from the mother and therefore FISH analysis of maternal lymphocytes may be an option (instead of amniocentesis) for some women who show undetectable uE3 on serum screening and have otherwise normal screening results. Some second-trimester maternal serum screening programs include a protocol for identifying pregnancies at high risk for Smith–Lemi–Opitz syndrome (SLOS).97 This screening is also based on MS-AFP, hCG and uE3. The Xp22 microdeletions are preferentially identified through that protocol because low uE3 is a key characteristic marker in the SLOS algorithm.98,99 In addition to Xp22 microdeletions, the SLOS screening also preferentially identifies trisomy 13, 18, 21, triploidy, unbalanced karyotypes, fetal death, other steroid defects and a variety of fetal anatomic abnormalities.99 There are other rare chromosome abnormalities that may be preferentially identified through

second-trimester serum screening. Trisomy 16 mosaic pregnancies show very high hCG and INH-A, often with moderately elevated MS-AFP but low uE3.100,101 Other chromosome abnormalities that are associated with disturbed placental function, growth restriction or some fetal malformations might result in abnormal maternal serum markers, such as trisomy 20 mosaicism,102 trisomy 9 mosaicism,103 and 22q11.2 microdeletion.104 Second-trimester screening will not preferentially identify 47,XXX, 47XXY, or 47,XYY over and above that expected on the basis of maternal age alone. Elevated maternal serum α-fetoprotein In the second trimester, the MS-AFP test is used to screen for NTDs (see Chapter 23) and prior to the widespread availability of targeted ultrasound to identify fetal abnormalities, many amniocenteses were performed to help rule out the presence of an NTD or other fetal anatomic abnormality. A normal ultrasound now generally provides a high level of reassurance. In a large study, Feuchtbaum et al.105 reported a twofold increased prevalence of fetal chromosomal abnormalities in the pregnancies of women who had “unexplained” elevated MS-AFP greater or equal to 2.5 MoM, relative to an unmatched popu-

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Prenatal Diagnosis of Chromosomal Abnormalities through Amniocentesis

lation prevalence. In that study, “unexplained” MS-AFP referred to cases in which this serum protein was elevated and the result was not attributable to the presence of a ventral wall or NTD. No significant excess was found when a cut-off of 2.0 MoM was used. The excess cases using the 2.5 MoM were mostly autosomal aneuploidies or triploidy and it is likely that many of these would have been associated with fetal anomalies identifiable by ultrasound. The excess risk for a serious chromosomal abnormality in women with unexplained (elevated) MS-AFP and normal ultrasound findings is therefore likely to be minimal. For cases in which an anomaly is identified by ultrasound, amniocentesis should be considered. Abnormal ultrasound findings A large number of studies have evaluated the risk for a chromosomal abnormality associated with the ultrasound identification of a fetal anomaly. Generally, the sonographic identification of anomalies is confined to a gestational age of 10–22 weeks, and the identification of some anomalies can be somewhat subjective in nature. Studies associating specific ultrasound findings with aneuploidy are often based on high-risk populations referred for ultrasound examinations because of maternal age, positive serum screening results or other concerns. Therefore, the risk figures presented in Table 6.10 should be considered crude estimates. They do, however, provide some indication of the magnitude of risk together with a guide to the most common chromosomal abnormalities seen. In Table 6.11 the results from four large studies documenting the cytogenetic abnormalities in cases with abnormal ultrasound findings are presented (see Chapter 25). Very high risk for fetal aneuploidy (>35 percent) Some of the highest rates of cytogenetic abnormality are to be found in AF specimens from pregnancies complicated by fetal cystic hygromas and nonimmune hydrops. Cystic hygromas are fluid accumulations in the lymphatics and are frequently associated with excess fluid in other tissues (nonimmune hydrops). Among second-trimester fetuses with cystic hygromas, only 37 percent show a normal karyotype.125 A 45,X karyotype is observed

203

in 43 percent of these cases; other abnormalities, including trisomies 21, 18, and 13, make up the remainder. Malone et al.126 found that 51 percent of first-trimester fetuses with cystic hygromas had abnormal kartyotypes; only 17 percent of fetuses with cystic hygromas survived and had a normal pregnancy outcome. Cystic hygromas may also occur in other genetic disorders such as Noonan syndrome. Studies of fetuses with cystic hygroma previously suggested a Noonan syndrome prevalence of 1–3 percent. A recent study by Lee et al.126a reported PTPN11 mutations (∼50 percent detection in Noonan syndrome) in 11 percent of fetuses in their series with isolated cystic hygromas. As prenatal Noonan syndrome molecular testing is clinically available and has significant prognostic and genetic counseling implications, this testing may be considered in those cases with a normal karyotype. A distinction has been drawn between cystic hygroma (bilateral, septated, cystic structures) and nuchal edema (subcutaneous fluid accumulation).155 Nuchal edema is visualized on firsttrimester ultrasonography as an increased nuchal translucency, and this finding can also be associated with a very high risk for fetal chromosomal abnormality.155 In the second trimester, distension of the nuchal skinfold also provides a marker for chromosomal abnormality.145 For both first- and second-trimester nuchal measurements, the extent of the enlargement can be combined with serum screening results and some other ultrasound findings to revise the maternal age-specific risk for aneuploidy for individual patients.156,157 Cardiac defects are among the most commonly encountered congenital anomalies.158,159 Approximately 19–48 percent of cases that would be apparent at birth might be detected prenatally through routine ultrasound screening,160 and a chromosomal abnormality is the cause in approximately 40 percent of the prenatally identified cases.115 The specific types of heart defects that are present in the common aneuploidies has been reviewed by Yates,161 and a list of the risks associated with specific cardiac defects has been developed by Allan et al.162 First-trimester markers associated with cardiovascular abnormality, and therefore aneuploidy, include tricuspid regurgitation120,121 and abnormal blood flow through the

204

Genetic Disorders and the Fetus

Table 6.10 Frequency and types of chromosome abnormalities in fetuses with ultrasound-detected fetal anomalies Anomaly identified by ultrasounda

Risk (%)b

Common chromosome

References

abnormalitiesd Abdominal wall defect Gastroschisis

0–2

None

Hunter and Soothill; 2002106 Stoll et al. 2008.107

Omphalocele

4.5–35

+18; +13; +21; 45,X; 3n; t(11p15.5)mat;

Kilby et al. 1998108 Stoll et al. 2008.107

dup(11p15.5)pat Agenesis of corpus callosum

10

+8; +13; +18; other

Gupta and Lilford, 1995.109

Choroid plexus cyst, isolated

0.7–3.3

+18; +21; 45,X; other

Gupta et al. 1995;110

Choroid plexus cyst, complex

3.6–12

+18; +21; 3n; 45,X

Beke et al. 2006.111 Gupta et al. 1995;110 Beke et al. 2006.111 Cleft lip, +/− cleft palate

21.6

+13; +18; del; +21; 3n; other

Clementi et al. 2000.112

Cleft palate

30.8

+13; +18; del; +21; 3n; other

Clementi et al. 2000.112

Club foot, isolated

3.4

various

Shipp and Banacerraf 1998;113 Malone et al. 2000.114

Cardiovascular anomalies Structural anomalies

40

+21; +18; +13; 45,X; other;

Stoll et al. 2001115

del(22)(q11.2q11.2) Echogenic focus, isolated

1.5–2.0

+21, others

Huggon et al. 2001;116 Sotiriadis et al. 2003117

Echogenic focus, complex

5–10

+21; +13; others

Bromley et al. 1998;118 Vibhakar et al.1999;119 Sotiriadis et al. 2003117

Tricuspid regurgitation

63

+21; +18; +13; 45,X; other

Ductus venosus blood flow

43

+21, +18, +13, other

Faiola et al. 2005;120 Falcon et al; 2006121 Borrell et al. 2003;122 Borrell 2003;123 Sonek 2006124

Cystic hygroma (1st trimester)

63

45,X; +21; +18; +13; other

Gallagher et al. 1999;125

Cystic hygroma (2nd trimester)

51

+21, 45,X, +18, +13, 3n

Malone et al. 2005126

Dandy–Walker malformation

60

3n; +18; +13; translocations

Ecker et al. 2000;127 Köble et al. 2000.128

Diaphragmatic hernia, complex

9.5

+18; +13; del(q36), +i(12p);

Duodenal atresia

33

+21

4n/2n

Witters et al. 2001;129 Klaassens et al. 2007130 Nicolaides et al 1992;131 Halliday et al; 1994;132 Hanna et al. 1996;133 Rizzo et al. 1996.134

Echogenic bowel

3–25

+21;3n;+18; 45,X; +13;

Penna and Bower 2000.135

others Femur, humerus, short

20 (c)

+21; +18;

Nyberg et al.136

Holoprosencephaly

55

+13; +18; del(13q); del(18p);

Peebles 1998.137

Hydrocephaly/ventriculomegaly

16

+21; +18; 3n; other

del(7q); other Nicolaides et al 1992;131 Halliday et al; 1994;132 Hanna et al. 1996.133

CHAP T E R 6

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205

Table 6.10 Continued Anomaly identified by ultrasounda

Risk (%)b

Common chromosome

References

abnormalitiesd IUGR

20

+18, 3n; +13; other; +21

Nicolaides et al 1992;131 Halliday et al;132 1994; Hanna et al 1996.133

Microcephaly

23

+13; del(7q34); +8 mos

Den Hollander et al. 2000.138

Nasal bone absence (1st trimester)

53

+21, +18, +13, 45,X, other

Cicero et al. 2006139

Nasal bone absence (2nd

71

+21

Sonek et al. 2006;140 Gianferrari et al.141

trimester) Neural tube defect, isolated

2.4

various

Neural tube defect, complex

6.5

+18; other

Kennedy et al. 1998.142 Kennedy et al. 1998.142 Sepulveda et al. 2004.143 Snijders et al.144

Nuchal translucency (1st trimester)

35 (c)

+21; +18; 45,X; +13; 3n;

Nuchal fold (2nd trimester)

40 (c)

+21; +18; 3n;

Benacerraf145

Oligohydramnios

14

3n; +13; other

Halliday et al. 1994;132 Hanna

other

et al. 1996.133 Polyhydramnios

12

+18; +21; +13; other

Halliday et al. 1994;132 Hanna

Pleural effusion

35

45,X; +21; +18; other

Waller et al. 2005146

Teratoma

nk

dup(1q)

Wax et al. 2000.147

Tetraphocomelia

nk

PCS

Van den Berg and Francke

Tracheo-esophageal fistula/

63

+18,+21; other

Nicolaides et al 1992;131 Hanna

et al. 1996.133

1993.148 et al 1996;133 Rizzo et al.

esophageal atresia

1996.134 Two-vessel cord, complex

5.5

+13; +18; other

Saller et al. 1990;149 Hanna et al. 1996.133

Urogenital anomalies Renal structural defect

nk

+18; +13; 45,X; 3n; +9 mos;

Wellesley and Howe 2001.150

del(10q); del(18q); del(22) (q11.2q11.2) Hydronephrosis/multicystic

12

+21; +18; +13; del; 45,X, del

kidneys Pyelectasis, isolated

(22)(q11.2q11.2) other 1.8

+21; other

Nicolaides et al. 1992;151 Wellesley and Howe 2001.150 Corteville et al. 1992;152 Wickstrom et al. 1996;153 Chudleigh et al. 2001.154

a

Complex and isolated anomalies are defined as with, or without, other abnormal ultrasound findings.

b

Percentage of cases with a chromosome abnormality.

c

Risks presented are based on fixed cut-offs to define presence or absence of the marker. Patient-specific risks are also

available. d

Listed in the approximate order in which the abnormalities might be encountered.

3n, triploidy; 4n/2n, tetraploid mosaicism; nk, not known; PCS, premature chromatid separation (diagnostic for Roberts syndrome, SC phocomelia syndrome).

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Genetic Disorders and the Fetus

Table 6.11 Ultrasound abnormalities and frequency of fetal aneuploidy Defect

Nicolaides et al. 1992131

Halliday

Hanna et al.

Rizzo et al.

Overall

et al. 1994132

1996133

1996134

frequencya

Isolated

Multiple

Isolated

Primary U/S

Primary U/S

No. Aneupl/

No. Aneupl/

No. Aneupl/

No. Aneupl/

Abn. No.

Abn. No.

Total (%)

Total (%)

Total (%)

Total (%)

Aneupl/

Aneupl/

Total (%)

Total (%)

Abdominal wall defect

1/30

41/86 (48)

3/45 (7)

38/196 (19)

7/16l (44)

90/373 (24)

Agenesis of corpus

–

–

–

0/2 (0)

8/19 (42)

8/21 (38)

1/49

33/72 (46)

0/21 (−)

21/514 (4)

–

55/656 (8)

Unspecified

0/4

101/152 (66)

8/42 (19)

10/60 (17)

20/34 (59)

Ventricular septal defect

–

–

–

8/21 (38)

9/13 (69)

Atrioventricular canal

–

–

–

2/2 (100)

8/11 (82)

Cystic hygroma

0/4

35/45 (73)

11/21 (52)

65/108 (60)

22/33 (67)

133/211 (63)

Diaphragmatic hernia

0/38

17/41 (41)

2/17 (12)

8/72 (11)

2/5 (40)

29/173 (17)

Duodenal atresia

1/6

9/17 (53)

3/10 (30)

10/45 (22)

8/15 (53)

31/93 (33)

Echogenic bowel

–

–

–

5/34 (15)

–

5/34 (15)

Facial cleft

0/8

31/56 (55)

1/7 (14)

–

3/11 (28)

35/82 (43)

Holoprosencephaly

0/7

15/51 (29)

3/9 (33)

9/19 (47)

6/12 (50)

33/98 (34)

Hydrocephaly

2/42

40/144 (28)

7/30 (23)

25/256 (9)

–

74/472 (16)

Hydronephrosis

–

–

–

8/110 (7)

–

8/110 (7)

Hydrops (nonimmune)

7/104

18/106 (17)

23/57 (40)

37/116 (32)

6/17 (35)

91/400 (22)

IUGR

4/251

133/424 (31)

8/37 (22)

71/389 (18)

–

216/1101 (20)

Limb anomalies

0/18

195/457 (43)

4/29 (14)

3/39 (8)

3/6 (50)b

205/549 (37)

callosum Choroid plexus cyst Congenital heart disease

166/339 (49)

Microcephaly

0/1

8/51 (16)

0/1 (0)

5/28 (18)

–

13/81 (16)

NTDc

–

–

1/33 (3)

4/57 (7)

2/6 (33)

7/96 (7)

Nuchal fold/thickness/

0/12

53/132 (40)

5/21 (24)

15/75 (20)

–

73/240 (30)

Oligohydramnios

–

–

1/14 (7)

14/97 (14)

–

15/111 (14)

Polyhydramnios

–

–

2/9 (22)

23/194 (12)

–

25/203 (12)

Renal anomalies

9/482

87/360 (24)

3/29 (10)

7/107 (7)

–

106/978 (11)

TF/EA

0/1

18/23 (78)

–

4/10 (40)

3/6 (50)

25/40 (63)

Two-vessel cord

–

–

–

5/72 (6)

–

5/72 (7)

edema

a

Combined isolated and/or multiple ultrasound abnormalities.

b

Club feet.

c

NTD excluding anencephaly and meningomyelocele.

U/S, ultrasound; Abn, abnormality; No. Aneupl/Total, number of aneuploidy cases divided by total cases with the abnormality; TF/EA, Tracheo-esophageal fistula/esophageal atresia.

ductus venosus identifiable by pulsed-wave Doppler ultrasonography.122–4 The association between tetralogy of Fallot, double-outlet right ventricle (DORV), and other conotruncal abnormalities with the deletion of 22q11 (DiGeorge/velocardiofacial syndrome) is noteworthy. The types of cardiac defects found with 22q11 deletion may not be limited to

conotruncal defects, and Manji et al.163 proposed that FISH testing using a 22q11 probe be carried out for all cases with prenatally detected heart defects (except hypoplastic left heart and echogenic focus). Moore et al.86 found 17 (3 percent) deletions of 22q11.2 by FISH among 540 fetuses with cardiac defects and apparently normal karyotypes by routine chromosome analysis.

CHAP T E R 6

Prenatal Diagnosis of Chromosomal Abnormalities through Amniocentesis

Other abnormalities identifiable on ultrasound associated with very high risk for aneuploidy are tracheoesophageal fistula/esophageal atresia, Dandy–Walker malformation, holoprosencephaly and pleural effusion.127 High risk for fetal aneuploidy (20–35 percent) A common purpose for amniocentesis is to identify the cause and full significance of intrauterine growth restriction (IUGR) identified by ultrasound. IUGR, in the absence of any other biochemical or screening tests, will occasionally signal the presence of pregnancies affected by trisomies 13 and 18, but the finding is not a strong indicator for trisomy 21. More severe IUGR is associated with an even greater chance for aneuploidy.164 Combined data from three large series131–133 suggest an overall risk of 20 percent for a cytogenetic abnormality in cases with IUGR. A broad range of abnormal karyotypes is possible. Comparable levels of risk are associated with an ultrasound finding of microcephaly. Other anomalies that can be considered to be associated with a high risk for fetal aneuploidy include facial clefts, duodenal atresia (“double bubble” anomaly), some limb anomalies, and omphalocele (but not gastroschisis) (see Chapter 25). There is also a high risk for aneuploidy when femur length, humerus length, or both, are shorter than that expected for the gestational age.136 These biometric measurements can be combined with serum screening tests and nuchal fold measurement to modify individual patient’s risk for aneuploidy.157 Moderate risk for fetal aneuploidy (10–19 percent) Hyperechogenic bowel is often a nonspecific finding seen in some fetuses with intestinal obstruction and meconium ileus secondary to cystic fibrosis.135 However, it may also be an indicator for fetal DS or other chromosomal abnormality. Estimates for the risk for fetal aneuploidy when “echogenic bowel” is observed have been somewhat variable, probably reflecting the variable criteria used to define hyperechogenicity and ascertainment bias. Moderate risks for fetal aneuploidy can also be assigned when there is ultrasound detection of

207

renal anomalies (including hydronephrosis), oligohydramnios or polyhydramnios, hydrocephaly/ ventriculomegaly, and diaphragmatic hernia (in association with other anomalies). Low risk for fetal aneuploidy (35), the clinical sensitivity of this test approaches 80 percent but is reduced for all prenatal patients to approximately 65–70 percent. Increased use of FISH as an initial evaluation would need either the cost of this test to be significantly lower or technical enhancements to the available probe sets. They also indicated that decisions based on prenatal testing should be accompanied by two of the following three pieces of information: (1) FISH results, (2) routine chromosome analysis, and (3) clinical information. While interphase FISH is utilized as a standard technique in clinical laboratories, the technologies utilized continue to change and evolve. One such technologic shift is the use of quantitative PCR (QF-PCR) as opposed to FISH for rapid analysis of AF. Leung et al. summarized this technology along with a number of studies utilizing QF-PCR.193 While this is a viable alternative, most laboratories still utilize interphase FISH. With respect to interphase FISH, different technologic changes have been proposed or implemented. Rather than utilizing standard hybridization, Gadji et al. have successfully used multicolor primed in situ labeling (PRINS) as an alternative approach.194 FISH has also been optimized for interphase analysis to allow results within 2 hours of collection rather than the typical 24 hours.195 Advances have also been made in the automation of the FISH protocol

Molecular Cytogenetics and Prenatal Diagnosis 333

and scanning of slides.196,197 For laboratories doing large numbers of analyses, this will make the process more efficient. As discussed earlier, in addition to the standard interphase analysis of aneuplodies, involving chromosomes 13, 18, 21, X and Y, interphase analysis is also routinely used for detection of the 22q11.2 microdeletion, as well as other chromosomes. Interphase FISH is also utilized for rapid prenatal diagnosis in translocation carriers using chromosome-specific subtelomeric probes. Unbalanced derivative chromosomes can be inferred by the detection of both deleted and duplicated regions of the chromosomes involved in the translocation.198

Chorionic villus samples Interphase FISH studies of noncultivated CVS have been reported less frequently than those with AF samples, possibly because it is relatively easy to obtain a complete karyotype analysis on direct CVS preparations within 24 hours of obtaining a specimen. Most of the reported interphase studies done on CVS have been small in both scope and limited number of patients analyzed (Table 8.4).199–207 Through 1995, approximately 60 or fewer patients had been examined in each study. These studies have demonstrated the feasibility of interphase FISH on direct CVS cells for rapid analysis. In 1996, Bryndorf et al. reported a study of 2,709 noncultivated CVS with interphase FISH.203 Their novel approach was to examine, with interphase FISH, noncultivated mesenchymal chorionic villus cells within 24 hours rather than using conventional chromosome analysis on cytotrophoblasts during the same time interval. This approach was taken because studies have shown that, although direct studies of cytotrophoblasts can be done in 24 hours, there is a 1–2 percent false-positive rate and a 0.04 percent false-negative rate associated with this approach.208 Bryndorf et al. also found that the technician time required by the two protocols was similar, taking about 1 hour. This study revealed that, on average, 99 percent of the nuclei had a hybridization pattern consistent with the sex of the fetus and that 94 percent of the nuclei demonstrated the appropriate pattern for fetuses with sex chromosome abnormalities. Cases needed to have >45 nuclei scored,

334

Genetic Disorders and the Fetus

Table 8.4 Selected interphase prenatal diagnosis by FISH: CVS studies Reference

Number studied 199

Evans et al.

Bryndorf et al.203

49

Number abnormala 1

2,709b

80

Cai et al.204

239

3

Quilter et al.205

100

12

Comments 10% failure rate 94% technically successful Successful in high-risk pregnancies 100% positive and negative predictive value FISH an accurate and less labor-intensive alternative to direct chromosome analysis of CVS

Fiddler et al.206

–

Useful and rapid fetal assessment before decisions about fetal reduction

Goumy et al.207

32

13

Emphasizes that interphase FISH is of great benefit in cases of known parental translocations and when hygroma is detected by ultrasonography

a

Detected by FISH.

b

Includes 39 abnormal placental specimens.

with an abnormal number of hybridization signals seen in >60 percent of the cells to permit an abnormal diagnosis to be made. An informative disomic sample was defined as having three signals in 1 kb. Different tissues can be studied involving the analysis of metaphase chromosomes or undivided interphase cells. For constitutional studies, FISH is extensively used with metaphase chromosomes to define structurally abnormal chromosomes. This can

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involve subtle deletions, duplications or cryptic rearrangement of chromosomal material. FISH is frequently used for the detection of microdeletions associated with contiguous gene syndromes. It has also been used extensively to define and characterize extrachromosomal material, whether present as interchromosomal or intrachromosomal duplications or supernumerary marker chromosomes. The appropriate probes for these studies include α-satellite DNA chromosome libraries and/or single-copy probes. In prenatal studies, FISH is also commonly used to study interphase cells in which metaphase chromosomes are not available for study, especially by design. The most frequent use has been in the “direct analysis” of noncultivated AF interphase cells for the rapid prenatal diagnosis of aneuploidy. The utilization of FISH will continue to decrease, in the future, due to the development of new technologies, such as array analysis. However, it still remains a robust technology to use in many circumstances, especially in cases where a directed diagnostic analysis can be undertaken.

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9

Prenatal Diagnosis and the Spectrum of Involvement from Fragile X Mutations Randi Hagerman,1 Vivien Narcisa1 and Paul Hagerman2 1

MIND Institute, University of California Davis, Sacramento, CA, USA, Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, USA

Introduction Mutations of the fragile X mental retardation 1 (FMR1) gene, including both premutation (55– 200 repeats) and full mutation (>200 repeats) CGG-repeat expansions, give rise to a broad spectrum of cognitive impairment ranging from mental retardation (now termed intellectual disability – ID) and autism to mild learning or emotional difficulties in the context of normal IQ. In addition, there arise late adult-onset neurological, cognitive, psychiatric and medical problems in some older premutation carriers. Clinical involvement in individuals with the full mutation (fragile X syndrome – FXS) is a consequence of transcriptional silencing of the gene and the resulting deficiency or absence of the FMR1 protein (FMRP), an RNA-binding protein that transports and regulates the translation of many messages into their respective proteins. The absence of FMRP leads in turn to dysregulation of a number of proteins that are important for synaptic maturation and plasticity.1 Since FMRP specifically downregulates the translation of a number of postsynaptic proteins, there is a significant upregulation of the production of these proteins in

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

the absence of FMRP.2 One important pathway that is upregulated in the absence of FMRP is the metabotropic glutamate receptor 5 (mGluR5) system, resulting in long-term depression (LTD) of synaptic activity and weakening of synaptic connections.3–5 The associated neuroanatomic phenotype includes long, thin (“immature”) synaptic connections, which are thought to be the cause of the ID in FXS. Recent research has lead to a number of treatment trials for FXS using various mGluR5 antagonists, which have been shown to reverse at least some of the neuroanatomic and clinical phenotype of FXS in the animal models.6,7 For CGG-repeat expansions in the premutation range, both clinical involvement and its pathogenesis (elevated FMR1 mRNA) are quite distinct from the FMRP-deficit model for full mutation alleles and FXS, although for alleles in the upper end of the premutation range, there may be participation of more than one pathogenic mechanism. The molecular pathogenesis for premutation-associated clinical involvement involves a toxic gain-of-function of the expanded-repeat FMR1 mRNA,8 which is produced at elevated levels in the premutation range9 in stark contrast to the reduced/absent levels of FMR1 mRNA in the full mutation range. Although most individuals with the premutation possess normal intellectual abilities, some do experience developmental problems that include attention deficit hyperactivity disorder (ADHD)

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Genetic Disorders and the Fetus

and/or social deficits ranging from social anxiety to autism spectrum disorder (ASD).10–13 These problems are more common in males (both children and adults) than in females, though the psychiatric problems, including anxiety and depression, are common in women.14 In addition, some adults with the premutation may develop clinical symptoms related to fragile X-associated tremor/ataxia syndrome (FXTAS),15,16 including neuropathy,17,18 autoimmune problems such as fibromyalgia and hypothyroidism,19 emotional difficulties including depression and anxiety14,20 and dementia.21,22 The abnormal mRNA triggers a cascade of events in neural cells that ultimately leads to clinical involvement described below. The complexity of clinical involvement and treatment, including the emerging targeted treatments that are becoming available for fragile X-related disorders, complicates the genetic counseling aspects of these disorders. This chapter examines the epidemiology, clinical involvement, genetic counseling, prenatal diagnostic procedures and treatment in the fragile X spectrum disorders.

Epidemiology One of the principal shortcomings at present is the paucity of quantitative information regarding the frequencies of allele expansion classes (premutation and full mutation) in the general population, or whether such numbers are even meaningful when comparing different ethnic or geographic populations. Furthermore, although FXS is generally regarded as the most common inherited form of cognitive impairment,23–25 there is little agreement as to either its prevalence or sex-specific differences in prevalence in the general population. Prevalence estimates are almost always based on population projections from groups of children with special education needs26 and as such, generally underestimate the number of individuals in the general population who have some degree of clinical involvement. This issue is particularly true for those without significant declines in IQ but who have other behavioral or more focal learning or socialization difficulties,27,28 as is the case for girls, most of whom have IQs that are in the normal range.27,29 Since the frequencies of expanded FMR1 alleles in a given population will strictly

define the upper limit for disease prevalence, knowledge of the true frequencies is a critical objective in epidemiologic studies. Such frequency information will also allow a more careful assessment of the range of involvement in FXS, since the disparity between frequency and prevalence may uncover more subtle extensions of the phenotype, features not identified through such basic measures as IQ. In a recent commentary on the issue of frequency estimates for expanded FMR1 alleles, Hagerman25 utilized data for the frequencies of premutation alleles from some of the more recent epidemiologic studies, particularly those performed in Israel (1/113,30 1/15231). (For a comprehensive review of epidemiologic studies prior to 2004, see Song et al.26) From other premutation data,30,31 the expected (average) frequency for full mutation males and females was estimated to be ∼1/2,400, remarkably close to the value (1/2,365) observed by Pesso et al.30 The expected frequency for premutation males was estimated to be ∼1/290. One important correlate to the frequency estimate for male carriers is that as many as ∼1/3,000 males over 50 years in the general population may suffer from FXTAS. Separate studies in Eastern Canada32,33 obtained lower frequencies for both female (1/259) and male (1/813) carriers. These latter results raise the possibility that founder effects contribute to the approximately twofold difference in absolute allele frequencies between the Canadian and Israeli populations. The studies cited above underscore several points.25 • The frequencies of premutation alleles in any given population, particularly those of females, are likely to represent the best metric of variation (e.g. founder effects) across populations, since they can be estimated with much greater accuracy than full mutation alleles. Based on current genetic models for FXS, the premutation allele frequencies can in turn be used to predict the number of carriers of full mutation alleles in those populations. • Based on the fact that premutation-to-full mutation transmission is strictly matrilineal, one expects approximately equal frequencies of full mutation alleles in males and females. Thus, screens for full mutation alleles in at-risk populations that yield appreciable differences in projected allele preva-

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lence or disease prevalence for males and females are likely to involve significant selection bias. • Observed disparities between allele frequencies and disease prevalence can actually be a useful tool for examining the range of more subtle phenotypes present among carriers of full mutation alleles. In an substantial epidemiologic study, Berkenstadt et al.34 have extended their earlier screening study30 to include a total of 40,079 women (69% tested during pregnancy, 31% preconception). Of this cohort, 36,483 women (91%) had no family history of FXS or associated clinical features. These authors observed a total of 260 carriers for an overall carrier frequency of 1/154; five of these individuals carried full mutation alleles, for an estimated frequency of 1/8,016 for the full mutation carrier frequency. However, four of these alleles were in the group of 3,596 women with a family history, for an overall frequency of 1/899 within that group. These numbers underscore a problem associated with screening based on foreknowledge of clinical involvement or even family history, since such knowledge may result in selection, knowingly or unknowingly, based on such history. A related issue with the screening study is the fact that the women being screened selfselected for participation and were, in addition, responsible for paying for the testing service. Thus, it is possible that either/both self-selection and cost may have biased the cohort being screened. In this regard, it would be instructive to perform a parallel, anonymous newborn screening study in the same population to see if such a selection bias exists. The Berkenstadt et al.34 study made several additional important observations. • There was no significant difference in carrier frequency between the groups defined by presence/ absence of family history (1/128 versus 1/157; p = 0.17), although the premutation expansion size was smaller in the absence of family history (58 versus 61 CGG repeats; p < 0.0005). • For these 260 carriers, and additional carriers identified through separate screening, invasive prenatal diagnosis in a total of 360 subsequent pregnancies (370 fetuses) yielded 30 full mutation pregnancies (8.1%). • Among all carrier pregnancies, the mutated allele was transmitted in 52% of the cases. Although the

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rate of expansion to a full mutation allele was lower in the group without family history, the difference could be accounted for by the smaller allele size distribution in that group.

Clinical involvement in those with the full mutation Most males with FXS have ID with a mean IQ in the 40s,27 although the floor effect of most IQ assessment measures does not accurately score IQs below 45.35 Only approximately 15% of males with FXS will have an IQ above 70, and these individuals have either significant mosaicism (significant percentage of cells with the premutation in addition to the full mutation) or a lack of methylation of their full mutation.36,37 Prenatal diagnosis using chorionic villi cannot reliably determine the methylation status of the fetus because methylation may not set in for the full mutation until later in gestation (see Chapter 5). Therefore, it is impossible to determine the level of cognitive involvement in a fetus with the full mutation beyond knowing the range of scores in the males and females. In females with the full mutation approximately 70% will have an IQ of 85 or lower.28 Although only 25% of girls with the full mutation will have an IQ lower than 70, those with a borderline IQ (70–85) have significant learning problems including executive function deficits, ADHD, language delays, impulsivity, visual spatial perceptual deficits and academic delays, particularly in math.27,38–42 These individuals will require significant interventions during their schooling.43 Approximately 25% of females with the full mutation will have a normal IQ without learning disabilities, although emotional problems, such as anxiety, are still common in this group.44 Some of the normal IQ women with the full mutation will have children with FXS. Overall, the IQ of individuals with FXS correlates with the level of FMRP, which is related to the activation ratio in females and the degree of mosaicism in males.36 However, these correlations were not done in fetuses but instead in children and adults with FXS. There are no studies of FMRP levels from fetuses and it is likely that these would not correlate to levels in the child or adult because of

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Genetic Disorders and the Fetus

changes in methylation.45 Methods used to measure FMRP include the Western blot,46 an immunocytochemical technique,47 hair root staining48 and recently an ELISA technique (Iwahashi et al., in press48a). Psychopathology is common in those with the full mutation and it includes ADHD in approximately 80% of boys and 25% of girls49,50; autism in 30% of boys and 10–20% of girls51–54; autism spectrum disorder (ASD) in 20–30% of boys and 10% of girls54,55; anxiety or mood disorder in 25–70% of boys and girls56; and psychosis in less than 10%.49 Often the behavioral problems, particularly impulsivity, anxiety, mood instability and aggression, cause the main problems for the family in rearing children with FXS. These difficulties often lead to medical intervention and diagnosis, which occur typically between 2.5 and 4 years of age.57,58 There are many medications that can help with these behavioral problems59; however, a multidisciplinary intervention plan is necessary including therapies such as speech and language therapy, occupational therapy, psychotherapy and special education supports.60,61 The physical features of FXS classically include a long face, prominent ears, hyperextensible finger joints, flat feet and macro-orchidism from adolescence onward. Approximately 30% of young children do not have these features so the diagnosis is often based on behavioral features, such as poor eye contact, hand flapping, hand biting, perseverative speech, autistic features, anxiety and ADHD symptoms.62 Many individuals are diagnosed with autism or ASD before the diagnosis of FXS, so all children with ASD should have FMR1 testing.62 The medical problems associated with FXS are relatively few and most are caused by the connective tissue problem that is intrinsic to FXS. The hyperextensible joints on occasion lead to dislocation, but this occurs in less than 5%. Hernias are more common (15%); in males, the weight of the large testicles combined with the loose connective tissue leads to hernias. Recurrent otitis media is the most common medical problem (85%) followed by strabismus (36%) and seizures (20%).49,63 The lack of FMRP leads to a seizure phenotype in the fragile X knock-out mouse that improves with the use of mGluR5 antagonists as a targeted treatment for FXS described below.64

Clinical phenotype in the premutation The premutation was originally thought to have no phenotype and males with the premutation were previously described as nonpenetrant males or normal transmitting males (NTMs). In 1991, premature ovarian failure (POF: menses stopping before age 40) was described in approximately 20% of premutation daughters of these males.65 Subsequent studies have found this problem to increase with increasing CGG repeat numbers in the premutation carriers, but after 120 repeats the prevalence of POF decreases somewhat.66 This problem has been renamed primary ovarian insufficiency (POI) because some women with this diagnosis may subsequently become pregnant. The cause of POI seems to be related to the toxicity of elevated FMR1 mRNA on ovum or the supporting cells of the ovum. For a number of years, psychologic problems in male and female carriers with the premutation have been reported with some controversy.67–69 Finally, with the identification of FXTAS, there was a clear neurologic phenotype involving an intention tremor, ataxia, neuropathy, autonomic dysfunction, and cognitive decline that occurred in some older males and occasional females with the premutation.70 FXTAS occurs in a greater incidence with increasing age such that 15% of males in their 50s but 75% of males in their 80s develop FXTAS.71 In females approximately 8% of carriers older than 50 years develop FXTAS, but their symptoms are less severe and cognitive decline is rare in females.19 Brain atrophy combined with white matter disease is part of the diagnostic criteria in FXTAS,72 with approximately 60% of males demonstrating a characteristic sign of increased T2 signal intensity in the middle cerebellar peduncles (MCP sign). The MCP sign is seen, however, in only 13% of females with FXTAS.73 Recent reports have expanded the phenotype in women with the premutation to include hypothyroidism (50%) and fibromyalgia (40%) in those with neurologic symptoms, suggesting that an autoimmune component occurs in some female carriers. For example, multiple sclerosis (MS) occurs in approximately 2–3% of carriers.19 Multiple sclerosis has been reported to occur in addition

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to FXTAS in one case, documented at autopsy.74 The elevated mRNA in the premutation leads to upregulation of a number of proteins including αB-crystallin, which is a primary antigen for MS.74 There is increasing evidence that a neurodevelopmental disorder is associated with a subgroup of children, particularly boys, with the premutation. Reports of ADHD, social anxiety and ASD are common in boys with the premutation10–12,58,75 and although most have a normal IQ, some have ID.76 A recent report found that premutation neuronal cell cultures demonstrate decreased branching and larger synaptic size compared to neurons without the premutation.77

Pathogenesis of the premutation-associated disorder, fragile X-associated tremor/ataxia syndrome Of the premutation-associated disorders, we understand the most about the pathogenic mechanisms associated with FXTAS; however, it is generally believed that similar mechanisms are at play for both the neurodevelopmental involvement and in the early menopausal features (fragile X-associated primary ovarian insufficiency – FXPOI).78,79 Therefore, the following paragraphs will focus on FXTAS. Neuropathology Gross neuropathologic features of the brains examined post mortem from individuals who had died with FXTAS reveal a general loss of brain volume, with prominent white matter disease involving pallor and spongioform changes accompanied by a loss of axons and myelin. The regions of pallor are associated with regions of high signal on T2-weighted MRIs in the same individuals.73,80–82 The primary neuropathologic finding is the presence of solitary, spherical (1–5 µm) ubiquitin-positive intranuclear inclusions in both neurons and astrocytes in broad distribution throughout the brain.81,82 The greatest concentration of inclusions is found in the hippocampal formation (up to 40% of nuclei bearing inclusions in some cases), with lower inclusion densities (2– 10%) in cortical neurons and the near absence of inclusions in Purkinje cells of the cerebellum,

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despite substantial Purkinje cell dropout. Inclusion counts are highly correlated with the number of CGG repeats within the premutation range.82 More recently, inclusions have also been observed in tissues outside the CNS, including both anterior and posterior pituitary, in the Leydig and myotubular cells of the testes,83 in ganglion cells of the adrenal medulla, dorsal root ganglia, paraspinal sympathetic ganglia, mesenteric ganglia and subepicardial autonomic ganglia.84 Finally, the presence of inclusions in neuronal nuclei within the hypoglossal cranial nerve nucleus may represent a neuropathologic correlate to the late-stage swallowing difficulties experienced by many FXTAS patients.82 Molecular pathogenesis Mounting evidence indicates that the pathogenesis of FXTAS involves a direct “toxic” gain-of-function of the FMR1 mRNA (Figure 9.1); that is, a novel RNA function that triggers the pathogenic pathway leading to FXTAS.70,78,85,86 First, the disorder appears to be confined to carriers of premutation alleles, where the gene is active. This observation indicates that FXTAS is not the result of the loss of FMRP, since FMRP levels are only moderately lowered within the premutation range yet profoundly lowered or absent in the full mutation range. Second, the absence of FXTAS among individuals with large, full mutation alleles (e.g. >500–1,000 repeats) indicates that the CGG repeat expansion, as DNA, is not contributing to disease formation. Third, the FMR1 mRNA is abnormal in at least two important respects: namely, that its production is substantially increased in the premutation range9,46,87 and that it possesses the expanded CGG repeat element. Additionally, several important features of the disorder (neurodegenerative changes and the presence of inclusions) have been recapitulated in both mouse and Drosophila models of FXTAS.88–90 Finally, consistent with the toxic RNA model for myotonic dystrophy,91,92 FMR1 mRNA is present within the intranuclear inclusions of FXTAS.87 One possible distinction between FXTAS and the myotonic dystrophy mechanism involves the role of the proteins that interact with the RNA. For myotonic dystrophy, the noncoding CUG repeat expansions, often exceeding several thousand

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Genetic Disorders and the Fetus

Figure 9.1 RNA “toxicity” model of fragile X-associated tremor/ataxia syndrome (FXTAS). Abnormal accumulation and/or interaction of proteins to the expanded CGG repeat FMR1 RNA is the trigger for FXTAS pathogenesis. Abnormal/excess protein–RNA interactions are thought to lead to cellular dysregulation through either protein

sequestration and solution depletion (e.g. myotonic dystrophy model) or altered activity of the bound proteins, possibly involving altered signaling pathways. The intranuclear neuronal and astrocytic inclusions found in patients with FXTAS contain the FMR1 mRNA and at least 30 proteins.

repeats, appear to operate by sequestering proteins such as MBNL1, thus preventing them from carrying out their normal functions. By contrast, the expansions in FXTAS are modest, with disease evident for expansions that are only two to three times normal. Thus, it is possible that the mechanism of RNA toxicity may involve a trigger/signaling event in which a bound protein signals downstream events. An example of such a protein is the double-stranded RNA protein kinase (PKR; OMIM *176871), which phosphorylates additional proteins (e.g. eIF2α), resulting in the shutdown of cellular protein synthesis; however, Handa et al.93 have presented evidence that PKR itself is not likely to be the transducer of the abnormal FMR1 mRNA. A great deal has been learned about the pathogenesis of FXTAS from the composition of the inclusions themselves. In particular, the inclusions are negative for staining for the presence of either α-synuclein or isoforms of tau protein,81,82 thus distinguishing FXTAS from the synucleinopathies (e.g. Parkinson disease) or tauopathies (e.g. Alzheimer disease). Further analysis of the protein complement of the inclusions by mass spectroscopy94 has revealed a number of proteins of interest as potential participants in the pathogenesis of FXTAS. There are at least two proteins whose function involves RNA interactions: the heterogeneous nuclear ribonuclear protein A2 (hnRNP A2)95 and

muscleblind-like protein 1 (MBNL1), which is implicated in myotonic dystrophy.92 Interestingly, hnRNP A2 and another nucleic acid-binding protein, Pur α, have been associated with the CGG repeat-coupled neuropathology in Drosophila,96,97 although the functional significance of these two proteins for FXTAS in humans remains to be determined. One of several neurofilament proteins found in the inclusions, lamin A/C (A and C isoforms), appears to be functionally impaired in individuals with FXTAS. In particular, expression of the expanded CGG repeat RNA in neural cell culture results in the disruption of the normal nuclear lamin architecture.98 Interestingly, specific mutations in the LMNA gene (OMIM *150330, producing lamin A/C) give rise to a peripheral neuropathy (Charcot–Marie–Tooth type 2B1), one of the prominent features of individuals with FXTAS.17,18 Thus, FXTAS may reflect, at least in part, a functional laminopathy.98 Although the inclusions are positive for ubiquitin immunostaining, the observations of Iwahashi et al.94 argue against aggregation models in which the accumulation of misfolded and/or aggregated proteins overwhelms the proteasomal degradation pathway.99–104 Only five or six proteins appear to be ubiquitinated in the purified inclusions. Furthermore, those proteins appear to be mono-

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ubiquitinated, which would make them less likely to be substrates for proteasomal degradation.105–107 Interestingly, the inclusions formed in cultured neural cells appear to be ubiquitin negative, although αB-crystallin is prominent, suggesting that ubiquitination may be a later event in the pathogenic process.98 Based on their earlier observation of shorter telomere lengths for chromosome 21 in people with Down syndrome and/or dementia,108 Jenkins et al.109 performed a similar examination of telomere length in peripheral blood leukocytes from individuals with FXTAS, using carriers without FXTAS/dementia and age-matched noncarriers as controls. Their primary finding was that telomere length was indeed shortened in individuals with FXTAS. However, more surprising was their finding that there was essentially no difference in the degree of shortening between those with FXTAS and premutation carriers without evidence of disease. This latter observation suggests that telomere shortening may precede the development of overt disease, a possibility that will need further study with younger carriers to assess whether telomere shortening might serve as a biomarker of risk for later clinical involvement. Finally, although to date research in humans on the cellular dysregulation in FXTAS has been restricted largely to the study of postmortem CNS tissue or transfected neural cells, we have recently examined skin fibroblasts from patients with FXTAS, where we have found both disorganization of the lamin A/C nuclear architecture and a cellular stress response that appears to parallel the dysregulation observed in postmortem CNS tissue (Arocena et al., unpublished results). Therefore, skin fibroblasts may provide a powerful tool to study several key aspects of the cellular pathogenesis in FXTAS.

Molecular prenatal diagnosis methodology After discovery of the FMR1 gene,110 prenatal diagnostic techniques utilizing PCR and Southern blot technology were validated in multiple laboratories (see Table 9.1). This technique, now considered the standard diagnosis for FXS, has replaced cytogenetic techniques.111 Immediate analysis of a

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sample can be carried out by PCR when it arrives in the laboratory to initially screen for the normal alleles in the parents. If allele status can be confirmed then a result can be given shortly after a sample’s arrival in the laboratory. However, in all other cases involving large (e.g. full mutation) alleles, cells must be cultured for Southern blot analysis, which takes additional time. Southern blot analysis is essential for the detection of size and/or methylation mosaicism, which cannot be adequately described using PCR-based methods; mosaicism has been documented in approximately 40% of expanded alleles.111 Although analysis of amniotic fluid (AF) or chorionic villus sampling (CVS) formerly required from 2 to 5 weeks, depending on the growth rate of the cells,111 Dobkin et al.112,113 developed a rapid Southern–PCR hybrid technique that reduced turnaround time by 2–4 weeks. Recent advances in PCR have increased both accuracy and efficiency of diagnosis. Fluorescence, methylation-specific PCR (ms-PCR) utilizes fluorescently labeled primers complementary to methylated and nonmethylated DNA. Treated DNA is then analyzed using high-resolution electropheretograms.114 Analysis of known normal, FXS and premutation samples found 100% concordance with the results using this method,110 including one prenatal case.115 A rapid PCR technique, recently developed for screening by Tassone et al.,116 uses a chimeric PCR primer to randomly target within an expanded CGG region. The benefit of this technique is the ability to identify all allele sizes with minimal amount of sample. This technique has been validated with blood spots and might be another promising technique for prenatal diagnosis. While alternatives for increasing the efficiency of PCR have been suggested, an alternative to Southern blot analysis also exists: methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) developed by Nygren et al.117 Although Nygren et al. suggest utilizing their technique with conventional PCR, their method, paired with one of the modified PCR techniques, may optimize the turnaround time for diagnosing FXS in the lab, both prenatally and postnatally. Willemsen et al.118,119 developed a monoclonal antibody technique to identify FMRP deficits in

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Genetic Disorders and the Fetus

Table 9.1 Molecular prenatal diagnosis studies for the full mutation Lab/Report

Initial report

a

AF

CVS

PUBS

Total

False negative

False positive

Hirst et al.

1991

0

1

0

1

0

0

Dobkin et al.a

1991

13

31

0

44

0

0

Rousseau et al.a

1991

1

10

1

12

0

0

Sutherland et al.a

1991

0

1

0

1

0

0

Murphy et al.a

1992

1

1

0

2

0

0

Howard-Peeblesa

1992

5

5

0

10

0

0

Maddalena et al.a Tarleton et al.a

1992

1

0

0

1

0

0

a

1992

0

1

0

1

0

0

Baranov et al.a

1993

0

0

1

1

0

0

Suzumori et al.a

1993

0

1

0

1

0

0

Yamauchi et al.a

1993

0

1

0

1

0

0

Ryynanen et al.a

1994

?

?

?

14

0

0

von Koskulla

1994

0

5

0

5

0

0

Suttcliffe et al.

Puissant et al.a

1994

?

?

?

2

0

0

Castellvi-Bel et al.a

1995

0

2

0

2

0

0

Grasso et al.a

1996

0

9

3

12

0

0

Appelman et al.a

1999

0

1

0

1

0

0

Drasinover et al.a

2000

?

?

0

5

0

0

Kallinen et al.a

2000

?

?

0

18

0

0

Pesso et al.a

2000

?

?

0

9

0

0

Wilkin et al.a

2000

0

1

0

1

0

0

Toledano-Alhadef et al.a

2001

?

?

0

5

0

0

Verma et al.150

2003

?

?

?

11

0

0

Charalsawadi et al.115

2005

1

0

0

1

0

0

Strom et al.151

2007

?

?

?

22

0

0

Berkenstadt et al.34

2007

?

?

0

30

0

0

a

Citations found in Jenkins & Brown, 2004.111

CVS cells from full mutation patients. Jenkins and colleagues also validated this method,111,120 which can demonstrate positivity the day the sample arrives. However, there may be significant variation in the color of the staining for FMRP as related to mosaicism. An individual with FXS who is high functioning may have an adequate staining for FMRP, even though the FMRP level may be reduced by 30–50%; these individuals may still be clinically affected by FXS but without intellectual deficit.44 In this regard, the immunohistochemical staining methodology for FMRP does not detect the premutation, and since the premutation can cause developmental problems in some instances and aging problems in a significant number of patients, which is important information to give to

the physician and the family, this technique is not utilized routinely in most laboratories. Preimplantation genetic diagnosis and polar body analysis An alternative to CVS and amniocentesis for prenatal diagnosis is preimplantation genetic diagnosis (PGD) utilizing polar body analysis.121–123 This technique removes the first and second polar bodies from a fertilized oocyte and analyzes them by PCR or fluorescent in situ hybridization (FISH). Conventional sequential PCR and FISH in PGD, however, introduce allele dropout (ADO), a phenomenon where only one allele amplifies.124–126 The instance where the normal allele amplifies rather than the mutated allele in a heterozygote leads to

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the misdiagnosis of healthy embryos intended for implantation123,126–129 (see Chapter 29). Several modified PCR techniques have been developed to avoid this inaccuracy in PGD. For X-linked disorders, including FXS, PGD has successfully been conducted using multiplex nested PCR, which simultaneously tests for multiple X-linked markers.121–128–130a A similar technique using multiplex fluorescent PCR (f-PCR) in single cells has been successful for PGD in five singlegene disorders, including fragile X.131 Multiplex PCR detects linked markers closely associated with the target gene and reduces the likelihood of ADO and maternal and paternal contamination. For multiplex PCR to be effective, it is essential that the parents are informative and distinct from one another for these markers.127,131 The multiplex nested PCR technique is used in several laboratories nationally and internationally with good results.130–133 Polar body analysis is the best current technique for PGD of FXS.

Neurobiologic advances and targeted treatment in the full mutation Recent advances in understanding the neurobiology of FXS have led to new targeted treatment endeavors in patients. In the knock-out (KO) mouse model of FXS, Huber and colleagues5 discovered enhanced LTD mediated by the metabotropic glutamate receptor 5 (mGluR5) activity. FMRP is normally inhibitory for this pathway and in the absence of FMRP in FXS, there is enhanced LTD and immature or weak synaptic connections documented in human and mouse studies.134,135 These findings led to trials of mGluR5 antagonists in animal models of FXS with subsequent reversal of the phenotype, including seizures, cognitive and behavioral deficits, and brain structural abnormalities.7,64,136,137 Recently, an mGluR5 antagonist, fenobam, has been trialled in 12 adults with FXS in a single dose to assess the pharmacokinetics and safety.138 The results demonstrated no significant side effects, reasonable pharmacokinetics, and improvement in behavior and a psychophysiologic measure of prepulse inhibition (PPI), which is known to be abnormal in FXS. These findings have encouraged larger and longer controlled trials of

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mGluR5 antagonists in FXS, which will take place in 2009. Additional targeted treatments in FXS include the use of minocycline which lowers the elevated levels of matrix metalloproteinase 9 (MMP9) in FXS.139 Treatment of newborn KO mice for a 1-month period with minocycline led to normalization of the synaptic defects with improvement in behavior and cognition.140 Subsequent human anecdotal treatment suggests improvements in behavior in children and adolescents with FXS, but minocycline has not been utilized in children under 7 because it can cause graying of the permanent teeth. Further studies are warranted to assess the benefits of minocycline treatment with study of the risks and side effects.

Genetic counseling The complexity of genetic counseling has intensified with the emergence of premutation involvement in varied manifestations, including aging problems and neurodevelopmental problems as described above (see Figure 9.2).141,142 In addition, the emergence of new targeted treatments that hold the promise of perhaps reversing the cognitive and behavioral problems of FXS is an exciting new development.139,143 Even the aging problems of the premutation have some treatment opportunities and if started early, such as the treatment of hypertension or hypothyroidism, there may be a beneficial effect in long-term outcome for neurologic function and FXTAS.144 Once an individual is diagnosed with a premutation or a full mutation through the obstetrics clinic or perhaps newborn screening, cascade testing will reveal a number of individuals in the family tree that may be affected with either the premutation or the full mutation and would likely benefit from treatment. Such genetic involvement is typically pervasive in multiple generations in the family pedigree24 (Figure 9.2). Early detection of premutation carriers has become increasingly important for family planning, especially for families with a known history of FXS. For a couple with this information, other options are available to them, such as PGD described above. Alternative strategies to PGD should be discussed with women as part of the

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40, 88 CGG FXPOI ataxia depression anxiety leg pain

29 yo HS drop-out alcohol & drug addiction, neuropathy

NT

15, 75 CGG OCD anxiety hypothyroid

64 CGG social anxiety, tremor, neuropathy, impotent, alcohol abuse

NT

31, 74 CGG FXTAS, FXPOI anxiety depression OCD, migraines, neuropathy,

NT

ADHD Dyslexia

died of gunshot

NT

FMR1 Neg Hepatitis-C

FMR1 Negative Full mutation Premutation Assessed at the M.I .N.D. I nstitute

Figure 9.2 Pedigree of family with FXS, premutation, FXPOI and FXTAS involvement. FXTAS, fragile X-associated tremor/ataxia syndrome; FXPOI, fragile X-associated primary ovarian insufficiency;

OCD, obsessive compulsive disorder; HS, high school; ADHD, attention deficit hyperactivity disorder; FMR1, fragile X mental retardation 1 gene; NT, not tested.

Genetic Disorders and the Fetus

30, 73 CGG FXTAS, died at 79 yo fibromyalgia, neuropathy addiction to pain meds

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genetic counseling session, including egg donation and adoption.141,142 In addition, the low yield of eggs after hormonal stimulation for IVF is a problem in premutation carriers presumably because of RNA toxicity.145 In 2006, the American College of Obstetrics and Gynecology recommended offering prenatal screening for women with known premutation or full status. They also recommend screening women with the onset of POI before the age of 40 to rule out fragile X-associated POI. The Canadian College of Medical Geneticists made similar recommendations and emphasized the need for genetic counseling and informed consent, and prior testing of women with a personal history of autism or ID.145a More widespread screening at obstetrics clinics is cost effective146 and uptake has been excellent in a number of studies (85– 100%)31,147,148 Newborn screening has been initiated in at least four centers in the US and the benefits of more intensive early interventions are now being studied149; at least three centers will be detecting both full mutation and premutation babies. The long-term developmental follow-up of babies with the premutation is important to study to better understand the unselected prevalence of developmental problems because previous studies may be biased toward a higher rate of clinical involvement.10,11,58,75 Clarification of the percentage of carriers who have developmental problems and further characterization of these difficulties will lead to better interventions. Perhaps early treatment of the developmental and emotional difficulties of premutation carriers will have a significant effect on their aging problems. New targeted treatments for FXS are also stimulating focused screening of at-risk populations. All children with ID of unknown etiology, as well as developmental delay and autism and ASD of unknown etiology, should be tested for fragile X. Early screening and diagnosis prior to conception, or prenatally, enable families to access early intervention therapies and strategies. With new pharmacologic treatments being developed and the success of early intervention, a positive prognosis for individuals with fragile X continues to be promising. Broader cascade testing throughout families with a proband diagnosed with a fragile X condition will identify many more individuals with

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a fragile X-associated disorder and encourage early treatment opportunities.143,144

Acknowledgments This work was supported by NICHD grants HD036071, HD02274, NIDCR grant DE019583, NIA grant AG032115, NINDS grant NS062412, and 90DD0596 from the Health and Human Services Administration on Developmental Disabilities.

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Prenatal Diagnosis by Microarray Analysis Joris Robert Vermeesch Center for Human Genetics, University of Leuven, Belgium

History of the prenatal karyotype Initially, chromosome studies were performed using simple staining techniques which only allowed the detection of entire groups of chromosomes. In 1966, Steel and Breg1 demonstrated that the chromosomal constitution of the fetus could be determined by the analysis of cultured amniotic fluid (AF) cells. One year later Jacobson and Barter2 performed the first prenatal diagnosis of a chromosomal abnormality. In the following years, several series of prenatal diagnoses with diverse chromosomal abnormalities were reported.2–4 The degree of precision was increased in the 1970s with the introduction of chromosome banding techniques. These enabled the detection of individual chromosomes and segments (bands) within chromosomes. Although chromosomal karyotyping allows a genome-wide detection of large chromosomal abnormalities and translocations, it has a number of inherent limitations: • it takes 4–12 days to culture the cells, visualize the chromosomes and perform the analysis • the resolution is limited to 5–10 Mb depending on (i) the location in the genome, (ii) the quality of the chromosome preparation and (iii) the skill and experience of the cytogeneticist

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

• it requires skilled technicians to perform a Giemsa-banded karyotype analysis, which increases employment costs and can lead to organizational difficulties in small laboratories. With the introduction of fluorescence in situ hybridization (FISH) (see Chapter 8), the detection of submicroscopic chromosomal imbalances became possible. In FISH, labelled DNA probes are hybridized to nuclei or metaphase chromosomes to detect the presence, number and location of small (submicroscopic) regions of chromosomes. Unfortunately, FISH can only detect individual DNA targets rather than the entire genome. To overcome this problem, multicolor FISH-based karyotyping (SKY, MFISH and COBRA FISH) was developed which enables simultaneous detection of all chromosomes. Another technology allowing the genome-wide analysis of copy number aberrations, termed comparative genomic hybridization (CGH), was introduced in 1992.5,6 In CGH, test and reference genomic DNAs are differentially labelled with fluorochromes and then cohybridized onto normal metaphase chromosomes. Following hybridization, the chromosomes are scanned to measure the fluorescence intensities along the length of the normal chromosomes to detect intensity ratio differences which subsequently are interpreted as genomic imbalances. Overall, the resolution at which copy number changes can be detected using these techniques is only slightly higher as compared to conventional karyotyping (>3 Mb) and experiments are labor intensive and time consuming.

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Array comparative genomic hybridization or molecular karyotyping One technology overcoming these limitations is molecular karyotyping. Molecular karyotyping, genome-wide array comparative genomic hybridization (array CGH)7,8 or hybridization of single nucleotide polymorphism (SNP) arrays9,10 enables the genome-wide detection of chromosomal imbalances. In array CGH, patient and reference DNA are differentially labelled and hybridized onto arrays containing genomic fragments. Subsequently, the intensities of the hybridized DNA to the targets are measured using a scanning device and the intensity ratios are plotted according to their position in the genome (Plate 10.1). While the first arrays were made with bacterial artificial chromosome (BAC) targets, other targets include cDNAs,11 PCR products12 and oligonucleotides.13–15 The resolution is therefore only dependent on the fragment lengths and the number of genomic fragments on the array16 and has been steadily increasing over recent years. In SNP arrays, genomic DNA is fragmented and adapters are linked to the fragments which are subsequently amplified by PCR. The amplified fragments are hybridized onto oligonucleotide arrays. The signal intensities across the array are then compared to intensity values obtained by reference samples (Figure 10.1). Array comparative genomic hybridization for the diagnosis of mental retardation and/or multiple congenital anomalies (MR/MCA) Molecular karyotyping has revolutionized the analysis of genomes in general and especially the analysis of the genomes of patients with mental retardation and developmental anomalies, because it enables the detection of submicroscopic chromosomal imbalances. These submicroscopic imbalances have been termed copy number variants (CNVs). In the last 5 years, more copy number changes have been linked to developmental disorders than in the 50 years before. The diseaseassociated CNVs can be categorized as recurrent and rare imbalances. Recurrent imbalances often result from nonallelic homologous recombination (NAHR)

between low copy repeats (LCR) flanking the commonly deleted or duplicated region. Many of these recurrent imbalances, also known as genomic disorders,17 were known before the advent of molecular karyotyping and were often recognized as a well-delineated syndrome. The first recurrent imbalance identified was at 17p12 associated with Charcot–Marie–Tooth disease type 1A (CMT1A, MIM #118220).18,19 Many of these genomic disorders were identified before the array era and have typically been screened by FISH (see Chapter 8). With the advent of molecular karyotyping, a series of novel recurrent imbalances responsible for a variety of phenotypes including MR/MCA have been identified (Table 10.1). Molecular karyotyping not only revealed the occurrence of recurrent imbalances but determined that many MR/MCA syndromes are caused by nonrecurrent imbalances that appear to be scattered at random across the genome. In general, it is assumed that deletions are generated by breaks in chromosomes that are subsequently healed by nonhomologous end joining.33 Although the precise underlying mechanism(s) remain(s) elusive, genomic architectural features have been associated with the generation of these copy number differences.33,34 A novel DNA repair mechanism called replication fork stalling and template switching (FoSTeS) has been proposed to explain nonrecurrent rearrangements based on junction analysis studies of PLP1 duplications.35 In this model, the LCR-associated genomic instability at the PLP1 locus negatively affects the smooth progression of the replication fork and, upon stalling, introduces switching from one active replication fork to another for which only microhomology is required. Another model generating duplication events is a break-induced replication (BIR) repair model that results in recombination36 and was suggested to cause MECP2 duplciations.37 These nonrecurrent imbalances are rare and vary in size in different patients. Several nonidentical but overlapping imbalances with similar MR/MCA phenotypes pinpoint regions that are copy number sensitive and cause developmental disorders. During the last few years, several novel loci have been identified to cause developmental anomalies. An example of pathogenic duplications is shown in Plate 10.2. A list of novel pathogenic CNVs is

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367

A RE

RE

RE

RE

1 RE digestion

Single g Primer Amplification 3

2

((Complexity p y reduction))

Fragmentation4 and Labeling 5

B

C

D

E

Figure 10.1 A, Schematic overview of the steps involved in SNP arrays. (1) Genomic DNA is digested by a restriction fragment. (2) Subsequently, adapters are ligated to the DNA fragments. (3) DNA fragments are PCR amplified. (4) Amplified fragments are labelled with a fluorochrome. (5) DNA fragments are hybridized on an array. (B) Scanned image of Affymetrix array. (C) For each single nucleotide polymorphism, 24 different oligonucleotides are spotted. Each oligo is a permutation

of the basic sequence. (D) On the left, the position of all permutations and on the right the colored image following scanning. One can observe the different haplotypes for this SNP. (E) Genome-wide view of all SNPs. Shown is a segment of a chromosome. On top, the intensity ratios across this chromosomal segment. The bottom line shows the copy number state of that segment. Note the presence of a duplication at position 30–32 Mb.

maintained at the DECIPHER website (see below: https://decipher.sanger.ac.uk/perl/ application?act ion=syndromes). Several studies screening individuals with mental retardation, multiple dysmorphic features and normal conventional karyotypes have demonstrated a high diagnostic yield in MR/MCA patients.38–45 In summary, at the 1 Mb resolution, 20–25 percent of selected individuals have deletions or duplications or a combination of both.

About half of these are conclusively causal for the disorder. The few studies at 100 kb resolution have also detected about 10 percent of pathogenic interstitial aberrations.39,46 The chromosome imbalances occur throughout the genome. In addition to the discovery of pathogenic imbalances in patients with MR/MCA, several studies have identified an association of copy number variants with several neuropsychiatric conditions (reviewed by Cook and Scherer47) such as autism spectrum dis-

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Table 10.1 Newly recognized interstitial microdeletion/duplication syndromes caused by NAHR between low copy repeats associated with mental retardation Name

Size (Mb)

1q21 microdeletion

0.5

MIM

Clinical features

Reference

Hypomegakaryocytic thrombocytopenia

Klopocki et al.20

and bilateral radial aplasia (TAR) 1q21 microdeletion &

1.35

Asymptomatic to severe developmental

microduplication

Mefford et al.21

delay and MCA. Susceptibility locus for neuropsychiatric disorders

3q29 microdeletion

1.6

609425

MR, mild FD including high nasal bridge and short philtrum

Willatt et al. 200522; Ballif et al.23 Lisi et al. 200824; Ballif

3q29 microduplication

1.6

611936

Mild/moderate MR, MC, obesity

7q11.23

1.5

609757

MR, speech and language delay, ASD

15q13.3 microdeletion

1.5

612001

MR, epilepsy, FD, digital abnormalities

Sharp et al.25

15q24 microdeletion

1.7

MR, growth retardation, MC, digital

Sharp et al.26

et al.23

and genital abnormalities 16p13.11 microdeletion

MR, MCA, ASD

Ullmann et al.27; Hannes et al.28

17p11.2 microduplication

3.7

610883

MR, infantile hypotonia, ASD

17q21.31 microdeletion

0.5

610443

MR, hypotonia, typical face

Sharp et al.29; Koolen et al.30; Shaw-Smith et al.31

22q11.2 distal microdeletion

611867

MR, growth delay, mild skeletal

Ben Sachar et al.32

abnormalities, FD

ASD, autistic spectrum disorder; FD, facial dysmorphism; MC, microcephaly; MCA, multiple congenital anomalies; MR, mental retardation; TAR, thrombocytopenia absent radius.

orders48,49 and psychiatric diseases such as schizophrenia.50–53 Rare recurrent CNVs discovered to be under negative selection through population genetic studies were utilized to identify schizophrenia susceptibility loci. Once the validity of the technique to detect chromosomal constitutional imbalances was demonstrated, it was rapidly introduced into genetic diagnostic laboratories as a routine technique in the genetic evaluation of patients with MR/MCA.54 This technology has also been used to focus on specific patient groups. Lu et al.55 reported imbalances in 17.1 percent of neonates with various birth defects. Thienpont et al.56 reported a frequency of 17 percent causal imbalances in patients with heart defects. Given the aforementioned studies, prenatal diagnosis using microarray analysis followed. Copy number variation/polymorphisms Besides the identification of disease-associated

CNVs, molecular karyotyping also uncovered large numbers of copy number variants in normal individuals. Thus far, SNPs were considered the main source of genetic variation. Hence, the discovery of an unexpected large number (12 percent of the genome) of apparently benign copy number variants, regions of 1–1000 kb that are present in different copy numbers in different individuals, was rightly called the discovery of the year in 2007, according to the journal Science. A number of array CGH studies have demonstrated the presence of polymorphic copy number variants.57–62 In the first large systematic study, Redon et al.63 mapped all CNVs using both array CGH and SNP genotyping arrays on the 270 individuals of the HapMap collection with ancestry from Europe, Africa and Asia; 1,447 submicroscopic copy variable regions in the human genome were uncovered. These nonpathogenic variations, including deletions, duplications, insertions and complex multisite variants, involve about 12

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percent of the genes, including a large number of genes known to be involved in genetic disorders and registered in OMIM. Recent fine mapping studies have revealed that those CNVs can cause intragenic variation, resulting in different splice variants, the use of different exons and even new gene products.64

The blurred boundary between benign and pathogenic copy number variants The consequence of the detection of multiple benign CNVs is that, at present, the clinical significance of a novel CNV often remains unclear. The traditional rules of thumb used when analyzing genomes by conventional karyotyping no longer apply. The identification of a large de novo cytogenetically visible imbalance was usually sufficient to confidently associate it with the disease phenotype. However, it is obvious that smaller imbalances carrying few or no genes may not be associated with a disease phenotype. Equally, it is becoming clear that de novo copy number variations arise frequently. Van Ommen estimated that copy number changes arise in every one in eight births.65 Hence, not all de novo copy number changes would be pathogenic. To determine which, if any, CNVs might be associated with disease phenotypes, collection of large numbers of patient genotypes and phenotypes is required. Several efforts are currently ongoing to collect this information. These efforts will eventually enable correlation between highly penetrant CNVs and disease states. The best known open source examples are the database of chromosomal imbalances and phenotype in humans using Ensembl Resources with the acronym DECIPHER which is organized at the Sanger Institute (https:// decipher.sanger.ac.uk/) and the European Cytogenetics Association Register of Unbalanced Chromosme Aberrations, ECARUCA, based in Nijmegen, The Netherlands (http://agserver01. azn.nl:8080/ecaruca/ecaruca.jsp).66 In addition, several large-scale collaborative efforts are under way to map population-embedded, apparently benign CNVs. These data are collected in the database of genomic variants (DGV, http://projects. tcag.ca/variation/). To fine map those imbalances

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increasingly higher resolution arrays are being used. Those efforts aim to identify CNVs with likely minor or no developmental consequences. While the mapping of apparently benign and pathogenic CNVs is an important endeavor, it is not sufficient to predict whether an imbalance will cause an abnormal phenotype. Apparently benign CNVs can cause autosomal recessive,65,67,68 autosomal dominant65,69 and X-linked disorders,70 and imprinted regions may only cause disease dependent on the parental origin.71 In addition, variable expressivity and penetrance may obscure the pathogenic relevance of CNVs. It appears not only that interindividual phenotypic variation is caused by benign CNVs, but even well-known diseasecausing CNVs may occasionally be tolerated and be part of the normal human phenotypic spectrum. For example, the 22q11 deletion as well as the duplication can cause both heart anomalies and midline defects such as cleft palate. If such an anomaly is detected on ultrasound, prenatal FISH testing for this condition is performed (see Chapter 8).72,73 However, both the familial inherited 22q11 deletion and duplication have now recurrently been reported.74,75 The parent carrying the 22q11 duplication is phenotypically normal. Detecting 22q11 duplication in a prenatal context will thus require decisions to be made without knowing the future phenotypic outcome. Similarly, subtelomeric imbalances are known to be a major cause of birth defects and mental retardation. However, in contrast to the view that these imbalances are always causal and result in phenotypic anomalies, several reports indicate that several subtelomeric imbalances, up to 10 Mb in size, may not result in obvious phenotypic anomalies.76,77 More recently identified recurrent imbalances with variable penetrance are the 16p13.1 and 1q21 regions. During the screening of patients with mental handicap and developmental anomalies, reciprocal deletions and duplications of the 16p13.1 region were recurrently observed.27,28 This 1.65 Mb rearrangement includes 15 genes. It was originially unclear whether these imbalances were causing the developmental problems in patients because of two reasons: first, the imbalance (deletion or duplication) was often observed to be inherited from an apparently normal parent; second, the phenotypes associated with either the deletion or duplication

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Genetic Disorders and the Fetus

are quite variable. An association study showed that the deletion is a risk factor for mental handicap while the duplication is more likely to be a benign variant. Law et al.78 reported the prenatal diagnosis of a de novo 16p13.11 microdeletion by array CGH. Because of the unclear clinical significance, the pregnancy was not terminated and an apparently healthy baby was born. 1q21 harbors two flanking regions where recurrent reciprocal rearrangements were detected in patients with MR/MCA. The deletions and duplications are mediated by NAHR of flanking low copy repeats. All 30 investigated patients with thromobocytopenia absent radius (TAR) syndrome carry a 200 kb deletion on chromosome 1q21.1.79 Analysis of the parents revealed that this deletion occurred de novo in 25 percent of affected individuals. Intriguingly, inheritance of the deletion along the maternal line as well as the paternal line was observed in the other patients. The absence of this deletion in a cohort of control individuals argues for a specific role played by the microdeletion in the pathogenesis of TAR syndrome. It is hypothesized that TAR syndrome is associated with a deletion on chromosome 1q21.1 but that the phenotype develops only in the presence of an additional, as yet unknown modifier (mTAR).20 Recently, the first prenatal diagnosis of TAR by array CGH was reported.79 Mefford and colleagues21 identified 20 inidivduals with a recurrent 1.35 Mb deletion distal from the TAR region from a screen of about 5,000 patients with mental retardation and/or associated congenital anomalies. The microdeletions arose de novo in six patients, were inherited from a mildly affected parent in three patients and inherited from an apparently unaffected parent in five. The absence of the deletion in about 5,000 control individuals represents a significant association with disease. In addition, the reciprocal duplication was also enriched in children with mental retardation or autism spectrum disorder (ASD) although too few cases have been observed to determine statistical significance. It seems likely that those recurrent rearrangements with variable penetrance and expressivity are a prelude to a large number of structural variants with diverse and complex phenotypes that will elude both traditional syndromic classification as

well as evading traditional Mendelian inheritance patterns. The elucidation of their association with disease will require genotyping and phenotyping large numbers of patients and controls. These imbalances pose special problems when introducing array CGH into the prenatal diagnostic setting.

Prenatal diagnosis by array comparative genomic hybridization There are no technical barriers to performing array CGH as a prenatal test. In a proof of principle experiment, Rickman et al.80 demonstrated the feasibility of performing array CGH for prenatal diagnosis on DNA extracted from AF cells. With the exception of a triploidy, 29/30 results were in complete concordance with the karyotype. The feasibility of using array CGH BAC and oligo arrays on uncultured amniocytes for the detection of chromosomal imbalances has been further illustrated.81–83 It should be noted that the quality of DNA isolated from AF is often suboptimal due to the presence of dead cells, small degraded DNA fragments and other unkown inhibiting factors. Cell-free fetal DNA (cff DNA) present in the supernatant of the AF can also be used to perform array CGH.84,85 An overview of all reports using prenatal arrays and the type of array used is presented in Table 10.2. Applying array CGH in a prenatal setting offers some advantages. Because array CGH can be automated and no cell culture is required, this would result in faster turnaround times. The increased resolution enables the detection of most (see below) chromosomal imbalances including aneuploidies and all known recurrent genomic imbalances causing MCA/MR which are currently (occasionally) tested for in a prenatal setting. Not surprisingly, the technology is implemented at several diagnostic centers86 and several reports have suggested that it is is ready for mainstream use.89,90 Chromosomal rearrangements missed by array CGH Inherent to the technique, balanced chromosomal rearrangements (inversions and balanced translocations) are not detected. When balanced rear-

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Table 10.2 Overview of prenatal diagnostic array published series of invasive prenatal diagnosis Reference

No. of samples

Type of array used

Sample details

Targeted BAC arrays

Cultured (46.6%) and

Other comments

tested van den Veyver

300

et al.86

& oligo arrays

uncultured (82%) AF, cultured (78.8%) & uncultured (17%) CVS

Bi et al.83

15

BCM chromosome

Uncultured AF + WGA

Proof of principle for oligo

microarray Version

array and uncultured AF,

6 oligonucleotide

using modified DNA

(V6 OLIGO) (details

extraction protocol, and

of array design in

WGA. Results are

paper)

included in the above paper

Targeted array Shaffer et al.87

151 prenatal

Targeted BAC array

Cultured AF or cultured

Targeted BAC array

Uncultured AF, cffDNA

cases Lapaire et al.88

10

CVS Proof of principle for

extracted from

extraction of cffDNA

supernatant

from fresh/frozen AF samples (no difference)

Miura et al.85

13

Custom BAC array Targeted, only chromosomes 13,

Uncultured AF, cffDNA

Proof of principle for DNA

extracted from

extracted from cffDNA in

supernatant (10 mL)

AF

18, 21, X & Y Rickman et al.80

30

Targeted BAC array

30 uncultured prenatal samples

Proof of principle for uncultured prenatal arrays, using as little as 1 mL of AF

Sahoo et al.81

98

Targeted BAC array

56 AF/42 CVS

Larrabee et al.84

28cffDNA, 8

Targeted BAC array

Frozen AF supernatant,

AF DNA

cffDNA extracted (28)

Proof of principle for cffDNA Higher noise with cffDNA

Cultured AF cells (8) Fetal sex and aneuploidy detected WGA, whole genome amplification. Other abbreviations defined in text.

rangements are detected prenatally on karyotypes, parents are usually tested and if a “normal” parent carries the same rearrangement, the translocation is considered benign. If the rearrangement is de novo, counseling is more challenging and the risk for developmental defects is estimated to be 6 percent.91 Array CGH analysis of patients with developmental anomalies and de novo translocations has revealed that about 45 percent of these

are actually unbalanced.92,93 Considering that de novo translocations occur about 1/1,000 births with 6 percent having an abnormal phenotype and half of these being detectable by array CGH, this would leave 0.003 percent pathogenic translocations undetected if no karyotype is performed. Neither triploidies (69,XXX and 69,XXY) nor tetraploidies are readily detected. However, the use of DNA from a patient with Klinefelter syndrome

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Genetic Disorders and the Fetus

(47,XXY) does result in aberant X and Y chromosome ratios, enabling the detection of XXX triploidies and all tetraploidies.94 An exception to this is the use of SNP arrays that would allow detection of polyploidy. Challenges of array CGH in a prenatal setting In addition to the limited predictive value of several novel recurrent imbalances, other challenges hamper the general and rapid introduction of this novel technology in a clinical diagnostic setting.95,96 The first challenge is determining which is the optimal platform/which resolution to use in prenatal diagnosis. On the one hand, several groups suggest the use of unbiased whole-genome arrays while others suggest that targeted arrays are the best for prenatal diagnosis. Whole-genome arrays offer the benefit of detecting all possible imbalances and are replacing the older targeted arrays in postnatal clinical practice. However, using higher resolution arrays results, at present, in the identification of an increasing number of variants of unknown clinical significance. In one study using 500K oligonucleotide arrays, a median of 30 CNVs per individual was observed.97 Targeted arrays including only loci known to be causal for developmental anomalies cover less of the genome but may result in fewer imbalances of unknown significance. In addition, for some deletions known to cause developmental anomalies, the consequences of the duplication may be less severe. For example, the 22q11 duplication is overall less penetrant than the 22q11 microdeletion. For both targeted and whole-genome arrays, predicting the phenotypic outcome when detecting an imbalance in a fetus with variable penetrance and expressivity is, at present, an impossible task. The second challenge to the introduction of array CGH is based on the discovery of “unwanted/ unexpected” findings. The outcome of array CGH provides a wealth of information and these findings can be classified in different categories: prenatal diagnosis of adult-onset conditions will occur; imbalances that cause congential anomalies that can be treated effectively or are relatively mild will be identified. It is at present unclear how individuals, clinicians and society should deal with those

findings and this may even disrupt the rationale and purpose of prenatal screening.98

Follow-up of abnormal conventional karyotype De novo marker chromosomes or apparently balanced translocations identified by conventional karyotyping during prenatal diagnosis pose genetic counselling challenges.99 In the absence of an abnormal ultrasound, it is often unclear whether the chromosomal rearrangement will lead to phenotypic abnormalities. With array CGH it is now possible to identify whether the marker chromosome carries dosage-sensitive euchromatic material or is composed solely of dosage-insensitive heterochromatin.100,101 Gruchy et al.100 retrospectively studied 20 small marker chromosomes (SMCs). The technique would have been less helpful in nine cases, that is, bisatellited SMCs, isochromosomes and translocation derivatives. On the other hand, it would have been helpful for the 11 remaining cases. It would have improved diagnostic accuracy for six SMCs whose chromosomal origin was ascertained by cytogenetics and FISH and for which prognosis was only based on literature and ultrasonographic data. Among five unidentified SMCs, array CGH would have been more reassuring for four containing only heterochromatin than normal ultrasonography alone and would have characterized the unidentified case associated with malformations. For apparently balanced translocations, array CGH can exclude the presence of a cryptic imbalance. Only a minority of the de novo translocations are associated with chromosomal losses and the majority of those translocations result in healthy individuals. Since about half of the MR/MCA patients with apparently balanced translocations do have deletions at one or both of the translocation breakpoints and about 90 percent of all carriers of complex translocations have deletions at either one or several breakpoints or even elsewhere in the genome,101a those imbalances can be readily identified by array CGH. Simovich et al. identified a microdeletion in a fetus with a de novo t(2;9) (q11.2;q34.3) referred for prenatal diagnosis because of an abnormal ultrasound with increased nuchal translucency.102

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Array CGH allows a more precise diagnosis in affected individuals with abnormal karyotpyes and therefore facilitates a more reliable prenatal diagnosis. Stumm et al. confirmed an interstitial deletion 12p by array CGH.103 Similarly, Peng et al. confirmed a small 2q interstitial deletion by array CGH.104 Brisset et al.105 identified a chromosome 9p with an extra chromosomal segment. Array CGH enabled the rapid identification of the origin of this segment and determined a de novo monosomy 9p24.3 and trisomy 17q24.3. This allowed more accurate counseling. In general, the confirmation of either the absence or presence of pathogenic copy number changes following the identification of a chromosomal anomaly in a conventional karyotype helps the counseling and is comforting to both the clinician and the patient.

Diagnosis of miscarriages by array comparative genomic hybridization Spontaneous abortions are common, with 10–15 percent of all clinically recognized pregnancies ending in early pregnancy loss. Cytogenetic analysis has shown that about 50 percent of firsttrimester miscarriages are caused by fetal chromosome abnormalities, most of which consist of numerical abnormalities (86 percent), including trisomies, monosomies and polyploidies. Structural abnormalities represent another 6 percent of anomalies found.106–108 Identification of the cause of a spontaneous abortion helps to estimate recurrence risks for future pregnancies and, when an anomaly is found, comforts parents. Over the years, routine analysis of products of conception (POC) has been performed by karyotyping of metaphase spreads following tissue culture. However, due to failure of culture growth, suboptimal chromosome preparations or possible maternal contamination,109,110 either no result or an erroneous result is obtained. Molecular karyotyping has been applied succesfully by several groups to detect fetal chromosome abnormalities in POC.110–115 The use of these clones as target DNA increases the resolution beyond the 3 Mb limit of normal banding and metaphase CGH. In Figure 10.2, a series of chromosomal

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imbalances identified in POCs are shown. Studies by Schaeffer et al.,116 Benkhalifa et al.117 and Shimokawa et al.118 have shown that it is now possible to detect submicroscopic variants that were previously not detected. In general, the application of array CGH in those cases also increases the diagnostic yield.

Preimplantation genetic diagnosis by array comparative genomic hybridization Preimplantation genetic diagnosis (PGD) was introduced over 15 years ago with the purpose of performing genetic testing before pregnancy, in order to establish only unaffected pregnancies and avoid the need for pregnancy termination.118a The first cases, reported in 1990, utilized PCR to determine the gender of embryos in couples at risk for X-linked diseases. Today PGD can be used for the detection of single gene defects, structural chromosome abnormalities and aneuploidy by employing FISH or PCR techniques (see Chapter 29). The main disadvantage of FISH is that only a limited number of loci can be interrogated. PGD aneuploidy screening by FISH only enables the detection of 7–10 chromosomes in a single cell. In addition, for the screening for chromosomal imbalances in translocation carriers, for each PGD cycle, novel probes have to be developed and their accuracy tested which is costly and time consuming. Single cell array CGH could enable detection of all chromosomal aneuploidies in a single blastomere and might provide a standardized assay for the detection of all segmental aneuploidies. Preimplantation genetic diagnosis requires the removal of one or more cells from an early embryo. DNA from a single cell is not sufficient to perform array CGH. However, several reports have demonstrated the feasibility of detecting chromosomal imbalances using array methods in a single cell as well as in single blastomeres.119–121 Using a novel array-based approach, screening of genome-wide copy number and loss of heterozygosity in single cells, Vanneste et al. established that during early human embryogenesis chromosomal instability is common, a feature thus far only observed in

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Genetic Disorders and the Fetus

Figure 10.2 Array CGH profiles using DNA from a mosaic monosomy X (A), a male product of conception with maternal contamination (B), a male mosaic for an isochromosome 7p (C), low-grade mosaicism of chromosome 22 versus DNA from an XXY cell line (D). For each panel, the X-axis represents the clones ordered from the short-arm telomere to the long-arm telomere and chromosomes are ordered from chromosomes 1 to 22, X and Y. The Y-axis shows log2 transformed intensity ratios at each locus. The chromosomes that are not present in equal ratios are indicated. Light blue dots represent known polymorphic clones. (Reproduced with permission from Robberecht et al., 2009126).

tumors.125 This study revealed not only mosaicism for whole chromosome aneuploidies and uniparental disomies in over 90 percent cleavage stage embryos derived from young fertile copules but also frequent segmental deletions, duplications and amplifications that were reciprocal in sister blastomeres, implying the occurrence of breakage– fusion–bridge cycles (Plate 10.3). The consequence of these observations may be that it will be impossible to use preimplantation genetic aneuploidy screening of cleavage-stage embryos as a diagnostic tool. Clearly, at present, this is not yet applicable in a diagnostic setting, but technologic advances make it likely that this technology will become available.

Conclusion Molecular karyotyping is challenging conventional karyotyping as the gold standard in prenatal diagnosis. Its introduction into prenatal diagnosis depends on both economic considerations and risk acceptance by society. Molecular karyotyping would detect the majority of anomalies identified by conventional karyotyping, but would miss chromosomal translocations. However, it is not the technical issues that hamper rapid clinical introduction, but rather the philosophical questions. As discussed above, the general introduction of genome-wide screening tools for all indications largely exceeds current practice and is associated

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with several questions. A compromise between the general introduction of array-based techniques and not introducing the technology at all in a prenatal setting would be the selected use of the technology.95,122 Routine ultrasound examination for fetal abnormalities is practiced in most centers. When an invasive test is indicated on the basis of an increased risk for Down syndrome (abnormal screening tests or advanced maternal age) without fetal abnormalities, FISH or PCR can be used as a stand-alone test. If ultrasound features are suggestive of a fetal abnormality, a more complete analysis may be warranted. This approach, within the context of clinical trials, has been suggested as the way forward.95 In the future, the major challenge for geneticists is to map the phenotypic consequences of the CNVs in relation to MR/MCAs. In the coming years, the blurred picture will become clearer and fully penetrant CNVs and disease-causing genes will be identified. Once this knowledge is gathered, it seems likely that we will be able to generate more comprehensive targeted arrays which interrogate those parts of the genome that are relevant to MR/MCAs. Such arrays are likely to become generally accepted and might replace current genetic testing tools. However, it remains prudent to move cautiously so that clinicians, parents and society can learn how to deal with the daunting amount of knowledge obtained from a single genome.

References 1. Steel MW, Breg WR. Chromosome analysis of human amniotic fluid cells. Lancet 1966;i:383. 2. Jacobson CB, Barter RH. Intrauterine diagnosis and management of genetic defects. Am J Obstet Gynecol 1967;99:796. 3. Jacobson CB, Barter RH. Some cytogenetic aspects of habitual abortion. Am J Obstet Gynecol 1967;97:666. 4. Nadler HL. Antenatal detection of hereditary disorders. Pediatrics 1968;42:912. 5. Kallioniemi A, Kallioniemi OP, Sudar D, et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 1992;258:818. 6. du Manoir S, Speicher MR, Joos S, et al. Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum Genet 1993;90:590.

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66. Feenstra I, Fang J, Koolen DA, et al. European Cytogeneticists Association Register of Unbalanced Chromosome Aberrations (ECARUCA): an online database for rare chromosome abnormalities. Eur J Med Genet 2006;49:279. 67. Olbrich H, Fliegauf M, Hoefele J, et al. Mutations in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and hepatic fibrosis. Nat Genet 2003;34:455. 68. Lesnik Oberstein SA, Kriek M, White SJ, et al. Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase. Am J Hum Genet 2006; 79:562. 69. Balikova I, Martens K, Melotte C, et al. Autosomaldominant microtia linked to five tandem copies of a copy-number-variable region at chromosome 4p16. Am J Hum Genet 2008;82:181. 70. van Esch H, Bauters M, Ignatius J, et al. Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males. Am J Hum Genet 2005;77:442. 71. Browne CE, Dennis NR, Maher E, et al. Inherited interstitial duplications of proximal 15q: genotype-phenotype correlations. Am J Hum Genet 1997;61:1342. 72. Davidson A, Khandelwal M, Punnett HH. Prenatal diagnosis of the 22q11 deletion syndrome. Prenat Diagn 1997;17:380. 73. Devriendt K, van Schoubroeck D, Eyskens B, et al. Polyhydramnios as a prenatal symptom of the digeorge/ velo-cardio-facial syndrome. Prenat Diagn 1998;18:68. 74. Portnoi MF, Lebas F, Gruchy N, et al. 22q11.2 duplication syndrome: two new familial cases with some overlapping features with DiGeorge/velocardiofacial syndromes. Am J Med Genet A 2005;137:47. 75. Yobb TM, Somerville MJ, Willatt L, et al. Microduplication and triplication of 22q11.2: a highly variable syndrome. Am J Hum Genet 2005;76:865. 76. Hengstschlager M. Fetal magnetic resonance imaging and human genetics. Eur J Radiol 2006;57:312. 77. Balikova I, Menten B, de Ravel T, et al. Subtelomeric imbalances in phenotypically normal individuals. Hum Mutat 2007;28:958. 78. Law LW, Lau TK, Fung TY, et al. De novo 16p13.11 microdeletion identified by high-resolution array CGH in a fetus with increased nuchal translucency. Br J Obstet Gynaecol 2009;116:339. 79. Uhrig S, Schlembach D, Waldispuehl-Geigl J, et al. Impact of array comparative genomic hybridization-derived information on genetic counseling demonstrated by prenatal diagnosis of the TAR (thrombocytopenia-absent-radius) syndrome-associated microdeletion 1q21.1. Am J Hum Genet 2007; 81:866.

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119.

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121.

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123.

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11

Molecular Genetics and Prenatal Diagnosis John A. Phillips III Department of Pediatrics Vanderbilt University School of Medicine and the Monroe Carell Jr. Children’s Hospital at Vanderbilt, Nashville, TN, USA

Molecular genetic techniques, including restriction endonucleases, DNA hybridization, Southern blots, polymerase chain reaction (PCR) amplification, DNA sequence analysis and multiplex ligation dependent probe amplification (MLPA), have been used to characterize the DNA alterations that cause a variety of genetic disorders. Use of these techniques facilitates prenatal detection of a rapidly increasing number of Mendelian and mitochondrial disorders (see Table 11.1). Future applications will be pertinent to almost every medical subspecialty. Consideration of some basic molecular aspects will facilitate comprehension of the ensuing discussion on prenatal DNA diagnostics.

Background: DNA and gene structure DNA structure Human chromosomes contain DNA and histone and nonhistone proteins. DNA is made up of three components: • bases, which are the purines adenine (A) and guanine (G) and the pyrimidines thymine (T) and cytosine (C) • a five-carbon deoxyribose sugar • a phosphate backbone.

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

380

The sugar phosphate backbone consists of a phosphate molecule attached to the 5′ carbon of the deoxyribose sugar with the 3′ carbon of the sugar attached to the phosphate of the next molecule. Phosphate bonding to the 3′ carbons gives direction to the DNA strand as bases are added 5′ to 3′. Hydrogen bonding between AT and GC bases on complementary DNA strands stabilizes the conformation of the double helix. The pairing of A/Ts is formed by two hydrogen bonds, whereas the pairing of G/C forms three hydrogen bonds. Notice that hydrogen bonds occur between the bases of complementary strands that run in antiparallel directions (i.e. 5′ to 3′ on one strand and 3′ to 5′ on the complementary strand). For example, 5′TCGA3′ on one strand corresponds to 3′AGCT5′ on the other, forming the double-stranded molecule. Units of three bases, called triplets, constitute a codon, which by convention is read from the “coding” DNA strand that has the same sequence as the mRNA. These codons are units of the genetic code that direct the cellular machinery to add specific amino acids during translation of the mRNA encoded by the gene. Codons are arranged in a linear sequence within the exons of genes.1,2 As the temperature increases, denaturation of the two DNA strands occurs, first between A/T pairs then between G/C pairs. The G/C base pairs melt at higher temperatures because their three hydrogen bonds make them more stable than A/T base pairs, which have two hydrogen bonds. Advantage is taken of these differences in melting temperature by a variety of methods used to detect DNA mutations.

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381

Table 11.1 Selected monogenic disorders detectable by DNA analysis Disease

Mode

OMIM no.

Reference 28

Aarskog–Scott syndrome

XL

305400

Abdominal aortic aneurysm

AD, AR

100070

29

Achondrogenesis, type II

AR

200610

30

Achondroplasia

AD

100800

31

Adams–Oliver syndrome

AD, AR

100300

32

Adenine monophosphate deaminase 1

AD

102770

33

Adenine phosphoribosyltransferase deficiency

AR

102600

34

Adrenal hypoplasia

XL

300200

35

Adrenoleukodystrophy, autosomal neonatal form

AR

202370

36

Adrenoleukodystrophy, X-linked

XL

300100

37

Adenomatous polyposis of the colon

AD

175100

38,196

Adenosine deaminase deficiency

AR

102700

39

Agammaglobulinemia, Bruton

XL

300300

40

Agammaglobulinemia, Swiss

XL

300400

41

Aicardi–Goutières syndrome 1

AR

225750

42–43

Alagille syndrome

AD

118450

44–45

Albinism, ocular, type 1

XL

300500

46

Albinism, oculocutaneous, type 1

AR

203100

47, 400–401

Albinism, oculocutaneous, type 2

AR

203200

48–49, 402

Albinism, oculocutaneous, type 3

AR

203290

50

Albright hereditary osteodystrophy-3

AD

600430

51

Aldolase deficiency

AR

103850

52

α1-Antitrypsin deficiency

AR

107400

53–58

Alport syndrome, autosomal

AR

203780

59

Alport syndrome, X-linked

XL

301050

60–62

Alzheimer disease, type 3

AD

104311

63

Alzheimer disease, type 4

AD

600759

64

Amyloidosis, type 1

AD

176300

65–67

Amyotrophic lateral sclerosis

AD

105400

68

Androgen insensitivity

XL

313700

69–71

Angelman syndrome

AD, de novo

105830

72

Angioneurotic edema, hereditary

AD

106100

73–74 75–76

Aniridia

AD

106200

Anophthalmos

XL

301590

77

Antithrombin III deficiency

AD

107300

78

Apert syndrome

AD

101200

79

Apolipoprotein E

AD

107741

80

Arachnodactyly, contractural

AD

121050

81

Arginase deficiency

AR

207800

82

Argininosuccinic aciduria

AR

207900

83

Arthrogryposis, multiplex congenita, distal, type 1

AD

108120

84 85

Arthrogryposis, multiplex congenita, distal, type 2

XL

301830

Ataxia telangiectasia

AR

208900

86–88

Azoospermia

AD

415000

89

Bardet–Biedl syndrome, type 2

AR

209900

90

Barth syndrome

XL

302060

91

Basal cell nevus syndrome

AD

109400

92

Beare–Stevenson syndrome

AD

123790

93

Beckwith–Wiedemann syndrome

AD

130650

94–95

382

Genetic Disorders and the Fetus

Table 11.1 Continued Disease

Mode

OMIM no.

Reference

Blepharophimosis, ptosis

AD

110100

96

Bloom syndrome

AR

210900

97

Branchio-oto-renal dysplasia

AD

113650

98

Breast cancer (BRCA1)

AD

113705

99

Breast cancer (BRCA2)

AD

600185

100

Bullous congenital ichthyosiform erythroderma

AD

113800

101

Cerebral autosomal dominant arteriopathy with subcortical infarcts

AD

125310

102–103

& leukoencephalopathy (CADASIL) Campomelic dysplasia

AR

211970

104

Camurati–Engelmann disease

AD

131300

105

Canavan disease

AR

271900

106–107

Carbamylphosphate synthetase 1 deficiency

AR

237300

108–111

Carnitine palmitoyltransferase deficiency

AR

255110

112

Cartilage hair hypoplasia

AR

250250

113

Cat-eye syndrome

AD, de novo

115470

114

Cerebrotendinous xanthomatosis

AR

213700

115

Ceroid lipofuscinoses

AR

256730

116

Charcot–Marie–Tooth disease

XL

302800

117

Charcot–Marie–Tooth disease, type 1A

AD

118200

118–119

Charcot–Marie–Tooth disease, type 4A

AR

214400

120

CHARGE syndrome

AD

214800

121

Chediak–Higashi syndrome

AR

214500

122

Chondrodysplasia punctata

XL

302950

123

Choroideremia

XL

303100

124

Chronic granulomatous disease

XL

306400

125–127

Citrullinemia

AR

215700

128

Clasped thumb and mental retardation

XL

303350

129

Cleidocranial dysplasia

AD

119600

130

Cockayne syndrome type II

AR

133540

131

Cornelia de Lange syndrome

AD

122470

132

Coffin–Lowry syndrome

XL

303600

133

Coloboma-renal syndrome

AD

120330

134

Congenital adrenal hyperplasia

AR

201910

135

Congenital adrenal hypoplasia

XL

300200

136

Congenital disorder of glycosylation type Ia

AR

212065

137–138

Congenital lipoid adrenal hyperplasia

AR

201710

139

Congenital nephrosis

AR

256300

140

Conotruncal heart malformations

AR

217095

141

Coproporphyria, hereditary

AD

121300

142

Corneal dystrophy, granular type

AD

121900

143

Cowden syndrome

AD

158350

144

Craniosynostosis, Shprintzen–Goldberg

AD

182212

145

Craniosynostosis, type II

AD

123101

146

CRASH (corpus callosum hypoplasia, retardation, adducted thumbs,

XL

303350

147

Creutzfeldt–Jakob disease

AD

123400

148

Cri du chat syndrome

AD

123450

149

Crouzon disease

AD

123500

150

spastic paraplegia & hydrocephalus) syndrome

Cystic fibrosis

AR

219700

151–154

Deafness, nonsyndromic

XL

304700

155

CHAPTER 11

Molecular Genetics and Prenatal Diagnosis

383

Table 11.1 Continued Disease

Mode

OMIM no.

Reference

Dentatorubral-pallidoluysian atrophy

AD

125370

156

Denys–Drash syndrome

AD

194080

157–158

Diabetes mellitus, maturity-onset diabetes of the young (MODY),

AD

125850

159

Diabetes mellitus, MODY, type II

AD

125851

160

Diabetes mellitus, non-insulin dependent

AD

138190

161

DiGeorge/velocardiofacial

AD

188400

162

Distal arthrogryposis type 2B

AD

601680

163

type I

Dyschromatosis symmetrica hereditaria

AD

127400

164–165

Dyskeratosis congenita

XL

305000

166

Dystonia, type I

AD

128100

167

Early-onset primary torsion dystonia (DYT1)

AD

128100

168

Ectrodactyly

AD

183600

169

Ectodermal dysplasia, anhidrotic

XL

305100

170–172 173

EEC syndrome

AD

129900

Ehlers–Danlos syndrome, type I

AD

130000

174

Ehlers–Danlos syndrome, type II

AD

130010

175

Ehlers–Danlos syndrome, type III

AD

130020

176

Ehlers-Danlos syndrome type IV

AD

130050

177 178

Ehlers–Danlos syndrome, type IV

AD

130050

Ehlers–Danlos syndrome, type VI

AR

225400

179

Ehlers–Danlos syndrome, type VII

AD

130060

180–181

Epidermolysis bullosa dystrophic

AR

226600

183

Epidermolysis bullosa simplex

AR, AD

226670/148040

182, 184–185

Epidermolysis bullosa simplex, Dowling–Meara

AD

131760

186

Epidermolysis bullosa simplex, Koebner

AD

131900

187–188

Epidermolysis bullosa simplex, Weber–Cochayne

AD

131800

189–190

Epidermolytic hyperkeratosis (EHK)

AD

113800

191

Exudative vitreoretinopathy, familial

AD

133780

192

Fabry disease

XL

301500

193

Factor V Leiden mutation

AD

227400

194

Factor XI deficiency

AR

264900

195

Familial dysautonomia

AR

256800

197

Familial hemophagocytic lymphohistiocytosis

AR

608898

198

Familial hepatic veno-occlusive disease with immunodeficiency

AR

235550

199 200–201

(VODI) Familial isolated growth hormone deficiency

AD

173100

Fanconi anemia

AR

227650

202–204

Fragile X syndrome

XL

309550

205–206

Friedreich ataxia

AR

229300

207

Fucosidosis

AR

230000

208–209

Fukuyama congenital muscular dystrophy

AR

253800

210

Fumarate hydratase deficiency

AD

136850

211

Galactosemia

AR

230400

212

Gaucher disease

AR

230800

213–216

Gerstmann–Straussler disease

AD

137440

217

Glanzmann thrombasthenia

AR

273800

218

Glaucoma, open-angle, type 1A

AD

137750

219

Glaucoma, primary open-angle

AD

137760

220

Glucocorticoid receptor deficiency

AD

138040

221

384

Genetic Disorders and the Fetus

Table 11.1 Continued Disease

Mode

OMIM no.

Reference

Glutaric aciduria type 1 (GA1)

AR

231670

222

Glutaric aciduria, type IIA

AR

231680

223

Glycerol kinase deficiency

XL

307030

224–225

Glycogen storage disease, type I/Ia

AR

232200

226

Glycogen storage disease, type II

AR

232300

227

Glycogen storage disease, type III

AR

232400

228

Glycogen storage disease, type IV

AR

232500

229–230

Glycogen storage disease, type V

AD

153460

231

Glycogen storage disease, type VI

AR

232700

232

Glycogen storage disease, type VII

AR

232800

233

Gonadal dysgenesis, 46,XY

XL

306100

234

Greenberg/HEM skeletal dysplasia

AR

215140

235 236–237

Greig syndrome

AD

175700

Hailey–Hailey disease (HHD)

AD

169600

238–239

Hallervorden–Spatz syndrome

AR

234200

240

Harlequin ichthyosis

AR

242500

241

Hemangioma, familial cavernous

AD

116860

242

Hemochromatosis

AR

235200

243

Hemophilia A

XL

306700

244–245

Hemophilia B

XL

306900

246–247

Hemoglobin C

AR

141900

248

Hemoglobin E

AR

141900

249

Hemoglobin O

AR

141900

250

Hemoglobin S

AR

141900

251–253

Hereditary hemorrhagic telangiectasia

AD

187300

254

Hereditary neuropathy with liability to pressure palsies

AD

162500

255

Hereditary chronic pancreatitis (HCP)

AD

167800

256, 420

Herlitz junctional epidermolysis bullosa

AR

226700

257–259

Hermansky–Pudlak syndrome

AR

203300

260

Hirschsprung disease

AD

142623

261–262

Holocarboxylase synthetase (HLCS) deficiency

AR

253270

263

Holoprosencephaly, type III

AD

142945

264–265

Holoprosencephaly and moyamoya disease

AD

142946

266 267

Holt–Oram syndrome

AD

142900

Homocystinuria

AR

236200

268

HSAS (hydrocephalus due to stenosis of aqueduct of Sylvius)

XL

307000

147

Hunter syndrome

XL

309900

269–270

Huntington disease

AD

143100

271–274 275

Hurler syndrome

AR

252800

Hydrocephalus

XL

307000

276

Hypercholesterolemia

AD

143890

277

Hyperhomocysteinemia (MTHFR deficiency)

AR

236250

278

Hyperkalemic periodic paralysis

AD

170500

279

Hyperlipoproteinemia, type I

AR

238600

280

Hyperoxaluria, type I

AR

259900

281

Hypertrophic cardiomyopathy

AD

192600

282

Hypochondroplasia

AD

146000

283 284–285

Hypoglycemia, persistent hyperinsulinemic, of infancy

AD

601820

Hypoparathyroidism

XL

307700

286

Hypophosphatasia, infantile

AR

241500

287–291

CHAPTER 11

Molecular Genetics and Prenatal Diagnosis

385

Table 11.1 Continued Disease

Mode

OMIM no.

Reference 292

Hypophosphatemic rickets

XL

307800

Ichthyosis

XL

308100

293–295

Incontinentia pigmenti, type I

XL

308300

296–297

Incontinentia pigmenti, type II

XL

308310

298

Isovaleric acidemia

AR

243500

299

Jackson–Weiss syndrome

AD

123150

300

Kallmann syndrome

XL

308700

301

Kell antigen

AD

110900

302

Kennedy disease

XL

313200

303

Kniest dysplasia

AD

156500

304

Krabbe disease

AR

245200

305 306

Lamellar ichthyosis

AR

242300

Langer–Giedion

AD

150230

307

Langer mesomelic dysplasia

AD (Pseudo)

31265

308

Leber congenital amaurosis

AR

204000

309

Leber hereditary optic neuropathy

XL

308900

310–311

Leprechaunism

AR

246200

312

Lesch–Nyhan syndrome

XL

308000

313–315

Leydig cell hypoplasia

AD

152790

316

Li–Fraumeni syndrome

AD

151623

317

Lissencephaly associated with mutations in TUBA1A

AD

602529

318

Long-chain 3-hydroxyl-CoA dehydrogenase deficiency

AD

143450

319

Long QT syndrome

AD

192500

320

Long QT syndrome

AR

220400

321

Lowe syndrome

XL

309000

322–323

Lujan–Fryns syndrome

XL

309520

324

Macular dystrophy, retinal, type I

AD

136550

325

Malignant hyperthermia

AD

145600

326

Maple syrup urine disease

AR

248600

327

Marfan syndrome

AD

154700

328–331

Maroteaux–Lamy syndrome

AR

253200

332

MASA (mental retardation, aphasia, shuffling gait & adducted

XL

303350

147 333

thumbs) McCune–Albright syndrome

AD

174800

Meckel–Gruber syndrome

AR

249000

334–335

Medium-chain acyl-CoA dehydrogenase deficiency

AR

201450

336

Medullary thyroid carcinoma

AD

155240

261

Menkes disease

XL

309400

337

Metachromatic leukodystrophy

AR

250100

338

Metaphyseal chondrodysplasia McKusick type

AR

250250

339 340

Metaphyseal chondrodysplasia, Schmid type

AD

156500

Methylmalonic acidemia

AR

251000

341–342

Methylmalonic aciduria and homocystinuria, cblC type

AR

277400

343

Migraine hemiplegic, familial

AD

141500

344

Miller–Dieker syndrome

AD

247200

345

Mitochondrial disorders

Mit

346–347

Morquio syndrome, type B

AR

253010

348–349

Mucopolysaccharidosis type IVA

AR

253000

350

Multiple endocrine neoplasia, type 1

AD

131100

351

Multiple endocrine neoplasia, type 2A

AD

171400

352–353

386

Genetic Disorders and the Fetus

Table 11.1 Continued Disease

Mode

OMIM no.

Reference

Multiple endocrine neoplasia, type 2B/3

AD

162300

354

Multiple epiphyseal dysplasia

AD

132400

355

Multiple exostoses

AD

133700

356–358

Muscular dystrophy, Duchenne and Becker

XL

310200

359–363

Muscular dystrophy, Emery–Dreifuss

XL

310300

364

Muscular dystrophy, facioscapulohumeral

AD

158900

365

Muscular dystrophy, Fukuyama

AR

253800

366

Muscular dystrophy, limb-girdle

AR

253600

367

Muscular dystrophy, oculopharyngeal

AD

164300

368

Myotonia congenita

AD

160800

369

Myotonia congenita

AR

255700

370

Myotonic dystrophy

AD

160900

371–375

Myotubular myopathy

XL

310400

376

Nail-patella syndrome

AD

161200

377

NARP syndrome (neurogenic weakness, ataxia, retinitis

Mit

516060

378

Nemaline myopathy

AD

161800

379

Nephrogenic diabetes insipidus

XL

304800

380

Nephrosis, congenital (Finnish)

AR

256300

381

Nesidioblastosis

AR

256450

284–285

Neurofibromatosis, type I

AD

162200

382–384

Neurofibromatosis, type II

AD

101000

385

Neuronal ceroid lipofuscinosis, juvenile

AR

204200

386

pigmentosa)

Niemann–Pick disease type A

AR

257200

387

Niemann–Pick disease type B

AR

607616

388 389

Niemann–Pick disease type C

AR

257220

Nijmegen breakage syndrome

AR

251260

390

Nonketotic hyperglycinemia

AR

238300

391–393

Noonan syndrome

AD

163950

394–395

Norrie disease

XL

310600

396–399

Omenn syndrome

AR

603554

403

X-linked Opitz G/BBB syndrome

XL

300000

404

Optic atrophy, type 1

AD

165500

405

Ornithine transcarbamylase deficiency

XL

311250

406–409

Osteo-arthrosis, precocious

AD

165720

410

Osteogenesis imperfecta, type I

AD

166200

411–413

Osteogenesis imperfecta, type II

AD

166210

414

Osteogenesis imperfecta, type III

AR

259420

415

Osteogenesis imperfecta, type IV

AD

166220

413, 416

Ovarian cancer

AD

167000

417

Ovarian failure, premature

AD

176440

418

Pallister–Hall syndrome

AD, de novo

260350

419

Pancreatitis, hereditary

AD

167800

256, 420

Pearson syndrome

Mit

557000

421

Pelizaeus–Merzbacher disease

XL

312080

422

Periventricular nodular heterotopia

XL

300049

423 424

Peroxisome biogenesis disorders (Zellweger syndrome)

AR

214100

Phenylketonuria

AR

261600

425–428

Pfeiffer syndrome

AD

101600

429

Pituitary dwarfism, type I (IGHD)

AR

262400

7

CHAPTER 11

Molecular Genetics and Prenatal Diagnosis

387

Table 11.1 Continued Disease

Mode

OMIM no.

Reference

Pituitary dwarfism, type III (panhypopituitary)

AD, AR

173110

7, 430

Polycystic kidney disease

AD

173900

431–435

Polycystic kidney disease

AR

263200

436

Polycystic liver disease

AD

174050

437

Porphyria, acute intermittent

AD

176000

438

Porphyria, congenital erythropoietic

AR

263700

439

Porphyria, cutanea tarda

AD

176100

440

Prader–Willi syndrome

AD, de novo

176270

441

Precocious puberty, male-limited

AD

176410

442

Primary hyperoxaluria type 1 (PH1)

AR

259900

443

Primary microcephaly with ASPM mutation

AR

605481

444

Progeria

AD

176670

445

Progressive familial intrahepatic cholestasis (PFIC 1)

AR

211600

446

Progressive familial intrahepatic cholestasis (PFIC2)

AR

601847

447

Propionic acidemia, type I

AR

232000

447–448

Propionic acidemia, type II

AR

232050

449

Protein C deficiency

AD

176860

450

Protein S deficiency

AD

176880

451

Pseudoachondroplasia

AD

177170

452

Pycnodysostosis

AR

265800

453

Pyridoxine 5′-phosphate oxidase deficiency

AR

603287

454

Pyruvate kinase deficiency

AR

266200

455

Pyruvate kinase-Amish mutation PKLR

AR

609712

456

Retinitis pigmentosa

AR

268000

457

Retinitis pigmentosa

XL

312600

458

Retinitis pigmentosa 1

AD

180100

459

Retinitis pigmentosa 4

AD

180380

460

Retinoblastoma

AD

180200

461–463

Retinoschisis, juvenile

XL

312700

464

Rh C genotyping

AD

111700

465

Rh D genotyping

AD

111680

466

Rh E genotyping

AD

111690

467

Rieger syndrome

AD

180500

468–469

Roberts syndrome

AR

268300

470

Rubinstein–Taybi syndrome

AD

180849

471

Russell–Silver syndrome

UPD

180860

472

SADDAN (severe achondroplasia with developmental delay and

AD

187600

473

Saethre–Chotzen syndrome

AD

101400

474

Salla disease

AR

269920

475

Sandhoff disease

AR

268800

476

acanthosis nigricans)

Sanfilippo syndrome, type B

AR

252920

477–478

Sanjad–Sakati syndrome

AR

241410

479

Severe combined immune deficiency

AR

601457

480

Sex-determining region Y

YL

480000

481–482

Sex reversal, dosage sensitive

XL

300018

483

Short-chain acyl-CoA dehydrogenase deficiency

AR

201470

484

Sickle cell anemia

AR

141900

251–253

Sideroblastic anemia

XL

301300

485

Simpson–Golabi–Behmel syndrome

XL

312870

486

388

Genetic Disorders and the Fetus

Table 11.1 Continued Disease

Mode

OMIM no.

Reference

Sjögren–Larsson syndrome

AR

270200

487–488

Sly syndrome

AR

253220

489

RSH/Smith–Lemli–Opitz

AR

270400

490

Spastic paraplegia

AR

270800

491

Spastic paraplegia

XL

312900

492

Spastic paraplegia type 3

AD

182600

493

Spastic paraplegia type 4

AD

182601

494

Spinal and bulbar muscular atrophy

XL

313200

495

Spinal muscular atrophy, types I/II/III

AR

253300

496–497

Spinocerebellar ataxia, type I

AD

164400

498

Spinocerebellar ataxia, type II

AD

183090

499–500

Spinocerebellar ataxia, type III

AD

109150

501

Spondyloepiphyseal dysplasia

AD

183900

502

Spondylometaepiphyseal dysplasia, Strudwick type

AD

184250

503

Steroid 11-hydroxylase deficiency

AR

610613

504

Stickler syndrome

AD

108300

505–506

Supravalvular aortic stenosis

AD

185500

507

Tay–Sachs disease

AR

272800

508–511

Thalassemia, α

AR

141800

251, 512

Thalassemia, β

AR

141900

252, 513

Thanatophoric dysplasia

AD

187600

514

Thyroid hormone β-receptor deficiency

AD

190160

515

Treacher–Collins syndrome

AD

154500

516–518

Trichorhinophalangeal syndrome, type I

AD

190350

519

Triose-phosphate isomerase deficiency

AD

190450

520–521

Tuberous sclerosis

AD

191100

522–524

Tyrosinemia, type I

AR

276700

525–526

Progressive Ullrich congenital muscular dystrophy

AR

254090

527

Usher syndrome, type I

AR

276900

528

Usher syndrome, type II

AR

276901

529

Usher syndrome, type III

AR

276902

530

van der Woude syndrome

AD

119300

531

Vas deferens, congenital bilateral aplasia

AR

277180

532

Velocardiofacial syndrome

AD

192430

533

Very long-chain acyl-CoA dehydrogenase deficiency

AR

201475

534

von Hippel–Lindau disease

AD

193300

535

von Willebrand disease

AD

193400

536–537

Waardenburg syndrome, type I

AD

193500

538–539

Waardenburg syndrome, type II

AD

193510

540

Werner syndrome

AR

277700

541

Williams syndrome

AD

194050

507

Wilms tumor

AD

194070

157–158

Wilson disease

AR

277900

542

Wiskott–Aldrich syndrome

XL

301000

543–544

Wolcott–Rallison syndrome

AR

226980

545

Wolf–Hirschhorn syndrome

AD

194190

546–547

Wolman disease

AR

278000

548

X-linked dilated cardiomyopathy (XLDC)

XL

302045

549

X-linked hypohidrotic ectodermal dysplasia

XL

305100

550

Xeroderma pigmentosum

AR

276700

551

CHAPTER 11

Gene structure A gene is a sequence of chromosomal DNA that is required for the production of a functional product, often a protein plus the regulatory sequences that control expression of the product (Figure 11.1). Human genes contain exons (which are contained in mature mRNA) and many also contain introns (which are removed in messenger RNA, or mRNA, synthesis). Because exons are the sequences that make up mature RNA, they include: (1) 5′ untranslated sequences, (2) internal exons whose codons encode for amino acids in the translated protein product, and (3) 3′ untranslated sequences. In contrast, introns or intervening sequences (IVS) contain only noncoding DNA sequences, which are transcribed but then removed from the mRNA by splicing before translation.3 Thus, mutations that are differences between the sequences of copies of a gene, called alleles, can occur within exons or introns of a gene. Nonsense mutations can lead to no protein being formed; missense mutations to formation of a protein with an altered amino acid sequence, and splicing mutations cause the mRNA to be incorrectly spliced and translated into proteins whose altered sequence often per-

Molecular Genetics and Prenatal Diagnosis

389

turbs their function. Mutations can also affect untranslated portions of exons or introns or other DNA sequences that flank the gene and are not contained in the exons. While these mutations in noncoding sequences can affect different steps in gene expression, or mRNA processing, they may not be detectable by analysis of the gene’s protein product. Many human genes occur in groups of related genes called gene families or clusters. Examples of gene clusters are the α- and β-globin and growth hormone gene clusters (Figure 11.2). The human α-globin gene cluster on chromosome 16 contains the paired α-globin loci and the Z locus. The human β-globin gene cluster on chromosome 11 contains the β, δ, γG, γA and € loci.3–5 The growth hormone gene cluster on chromosome 17 contains five loci that are evolutionarily related.6,7 Frequently, pseudogenes that are inactive but stable sequences derived by mutation of an ancestral active gene are also found in gene clusters (ψα and ψβ in Figure 11.2). These pseudogenes have homology to and resemble related, active genes but have acquired DNA sequence alterations that prevent their expression.

Figure 11.1 Schematic representation of the structure of a typical gene showing its exons and introns as well as selected sequences that are important in various steps of its expression.

390

Genetic Disorders and the Fetus

Figure 11.2 α-Globin (AG), β-globin (NAG) and growth hormone (GH) gene clusters. For every gene shown, the black boxes represent coding regions (exons), small white boxes represent introns, and hatched boxes are the 5′ and 3′ untranslated regions. The large white boxes

The nature and mechanisms of human gene mutation CpG dinucleotides are hot spots for nucleotide substitutions In the genomes of eukaryotes (higher organisms that have a well-defined nucleus), 5-methylcytosine (5MeC) occurs predominantly in CpG dinucleotides, the majority of which appear to be methylated.8,9 Methylation of such cytosines results in a high level of mutation because of the propensity of 5MeC to undergo deamination to form thymine (Figure 11.3). Deamination of 5MeC probably occurs with the same frequency as either cytosine or uracil. However, whereas uracil DNA

represent pseudogenes, which are not expressed because of DNA sequence alterations. The numbers below the areas of the coding sequences represent the corresponding amino acid encoded by that sequence.

glycosylase activity in eukaryotic cells is able to recognize and excise uracil, thymine is a “normal” DNA base that is thought to be less readily detectable as a substitution for C and hence escapes removal by cellular DNA repair mechanisms. One consequence of the hypermutability of 5MeC is the paucity of CpG in the genomes of many eukaryotes, and heavily methylated vertebrate genomes exhibit “CpG suppression.”9 For example, in vertebrate genomes, the frequency of CpG dinucleotides is 20–25 percent of the frequency predicted from observed mononucleotide frequencies.10 The distribution of CpG in the genome is also nonrandom; about 1 percent of the vertebrate genome consists of a fraction that is CpG rich and that

CHAPTER 11

Figure 11.3 The structure of cytosine,

Molecular Genetics and Prenatal Diagnosis

391

5Me

C cytosine, and thymine. Note the tendency for CRT substitutions.

accounts for 15 percent of all CpG dinucleotides.11 In contrast to most of the scattered CpG dinucleotides, these “CpG islands” represent unmethylated domains that, in many cases, coincide with transcribed regions. The evolution of the heavily vertebrate genome has been accompanied by the progressive loss of CpG dinucleotides or “CpG suppression” as a direct consequence of their methylation in the germline.9 The CpG hypermutability seen in inherited disease implies that these sites are methylated in the germline, resulting in 5MeC s that are prone to deamination. Evidence that 5MeC deamination is directly responsible for these mutational events is that several cytosine residues known to have undergone a germline mutation in the LDL receptor gene (hypercholesterolemia) and the tumor protein 53 (TP53) gene (various types of tumors) are indeed methylated in the germline.12 Finally, recurring mutations of a single cytosine, which resides in a CpG dinucleotide, is found in achondroplasia.13 Achondroplasia is transmitted as an autosomal dominant trait and is the most common form of short-limbed dwarfism. Essentially, almost all cases of achondroplasia are due to a single base change at nucleotide 1138 in the fibroblast growth factor receptor 3 gene (FGFR3). The mutation rate at nucleotide 1138 is at least 2–3 orders of magni-

tude higher than other known CpG spots in the human genome. The mutation results in the substitution of glycine at codon 380 by an arginine. The net effect of the amino acid substitution is to reduce the rate of endochondrial bone growth. Non-CpG point mutation hot spots Because a variety of DNA sequence motifs are known to play an important role in the breakage and rejoining of DNA and could be potential determinants of single base pair (bp) changes, the DNA sequence environment of mutations has been analyzed. Some trinucleotide and tetranucleotide motifs are significantly over-represented within 10 bp on either side of the mutation hot spots. These motifs include TTT, CTT, TGA, TTG, CTTT, TCTT, and TTTG. In addition, Cooper and Krawczak14 screened a region of 610 bp around 219 non-CpG base substitution sites with known sequence environment for triplets and quadruplets that occurred at significantly increased frequencies. Only one trinucleotide (CTT) was found to occur at a frequency significantly higher than expected. Interestingly, CTT is the topoisomerase I cleavage site consensus sequence described by Bullock et al.15 and it was observed 36 times in the vicinity of a point mutation, whereas the expected frequency was 20.

392

Genetic Disorders and the Fetus

Strand difference in base substitution rates reveals some asymmetry, suggesting a strand difference for single base pair substitutions. For example, the relative dinucleotide mutability of CT to CC and AG to GG differs by almost fivefold. This finding suggests that, at least within genecoding regions, the two strands are differentially methylated and/or differentially repaired. Holmes et al.16 demonstrated in vitro the existence of a

strand-specific correction process in human and Drosophila cells whose efficiency depends on the nature of the mispair. Single nucleotide substitutions that affect mRNA splicing are nonrandomly distributed, and this nonrandomness can be related to the resulting phenotype.16 Naturally occurring point mutations that affect mRNA splicing fall into different categories (Figure 11.4). First, mutations can occur in 5′

Figure 11.4 Normal (A) and abnormal (B–D) patterns of splicing. Genes are shown above and resulting mRNA products below. A, Normal splicing. B, Exon skipping due to defects in either 3′ splice site of IVS1 or 5′ splice site IVS2. C, Exon skipping due to terminator mutation in exon 2. D, Alternative splicing that deletes a portion of

exon 1 due to the use of a cryptic 5′ splice site (left) and includes a portion of IVS2 due to the use of a cryptic 3′ splice site (right). Rectangles, exons; horizontal lines, introns; solid and open circles, 5′ and 3′ splice sites, respectively.

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or 3′ consensus splice sites. These mutations usually reduce the amount of correctly spliced mature mRNA and/or lead to the use of alternative splice sites in the vicinity. The use of alternative splice sites can cause production of mRNAs that either lack a portion of the coding sequence (“exon skipping”) or contain additional sequences of intronic origin (“cryptic splice site utilization”). Second, mutations within an intron or exon that may serve to activate cryptic splice sites can lead to the production of abnormal mRNAs. Third, mutations within a branch-point sequence can reduce normal splicing. Fourth, changes in other intronic sequences that are binding sites for proteins regulating splicing can cause alternative splicing.17 Last, base changes occurring in exon sequences called exon splicing enhancers (ESEs) may also disrupt normal splicing.18,19 ESEs are 6–8 nucleotide sequences found within exons and, in concert with the SR proteins, enhance the use of adjacent splice sites. ESEs can be disrupted by single nonsense, missense or silent point mutations. Disruption of ESEs has been identified in a number of genes and may be a very common mechanism for the alteration of normal gene splicing. Transcription of an mRNA is initiated at the cap site (+1), so named because of the posttranscriptional addition of 7-methylguanine at this position to protect the transcript from exonucleolytic degradation. Mutations at the cap site can either reduce transcription or cause initiation of transcription at a different but incorrect site. In the latter case, the transcript can be incomplete and/or unstable. A number of mutations in Met (ATG) translational initiation codons have been reported, with a preponderance of Met-to-Val substitutions. Whether the mutant mRNA is translated depends on a complex interplay of the different structural features of an mRNA that serves to modulate its translation.20 It was previously thought that an AUG codon was an absolute requirement for translational initiation in mammals. However, some exceptions are now known – for example, ACG, CUG. The scanning model of translational initiation predicts that the 40S ribosomal subunit initiates at the first AUG codon to be encountered within an acceptable Kozak consensus sequence context (GCC A/G CCAUGG).20 Another type of

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mutation that interferes with correct initiation is the creation of a cryptic ATG codon (in the context of a favorable Kozak consensus sequence shown above). The first reported example of a mutation in a termination codon was that in the α2-globin gene causing hemoglobin Constant Spring, an abnormal hemoglobin that occurs frequently in Southeast Asia.21 The associated α-globin chain is 172 amino acids in length, rather than the normal 141 amino acids, as a result of a TAA-to-CAA transition in the translation termination codon that causes a readthrough mutation. All polyadenylated mRNA in higher eukaryotes possess the sequence AAUAAA, or a close homolog, 10–30 nucleotides upstream of the polyadenylation site. This motif is thought to play a role in 3′ end formation through endonucleolytic cleavage and polyadenylation of the mRNA transcript. Several single bp substitutions are now known in the cleavage/polyadenylation signal sequences of the α2- and β-globin genes, and all of these cause a relatively mild form of thalassemia. Single base changes that result in new stop codons are known as nonsense mutations and are often associated with severe phenotypic consequences. A mRNA carrying a premature stop codon is usually rapidly degraded by a form of RNA surveillance known as nonsense-mediated mRNA decay.22 However exon skipping in exons that contain nonsense mutations has also been reported by Deitz et al.23 For example, exon B was deleted or skipped from fibrillin transcripts of a patient with Marfan syndrome whose exon B of the fibrillin gene contained a TAT-to-TAG nonsense mutation. Regulatory mutations Most mutations causing human genetic disease lie within the coding regions of exons. A different class of mutation is represented by those affecting regulatory regions of genes. These mutations disrupt the normal processes of gene activation and transcriptional initiation and serve to either increase or decrease the level of mRNA/ gene product synthesized rather than altering its nature. Single nucleotide substitutions in the promoter region 5′ to the β-globin gene produce β-

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thalassemia by causing a moderate reduction in globin synthesis. The known naturally occurring mutations are clustered around two regions that are implicated in regulation of expression of the human β-globin gene. One is a CACCC motif located 291 to 286 and the other is the TATA box found at about 230 bp upstream to the transcriptional initiation site. In addition to mutations in the remote promoter element known as the “locus control region” (LCR), Gastier et al.24 reported a mutation at 2530 that causes reduced β-globin synthesis. This mutation causes a ninefold increase in the binding capacity of BP1, a protein that may function as a repressor of expression of the βglobin gene. Gene deletions Gene deletions cause many different inherited conditions in humans, and these may be broadly categorized on the basis of the length of DNA deleted. Some deletions consist of only one or a few base pairs, while others may span several hundred kilobases.14 The term homologous recombination describes recombination occurring at meiosis or mitosis between identical or similar DNA sequences. It can involve recombination of homologous but nonallelic DNA sequences. This type of recombination is one cause of deletions of the α-globin genes that underlie α-thalassemia. Repetitive DNA sequences can also produce this type of deletion by promoting unequal crossovers through homologous unequal recombination. For example, the Alu repeat is the most abundant repetitive element in the human genome and Alu repeats are involved in recombinations that cause deletions of the LDLR and complement component 1 inhibitor genes (CINH). Hemoglobin (Hb) Lepore is the classic example of a gene fusion. Hb Lepore is an abnormal molecule, which has the first 50–80 amino acid residues of δ-globin at its N terminal and the last 60–90 amino acid residues of β-globin at its C terminal. Nonhomologous or illegitimate recombination occurs between two sequences that show minimal sequence homology. This kind of recombination underlies some gross DNA rearrangements, in

which the breakpoints share only a few nucleotides of homology. The endpoints of some deletions are found to be marked by short repeated sequences. These short tandem repeats have been shown to be prone to slipped strand mispairing during DNA replication. This occurs when the normal pairing between the two complementary strands of a double helix is altered by the staggering of the repeats on the two strands, leading to incorrect pairing of repeats and a deletion of the intervening sequence on the newly synthesized strand. This slipped strand mispairing mechanism has been shown to generate deletions in the Kearns–Sayre syndrome (KSS), a mitochondrial encephalomyopathy characterized by external ophthalmoplegia, ptosis, ataxia, and cataracts.25 About one-third of the cases of KSS are due to a common 4,977 bp deletion that is associated with two perfect 13 bp direct repeats. The deletion result is the elimination of the intervening sequence between two perfect 13 bp repeats. Because the mitochondrial genome is recombination deficient, it has been postulated that the common deletion occurs by the replication slippage mechanism. Gene conversion Gene conversion is the alteration of one allele so that it acquires one or more changes from the other allele.26 The result is similar to that of a double cross-over event. The difference between the two processes is that the modification of one allele (the target) after gene conversion is nonreciprocal because the other allele (the source) is left unchanged. Pseudogenes have been shown to be sources of deleterious mutations by gene conversion. The majority of the point mutations occurring in steroid 21-hydroxylase deficiency arise by gene conversion between the functional 21-hydroxylase gene, CYP21B, and the closely related pseudogene, CYP21A. The two genes occur on tandem DNA sequences, and the point mutations are copied from the pseudogene into the functional gene. Approximately 75 percent of the mutations in the functional gene have been transferred from the pseudogene by gene conversions. About 20 percent of the mutations are the result of an unequal cross-over during meiosis, which deletes a 30 kb gene segment that encompasses the 3′ end of the pseudogene and the 5′ end of

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the functional gene, producing a nonfunctional chimeric pseudogene.27 Expansion of unstable repeat sequences Trinucleotide repeat expansions are the molecular basis for a growing number of human genetic diseases, including fragile X syndrome, myotonic dystrophy, Huntington disease, spinal bulbar muscular atrophy, Friedreich ataxia, and many other inherited ataxias. Trinucleotide repeats undergo a unique process of dynamic mutation, whereby the polymorphic repeat sequences are unstable and expand beyond the normal size range. The effects of the expansion are varied and may result in a loss of gene expression, a gain of function or abnormal RNA processing. Genetic anticipation, mosaicism and phenotypic heterogeneity are common features of these disorders. Summary There are various kinds of mutations in the human genome and many potential mechanisms for their production. Single base pair substitutions account for the majority of gene defects. Among these, the hypermutability of CpG dinucleotides represents an important and frequent cause of mutation in humans. Point mutations can affect protein function and stability, transcription, translation, mRNA stability, and mRNA splicing and processing. Mutations in regulatory elements are of particular significance because they often reveal DNA domains that are bound by regulatory proteins. Mutations that affect mRNA splicing likewise contribute to our understanding of sequences important in transcript splicing. Additional splicing mutations whose phenotype results primarily or exclusively from ESE disruption may be even more prevalent. There is a growing list of disorders caused by abnormal copy number of trinucleotide repeats within the 5′ or 3′ untranslated regions or coding sequences of genes. However, the mechanisms by which the numbers of these trinucleotide repeats expand or contract during meiosis and mitosis are not completely understood. The study of mutations in human genes is vital in understanding the pathophysiology of hereditary disorders, providing improved diagnostic tests, and designing appropriate therapeutic approaches.

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Molecular genetic techniques used in prenatal diagnosis To determine if a fetus has inherited a mutation that will cause a genetic disease, one must overcome several problems related to DNA and gene structure and the variety of different possible mutations. First, the amount of DNA that is present in each cell by itself poses a problem. Because the haploid DNA complement of each cell is ∼3 × 109 base pairs, the average gene compromises only 1/100,000 of the total nuclear DNA. Second, the presence of related DNA sequences such as those found in gene clusters can make the process of detecting a mutation in one member of the gene cluster difficult. Third, the variety of mutations at different loci (locus heterogeneity) and different alleles at the same locus (allelic heterogeneity) that occurs in different diseases must be considered in all DNA diagnostic tests. Although these and other factors pose problems for prenatal diagnosis, the current and growing variety of molecular genetic techniques enables detection of a growing list of mutations that cause heritable disorders. The basic tools of gene diagnosis are restriction endonucleases, gene segments, oligonucleotides, and DNA polymerase. Restriction endonucleases are used to produce genomic DNA fragments that are suitable for size analysis and can be separated by size using gel electrophoresis. The fragments resulting from endonuclease cleavage are denatured and transferred to a membrane to form a Southern blot. Gene segments and oligonucleotides can be labeled and used as probes that anneal to and detect specific genomic fragments. Oligonucleotides and DNA polymerase can be used to amplify selected genomic segments using PCR amplification. These amplified segments or amplicons can be analyzed by DNA sequencing or a variety of other methods to detect mutations. Applications of these tools to directly or indirectly detect gene alterations are illustrated by the following examples and the genetic diseases are listed in Table 11.1. Specimens for prenatal diagnosis Specimens submitted for prenatal testing of the fetal DNA most often include chorionic villi

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or cultured chorionic villi or amniotic fluid cells. Direct detection of mutations Many mutations can be directly detected because they alter a restriction endonuclease site, change the size of a fragment or alter a PCR product size and/or sequence. The use of a variety of methods detecting these specific changes is discussed and illustrated below. Restriction endonuclease analysis and Southern blots The basic tools of molecular genetics for the past two decades have been restriction endonucleases, Southern blots and probes.552–554 Restriction endonucleases are bacterial enzymes that protect bacteria from invasion by foreign DNA.554a They prevent viral infection by recognizing specific nucleotide sequences of four or more bases and cleave the double-stranded viral DNA at that site. Molecular geneticists take advantage of the thousands of restriction endonuclease sites in human DNA and use restriction enzymes to cleave long strands of DNA into fragments of reproducible size.555 Despite the morphologic differences between cells from different organs within the human body, DNA contained within each cell (amniotic fluid cells, chorionic villi, leukocytes, fibroblasts, solid tissue, etc.) is identical. After purification from cells or tissue, the DNA is quantitated and microgram quantities are digested with a restriction endonuclease specific for the analysis and for which fragments of reproducible size have been well characterized. The digested DNA is subjected to agarose gel electrophoresis to separate the DNA fragments by size556 (Figure 11.5). After electrophoresis, the DNA fragments can be visualized by a variety of techniques, including gel staining with ethidium bromide and exposure to ultraviolet light. Because thousands of DNA fragments of varying size are generated from the digestion, a smear spanning the length of the gel, as opposed to discrete bands, is seen. DNA hybridization is then done to identify the specific DNA fragment of interest. Originally described by Southern in 1975,556 this method involves treatment of the

gel with alkali to render the patient’s DNA single stranded (denatured). Next, the DNA is transferred from the gel to a filter membrane either by diffusion, using a salt solution passed through the gel and the filter into blotting paper, or by vacuum or electrophoretic transfer. Once transferred, the DNA fragments are firmly attached to the membrane by either baking or chemical treatment. The filter-bound DNA is then placed in a hybridization solution containing a singlestranded radiolabeled or otherwise tagged DNA, cDNA or RNA probe. During incubation, the single-stranded probe anneals to single-stranded DNA fragments with complementary DNA sequences to form hybrid molecules. After hybridization, the excess probe is removed from the membrane and the membrane is exposed to film by autoradiography, and genomic DNA fragments containing sequences that are complementary to the probe appear as bands on the autoradiograph. The sizes and number of bands seen are determined by the number and locations of restriction endonuclease sites in the segment under study. Applications of Southern blotting to detect chromosome fragments, deletions, rearrangements and point mutations will be illustrated by the following examples.

Detection of gene deletions Familial isolated growth hormone (GH) deficiency type 1A (IGHD 1A) is an endocrine disorder that is caused by deletion of the GH genes. This disorder has an autosomal recessive mode of inheritance, and affected individuals have severe growth retardation due to complete deficiency of GH. Most cases respond only briefly to GH replacement therapy due to their tendency to develop high titers of anti-GH antibodies.7 Restriction analysis of the GH gene (GH1) is complicated by the fact that it is one of the five GH-related genes (5′-GH1:CSHP1:CSH1:GH 2:CSH2-3′) contained in the GH gene cluster (see Figure 11.2). Although these other genes share extensive sequence homology, only the GH1 locus encodes GH. The GH1 gene is flanked by consistent BamHI sites that are 3.8 kb apart. Although the CSHP1, CSH1, GH2 and CSH2 genes are sufficiently homologous to hybridize to the GH1 probe, they all are contained in BamHI-derived

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Figure 11.5 Restriction endonuclease analysis of genomic DNA. Genomic DNA is digested with one or more restriction endonucleases and the resulting DNA fragments are separated by size through agarose gel electrophoresis and blotted to nitrocellulose. The

nitrocellulose-bound DNA fragments are then hybridized to a radiolabeled gene probe, and the resulting radio-active double-stranded DNA molecules are visualized by autoradiography.

fragments that differ in size from that of GH1. Autoradiograms of DNAs from IGHD 1A subjects lack the 3.8 kb fragments that normally contain the GH1 genes (Figure 11.6). In addition, the intensity of the 3.8 kb bands in DNA from the

heterozygous parents is intermediate between that of controls and their affected children. These results show that IGHD 1A subjects are homozygous and their parents are heterozygous for GH1 gene deletions. Because these deletions

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Figure 11.6 Autoradiogram patterns of DNA from a child (solid symbol) with isolated growth hormone deficiency, his parents (half solid symbols), and two controls (C1 and

C2) after digestion with BamHI and hybridization to the GH cDNA probe. Note that the GH gene (hGH-N) is deleted in the child.

preclude production of any GH, affected individuals tend to be immunologically intolerant to exogenous GH. Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder

characterized by recurrent focal neuropathy. Patients with HNPP typically have a deletion of the PMP gene on chromosome 17p11.2-12 due to unequal crossing over during germ cell meiosis.557 Diagnosis can be accomplished using fluorescent

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in situ hybridization (FISH)588 or by quantitative PCR multiplex assay.559,560

Detection of gene expansions The molecular defect in patients with fragile X syndrome can be detected by Southern blot analysis. In contrast to IGHD-1A, in which there is a deletion of genetic information resulting in the disease state, in fragile X syndrome, there is an expansion of a repetitive trinucleotide (CGG) DNA segment in the FMR-1 gene.205,206,561,562 Fragile X syndrome is the most common cause of inherited mental retardation (see Chapter 9) and the fragile site at band Xq27.3 can be determined in lymphocytes of affected males when cultured in folate-deficient media.563 The FMR-1 gene mapped to this location encodes an mRNA-binding protein with domains

Figure 11.7 Autoradiogram patterns obtained from a fragile X syndrome family after digestion of DNA with restriction endonucleases EcoRI and EagI. Normal females demonstrate bands corresponding to DNA fragments 5.2 and 2.8 kb in length and represent the inactive and active X chromosomes, respectively. Normal males have a single

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that function to mediate nucleocytoplasmic shuttling of RNA564 which is prominently expressed in brain and testis.565 In the majority of patients with fragile X syndrome, the disease results from a massive postzygotic expansion of the CGG repeat located in the 5′ untranslated region of the FMR-1 gene (Figure 11.7).566 When the repeat number exceeds about 230, the DNA becomes abnormally methylated and the gene becomes nonfunctional. Thus, the repeat expansion and methylation result in the full mutation observed in affected patients. Varying degrees of methylation can lead to a “mosaic” male. Although some FMR-1 protein may be produced from the unmethylated alleles, phenotypically, the majority of these males have moderate to severe mental retardation.567 In the normal population,

band corresponding to DNA fragments 2.8 kb in length. Additional bands in carrier females represent “premutation” alleles. Full-mutation alleles observed in affected males and females are characterized by smears as opposed to discrete bands

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the CGG repeat number is polymorphic and varies from 6 to 50, with the most common alleles having 29–30 repeats. Carriers have premutations that are unmethylated, transcriptionally active, and 50–200 repeats. However, the fragile site analysis568 has been replaced by the more sensitive molecular analysis, which facilitates identification of the majority of carriers. Premutations are unstable and at risk for expansion into full mutations when maternally transmitted. The risk for expansion increases as the repeat number increases.569,570 It appears very low (50 years of age) may develop a neurodegenerative disorder (FXTAS), characterized by cerebellar ataxia and/or intention tremor, cognitive decline, and other features.574

Detection of gene rearrangements Hemophilia A is an example of a disease that can result from a gene rearrangement. Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of coagulation factor VIII; it affects approximately 1 in 10,000 males. The disease results from mutations in the factor VIII gene that are heterogeneous in both type and position within the gene. However, intragenic inversion mutations have been found to be common to 45 percent of patients with severe disease (factor VIII activity levels 95 °C. However, they can differ in their half-life and their efficiency of base incorporation, making one polymerase

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Figure 11.8 The size of genomic DNA fragments obtained after MstII digestion of the β-globin gene. The locations of the βS mutation (center arrow), which eliminates an

MstII site that normally occurs at codons 5–7 of the βA-globin gene and probe sequences used in Southern blot analysis are indicated.

more suitable than another depending on the type of assay that is to be performed. The PCR reaction begins with an initial denaturation phase at 94 °C to render the template DNA single stranded by breaking the hydrogen bonds between the complementary purine and pyrimidine bases of the two strands of the double helix (Figure 11.10). The initial denaturation phase is followed by 20–30 repetitive cycles of short denaturing, annealing, and extension periods. After the denaturing period at 94 °C, the primers are allowed to anneal to opposite strands of the DNA template by lowering the temperature from 94 to 65 °C. The annealing temperature used is a function of the GC content of the oligonucleotide primers. The greater the number of complementary G/C base pairs in the sequence, the higher the annealing temperature. During the extension period at 72 °C, DNA polymerase directs DNA synthesis. After this primer-directed synthesis, the strands of DNA are again denatured at 94 °C to begin yet another round of amplification. With each amplification cycle, the number of copies of that specific region of DNA synthesized doubles. Potentially, after 30 cycles, 106 to 109 copies are achieved. The amplification yields enough DNA for subsequent analysis using simple gel electrophoresis or gel electrophoresis coupled with restriction endonuclease

digestion, allele-specific hybridization or DNA sequencing.

Direct detection of chromosome fragments An example of the utility of PCR amplification is detection of Y chromosome fragments in Turner syndrome. Kogan et al. used PCR and Y-specific oligonucleotide primers to determine whether a chromosomal fragment was derived from an X or Y (see Fig. 11.10).245 Note that the affected subject, who was mosaic and had a 45,X/46X +frag karyotype, has easily detectable Y chromosomal material in DNA derived from peripheral blood. Such studies can be done in hours, require small amounts of blood, amniotic fluid or chorionic villi, and avoid the use of Southern blots or probes. This technique has been applied to determine the sex of fetuses at risk for X-linked disorders.

Direct detection of gene deletions Cystic fibrosis (CF) is an autosomal recessive disorder characterized by chronic lung disease and pancreatic insufficiency (see Chapter 17). The genetic abnormality underlying CF was discovered in 1989 by characterization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.153,585,586 About 70 percent of abnormal CFTR genes have a 3 bp deletion in exon 10, causing the

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Figure 11.9 Schematic representation of PCR amplification, showing complementary genomic strands to be amplified and their corresponding primers after annealing. Different temperatures are used to denature (94 percent), anneal (45 percent) and elongate (72 percent), enabling rapid accumulation of multiple copies with succeeding cycles of amplification.

loss of phenylalanine at codon 508 (ΔF508). Detection of the ΔF508 mutation by PCR amplification of exon 10 followed by polyacrylamide gel electrophoresis of the amplified products and staining with ethidium bromide is shown in Figure 11.11. Using this procedure, the altered mobility from the 3 bp deletion in exon 10 resulting in the loss of phenylalanine at codon 508 (ΔF508) in the CFTR gene can be seen easily.587 This and other tests for specific CFTR gene mutations are usually done to confirm that a patient has CF or for carrier screening. The heteroduplexes seen in heterozygotes in Figure 11.11 are sensitive indicators that the allelic

PCR products of the CFTR alleles differ in size. Such differences in the size of the forward and reverse strands from two different alleles cause “bubbles” to form in the heteroduplex that affect its migration. PCR can also be used to detect large deletions associated with the allelic disorders Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). The combined incidence of these X-linked disorders is 1 in 3,500 boys. Both disorders are characterized by progressive muscle wasting, with DMD being more severe and associated with early death.588 The gene that is defective in DMD and BMD is dystrophin, which has been mapped to Xp21, spans 2,000 kb, and contains 79 exons.360,589–591 About 65 percent of dystrophin gene mutations are intragenic deletions clustered in two hot spots containing the 59 terminal and exons 44–53.592,593 With the ability to identify deletions in 65 percent of the affected patients, accurate direct DNA testing can be used for these cases. By using full-length dystrophin cDNA clones to probe Southern blots, it is possible to directly detect deletions and duplications. The cDNA probes detect the site of the mutation itself, so meiotic recombination events are irrelevant. Therefore, the chance of diagnostic error is greatly reduced. The deletions are simply detected by examination of Southern blots for the presence or absence of each exon containing genomic restriction fragments that hybridize to the cDNA probe. However, the Southern blotting technique requires isotope and is tedious and time consuming. A deletion screen can be quickly performed using a multiplex PCR.594,595 The technique facilitates amplification of specific deletion prone exons within the DMD gene up to a millionfold from nanogram amounts of genomic DNA. When any one of the coding sequences is deleted from a patient’s sample, no ethidium bromide-stained amplification product, corresponding to the specific exon, is present on the gel (Figure 11.12). Multiplex PCR, using primer sets for about 20 different exons, now detects approximately 98 percent of the deletions in the dystrophin gene. In contrast to Southern blotting, which may require several cDNA hybridizations and take several weeks to obtain results, the PCR can be completed in 1 day. This makes the technique ideal for prenatal diagnosis, when time is critical.

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Figure 11.10 Polyacrylamide gel analysis of PCR product of DNA from a family similar to that shown in Figure 11.6 after amplification using Y chromosome-specific primers.

The identification of a deletion in a patient with DMD not only confirms the diagnosis but also allows accurate carrier detection in the affected family. Carrier status is determined by gene dosage, which shows whether a female at risk exhibits no reduction or 50 percent reduction in hybridization intensity in the bands that are deleted for the affected male. A 50 percent reduction (single-copy intensity) for the deleted band or bands on the autoradiograph indicates a deletion on one of her X chromosomes and she would therefore be a carrier. Dosage determinations can be made from Southern blots or using a quantitative PCR.596

Detection of gene expansions Similar to fragile X syndrome, the cause of Huntington disease (HD) is an expansion of a trinucle-

otide repeat.597 HD is a late-onset autosomal dominant neurodegenerative disorder characterized by involuntary choreic movements, psychiatric disorders, and dementia; it has an incidence of 1 in 10,000. In HD, the polymorphic repetitive segment is a CAG repeat located in exon 1 that encodes a polyglutamine tract near the N terminal of the huntingtin protein. Huntingtin contains 3,144 amino acids, is widely expressed throughout the brain and nonneural tissues598 and is located primarily in the cytoplasm, but a nuclear function has also been observed.599 Structural analysis of the HD gene promoter region is consistent with the gene being a housekeeping gene. Although on a cellular level mutant huntingtin is widely expressed in both neural and nonneural tissue, there is regional specific neuronal loss in the neurons in the caudate and putamen.

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Figure 11.11 Polyacrylamide gel analysis of PCR products obtained from blood spots collected on newborn screening forms from a family with CF. Upper symbols indicate heterozygous control subjects (C) and parents; lower symbols represent (left to right) carrier son,

affected daughter and unaffected daughter. Fragment sizes are shown on right. Additional upper fragments seen in heterozygotes are due to heterodimer formation, which occurs when different strands of 95 and 98 bp PCR products anneal.

Figure 11.12 Multiplex DNA amplification of Duchenne muscular dystrophy exons 8, 13, 19, 45 and 47. Lane 1: normal control; lane 2: DMD patient deleted for exons 8 and 13; lane 3: DMD patient deleted for exons 45 and 47.

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Huntington disease is caused by a toxic gain-offunction mechanism. The gain of function could be due either to an overactivity of the normal function or perhaps to the introduction of a novel function of the protein. The pathogenic process relation to the expansion may involve a novel interaction with other proteins or multimerization of the protein, leading to large insoluble aggregates. In HD brains, intranuclear inclusions of the truncated mutant protein aggregates have been identified. These alterations are ultimately associated with, but not necessarily causative of, cell death. Unlike fragile X, however, the HD allele is transcribed at equal amounts as the normal allele.600 Normal alleles are defined as alleles with 99 percent of meiosis. The most common normal allele lengths contain 17 and 19 CAG repeats. Mutable normal alleles are defined as alleles with 27–35 CAG repeats, and this repeat range is often referred to as the meiotic instability range.601,602 These alleles have yet to be convincingly associated with an HD phenotype; however, they can be meiotically unstable in sperm, and pathologic expansion of paternally derived alleles in this size range has been described. Approximately 1.5–2 percent of the general population carries alleles in this size range. The likelihood that transmission of an allele in this range will expand into an HD allele depends on several factors, which include sex of the transmitting individual, the size of the allele, and the molecular configuration of the region surrounding the CAG repeat and its haplotype. This risk may be as high as 10 percent for paternal alleles carrying a CAG repeat of 35. HD alleles with reduced penetrance are defined as alleles with 36–39 CAG repeats. Repeat sizes in this range are often referred to as being in the reduced penetrance range.603 Alleles in this size range are meiotically unstable and are associated with the HD phenotype in both clinically and neuropathologically documented cases. However, in rare cases, these alleles may also manifest reduced penetrance and have been found in elderly asymptomatic individuals. Huntington disease alleles with full penetrance are defined as alleles with 40 CAG repeats (Figure 11.13). Although a large repeat number contributes significantly to the age of onset in juvenile-

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onset patients, the repeat number is less correlated with the age of onset in the elderly and thus implies that other genetic or environmental factors may contribute to the age of onset in that group.602 Careful interpretation of data is necessary when a fetus is at risk for juvenile-onset HD associated with very large expansions that may not be easily identifiable by routine analysis. Additional techniques such as Southern blot or extra-long PCR may be utilized in these cases.603a Interestingly, the expansion of HD alleles demonstrates a sexof-parent effect, with 69 percent of paternal transmission demonstrating expansion compared with 32 percent of maternal transmission. In addition, 21 percent of paternal-transmitted expansions increase by five repeats as compared to 90 percent of those reported.604 The K329E mutation does not create or remove a restriction endonuclease site but can be detected by NcoI digestion of PCR amplification products containing a segment of the MCAD gene and a modified oligonucleotide primer605 (Figure 11.16). Note that the modified forward primer with a mismatch 5 bases upstream from its 3′ end contains a portion (CCATG) of the NcoI recognition site (CCATGG). When normal MCAD alleles are amplified by the CCATGA, amplified products are not cut by NcoI, thus producing the uncut 63 bp fragments. However, amplified products derived from alleles containing the K329E mutation yield CCATGG products that are cleaved by NcoI to produce 43 and 20 bp fragments (Fig. 11.17). A third example using PCR and restriction endonuclease analysis is the detection of the spinal muscular atrophy deletion. The autosomal recessive disorder proximal spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by degeneration of α motor neurons in the spinal cord, which results in progressive proximal muscle weakness and paralysis. SMA is the second most common fatal autosomal recessive disorder

(after cystic fibrosis), with an estimated prevalence of 1 in 10,000 livebirths.606 Childhood SMA is subdivided into three clinical groups on the basis of age of onset and clinical course: type I SMA (Werdnig–Hoffmann) is characterized by severe, generalized muscle weakness and hypotonia at birth or within the first 3 months. Death from respiratory failure usually occurs within the first 2 years. Type II children are able to sit, although they cannot stand or walk unaided, and they survive beyond 4 years. Type III SMA (Kugelberg–Welander) is a milder form, with onset during infancy or youth; patients learn to walk unaided. The survival motor neuron (SMN) gene comprises nine exons and has been shown to be the primary SMA-determining gene.607 Two almost identical SMN genes are present on 5q13: the telomeric or SMN1 gene, which is the SMA-determining gene, and centromeric or SMN2 gene. The SMN1 gene exon 7 is absent in about 95 percent of affected patients, while small, more subtle mutations have been identified in the remaining affected patients. Although mutations of the SMN1 gene are observed in the majority of patients, no phenotype genotype correlation was observed because SMN1 exon 7 is absent in the majority of patients independent of the type of SMA. This is due to the

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Figure 11.15 Polyacrylamide gel analysis of PCR products of DNA from a subject with sickle cell anemia (solid), his parents (hatched), and a control (open symbol) after MstII digestion.

fact that routine diagnostic methods do not distinguish between a deletion of SMN1 and a conversion event whereby SMN1 is replaced by a copy of SMN2. There have been several studies that have shown that the SMN2 copy number influences the severity of the disease.608–610 The copy number varies from 0 to 3 copies in the normal population, with approximately 10 percent of normals having no SMN2. However, patients with milder type II or III have been shown to have more copies of SMN2 than do patients with type I. It has been

proposed that the extra SMN2 in the more mildly affected patients arise through gene conversions, whereby the SMN2 gene is copied either partially or totally into the telomeric locus. Five base-pair changes exist between SMN1 and SMN2 transcripts, and none of these differences change amino acids. Because virtually all individuals with SMA have at least one SMN2 gene copy, the obvious question that arises is why individuals with SMN1 mutations have an SMA phenotype. It has now been shown that the SMN1 gene produces

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Figure 11.16 Detection of the K329E MCAD mutation. Normal MCAD alleles yield CCATGA PCR products that are not cut by NcoI and are 63 bp, while K329E alleles yield CCATGG products that are cleaved into 43 and 20 bp fragments.

predominantly full-length transcript, whereas the SMN2 copy produces predominantly an alternatively transcribed (exon 7 deleted) product. The inclusion of exon 7 in SMN1 transcripts and exclusion of this exon in SMN2 transcripts are caused by a single nucleotide difference at 16 in SMN exon 7. Although the C-to-T change in SMN2 exon 7 does not change an amino acid, it does disrupt an ESE, which results in the majority of transcripts lacking exon 7.18,19 Therefore, SMA arises because the SMN2 gene cannot compensate for the lack of SMN1 expression when SMN1 is mutated. However, the small amounts of full-length transcripts generated by SMN2 are able to produce a

milder type II or III phenotype when the copy number of SMN2 is increased. Recent evidence supports a role for SMN in small nuclear ribonuclear protein (snRNP) biogenesis and function.611 Based on recent reports, SMN has been shown to be required for premRNA splicing. Immunofluorescence studies using a monoclonal antibody to the SMN protein have revealed that the SMN protein is localized to novel nuclear structures called “gems,” which display similarity to and possibly interact with coiled bodies, which are thought to play a role in the processing and metabolism of small nuclear RNAs. snRNPs and possibly other splicing compo-

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Figure 11.17 DNA fragments generated after PCR amplification, digestion with NcoI, and electrophoresis in a polyacrylamide gel. Both parents’ (denoted by hatches) DNAs are heterozygous and yield fragments of 63 and 43 bp. Both of their affected children’s samples yield patterns containing only 43 bp fragments that are identical to the positive control.

Figure 11.18 Restriction enzyme digestion of PCR product distinguishes SMN1 from SMN2. Lanes 1 and 3: SMA patients deleted for SMN1; lanes 2 and 4: normal controls with SMN1 present.

nents require regeneration from inactivated to activated functional forms. The function of SMN is in the reassembly and regeneration of these splicing components. Mutant SMNs, such as those found in patients with SMA, lack the splicingregeneration activity of wild-type SMN. SMA may be the result of a genetic defect in spliceosomal snRNP biogenesis in motor neurons. Consequently, the motor neurons of patients with SMA have an impaired capacity to produce specific mRNAs; as a result they become deficient in pro-

teins that are necessary for the growth and function of these cells. The molecular diagnosis of the SMA consists of the detection of the absence of exon 7 of the SMN1 gene (Figure 11.18). Although this is a highly repetitive region and there is the almost identical centromeric SMN2 copy of the SMN1 gene, there is an exonic base pair difference and one can distinguish SMN1 from the SMN2 by restriction enzyme digestion. The absence of detectable SMN1 in patients with SMA is being used as a powerful diagnostic and prenatal test for SMA. As the carrier frequency of SMA approximates 1/50, the American College of Medical Genetics has proposed preconception/pregnancy carrier testing.611a This will lead to the increased identification of carrier couples and additional opportunities for prenatal diagnosis. Multiplex ligation-dependent probe amplification (MLPA) to detect deletions/duplications Multiplex ligation-dependent probe amplification (MLPA) is a variation of the PCR chain reaction that can amplify multiple targets with only a single set of primers. Each probe consists of two oligonucleotides which anneal specifically to adjacent target sites on the DNA. One oligonuclotide probe contains the sequence recognized by the forward primer, the other the sequence recognized by the reverse primer. Only when both probes are hybridized to their respective complements that make up the 5′ and 3′ ends of the target site can they be ligated into a complete probe. The advantage of separating the probe into two parts is that only the oligonucleotides that annealed to the target and were subsequently ligated are amplified. Since the unbound probe oligos do not present a doublestranded target for ligation, and remain single stranded, they cannot be PCR amplified. Since the probes are split in this way, the primer sequences hybridize to the template DNA at either end, and the amplification product amount is dependent on the number of target sites present in the sample DNA. Each complete probe has a unique length that can be determined by the addition of sequences between the primer and recognition sequences, so that its resulting amplicons can be separated and identified by differences in length.

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Figure 11.19 Multiplex ligation-dependent probe amplification (MLPA) analysis of an individual with familial pulmonary arterial hypertension (FPAH) who has an exon 2 deletion of a BMPR2 allele. Note the MLPA tracing for the individual with FPAH (top) shows a

reduction of the exon 2 peak (asterisk) as compared to that of a normal control (bottom). This approximate 50 percent reduction in peak intensity is indicative of a heterozygous deletion of exon 2 at the genomic DNA level.

This avoids the resolution limitations of multiplex PCR. Since the forward primer used for probe amplification is fluorescently labeled, each amplicon generates a fluorescent peak which can be detected by a capillary sequencer. Comparing the peak pattern obtained on a given sample with that obtained on various reference samples, the relative quantity of each amplicon can be determined. This ratio is a measure for the ratio in which the target sequence is present in the sample DNA. An example of the use of MLPA of genomic DNA to detect an exon 2 deletion of the BMPR2 gene in an individual with familial pulmonary arterial hypertension (FPAH) is shown in Figure 11.19.612 Similarly, exonic or whole gene duplications can also be detected.612a

Chapter 12). A deficiency in hexosaminidase A is the underlying cause of the disease. Three mutations in the hexosaminidase A (Hex A) gene – a GRC substitution in exon 12, a GRA substitution in exon 7, and a 4 bp insertion in exon 11 – represent approximately 95–99 percent of abnormal Hex A genes in Ashkenazi Jews.508,613,614 Because both the GRC and the GRA substitutions alter a restriction endonuclease site, they can be easily detected by restriction endonuclease digestion of amplified products after gel electrophoresis. The 4 bp insertion in exon 11 of the Hex A gene can be easily detected by PCR amplification coupled with dot-blot hybridization using allelespecific oligonucleotides (ASOs) to distinguish the normal and mutant alleles (Figure 11.20). This method is similar to Southern blot analysis. The PCR amplification products of both alleles are denatured and applied to the filter membrane and blotted by vacuum. ASOs corresponding to the normal (5′GAACCGTATATCCTATGGC3′) and mutant (5′GAACCGTATATCTATCCTA3′) alleles

Direct detection using allele-specific oligonucleotides Tay–Sachs disease is an autosomal recessive disease characterized by the accumulation of GM2 ganglioside primarily in the lysosomes of neurons (see

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Figure 11.20 Schematic of an autoradiogram obtained after PCR amplification of exon 11 of the Hex A gene coupled with hybridization to detect a 4 bp insertion. The

presence or absence of a signal after hybridization with normal and mutant allele ASOs is used to determine the genotype of each individual (see text).

are radiolabeled with 32P and hybridized to small circular areas or dots on the membrane. After the removal of excess unbound probe by washing, the radiolabeled probe that has annealed to complementary sequences in the PCR-amplified products will appear as blackened circles, and three patterns can be observed on the autoradiogram. First, PCR-amplified products from homozygous normal individuals will yield a positive signal only after hybridization with the ASO complementary to the normal allele. Second, PCR products from affected individuals homozygous for the mutation will yield a positive signal only after hybridization with the ASO complementary to the mutant allele. Third, samples from heterozygotes for the mutation yield a positive signal after hybridization with both of these probes. PCR amplification coupled with ASO hybridization is a highly sensitive technique and is capable of detecting as small as a single base difference. This technique is most useful in instances in which the mutation does not either create or destroy a restriction endonuclease recognition site, no modified primer is easily generated, and direct DNA sequencing of

PCR products is too labor intensive. This method is adaptable for high-volume throughput615 and is used for the detection of many genetic diseases, including α1-antitrypsin deficiency, β-thalassemia, cystic fibrosis, Lesch–Nyhan syndrome, and phenylketonuria.53–58,151–154,252,313–315,425–428,513,616–618 DNA sequence analysis of amplified DNA One of the most widespread uses of PCR is to generate amplified products for direct DNA sequence analysis. DNA sequence analysis of amplified products enables the base-by-base sequence determination of the amplified fragment, which is then a reflection of the sequence in the patient’s genomic DNA. All types of mutations can be detected using this method, including missense, nonsense, insertions, and deletions. Sanger or dideoxy DNA sequencing uses randomly incorporated 2′, 3′ dideoxynucleotides of A, C, G or T containing no 3′ OH group, thereby inhibiting 3′ extension of the growing chain.619 When one of the dideoxynucleotides is incorporated, the 3′ end of the reaction is no longer a substrate for chain

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elongation, the growing DNA chain is terminated and DNA fragments of varying length are produced. Original cycle sequencing used 32P endlabeled primer and a separate reaction mix was required for each terminating dideoxynucleotide. Current methods of cycle sequencing of doublestranded DNA, which rely on the principle of PCR to produce products by chain elongation, use fluorescence-labeled dideoxynucleotides, each A, C, G and T labeled with a unique dye. The reaction mix can now be performed in a single tube, and the reaction products are detected fluorescently as they pass an exciting source and an emission detector after electrophoretic separation, either by vertical polyacrylamide or capillary gel electrophoresis. Instrument computer software now is capable of separating the fluorescent dye colors and assigning a base (A, C, G or T) to each terminating dye peak in the chromatogram (Figure 11.21). The sequence of the PCR product can then be read from the chromatogram and compared with a known or wild-type sequence.

Indirect detection of mutations In some inherited disorders, the gene responsible for the disease has not yet been elucidated. However, the chromosomal locations of many of these genes have been mapped. As a result, the

Figure 11.21 Automated DNA sequencing using fluorescence-labeled dideoxynucleotides. This shows a typical output of sequence data from an ABI377

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transmission of a defective gene can be tracked in a family through the use of DNA polymorphisms. The DNA polymorphisms identified in each family act to flag the abnormal gene so that, in the case of prenatal testing, the normal, carrier or affected status of the fetus can be determined. This process is referred to as DNA linkage analysis. This technique is most useful for diseases in which the gene has not yet been isolated but is quite helpful for diseases in which the gene has already been identified but for which a multitude of different mutations have been identified, thus preventing the feasibility of direct mutation analysis for each family.

DNA polymorphisms The majority of variation occurring between individuals at the DNA level is the normal variation associated with DNA polymorphisms, which occur about every 250–500 nucleotides in noncoding regions of the genome. By definition, DNA polymorphisms are changes within the DNA present in the population at frequencies of greater than 1 percent.620,621 DNA polymorphisms, in some cases, can lead to differences in the number and location of restriction enzyme recognition sites (Figure 11.22). The resulting differences in DNA fragment sizes are referred to as restriction fragment length

automated DNA sequencer. Note the individual in A has a T, whereas the individual in B has an A substitution.

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Figure 11.22 Schematic representation of a restriction fragment length polymorphism. Note that the substitution of a T for a C on the white chromosome eliminates its middle CG MspI recognition site and yields

a single, larger fragment, which can be used as a chromosome-specific marker after Southern blotting, as shown on the right.

polymorphisms (RFLPs) and are easily detected by PCR. An example of how a DNA polymorphism produces an RFLP is shown in Figure 11.22, in which the black chromosome has three MspI recognition sites (CCGG). Cleavage of DNA from this chromosome segment results in two DNA fragments of different sizes. The electrophoretic pattern of DNA from an individual homozygous for this RFLP pattern is shown on the extreme right as it would appear on a Southern blot after hybridization with a probe contained within this region. In contrast, the homologous (white) chromosome has a polymorphic C-to-T substitution so that the middle MspI site (CCGG) is replaced by CTGG and now is no longer recognized by the enzyme. Thus, after digestion with MspI, a single DNA fragment is obtained and the pattern of an individual homozygous for the lack of this site is shown under the two white chromosomes in the right panel. In the middle of the panel, the pattern of a heterozygote (i.e. an individual having both a black and a white chromosome) is shown. In contrast to homozygous individuals, with two black or two white chromosomes, it is only in the heterozygous

individual that the two chromosomes can be distinguished and their transmission followed to the offspring. A second type of common DNA polymorphism is dinucleotide, trinucleotide and tetranucleotide repeats, also called microsatellites. Microsatellites are small simple repeats of 1–6 bases (i.e. AGAG, CAGCAG, CGGGCGGG) and are found throughout the genome. As such, they provide a source of highly polymorphic markers and their high frequency in the genome has facilitated the identification of many genes, the positioning of genes on the chromosomes in relation to each other, and traditional linkage studies for diagnostic analysis. In addition, because of their highly polymorphic nature, they can also be used for identity testing associated with paternity and forensic studies. A third type of common DNA polymorphism is single nucleotide polymorphism (SNP). SNPs occur about every 1,300 nucleotides and extensive data on the location and characterization of SNPs throughout the genome are available at www.ncbi. nlm.nih.gov/SNP. The SNPs have been most useful in mapping complex genetic disorders. The SNPs, which do not alter restriction enzyme sites, are

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detected by DNA sequencing. SNP microarrays are also utilized for copy number analysis and the identification of cryptic chromosomal abberations (see Chapter 10).

Linkage analysis using RFLPS The transmission of mutant genes can be indirectly detected by genetic linkage analysis using DNA polymorphisms as markers. When a gene and a DNA polymorphism are close to each other on a chromosome, they are said to be linked. The closer genes are physically, the more likely they will cosegregate. Rough estimates indicate that an RFLP and gene that are 106 bp apart have 99 percent probability of segregating together without being separated by genetic recombination. When the disease gene and a specific polymorphism are on the same chromosome, the polymorphism and disease are in coupling. When the disease gene and the polymorphism are on opposite chromosomes, the disease and the polymorphism are said to be in repulsion. For linkage analysis to be informative for a family, various family members must be heterozygous for the polymorphic markers linked to the disease gene so that the linkage phase can be determined and the polymorphism in coupling with the abnormal gene and the polymorphism in repulsion with the abnormal gene can be determined. Once the linkage phase has been determined, the transmission or lack of transmission of disease genes to the fetus can be inferred.

Indirect analysis for an X-linked disease Although common inversion mutations in the factor VIII gene have been described, most patients with hemophilia A have “private” or family-specific mutations. This heterogeneity makes direct detection of all mutations for each family impractical. Linkage analysis using RFLPs that lie either within the factor VIII gene or outside the factor VIII gene but closely linked provides an alternative method to determine the carrier status of at-risk females and ultimately at-risk fetuses. For example, a polymorphic BclI recognition site lies within intron 18 (IVS18) of the factor VIII gene.194,245 Because half of females are heterozygous for this BclI IVS18 RFLP, its analysis often enables inference of the factor VIII status of their offspring.

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For example, the PCR reaction is used to amplify a 143 bp fragment containing the polymorphic BclI site within IVS18. The amplified products are digested with BclI and the products are subjected to gel electrophoresis (Figure 11.23). Individual II-1 is the daughter of an affected male and is therefore an obligate carrier of hemophilia A. Digestion of the PCR products generated from her DNA with BclI shows that she is heterozygous for this PCR RFLP because 99 and 143 bp fragments are seen in which the BclI recognition site is (1 allele) and is not (2 allele) present, respectively. Because she donated her 1 allele to her affected son, her abnormal factor VIII gene is coupled with the 1 allele. Because this is an intragenic PCR RFLP, the likelihood of genetic recombination between the mutation in the gene and this site in II-1 is negligible. Thus, the diagnostic accuracy is 99 percent. Examination of the PCR products from II-I’s DNA after BclI digestion also shows a 1,2 (heterozygous) pattern. These results suggest that III-1 is a carrier, because she inherited the 2 allele from her father and her mother’s 1 allele, which is contained in her abnormal factor VIII gene. DNA extracted directly from chorionic villi or cultured chorionic villi or amniocytes can be used to determine the carrier status of the fetus (IV-1). In this case, the pattern of the fetal DNA indicates a 1 allele. Because the 1 allele is contained in the abnormal factor VIII gene, if the fetus is male, it would be predicted to be affected. However, if the fetus is female, it would have received a 1 allele from both parents and would be a carrier of hemophilia A. Thus, the fetal sex must be known to interpret the results.

DNA linkage analysis for an autosomal recessive disease Carbamyl phosphate synthetase I deficiency (CPSID) is an autosomal recessive disease of ureagenesis (see Chapter 14).622 Common mutations in the CPSI gene have been described623 but most are thought to be family specific. For this reason, linkage analysis is used for the prenatal diagnosis of CPSID. In this case, PCR is used to amplify several polymorphic dinucleotide repeat markers linked to the CPSI gene.109 To separate the fragments that differ by as few as two bases, one of the two oligonucleotide primers used in a PCR can be

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Figure 11.23 DNA fragments generated after PCR amplification of intron 18 of the factor VIII gene and restriction endonuclease digestion with BclI. Digested PCR-amplified products are subjected to polyacrylamide gel electrophoresis and stained with ethidium bromide for visualization. Those that do not contain the

polymorphic BclI recognition sequence are referred to as the 1 allele and remain 142 bp in length. PCR products of alleles containing the BclI recognition sequence will generate a 99 and 43 bp (not shown) fragment. In this family, the abnormal factor VIII gene is linked to the 1 allele (see text).

fluorescently labeled, and amplified fragments can be separated on a 4.25 percent polyacrylamide denaturing gel on an ABI 377 instrument. Figure 11.24 illustrates the results of a prenatal analysis in a CPSID family. In this case, both the father (120/122) and the mother (110/128) are heterozygous and informative for marker D2S143, which lies 3 × 106 bp 3′ to the CPSI gene. The genotype of the affected child is 110/120, indicating that the abnormal paternal CPSI gene is coupled with the 120 bp allele while the abnormal maternal CPSI gene is coupled to the 110 bp allele. The fetal DNA yields a 110/120 pattern, which is identical to the DNA from the previously affected child. The fetus is predicted to be affected with CPSID with an accuracy of 94 percent.

needed from multiple family members, often including an affected patient, a normal sibling or, alternatively, spouses or grandparents. These samples are needed to establish the linkage phase between the mutation and the RFLP being used as a marker. In addition, for the studies to be informative, certain members of the family will have to be heterozygous for the markers used to enable inference of the coupling phase. Occasionally, specimens must be obtained from deceased family members. In these cases, not only is it difficult to acquire archived material, but also the quality of the DNA recovered may not be suitable for analysis. The distance between the RFLP being used as a marker and the mutant gene is critical. The greater the distance between the two, the greater the chance that an erroneous diagnosis will be made. This is because the probability of recombination increases with increasing genetic distance between the RFLP and the gene being analyzed. A single recombination between the gene and RFLP during meiosis will cause a reversal of the linkage phase

Diagnostic pitfalls associated with linkage analysis One can use such RFLPs to detect the transmission of any mutation within a family through linkage analysis without actually knowing the nature of the mutation. However, to do this, DNA samples are

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Figure 11.24 A chromatogram of fluorescently labeled PCR products for marker D2S143, illustrating prenatal testing for carbamyl phosphate synthetase I deficiency.

The pattern generated from the fetal DNA is the same as the one generated from the affected child.

and cause an error in the inferred genotype of the fetus (Figure 11.25). The map distance between two loci that corresponds to a 1 percent chance of recombination is called a centimorgan (cM). An estimate of the relationship between the map distance and physical distance is that two loci separated by a distance of 106 bp have about a ∼1 percent chance of recombination (Figure 11.26).624 In the case of a second CPSI family depicted in Figure 11.27, marker D2S355 was used. This marker lies 4 × 106 bp or ∼4 cM 5′ to the CPSI gene. In this case, one of the PCR primers was radiola-

beled with 32P, resulting in the radiolabeling of all PCR products. Both the mother and father are informative for this analysis, 1/3 and 2/4, respectively. Both affected siblings had inherited the same maternal 1 allele but they had received alternate paternal alleles of 2 or 4. These results suggest that a recombinational event between the CPSI gene and marker D2S355 had occurred in one of the paternal gametes. Correct identification of the biologic father is essential for linkage studies to be accurate. False assignment of biologic paternity can cause errone-

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Figure 11.25 Recombination event during meiosis in which sister chromatids exchange homologous segments. Note that the recombinant chromatids have reversed the coupling phase of the AB (ab) and ab (aB) alleles.

Figure 11.26 Schematic representation of intragenic and paragenic RFLPs, showing the correlation between physical distance and recombination.

ous assignments of linkage phase between mutant alleles and DNA polymorphisms and can result in errors in prenatal diagnoses. This problem is serious for autosomal dominant and recessive disorders involving families with a single affected individual. Although in X-linked recessive disorders, false paternity of males does not affect the accuracy of prenatal diagnosis of females who are carriers, false paternity can result in errors in determining the carrier status of females.

Genetic heterogeneity (occurrence of the same phenotype from different genetic mechanisms) presents a possible source of error in all linkage studies, especially those involving small kindreds. The major assumption made, when performing the indirect linkage analysis, is that the disease is linked to the polymorphic loci. Because many mendelian disorders are caused by heterogeneous mutations at the same as well as different loci, phenotypes that appear clinically identical can be

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Figure 11.27 Autoradiogram generated from PCR products for marker D2S355 from a family with carbamyl phosphate synthetase I deficiency. Although both

daughters are thought to have this disease, they have inherited different paternal alleles, indicating that a recombination event has occurred.

caused by mutations at nonlinked loci. For example, referring to the family in Figure 11.27, while it is possible that a recombination event has occurred in one paternal gamete, it is also possible that the mutation causing the disease in these children is not found at the CPSI locus and could also explain the lack of genotypic concordance observed at the CPSI locus between the two affected sibs.

analysis, in which only one small cultured flask is needed. Alternatively, the CVS sampling procedure provides an advantage regarding DNA yield. Cultures are usually not needed because 10–60 mg of fetal DNA are usually obtained from each CVS tissue sample.625,626 Although CVS material may be useful, because it offers prenatal testing earlier in gestation and can provide an adequate amount of material without culturing, maternal contamination can be a problem and can lead to erroneous results. A maternal blood sample for maternal cell contamination (MCC) studies is routinely obtained to ascertain if MCC is present. If detected, the final results are delayed because the back-up flasks representing the cultured tissue must be analyzed. Only through culturing of CVS tissue can maternal cells not teased away at the time of collection be outgrown by the rapidly dividing fetal cells.

General laboratory issues Sampling problems Fetal DNA necessary for prenatal diagnostic studies can be isolated from cultured amniotic fluid cells or chorionic villus biopsies (CVS) or directly from CVS tissue. The amount of DNA needed for different studies varies and could be the limiting factor in an analysis. For Southern blot analysis, 3–4 culture flasks are requested as opposed to PCR

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Informed consent Legal, ethical and policy issues concerning DNA analysis are discussed in detail in Chapters 34 and 35. Because of the implications of genetic test results, for both individuals and their families, informed consent should be obtained before genetic testing. In the case of most genetic tests, the patient or subject should be informed that the test might yield information regarding a carrier or disease state that requires difficult choices regarding their current or future health, insurance coverage, career, marriage or reproductive options. The objective of informed consent is to preserve the individual’s right to decide whether to have a genetic test. This includes the right of refusal if the individual decides the potential harm (stigmatization or undesired choices) outweighs the potential benefits of the test.627 When obtaining samples for genetic tests, it is recommended that the following be clarified: • description of current test including its purpose, limitations (e.g. possibility of false-positive and false-negative results and predictive value) • possible outcomes of the test • how the results will be communicated • anticipated use of samples, including whether samples will be used only for the purpose for which they were collected and then be destroyed. If samples will be retained after testing, the scope of permission needs to include using samples or results in counseling and testing relatives, the possibility of future test refinements, and subjects’ expectations that their samples will be analyzed using these new tests, and that the results will be communicated to them. The duration of storage of samples should also be clarified.627 Privacy and confidentiality (see also Chapter 34) Consent forms related to genetic molecular diagnosis should respect the patient’s right to privacy.627 In some cases, the need to obtain a detailed family history of medical and genetic information can pose problems, especially when information needed on certain relatives is not offered voluntarily. Usually, requests for medical information from relatives are made by the interested family member. Because samples from relatives often need to be

analyzed to infer the genotype, the inclusion of results of these relatives in the laboratory reports is both necessary and problematic. Logical inferences that can be made regarding the disease status or nonpaternity of patients or their relatives pose serious problems regarding confidentiality. Careful attempts to address these issues should be covered in the consent process before sampling. If samples will be retained after testing, the scope of permission to use samples or results in counseling and testing relatives should be made clear. Time required for sample analysis In studies in which PCR analysis coupled with restriction endonuclease and/or gel electrophoresis is performed, results can be obtained within 48 hours of receipt of the sample. In PCR studies requiring more extensive analysis such as allelespecific oligonucleotide hybridization or DNA sequencing, results are usually obtained within 1–2 weeks. Other studies requiring Southern blot analysis may take as long as 2 weeks. If direct CVS tissue is not used in the analysis, an additional 2–3 weeks may be required to culture enough cells before sending for DNA analysis. Quality control In the clinical molecular diagnostics laboratory, quality control measures must be established to ensure the accuracy of the results. Of vital importance is an in-depth procedure manual listing each assay. Each procedure should contain specimen requirements, specimen processing, controls, recipes and storage conditions for all materials and reagents, and a step-by-step procedure with interpretation of the data. In addition, documentation of scheduled calibration as well as routine and preventive maintenance checks on all equipment and instrumentation is required. Further, documentation of personnel competency and laboratory proficiency in all assays is essential. In laboratories in which PCR analysis is used, quality control is especially challenging in preventing the contamination of highly abundant PCR products in the “prePCR” area of the laboratory. Several preventive measures can be taken, the most important of which is physical separation of pre- and post-PCR areas of the laboratory with designated equipment for each. Lastly, it is imperative that laboratories

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participate in proficiency testing. Proficiency testing for DMD, cystic fibrosis, factor V Leiden, prothrombin, fragile X, hemochromatosis, Huntington disease, myotonic dystrophy, multiple endocrine neoplasia type 2 (MEN2), spinal muscular atrophy, spinocerebellar ataxia, MTHFR, BRCA1 and BRCA2, hemoglobin S/C, and Prader– Willi/Angelman is offered through the College of American Pathologists (CAP). These proficiency specimens are sent to participants twice per year. For rarer disorders not offered through the CAP, blinded samples distributed to the technologists is an alternative means.

New directions Preimplantation genetic diagnosis (PGD) refers to genotype analysis of an oocyte before fertilization by study of the polar body628 or embryonic blastomere (see full discussion in Chapter 29).629,630 The list of disorders successfully detected or excluded has grown rapidly (see Chapter 29). Although PGD offers great hope for couples at risk for a child with a genetic disease, erroneous results from amplification inefficiency, cell preparation or cross-contamination, and differential amplification of alleles in the heterozygous state continue to pose great challenges for investigators using these procedures.631–642

Conclusion It has been more than 30 years since the first prenatal diagnosis using DNA analysis was done for sickle cell anemia.643 Since that time, accurate tests using different methods of DNA analysis have been developed for many inherited diseases (see Table 11.1). Because of the Human Genome Project, the number of applications has increased dramatically. This initiative in gene mapping and sequencing will identify all DNA markers and genes contained in the human genome. These maps are providing countless DNA segments, oligonucleotides, and PCR primers that can be used to detect mutations underlying many inherited disorders, both single and polygenic, as well as acquired gene rearrangements associated with neoplasia and aging. There is no question that the gene discoveries have had their largest impact on improved genetic

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testing. The application of DNA-based assays, for several of the genetic diseases described in this chapter, has significantly improved the accuracy of diagnosis and has provided families with more accurate risk estimates. Today, through genetic counseling, at-risk family members are able to make family planning decisions with information that was not available a short time ago. It is to be hoped that in the near future, we will observe a direct effect of the testing on therapy. The type of therapy will often be determined by the specific gene mutation. Molecular therapies (such as antisense oligonucleotides, antibiotics, chimeric RNA/ DNA, etc.) will be applied according to and require knowledge of the exact mutation. At least four ideas are important in understanding diagnostic applications that use DNA analysis. 1. When DNA changes in a gene are detected, one must determine if these represent DNA variations not associated with disease or mutations that affect expression of the gene. This is particularly important for missense mutations that have not previously been reported in the literature. 2. Different mutations found in the same gene from different patients are examples of allelic heterogeneity. In a growing number of disorders, including retinitis pigmentosa, defects at many different genes or locus heterogeneity can cause the disease in different families. Allelic and locus heterogeneity can often explain clinical variation at a molecular level, and both must be considered in molecular studies done for prenatal diagnosis to maximize its accuracy. 3. Gene diagnosis is applicable to many clinical disorders, both genetic and acquired. Requisites are a portion of the gene involved or a segment of DNA that lies close to the gene. 4. It is imperative that the results of the genetic tests be accurately conveyed to the affected individual or family members at risk. This type of communication often requires the expertise of a clinical geneticist or genetic counselor.

References 1. Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature 1953;171:737. 2. Lewin B, Genes IX. New York: Oxford University Press, 2007.

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tion in an exon of the CPS I gene causes a 9 base pair deletion due to aberrant splicing. J Clin Invest 1993;91: 1884. Summar ML. Vanderbilt University School of Medicine Nashville, TN. Personal communication, 1997. Rodeck CH, Morsman JM. First-trimester chorion biopsy. Br Med Bull 1983;39:338. Humphries SE, Williamson R. Application of recombinant DNA technology to prenatal detection of inherited defects. Br Med Bull 1983;39:343. American College of Medical Genetics. Statement on storage and use of genetic materials. Am J Hum Genet 1995;57:1499. Coutelle C, Williams C, Handyside A, et al. Genetic analysis from DNA from single human oocytes: a model for preimplantation diagnosis of cystic fibrosis. BMJ 1989;299:22. Navidi W, Arnheim N. Using PCR in preimplantation genetic disease diagnosis. Hum Reprod 1991;6:836. Handyside AH, Lesko JG, Tarin JJ et al. Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N Engl J Med 1992;327:905. Kristjansson K, Chong SS, van den Veyver IB, et al. Preimplantation single cell analysis of dystrophin gene deletions using whole genome amplification. Nat Genet 1994;6:19. Liu J, Lissens W, Selber SJ, et al. Birth after preimplantation diagnosis of the cystic fibrosis DF508 mutation by polymerase chain reaction in human embryos resulting from intracytoplasmic sperm injection with epididymal sperm. JAMA 1994;272:1858. Snabes MC, Chong SS, Subramanian SB et al. Preimplantation single cell analysis of multiple genetic loci by whole-genome amplification. Proc Natl Acad Sci USA 1994;91:6181. Ao A, Ray P, Harper J, et al. Clinical experience with preimplantation genetic diagnosis of cystic fibrosis. Prenat Diagn 1996;16:137. Soussis I, Harper JC, Handyside AH, et al. Obstetric outcome of pregnancies resulting from embryos biopsied for preimplantation diagnosis of inherited disease. Br J Obstet Gynaecol 1996;103:784. Eldadah ZA, Grifs JA, Dietz HC. Marfan syndrome as a paradigm for transcript-targeted preimplantation diagnosis of heterozygous mutations. Nat Med 1995;8:798. Gibbons WE, Gitlin SA, Lanzendorf SE. Strategies to respond to polymerase chain reaction deoxyribonucleic acid amplification failure in a preimplantation genetic diagnosis program. Am J Obstet Gynecol 1995;175:1088. Dreesen JCFM, Bras M, Coonen E, et al. Allelic dropout caused by allele specific amplification failure in single-

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cell PCR of the cystic fibrosis DF508 deletion. J Assist Reprod Genet 1996;13:112. 639. Gitlin SA, Lanzendorf SE, Gibbons WE. Polymerase chain reaction amplification specificity: Incidence of allele dropout using different DNA preparation methods for heterozygous single cells. J Assist Reprod Genet 1996;13:107. 640. Ray PF, Winston RML, Handyside AH. Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis. J Assist Reprod Genet 1996;13:104.

641. Sermon K, de Rycke M. Single cell polymerase chain reaction for preimplantation genetic diagnosis: methods, strategies, and limitations. Methods Mol Med 2007;132:31. 642. Kuliev A, Verlinsky Y. Preimplantation genetic diagnosis: technological advances to improve accuracy and range of applications. Reprod Biomed Online 2008; 16:532. 643. Kan YW, Dozy AM. Antenatal diagnosis of sickle cell anemia by DNA analysis of amniotic fluid cells. Lancet 1978;2:910

12

Prenatal Diagnosis of Disorders of Lipid Metabolism Bryan G. Winchester Biochemistry Research Group, UCL Institute of Child Health at Great Ormond Street Hospital, University College London, London, UK

Most disorders of lipid metabolism are the result of defects in enzymes or nonenzymatic proteins located in lysosomes1–4 (Tables 12.1, 12.2) or peroxisomes (see Chapter 13). The neuronal ceroid lipofuscinoses (NCLs) are included because they are characterized by the lysosomal accumulation of autofluorescent pigments that stain positively for lipid with Sudan Black B.5 In addition, mutations in plasma lipoproteins or lipoprotein receptors result in changes in the concentration of certain lipids in the blood and tissues, which can contribute to diseases, such as coronary heart disease6 (see Table 12.3). Although defects in the metabolism of the different classes of lipids may appear initially to give rise to distinct clinical presentations and biochemical abnormalities, the metabolic pathways for these compounds are interconnected. Therefore, a defect in one pathway can have an impact on another to produce secondary effects and atypical symptoms. Lipids play important roles in many cellular processes, including development, differentiation and intracellular signaling.7–9 Consequently, defects in their metabolism will affect many systems and give rise to a wide range of symptoms, including developmental delay and other neuropathies. Although most defects in the lysosomal metabolism of lipids are untreatable, a few disorders in which there is little or no involvement of the

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

central nervous system (CNS) can be treated by enzyme replacement therapy, by direct administration of recombinant human enzyme or by bone marrow transplantation.10,11 The use of drugs to decrease the rate of synthesis of glycosphingolipids and hence their rate of delivery to lysosomes for catabolism, substrate deprivation therapy, is an alternative therapeutic strategy.12 Another pharmacologic approach is the use of potent, reversible active site inhibitors as molecular chaperones to stabilize mutant enzymes, thereby enhancing the residual enzyme activty.13 The advantage of these pharmacologic approaches is that low molecular weight drugs can cross the blood–brain barrier. The levels of cholesterol and other lipids in the lipoprotein-associated disorders can sometimes be controlled by drugs. In contrast, a vector containing a normal copy of the CLN2 gene has been injected directly into the brains of children with late infantile ceroid lipofuscinosis in an attempt to deliver therapeutic enzyme to the central nervous system.14 The possibility of newborn screening and early treatment for the lysosomal storage diseases is undoubtedly going to have a major impact on the uptake of prenatal diagnosis for these disorders. However, with the current limited treatment for most of the lipidoses, genetic counseling with the option of prenatal diagnosis is still very important for families affected by one of these disorders. Reliable prenatal diagnosis depends on accurate diagnosis in the index case and a robust test for assaying the fetal material from the pregnancy at risk. The estabishment of the mutation(s) in the index case

445

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Genetic Disorders and the Fetus

Table 12.1 Lysosomal disorders of lipid metabolism Disorder

Major storage products

Measured defect

Prenatal diagnosis

GM1 gangliosidosis

GM1 ganglioside,

Acid β-galactosidase

CVS/AFC

Acid β-galactosidase and

CVS/AFC

glycoproteins, oligosaccharides Galactosialidosis

Glycoproteins, oligosaccharides

sialidase GM2 gangliosidosis B variant (Tay–Sachs/B1

GM2 ganglioside

Hexosaminidase A

CVS/AFC

GM2 ganglioside, GA2,

Hexosaminidase A and B

CVS/AFC

GM2 activator protein

Cultured CVS mutations

variant) O variant (Sandhoff)

globoside AB variant

GM2 ganglioside, GA2

Lipid loading/Ab mutations Fabry disease

Trihexosylceramide

α-galactosidase

CVS/AFC

Gaucher disease

Glucosylceramide

β-glucosidase

CVS/AFC

SAP-C deficiency

mutations/Ab

LIMP-2 deficiency

mutations/Ab mutations

NA

Arylsulfatase A or SAP-B

CVS/AFC, cultured CVS

Metachromatic

Sulfatide

leukodystrophy

(SAP-1) mutations

Multiple sulfatase deficiency

Sulfatide, mucopolysaccharides

Most sulfatases

CVS/AFC

Krabbe disease

Galactosylceramide, psychosine

Galactocerebrosidase

CVS/AFC

Niemann–Pick disease Types A and B

Sphingomyelin, cholesterol

Sphingomyelinase

CVS/AFC

Type C (NPC1 and NPC2)

Cholesterol, sphingomyelin,

Cholesterol esterification

Cultured CVS

Acid ceramidase

CVS/AFC

glycolipids Farber disease

Ceramide

Lipid loading Wolman disease and cholesteryl ester storage

Cholesteryl esters and

Acid lipase

CVS/AFC

Lipid loading in fibroblasts

Cultured CVS mutations

triglycerides

disease Prosaposin deficiency

Glycosphingolipids

AFC, cultured amniotic fluid cells; CVS, chorionic villi samples; SAP, sphingolipid activator protein; Ab, antibody detection. NA, not attempted yet. Where enzyme is indicated as measured defect, prenatal diagnosis can be carried out by measuring the enzymatic activity and/or detection of specific mutations.

and confirmation of the genotype of the parents is standard practice for prenatal diagnosis now that the genes encoding the lysosomal enzymes and proteins involved in glycosphingolipid metabolism and most of the neuronal ceroid lipofuscinoses have been characterized. DNA analysis is the ony

reliable method for the detection of carriers among family members. Increasingly, detection of the father’s and mother’s mutant alleles in the fetal material is being used to support a prenatal diagnosis based on a functional biochemical assay or used alone to make the diagnosis. If this approach

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Table 12.2 Neuronal ceroid lipofuscinoses Gene

Clinical type

Protein

Storage morphology

Prenatal diagnosis

(proteins) CLN 1

Infantile, late infantile

PPT1

GROD (Saps A & D)

Enzyme assay, DNA

juvenile and adult

(histology)

CLN2

Late infantile

TPP1

CL (subunit c)

Enzyme assay, DNA

CLN3

Juvenile plus slower

CLN3, lysosomal

FP (subunit c)

DNA, histology

CLN4

Adult

Not known

CL/FP/GROD (subunit c)

‡

CLN5

Late infantile, Finnish

CLN5, soluble lysosomal

FP/CL (subunit c)

DNA, histology

CLN6

Late infantile

FP/CL (subunit c)

DNA, histology

MFS8

FP/CL (subunit c)

DNA, histology (MFSD8)

CLN8, transmembrane

CL (subunit c)

DNA, histology

(histology) onset

variant

membrane protein

protein CLN6, transmembrane protein of ER

CLN7

Late infantile, Turkish variant

CLN8

Late infantile or Northern epilepsy

CLN9(not

protein of ER

Juvenile

Not known

CL (FP, GROD)

‡

Congenital late

Cathepsin D

GROD (Saps A & D)

DNA

identified) CLN10

infantile GROD, granular osmiophilic deposits; CL, curvilinear profiles; FP, fingerprint profiles; MFS8, lysosomal transmembrane protein of MFS facilitator family; Saps A & D, saposins A and D; subunit c, subunit c of mitochondrial ATP synthase; TPP1, tripeptidyl peptidase 1; PPT1, palmitoyl protein thioesterase 1; ER, endoplasmic reticulum. ‡

Prenatal diagnosis not reported.

is used, it is essential that the relationship between the genotype and phenotype is clearly understood and explained to the parents. Whatever experimental technique is used, it is essential that the laboratory carrying out the prenatal diagnosis is experienced in handling fetal biopsies and carrying out and interpreting the assay procedure. This chapter describes the molecular basis and any genotype/phenotype correlation for each disorder and how this information is used for accurate diagnosis of patients, carrier detection and prenatal diagnosis. Detailed information regarding each disorder can be found in Scriver et al.,15 Blau et al.,16 Applegarth et al.17 and Zimran.18

Lysosomal storage diseases: lipidoses The biochemical defects in lysosomal storage diseases were initially delineated in the 1960s, but our understanding of the molecular and cellular bases

of these diseases continues to grow (Figure 12.1). Reliable methods for diagnosing patients and prenatal diagnosis in pregnancies at risk are available for all the lipidoses. The genes encoding the lysosomal proteins affected in the lipidoses have been cloned and disease-causing mutations, often family specific, have been identified. This has permitted accurate early diagnosis and more reliable carrier detection, particularly for the X-linked disorders (e.g. Fabry disease, Hunter disease (MPS II) and Danon disease). It also raises the possibility of preimplantation diagnosis of affected embryos19 (see Chapter 29). For most lysosomal disorders, mutation analysis has provided some insight into the causes of the clinical variability, although other genetic and environmental factors can clearly affect the severity and age of onset. Molecular genetics has also revealed that the so-called pseudodeficiencies of lysosomal enzymes are due to mutations or polymorphisms (pseudodeficiency alleles) that drastically decrease the activity of an enzyme

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Genetic Disorders and the Fetus

Table 12.3 Monogenic lipoprotein-associated disorders Disorder (inheritance)

Main features

Observed plasma

Gene responsible

Key references

486

lipoprotein pattern Apolipoprotein disorders Apo-A deficiencies Type I (AR)

CHD, corneal clouding

↓HDL

apo-A-I and C-III

Type II (AR)

CHD, corneal clouding

↓HDL

apo-A-I, C-III and A-IVa,

487

↓HDL

apo-A-I

488, 489

↑TG

apo-A-V

490, 491

↓apo-B

apo-B

464, 472

↑LDL

apo-B

464

↑Chylomicrons,

apo-C-II

492, 493

apo-E

494

↑Chylomicrons

Lipoprotein lipase

492

Increased risk for CHD

Altered HDL

HTGL

492

Corneal clouding,

↓HDL

LCAT

495

↑LDL

LDL receptorb

465–467

Normal

Altered HDL

CETP

488

Fat malabsorption,

↓apo-B

Large subunit of

464, 496, 497

Type III (AR) Hypobetalipoproteinemia (AD)

Fat malabsorption, retinal degeneration,

lipoproteins

anemia,

(chylomicrons,

neuromuscular

VLDL, LDL)

weakness. Mild Familial ligand-defective apo-B, FLBD (AD) Apolipoprotein C-II deficiency (AR)

Mild increased risk for CHD Acute pancreatitis, anemia, eruptive

VLDL

xanthomas Type III Hyperlipoproteinemia

Cutaneous xanthomas, atherosclerosis

↑Chylomicron remnants, VLDL

(dysbetalipoproteinemia) (AR &AD) Enzyme disorders Familial lipoprotein lipase deficiency (AR)

Abdominal pain, HSM, pancreatitis, cutaneous xanthoma

Hepatic triglyceride lipase (AR) Familial LCAT deficiency and fish-eye disease (AR)

anemia, proteinuria, uremia

Receptor/transport disorders Familial hypercholesterolemia

CHD, tendon xanthomas

(AD) Cholesteryl ester transfer protein deficiency (NK) Abetalipoproteinemia (AR)

retinal degeneration,

lipoproteins

microsomal

anemia,

(chylomicrons,

triglyceride transfer

neuromuscular

VLDL, LDL)

protein

weakness Other Tangier disease (AR) ATP-binding cassette transporter 1 ABC1

Corneal clouding,

↓HDL

ABC1

498–501

orange tonsils, neuropathy

ABC1, ATP-binding cassette transporter 1; AD, autosomal dominant; apo, apolipoprotein; AR, autosomal recessive; CETP, cholesterol ester transfer protein; CHD, coronary heart disease; HDL, high-density lipoproteins; HSM, hepatosplenomegaly; HTGL, hepatic triglyceride lipase; LCAT, lecithin cholesterol acyltransferase; LDL, low-density lipoproteins; VLDL, very low density lipoproteins. a

A-I, C-III and A-IV are adjacent genes.

b

Homozygotes are more affected.

GM1

NeuAc I α2->3 Galβ1->3GalNAc β1->4Galβ1->4Glcβ1-> Cer GM1gangliosidosis

Sialidase

b-Hexosamindase A,B

SAP-B

Lactosylceramide

Galα1 ->4 Galβ1-> 4Glcβ1-> Cer CTH

Fabry

GM1-b-galactosidase GalCer-b-galactosidase SAP-B and -C

Sulphatide

CMH or glucocerebroside Glcβ1->Cer Arylsulphatase A SAP-B Gaucher disease b-Gluco-cerebrosidase SAP-C b-Galactosylceramidase

Niemann-Pick

Ceramide

Sphingomyelinase

SAP-A and -C Krabbe

Ceramidase SAP-C and-D

Farber

Sphingosine

449

Figure 12.1 Lysosomal catabolism of some glycosphingolipids.

Cer-Phosphorylcholine Sphingomyelin

Prenatal Diagnosis of Disorders of Lipid Metabolism

SAP-B

Gal(3-SO3H)β1->Cer

Galβ1->1 Cer

Sandhoff disease

a-Galactosidase Galβ1-> 4Glcβ1-> Cer

Sialidosis

Metachromatic leucodystrophy

GalNAclβ1->3 Galα1->4 Galβ1-> 4Glcβ1-> Cer

C H A PTER 1 2

GM3

Globoside

b-Hexosamindase A,B GM2-activator

b-Hexosamindase A GM2-activator NeuAc I α2->3 Galβ1->4Glcβ1-> Cer

GM1-gangliosidosis

GalNAcβ1->4Galβ1->4Glcβ1-> Cer

GalNAcβ1->4Galβ1->4Glcβ1->Cer Tay-Sachs AB variant Sandhoff

Galβ1->3GalNAcβ1->4Galβ1->4Glcβ1-> Cer

GM1-b-galactosidase SAP-B GM2-activator GA2

NeuAc I α2->3

GM2

GA1

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Genetic Disorders and the Fetus

without actually causing disease.20,21 The decrease in enzymatic activity due to a pseudodeficiency allele or a disease-causing mutation cannot be distinguished by enzymatic assay, but a simple DNA test for the presence of the pseudodeficiency allele can usually resolve the problem.22 The lysosomal catabolism of membrane-bound glycosphingolipids is brought about by soluble hydrolases in the lumen of lysosomes.4,23 Nature has developed two strategies for coping with this heterologous system: association of the enzymes with the lysosomal membrane and the use of nonenzymic protein detergents and co-factors. βGlucocerebrosidase, which cleaves the βglucosylceramide core linkage found in most extraneural glycosphingolipids, associates with the membrane, enabling it to interact directly with its predominantly hydrophobic substrate.24 If the glycan of a glycosphingolipid is longer than a tetrasaccharide, the monosaccharide at the nonreducing terminal can be released by an exoglycosidase alone, but the degradation of shorter glycans requires the assistance of a nonenzymic protein co-factor or sphingolipid activator protein (saposin, or SAP).25,27 To date, two genes are known to encode sphingolipid activator proteins. One encodes the GM2 activator protein, which facilitates the action of hexosaminidase A on ganglioside GM228 and is deficient in the AB-variant of GM2-gangliosidosis. The other gene encodes prosaposin (or sap-precursor),29 which is proteolytically processed sequentially from the N-terminal end to four homologous saposins, A–D, with specificities for different sphingolipids. A deficiency of prosaposin leads to the accumulation of a range of glycosphingolipids.30 The activities of SAP-B, the GM2 activator protein, and β-glucocerebrosidase are also stimulated by acidic lipids, such as phosphatidylserine or phosphatidylinositol and bis(monoacyl)glycerophosphate. In the laboratory most lipid hydrolases are assayed using water-soluble synthetic substrates, obviating the need for detergents, or by using natural substrates in the presence of added detergent. Consequently, a deficiency of a sphingolipid activator protein can be missed using such assays. If there is strong clinical indication of a lipidosis but the enzyme activity appears to be normal, a deficiency of a sphingolipid activator protein should be

considered. In contrast, multiple deficiencies of lysosomal hydrolases can arise because of defects in the post-translational modification of lysosomal enzyme precursors. In mucolipidosis II (I-cell disease) and III, the soluble lysosomal hydrolases fail to acquire the lysosomal recognition marker, mannose-6-phosphate, and are diverted from the lysosomes to the extracellular compartment in many cell types, including fibroblasts, white blood cells, cultured amniotic fluid cells (AFC) and chorionic villi (CV).31 The assay of two or more relevant enzymes can distinguish between mucolipidosis II/ III and a genuine single enzyme deficiency. Similarly, in multiple sulfatase deficiency (mucosulfatidosis), the activities of all lysosomal sulfatases, including arylsulfatase A, which is deficient in metachromatic leukodystrophy, are defective. This is due to a defect in the enzyme that catalyzes the modification of a common active site cysteine, which is essential for sulfatase activity.32,33 Enzyme replacement therapy is now available for six lysosomal storage diseases including two lipidoses, the non-neuronal form of Gaucher disease, type 134 and Fabry disease.35,36 This has led to a decrease in requests for prenatal diagnosis of these disorders. Enzyme replacement therapy for Niemann–Pick B using recombinant human sphingomyelinase is imminent. Substrate deprivation using Miglustat (N-butyldeoxynojirimycin) is a licensed alternative treatment for Gaucher disease type 1.37,38 It has also been used to decrease the secondary accumulation of GM2- and GM3-gangliosides in Niemann–Pick C disease with encouraging clinical benefits.39 Hematopoietic stem cell transplantation is considered an option for metachromatic leukodystrophy, globoid cell leukodystrophy and Gaucher disease type 3.40,41 As early commencement of treatment is beneficial, considerable effort is going into developing methods for newborn screening of lysosomal storage diseases.42–46 Early diagnosis16,47–51 and therapy of the lipidoses will significantly impact genetic counseling and prenatal diagnosis.52,53 GM1-gangliosidosis/MPS IVB A deficiency of acidic β-galactosidase (EC 3.2.1.23) is the underlying defect in two autosomal recessive, lysosomal storage diseases: GM1-gangliosidosis and Morquio disease type B (mucopolysaccharido-

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sis IVB)54–56 (see Chapter 14). These disorders represent the two extremes in a spectrum of clinical phenotype resulting from mutations in the βgalactosidase gene (BGAL). β-Galactosidase has a relatively wide specificity and acts on β1-4 galactosidic linkages in N-glycans and keratan sulfate and β1-3 and β1-4 galactosidic linkages in glycolipids. Therefore, a deficiency of the enzyme leads to a mixture of storage products, the composition of which depends on the underlying mutations. A secondary deficiency of β-galactosidase can arise from defects in the protective protein-cathepsin A (galactosialidosis).57 The hydrolysis of GM1-ganglioside and lactosylceramide are stimulated in vitro by saposin B and saposins B and C, respectively,58 but mutations in saposin B do not give rise to the GM1-gangliosidosis or Morquio disease phenotype. A second, genetically distinct lysosomal βɶ -galactosidase, galactocerebrosidase, which acts on galactosylceramide and galactosylsphingosine, is deficient in globoid cell leukodystrophy. Historically, GM1-gangliosidosis has been classified into three forms, infantile type 1, late infantile/ juvenile type 2 and adult/chronic type 3, with the majority of patients having type 1. GM1-ganglioside and its asialo derivative GA1 accumulate in the brain in all three types, and galactose-terminated oligosaccharides are excreted in the urine of types 1 and 2. Some glycosaminoglycan derived from keratan sulfate is excreted in the urine of patients with type 1, who have severe skeletal dysplasia, but it is not believed to contribute to the pathology. The amount of residual enzymic activity and the level of storage material correlate with the severity and rate of neurologic deterioration. In contrast, keratan sulfate is the major storage product in patients with Morquio B, but it is different from that excreted by patients with GM1-gangliosidosis type 1. Patients with Morquio B have extensive skeletal dysplasia but normal intelligence. There is no CNS involvement consistent with lack of storage of GM1-ganglioside. However, the biochemical and clinical distinction between the GM1-gangliosidosis and Morquio B disease is disappearing, as more cases are investigated in depth. More than 100 different mutations have been found in the BGAL gene,59 including nonsense, frame-shift and splice-site mutations, duplications, insertions and a predominance of missense muta-

451

tions. GM1-gangliosidosis is extremely heterogeneous and there is no obvious relationship between the type and position of the mutation and the phenotype. Most mutations give rise to no activity in expression studies, and combinations of such mutations give rise to the severe infantile form of the disease. Mutations with measurable residual activity are associated with the juvenile, adult and Morquio B variants60 in either homozygotes or compound heterozygotes. The second allele can modify the rate of progression of the disease in adult GM1-gangliosidosis, and individuals homozygous for the mild mutations may be asymptomatic.61 A common mutation and mutations in a specific domain of β-galactosidase are associated with the Morquio B phenotype.60 A pseudodeficiency allele occurs in the GLB1 gene.62 Currently, there is no effective treatment for GM1-gangliosidosis but substrate depletion63 and molecular chaperone64 therapy using drugs that can cross the blood–brain barrier have been shown to have beneficial effects in the mouse model of GM1-gangliosidosis. In contrast, Morquio B disease in which there is no neurologic involvement should be amenable to enzyme replacement therapy or haematopoeitic stem cell therapy. Definitive diagnosis of GM1-gangliosidosis and Morquio B disease is based on demonstrating a deficiency of acidic β-galactosidase activity in leukocytes or cultured skin fibroblasts, typically using the synthetic substrate 4-methylumbelliferyl-β-dgalactopyranoside.55 Carriers can be identified by testing for the mutations in the index case. Prenatal diagnosis of GM1-gangliosidosis and Morquio B disease can be achieved by assaying the βgalactosidase activity directly in CV samples (CVS), cultured CV cells (CCV) and in AFC.52,53,55 Galactosialidosis can be diagnosed by demonstating a deficiency of cathepsin A (protective protein) and a secondary deficiency of β-galactosidase and αneuraminidase in cultured fibroblasts or CV samples.65,66 It is also a cause of hydrops fetalis and there is vacuolation of fetal blood.67 GM2-gangliosidoses The GM2-gangliosidoses are characterized by massive accumulation of GM2-gangliosides and related lipids in lysosomes, predominantly in neurons, due to a deficiency of β-N-acetyl-d-

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Genetic Disorders and the Fetus

hexosaminidase (EC 3.2.1.52) activity.56,68 Three gene products are involved in the lysosomal catabolism of GM2-gangliosides: the α- and β-subunits of β-N-acetyl-d-hexosaminidase (HEXA and HEXB) on chromosomes 15 and 5 respectively, and the GM2-activator protein (GM2A) also on chromosome 5.68,69 The monomeric subunits of βN-acetyl-d-hexosaminidase have inactive catalytic sites but combine to form active dimers, known as hexosaminidase A (αβ), hexosaminidase B (ββ), and hexosaminidase S (αα). All these forms of hexosaminidase are specific for the hydrolysis of terminal, nonreducing β-glycosidically linked N-acetylglucosamine or N-acetylgalactosamine. However, they have different substrate specificities because of differences in the specificities of the catalytic sites on the α- and β-subunits. The αsubunit catalytic site can act on neutral or negatively charged glycolipids, oligosaccharides, glycosaminoglycans, and synthetic substrates. In contrast, the β-subunit acts preferentially on neutral, water-soluble, natural and synthetic substrates. To be degraded in vivo, lipophilic GM2gangliosides must combine with the GM2-activator protein, which lifts the gangliosides out of membranes and presents the hydrophilic oligosaccharide moiety to the water-soluble enzyme.58,70 Only hexosaminidase A (αβ) can act on the GM2ganglioside/GM2-activator protein complex. In addition to GM2-ganglioside, a range of other glycolipids and oligosaccharides accumulate in the GM2-gangliosidoses, depending on which gene is mutated. GM2-gangliosidosis can arise from a defect in any of the three genes, HEXA, HEXB or GM2A.69 All three genes have been cloned and the identification of a wide range of different mutations in each gene has provided a basis for much of the clinical variation in GM2-gangliosidosis. The crystal structures of human hexosaminidase71,72 and the GM2activator protein73 have been elucidated, allowing the molecular modeling of hexosaminidase A. These structures show how the active dimers are formed, the molecular basis of their substrate specificities, and how point mutations in the genes cause the different forms of GM2-gangliosidosis. The different forms or variants of GM2-gangliosidosis are very similar clinically, but all present with a wide range of severity and age of onset. No effec-

tive therapy is available for treating these disorders but treatment of mice with Sandhoff disease with N-butyldeoxynojirimycin (Miglustat) and its galactose analog delayed symptom onset and increased life expectancy74 and reduced brain ganglioside accumulation,75 respectively. Both carbohydrate-based76 and noncarbohydrate-based77 inhibitors of β-hexosaminidase enhanced the residual β-hexosaminidase activity in fibroblasts of adult Tay–Sachs and Sandhoff patients. These preclinical results suggest that substrate deprivation and chaperone therapy may be helpful in the GM2gangliosidoses. However, although Miglustsat was well tolerated in an open label trial with 30 patients with late-onset Tay–Sachs disease, no clinical benefit was seen over 2 years.

Tay-Sachs disease: mutations in HEXA gene (α subunit) Mutations in the HEXA gene lead to a deficiency of hexosaminidase A (αβ) and hexosaminidase S (αα) but the hexosaminidase B (ββ) activity is normal. Patients with a deficiency of hexosaminidase A are called B variants because hexosaminidase B is present. More than 100 different mutations have been reported in the HEXA gene database at www.medgen.mcgill.ca. Combinations of null alleles, such as all the nonsense mutations and the deletions and insertions that produce frame-shifts and most of the splice-site mutations, give rise to the severe infantile form of GM2gangliosidosis or classic infantile Tay–Sachs disease. The incidence of infantile Tay–Sachs disease is high in certain ethnic groups because of founder effects; it has been estimated to be 1 in 2,500 livebirths in Ashkenazi Jews67 with three mutations accounting for more than 98 percent of mutant alleles.78 Many other combinations of null alleles cause infantile Tay–Sachs disease in individual non-Jewish families. If the family is consanguineous, the patients are generally homozygous for a rare mutation; if not, they are usually compound heterozygotes for a recurrent mutation and a rare mutation. Typical patients with Tay–Sachs disease present between 3 and 6 months of age with loss of interest in surroundings, hypotonia, poor head control, apathy, and an abnormal startle response to sharp sounds.68 Deafness, blindness, seizures and gener-

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alized spasticity are usually evident by 18 months of age. Bilateral cherry-red spots in the macula caused by perimacular lipid deposition and macrocephaly are almost always present. Death from respiratory infection usually occurs between 3 and 5 years. The number of Jewish cases has dropped because of screening programs and most patients diagnosed now are non-Jewish juvenile patients, and adult patients with deficiency of hexosaminidase A have been described.68,79 Juvenile patients usually present between 2 and 8 years of age with ataxia and progressive psychomotor retardation. Loss of speech, progressive spasticity, athetoid posturing of hands and extremities, and minor motor seizures become evident. Neuronal storage of GM2-ganglioside similar to classic Tay–Sachs disease can be found. A number of adult patients with spinocerebellar degeneration (ataxia, muscle atrophy, pes cavus, foot drop, spasticity, and dysarthria) with or without psychoses have been demonstrated to have a defect in hexosaminidase A.80,81 Some of these patients were originally considered to be healthy people with low hexosaminidase A activity.82 Any infant, child or adult with psychomotor retardation and regression with no known cause should be a candidate for enzymatic testing for hexosaminidase A levels. The tests are simple and reliable and will result in a diagnosis of a small, but significant, number of people. Most patients are readily diagnosed using the fluorogenic substrate 4-methylumbelliferyl-2acetamido-2-deoxy-β-d-glucopyranoside (MU-βGlcNAc),but others can be diagnosed only using the natural substrate or a sulfated fluorogenic substrate, 4-methylumbelliferyl-6-sulfo-2-acetamido2-deoxy-β-d-glucopyranoside (MU-β-GlcNAcS).83 For prenatal diagnosis, the sulfated substrate has become the substrate of choice. The diagnosis of patients with a defect in the α-chain of hexosaminidase A requires accurate determination of hexosaminidase A in the presence of hexosaminidase B. Methods for differentiating the two isozymes have been developed because both hexosaminidase A and B hydrolyze MU-β-GlcNAc. They include heat denaturation (hexosaminidase A is unstable),84 pH-inactivation of hexosaminidase A85 and separation of hexosaminidases A and B on small ion exchange columns.86 Many laboratories use the heat denaturation method, which has proved to be

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useful for diagnosis in most cases. Most patients of all age groups have a severe deficiency of hexosaminidase A, usually 0–10 percent of the total hexosaminidase activity, compared with 58–70 percent of the total hexosaminidase activity in controls. Some juvenile patients have been reported to have up to 25 percent hexosaminidase A activity87 but with hindsight these may have been B1 variants. B1 variant Some patients have near normal levels of hexosaminidase A activity when measured with the neutral, synthetic substrate, MU-β-GlcNAc but a marked deficiency of hexosaminidase A activity with either the natural substrate or MU-βGlcNAcS.88 These patients, who were probably undiagnosed in the past, are called B1 variants to differentiate them from classic Tay–Sachs patients, the B variant. This change in specificity of the enzyme was shown to be due to a mutation, R178H (DN allele), which inactivates the α-subunit89 but does not affect the association of the α- and βsubunits or the activity of the β-subunit. As a result, the mutant dimeric hexosaminidase A behaves like hexosaminidase B and hydrolyzes uncharged substrates predominantly. Homozygotes for this B1 mutation have the juvenile disease but compound heterozygotes for the B1 mutation and a null allele have a more severe phenotype with late infantile onset.90,91 Two other mutations, which occur in the same codon, R178C and R178L, produce a more severe, acute B1-like phenotype. Arginine 178 is in the active site cleft of the αsubunit and another mutation in the α-subunit active site, D258H, also results in the B1 variant phenotype.92 Patients with the B1 variant may be identified by the determination of the apparent activation energy of hexosaminidase using the neutral substrate 3,3′-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide.93 Pseudodeficiency Two benign mutations in the HEXA gene lead to a pseudodeficiency of hexosaminidase A,94,95 in which the α-subunit loses activity toward synthetic substrates but retains activity toward GM2ganglioside and does not, therefore, cause disease. The loss of activity toward the synthetic substrates

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is due to a decrease in stability rather than a change in substrate recognition which is suprising because the mutations are in the active site of the enzyme. These two mutations are responsible for most falsepositive results in the enzyme-based screening for Tay–Sachs carriers. Fortunately, their frequency in the Ashkenazi Jewish population is very low. Hexosaminidase S Hexosaminidase S (αα), which is also deficient in the B variant, is more active than hexosaminidase A toward sulfated glycolipids such as SM2 in the presence of the GM2-activator protein and sulfated oligosaccharides.96 Mice with the double knockout of Hexa and Hexb show signs of mucopolysaccharidosis as well as GM2-gangliosidosis. This suggests that β-hexosaminidase has a role in the degradation of glycosaminoglycans.97 Patients with deficiencies of both the α- and β-subunits of hexosaminidase have not been reported. Carrier detection Reliable Tay–Sachs disease carrier identification in serum samples has led to the mass screening of Ashkenazi Jewish communities around the world.98 As a result of the success of the Tay–Sachs carrier testing program in the Ashkenazi Jewish population, most patients diagnosed with Tay–Sachs disease today are not Jewish. The carrier frequency is about 1 in 25 in Ashkenazi Jews and about 1 in 150 in the general population. Accurate heterozygote detection is possible by demonstrating intermediate levels of hexosaminidase A activity in serum and leukocytes. The preferred method of carrier identification in the Jewish population is by mutation analysis because three mutations account for 98 percent of the mutant alleles.99 Mutation analysis has other advantages because it can identify mutations causing infantile and adult forms, and the so-called pseudodeficiency mutations.95,100 After a mutation has been identified in a family, other family members can be tested by rapid, accurate DNA analysis and genetic counseling offered.101 Serum and plasma are not suitable for carrier detection in pregnant women but carriers can be identified accurately by studies of hexosaminidase A in mixed leukocytes.98Also some noncarrier women taking oral contraceptives have been found to have reduced hexosaminidase A so leukocyte

studies are again recommended. Up to 10% of Tay–Sachs carriers may have a normal percentage of hexosaminidase A with low total hexosaminidase because of the high frequency of common polymorphisms (delTG (+) 619A>G) in the HEXB gene in the Ashkenazi Jewish population. Therefore where the total hexosaminidase activity is reduced, further testing in leukocytes is required.102 Prenatal diagnosis Tay–Sachs disease was among the first lysosomal storage diseases to be diagnosed prenatally using CVS.103 In noncultivated CV, measurement of hexosaminidase A directly using MU-β-GlcNAcS is the most accurate method of diagnosing a fetus affected with Tay–Sachs disease. Most studies can be completed within hours of sampling.104 Cultured CV cells can be used to confirm the preliminary studies on direct CVS. The assay is also reliable in AFC. Prenatal diagnosis of Tay–Sachs disease is available for couples with previously affected children and couples identified as at risk in carrier testing programs, with the latter group in the majority. When both mutations are known, DNAbased diagnosis is very reliable, highly specific and can exclude the pseudodeficiency alleles.99 The preferred strategy is to simultaneously carry out enzymatic analysis of the AF supernatant or CV and molecular DNA-based testing of an AF cell-pellet or CV.105 Prenatal diagnosis may be carried out when it is not known definitely that both parents are carriers because of inconclusive heterozygote screening or to reassure an obligate carrier with a new partner.

Sandhoff disease: mutations in β-subunit (GM2-gangliosidosis 0 variant) Mutations in the β-subunit (HEXB gene) lead to a combined deficiency of β-hexosaminidase A and B or Sandhoff disease (GM2-gangliosidosis 0 variant). Over 25 mutations have been reported in the HEXB gene (database at www.medgen.mcgill. ca).69,71 Most are associated with the severe infantile form of the disease, which is clinically identical to classic infantile Tay–Sachs disease, with the possible exception of the presence of hepatomegaly in some cases.68 There is no ethnic predilection for this autosomal recessive disease. Juvenile and adult cases also have been described.68,106–110

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GM2-ganglioside and its asialo derivative (GA2) accumulate in the brain and globoside, a major red blood cell glycosphingolipid, accumulates in the visceral organs.68 There is less than 10 percent of the total, normal, hexosaminidase activity, measured with MU-β-GlcNAc substrate in serum, plasma, leukocytes, fibroblasts, or tissues of affected children.68,69,110 A significant amount of residual activity is found if MU-β-GlcNAcS is used for the diagnosis of Sandhoff disease because of the presence of excess α-chains that combine to form hexosaminidase S (αα), which is able to hydrolyze the MU-β-GlcNAcS. Carriers have a lower total hexosaminidase activity but a higher percentage of hexosaminidase A than controls. Leukocytes and plasma can be used for carrier identification111,112 but, as in Tay–Sachs disease, plasma is not suitable for carrier detection in pregnant women or women taking oral contraceptives. Reliable carrier detection is achieved by DNA analysis. Prenatal diagnosis is possible by measuring the total hexosaminidase activity with MU-β-GlcNAc in CV directly, CCV cells, and AFC.113 A mutation in the β-subunit causing a pseudodeficiency of hexosaminidase A and B can cause problems with enzymic diagnosis, especially when it occurs in the same family as a Sandhoff mutation.114 The problem can be resolved by DNA analysis, which is the preferred method, in combination with a functional enzyme assay for prenatal diagnosis if the mutations in the index case and parents are known. Variant AB A deficiency of the GM2-activator protein due to mutations in GM2A gene (variant AB) prevents the formation of the GM2-ganglioside/GM2-activator protein complex and a loss of hexosaminidase A activity toward GM2-ganglioside.27,28 Five mutations in the GM2A gene leading to a deficiency of the GM2-activator protein have been discovered to date (in the GM2A gene database at www.medgen. mcgill.ca).58,69,71 They all occur in the homozygous state and lead to a severe infantile form of GM2gangliosidosis. The activities of hexosaminidases A and B toward the synthetic soluble substrates are unaffected, making diagnosis of this variant difficult both prenatally and postnatally. GM2-activator activity can be measured in vitro by its ability to stimulate hydrolysis of GM2-ganglioside by puri-

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fied hexosaminidase A88 or by the hydrolysis of radiolabeled GM2-ganglioside in cultured cells.115 A deficiency of the GM2-activator protein can also be demonstrated by an ELISA method.116 Identification of the mutation in the index case is essential for reliable carrier detection and can be helpful for prenatal diagnosis. Fabry disease Fabry disease is an X-linked lipidosis resulting from a deficiency of α-galactosidase A (EC 3.2.1.22).117–120 It is characterized biochemically by the progressive accumulation within lysosomes of glycosphingolipids with terminal α-galactosyl residues: globotriaosylceramide and to a lesser extent galabiosylceramide and blood group AB- and B-related glycolipids. Storage occurs predominantly in the endothelial, perithelial and smooth muscle cells of blood vessels, but there is deposition in many other cell types. Fine sudanophilic, periodic acid–Schiff (PAS)-positive granules and foamy storage cells are found in tissues of patients, and bone marrow samples show granular material in the histiocytes. The levels of storage products in the urine and plasma are elevated in most but not all patients with Fabry disease. The elevation reflects the clinical severity and progression of the disease and may be used to monitor the progress of the disease and conversely treatment.121,122 Male hemizygotes with Fabry disease usually present with pain in the extremities, lack of sweating, unexplained proteinuria, attacks of fever, corneal atrophy, and the presence of purple skin lesions.117–120 Similar purple skin lesions have been found in patients with fucosidosis, GM1-gangliosidosis, sialidosis, galactosialidosis, and Schindler disease (α-galactosaminidase deficiency). Although most patients present in the second decade of life, some present before 5 years of age and others in the fourth decade of life. As the disease progresses, there are symptoms and signs related to easy fatigability (due to storage in skeletal muscle), poor vision (corneal opacities, tortuosity of retinal and conjunctival vessels, and cataracts), and high blood pressure (due to continued vascular storage). The storage can lead to cardiac or renal failure in the third or fourth decade. There is negligible residual α-galactosidase activity123 and mostly no detectable α-galactosidase protein124 in male hemizygotes

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with the typical (“classic”) clinical presentation. A group of atypical patients, who lack the typical early symptoms, present with a late-onset cardiomyopathy or cardiomegaly (“cardiac variant”). These and other patients, who may be asymptomatic or mildly affected, do generally have residual activity Paradoxically, several male patients with classic clinical symptoms have been reported with normal activity in vitro. Fabry disease has also been found in patients with left ventricular hypertrophy or hypertrophic cardiomyopathy,125 end-stage renal disease on haemodialysis126 and in young stroke patients127 who have no other known disease. The α-galactosidase activity in female heterozygotes for all variants ranges from near zero to normal due to random inactivation of the Xchromosome123 and heterozygotes can only be detected reliably by molecular genetic techniques. Only a few female heterozygotes are asymptomatic and some are as severely affected as typical hemizygotes.128–130 Their symptoms may be confined to a single organ because of the pattern of the Xinactivation (e.g. in some female patients the characteristic corneal and retinal changes may be the only indication). The extreme of this mosaicism is seen in two identical female twin carriers who showed very different phenotypes because of uneven X-inactivation.131 It has been proposed that X-inactivation may be a major factor determining the severity of clinical involvement in Fabry heterozygotes because a statistically significant difference was found between the severity score values of heterozygotes with random and nonrandom X-chromosome inactivation.132 In contrast, evidence has been obtained that heterozygotes show random X inactivation and that the occurrence and severity of disease manifestations in the majority of Fabry heterozygotes are not related to skewed X-inactivation.133 The major cause of death in patients with typical Fabry disease is renal failure, and hemodialysis and renal transplantation were life-saving procedures134 prior to the availability of enzyme replacement therapy in 2001. Although transplantation improves renal clearance, no consistent improvement of other symptoms was observed.135 Fabry disease is amenable to enzyme replacement therapy because of the lack of major CNS involvement, and two forms of recombinant human α-galactosidase

are in use for enzyme replacement therapy.35,36 Although significant clinical benefits have been reported for both drugs in trials and on prescription, prospective studies are needed to assess the long-term treatment of adults and early treatment of children136 and the comparative benefits of the two drugs, which are used at different doses.137 The introduction of newborn screening for Fabry disease138,139 and the availability of enzyme replacement therapy and possibly substrate deprivation140,141 or enzyme stabilization by product142 or substrate analogs143 will affect the demand and necessity for prenatal diagnosis of Fabry disease. The GLA gene has been fully characterized144,145 and over 500 different mutations have been reported.146,147 Most mutations are private and all except a few missense mutations give rise to null alleles and the classic phenotype in hemizygotes. The “cardiac variants” have missense mutations that give rise to residual α-galactosidase A activity.148 Other atypical patients with a slower course of the disease or limited range of symptoms have missense mutations,149 suggesting that there is a spectrum of phenotypes depending on the amount and distribution of the residual α-galactosidase A activity. Some of the mutations found in these variants are also found in patients with the classic phenotype,150 and intrafamilial variation is found with some null alleles,151 suggesting that other genetic factors affect the phenotype.152 Manifesting females with decreased α-galactosidase A activity but no proven mutations in the GLA gene are also known,153 and 0.5 percent of normal individuals have a mutation that gives rise to elevated plasma αɶ -galactosidase A.154 It is important to be aware of these genetic variations when making an enzymatic diagnosis of Fabry disease. The three-dimensional structure of recombinant human α-galactosidase A protein has been elucidated.153 Using this model, it has been calculated that missense mutations in classic Fabry patients produce large structural alterations in the core or active site cleft of the enzyme whereas the changes due to missense mutations in the variants were small or localized on the surface of the molecule away from the active site.156 This model will be useful in predicting the effect of novel mutations and in formulating rational approaches for chaperone therapy in individual patients.157

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The definitive diagnosis of Fabry disease is based on demonstrating a deficiency of α-galactosidase A activity in leukocytes,158 serum or plasma,159 or cultured skin fibroblasts.160 The fluorogenic substrate, 4 - methylumbelliferyl - α - d - galactopyranoside (MU- αɶ -Gal) is widely used as the substrate. α-Nacetylgalactosaminidase (also called α-galactosidase B) also acts on this synthetic substrate and a specific inhibitor, N-acetylgalactosamine,123,161 is added to the assay to eliminate this activity, which could mask a deficiency of α-galactosidase A. Heterozygote detection in family members is now carried out by DNA analysis.120,123 Prenatal diagnosis of Fabry disease can be made in noncultivated CV, CCV or AFC by measuring α-galactosidase A activity and/or mutation analysis if the family mutation is known.123,162 Fetal sex determination is performed, for example, by FISH and subsequent chromosome analysis to support the diagnosis of an affected male. Heterozygous females can be detected by mutation analysis of the fetus. As it is not possible to reliably predict the course and severity of disease in a female heterozygous for a particular mutation, the testing of female fetuses has not been offered widely. However, as most, if not all, female heterozygotes will develop symptoms of Fabry disease,133 it has been suggested that families should be counseled on this issue and that diagnosis of heterozygosity for Fabry disease in female fetuses should be provided if the parents request the test.163 Preimplantation genetic diagnosis by mutation analysis has been accomplished (see Chapter 29). Gaucher disease Gaucher disease is the most prevalent lysosomal storage disease, with an overall frequency of about 1 in 40,000 to 50,000 livebirths164 but a carrier frequency of about 1 in 15 in the Ashkenazi Jewish population.165 It results from a deficiency of acidic β-glucosidase (β-glucocerebrosidase; EC 3.2.1.45), which catalyzes the lysosomal hydrolysis of the βglucosidic linkage in glucosylceramide and its deacylated derivative, glucosylsphingosine, in the presence of saposin C.165–167 A deficiency of acidic β-glucosidase can also arise from a defect in saposin C168,169 or LIMP-2 (SCARB2), a protein involved in the transport of acidic β-glucosidase to the lysosome.170,171

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The deficiency of β-glucosidase leads to the accumulation of these glycolipids in cells of the monocyte/macrophage system, and large lipidladen histiocytes (Gaucher cells) are found in tissues from most patients. There is marked elevation in the spleen (10–1000-fold), liver and bone marrow of the major storage product, glucosylceramide, which is widely distributed normally at low levels as an intermediate in the biosynthesis and catabolism of glycosphingolipids. This results in enlargement of the liver and spleen and storage in bone marrow in most patients. Plasma and erythrocyte glucosylceramide is increased.172 High concentrations of glucosylsphingosine, which is not normally present in detectable amounts, are found in the liver and spleen of all patients with Gaucher disease, but in the brain only of patients with the neuronopathic forms of the disease.165,173 It is the effect of the brain-specific storage products on neuronal loss rather than the accumulation of glucosylceramide that causes the neuronopathic forms.174 The structures of the storage products reflect their tissue of origin, with only the brain storage products in the neuronopathic forms of the disease being of neural origin. Three main clinical phenotypes of Gaucher disease are recognized on the basis of the absence (type 1) or presence and rate of progression of neurologic involvement (acute type 2 and chronic type 3).165–167,175 Type 1 is the most common subtype and is particularly prevalent in Ashkenazi Jews, in whom the predicted prevalence is 1 in 800.165 Patients usually present with splenomegaly and thrombocytopenia, resulting in easy bruising and possibly bone pain, but without neurologic disease. The age of enzymatic diagnosis ranges from less than 2 to 84 years of age. Most of the health problems of these patients result from continued spleen enlargement and moderate to severe bone deterioration caused by the replacement of healthy bone marrow with marrow filled with Gaucher cells. Some patients have a more severe type of Gaucher disease resulting in liver disease and lung infiltration. Although many patients with type 1 Gaucher disease live a full life, some have a rapid rate of glucosylceramide accumulation resulting in death in the second or third decade of life. Pathologic changes have been observed in brain samples from the few adult patients who came to autopsy.176

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There is wide variation in the age of onset and severity, even within families, making prediction of the clinical course very difficult even with genotyping. The acute neuronopathic form of Gaucher disease, type 2, is very rare (∼1 in 500,000 livebirths or 1% of the patients in the International Collaborative Gaucher Group (ICGG)),177 with rapidly progressing visceral and CNS disease.178,179 Patients usually present in the first few months of life with hepatosplenomegaly, slow development, strabismus, swallowing difficulties, laryngeal spasm, opisthotonos, and a picture of “pseudobulbar palsy.”165 Most cases have continual problems with respiration and chronic bronchopneumonia, which result in death by 18 months of age (mean age, 9 months). Some patients die at birth from fetal hydrops.180,181 This perinatal and lethal variant may be more common than thought originally.182 The subacute neuronopathic form of Gaucher disease, type 3, is characterized by a later age of onset of neurologic symptoms and a more chronic course than type 2.178,179 Although rare, with an incidence of approximately 1 in 100,000 livebirths or 5% of patients in the Gaucher Registry,177 a large number of cases has been reported in the Norrbotten region of Sweden.183 These patients are homozygous for the L444P mutation, which is polymorphic in this population. Children generally present in early childhood with hepatosplenomegaly similar to type 1 Gaucher disease. However, by early adolescence, dementia, seizures, and extrapyramidal and cerebellar signs become evident. They all have a horizontal gaze palsy.184 In some cases, the degree of splenomegaly is very minimal. In one family one second cousin had type 2 Gaucher disease and the other had type 3, with no evidence of spleen enlargement or glucosylceramide storage.185 A rare variant of type 3, with the genotype D409H/D409H, has been described with hydrocephalus and calcification of heart valves with only mild to moderate involvement of liver, spleen, and bones.186 The age of onset of the neurologic signs can vary greatly from the neonatal period to adulthood187 and the range of neuronopathic variants will increase as different populations are investigated.167

Saposin C and LIMP-2 defect variants A deficiency of saposin C can lead to nonneuronopathic169 or neuronopathic type 3168 Gaucher

disease. A defect in LIMP-2 results in progressive myoclonic epilepsy without intellectual impairment and nephrotic syndrome170 or myoclonus epilepsy and glomerulosclerosis.171 The gene for acidic β-glucosidase, GBA, has been fully characterized188,189 and more than 300 mutations, mostly missense, have been described.165,190,191 The existence of a pseudogene with a high degree of homology close to the functional gene causes problems in the detection of pathogenic mutations in the functional gene.192,193 Recombinant alleles are found in 20 percent of patients.194 Genotyping is providing some insight into the molecular basis of the different phenotypes.165,167,190,191 Four common mutations (N370S, c.84–85insG, IVS211GRA, and L444P) account for more than 93 percent of the mutations in type 1 Jewish patients but only 49 percent in non-Jewish type 1 patients.194 The most common mutation, N370S, produces sufficient enzyme with residual activity to protect against neurologic disease, and individuals homozygous for N370S may even be asymptomatic.164,190,195 The null alleles, c.84–85insG and IVS211GRA, are never found homoallelically and are found only rarely in non-Jewish type 1 patients. Other mutations found in combination with null alleles in patients with type 1 disease are deduced or have been shown to produce residual activity. The L444P mutation, which is also found in the form of complex alleles, is associated generally but not always with neuronopathic disease. A combination of a complex allele and the L444P mutation usually gives rise to type 2 disease, whereas homozygosity for the L444P mutation is generally but not exclusively associated with type 3 disease. On account of the complex and recombinant alleles and additional sequence changes in other recurrent alleles, it is necessary to sequence the whole GBA gene for reliable mutation analysis in Gaucher patients. Specific tests can then be developed for genetic testing of family members. Diagnosis of all the types of Gaucher disease is based on demonstrating a deficiency of acidic βglucosidase activity in leukocytes, platelets, cultured skin fibroblasts or dried blood spots.196–199 A great variety of substrates and conditions for assay have been described, but the fluorogenic substrate MUβ-Glc is widely used with bile salt detergents such as sodium taurocholate plus oleic acid or Triton

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X-100 included in the assay. The presence of isoenzymes of β-glucosidase necessitates careful control of the assay conditions, particularly pH. Fluorescent derivatives of glucosylceramide and the radiolabeled natural substrate can also be used. Patients usually have less than 15 percent of normal activity, with no significant difference between clinical subtypes. The residual β-glucosidase activity in patients with type 1 Gaucher disease is stimulated by the sphingolipid activator protein SAP-C and phosphatidylserine, whereas samples from patients with type 2 disease are not.200 Diagnosis should always include the demonstration of a deficiency of acidic β-glucosidase because novel sequence changes in the gene might not be pathogenic. Identification of carriers by enzymic assay is unreliable and when the mutations in the family are known, heterozygotes should be identified by DNA testing. On account of the high incidence of type 1 Gaucher disease and the prevalence of a small number of mutations in Ashkenazi Jews, carrier screening by mutation analysis has been incorporated into many Jewish genetic disease screening programs. Five mutations account for approximately 97 percent of the carriers in this population.165 Only about 75 percent of the mutations in the non-Jewish population can be detected by this approach because of the large number of private mutations. Therefore, assessment of the risk of Gaucher disease by mutation analysis for reproductive decision making is accurate if both parents are Ashkenazi Jews but less informative if one parent is non-Jewish.201 Prenatal diagnosis of Gaucher disease can be achieved by measuring the acidic β-glucosidase activity directly in CV, in CCV cells and AFC using natural or synthetic substrates in the presence of bile salts.177,202,203 If the mutations in the parents are known, DNA analysis can be used to confirm the diagnosis, but it must be remembered that a precise genotype/phenotype correlation does not exist, especially for type 1. Prediction of phenotype is complicated if one parent is affected with type 1 and the other is a carrier.177 Prenatal testing may be undertaken for families who have had an affected child or for couples identified to be at risk by population screening for carriers of Gaucher disease.204 The lack of a reliable genotype/phenotype correlation for nonneuronopathic Gaucher

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disease and the advent of treatment are important considerations for prenatal screening for Gaucher disease.205 The availability of treatment for some forms of Gaucher disease has important implications for counseling and reproductive decision making. The principal cause of visceral storage in Gaucher disease is the accumulation of β-glucosylceramide in macrophages. It is possible to deliver replacement enzyme to these cells and to disperse the storage either by bone marrow transplantation (BMT)206,207 or by direct intravenous administration of recombinant human β-glucosidase that has been modified for targeting to macrophages.34,208 Although BMT represents a permanent treatment, it has been superseded by enzyme replacement therapy (ERT) for type 1 because of its considerable risk and the difficulty of finding matched donors. It may have some value in the treatment of type 3.209 ERT for type 1 is safe and effective in decreasing or preventing the visceral aspects of type 1 Gaucher disease, but many patients on treatment may still have appreciable symptoms.210 An 8-year multicenter study of 884 children with Gaucher disease type 1 on ERT (alglucerase or imiglucerase) assessed the effects of treatment on hematologic and visceral symptoms and signs, linear growth and bone disease.210a In this longitudinal study most clinical parameters became normal or nearly normal. ERT will also clear the visceral disease in type 3, but there is no clear evidence that it can reverse the CNS disease. Inhibitors of glucosyltransferase can cross the blood–brain barrier, and on the basis of favorable results in a cell model of Gaucher disease,211 in animal models of other sphingolipidoses,212 and clinical trials,37,213 substrate deprivation therapy using N-butyldexynojirimycin (Miglustat) is now a licensed treatment for Gaucher disease type 1.214,215 There is some evidence for a reduction in the incidence of bone pain and improvement in the bone marrow in type 1 Gaucher patients treated with Miglustat.216 Although no clinical benefit was seen in a trial of Miglustat with type 3 patients, another type 3 patient showed neurologic improvement when treated with a combination of replacement enzyme (imiglucerase) and Miglustat.217 Clinical trials are in progress to evaluate the use of molecular chaperone therapy in Gaucher type 1 patients.218

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Metachromatic leukodystrophy Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder resulting from a defect in the release of the sulfate moiety from sulfatide (3-sulfo-galactosylceramide) (see Figure 12.1).219,220 The hydrolytic release of the sulfate moiety is catalyzed by the lysosomal enzyme sulfatide sulfatase or arylsulfatase A (ASA, EC 3.1.6.1),221,222 in the presence of saposin B. Therefore, MLD can arise from a defect in either arylsulfatase A or, more rarely, in saposin B, with only 10 cases reported.223,224 Sulfatide occurs mainly in the myelin sheath of the central and peripheral nervous systems and to a lesser extent in gallbladder, kidney and liver. The defect in MLD leads to the accumulation of sulfatide in the lysosomes of cells of these tissues and the deposition of storage granules, which appear metachromatic and stain strongly positive with PAS and Alcian blue. The disruption of the turnover of myelin ultimately leads to demyelination in the central and peripheral nervous systems, which is responsible for the predominantly neurologic symptoms of MLD. There is great variation in the severity and age of onset of MLD, but most patients have the late infantile form. These children present between 1 and 2 years of age with genu recurvatum and impairment of motor function.219,220,225,226 Examination reveals reduced or absent tendon reflexes. Within a span of months or years, nystagmus, signs of cerebellar dysfunction, dementia, tonic seizures, optic atrophy, and quadriparesis develop. Death usually comes before 10 years of age. Patients with the juvenile form usually present between 5 and 12 years of age with ataxia and intellectual deterioration. These patients continue to have psychomotor deterioration and usually die 4–6 years after diagnosis. Adult patients present with psychoses, ataxia, weakness, and dementia after 18 years of age.227 Some patients are noted to have emotional lability, apathy or change in character. The neurologic deterioration continues until death occurs in the fourth or fifth decade of life. Some are initially misdiagnosed as having multiple sclerosis. Decreased nerve conduction velocities and detection of demyelination by MRI or CT scan are useful diagnostically. Although saposin B stimulates the hydrolysis of many glycolipids, the clinical symptoms of patients with a deficiency of saposin

B are predominantly those associated with MLD, with a few exceptions.25,27,224 The blood–brain barrier is a major obstacle for delivery of replacement enzyme to the central nervous sytem in MLD.228 Bone marrow transplantation is considered an option for adult patients and those with mild neurologic manifestations.220,229–231 It is assumed that sufficient bone marrow-derived monocytes can cross the blood– brain barrier to form perivascular microglia, which can secrete replacement enzyme for the deficient glial cells. The outcome for the juvenile and lateinfantile forms of MLD is variable. BMT did not prevent deterioration in a 2-year-old boy with a deficiency of saposin B.232 A phase I/II trial is in progress for ERT based on encouraging results in a mouse model of MLD.233 The genes for arylsulfatase A234 and SAP-B235 have been cloned. More than 60 mutations have been identified in the ASA gene, many of which are private mutations, indicating a genetic basis for the clinical heterogeneity of MLD. Three recurrent mutations occur in European patients with a high frequency, and other mutations are associated with ethnic groups.215 The functional significance of many mutations has been assessed by in vitro expression studies. Patients with infantile MLD have two null alleles, whereas juvenile or adult patients have at least one allele with residual enzymic activity.236 There is a good inverse correlation between residual enzymic activity and severity of disease. At least six different mutations have been identified in the saposin B portion of the prosaposin gene, which encodes a common precursor for the four specific saposins.25 Confirmation of a clinical diagnosis of MLD is made by demonstrating a deficiency of ASA in leukocytes or cultured cells,237–239 using a synthetic, colorimetric substrate, nitrocatechol sulfate (NCS). Serum240 and other tissues have also been used. Support for the diagnosis can be obtained by the detection of metachromatic granules in urine by staining with toluidine blue and by quantitative measurement of excreted sulfatide.241,242 However, the enzymic diagnosis of MLD is complicated by two factors.51,219 First, a defect in saposin B cannot be detected using nitrocatechol sulfate because its hydrolysis is not dependent on the presence of a saposin. These patients have normal ASA activity

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using this substrate and with the natural radiolabeled substrate if a detergent is included in the assay. The detection of metachromatic granules or increased secretion of sulfatide in the urine provides support. The profile of excreted glycolipids can also give a clue because glycolipids, such as globotriaosylceramide and digalactosylceramide, should be present in addition to sulfatide because of the broad specificity of saposin B.243 Diagnosis can be confirmed by an ELISA for saposin B244 or a sulfatide loading test in cultured cells.245 If the test is being carried out on a patient from a family with a known mutation in saposin B, diagnosis can be confirmed by DNA analysis.The second serious complication with the enzymic diagnosis of MLD is that a significant number of healthy people have ASA levels near those found in affected patients. This is due to homozygosity for a benign pseudodeficiency allele (Pd allele), which gives residual enzymic activity of 5–15 percent of the normal activity.20 These individuals do not excrete excessive sulfatide or show any clinical symptoms of MLD.171 About 1–2 percent of the European population are homozygous for the Pd allele, with a carrier frequency of about 1 in 7 in most ethnic groups. The Pd allele can lead to the incorrect identification of patients and carriers in some families. The molecular basis of the Pd allele has been shown to be a mutation in the polyadenylation signal that results in the production of only about 10 percent messenger RNA.20 It is usually, but not always, found cis with another polymorphism that abolishes a glycosylation site on the protein but is believed not to affect the catalytic properties of the enzyme. A simple DNA test is available for the detection of the Pd allele and it is essential to carry out this test if a low level of ASA activity is found.22 Compound heterozygotes for the Pd allele and an MLD allele will have ASA activity lower than Pd homozygotes but they do not have the neurologic problems associated with MLD or excrete excessive sulfatide.246 However, detection of homozygosity for the Pd allele in a symptomatic patient by DNA analysis does not preclude diagnosis of MLD, because disease-causing mutations in the ASA gene occur on chromosomes carrying the Pd allele.20,51,247– 250 It has been estimated that one-fifth of MLD mutations occur on a Pd background.21 Identifica-

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tion of carriers in families with MLD must include DNA tests for the Pd allele and the mutations in the index case. It is important to genotype the parents of an affected child to establish whether the disease-causing mutations are on a Pd background. With this knowledge it is possible to make an accurate prenatal diagnosis of MLD in a subsequent pregnancy by demonstrating a deficiency of ASA in CV or CCV cells or AFC together with DNA analysis for the Pd allele and the MLD mutations. When both parents are heterozygous for an allele containing both the Pd allele and the MLD mutation, as occurs in consanguineous couples, it is essential to analyze the fetal DNA for both mutations. The sulfatide loading test carried out with CCV or AFC is also very helpful in resolving difficult situations. Multiple sulfatase deficiency The lysosomal sulfatases, including arylsulfatase A, undergo a specific post-translational modification to generate the active site, the conversion of an active site cysteine to Cαformylglycine (FGly).32 The enzyme catalyzing this reaction (FGlygenerating enzyme, FGE) has been purified and its gene (SUMF1) identified.251,252 Mutations in this gene lead to a multiple deficiency of lysosomal and other sulfatases, called multiple sulfatase deficiency or multiple sulfatidosis (MSD).33,253,254 This results in the disruption of the lysosomal catabolism of sulfated glycolipids and glycosaminoglycans and cholesterol sulfate. The clinical presentation ranges from a severe neonatal form to less severe phenotypes with mild neurologic involvement. The neonatal form resembles mucopolysaccharidosis with a coarse face, cataract and hydrocephalus.255–257 Children with MSD present with clinical features similar to those of late infantile metachromatic leukodystrophy, but features such as coarse facies, low-level dysostosis multiplex and stiff joints reminiscent of a mucopolysaccharidosis contribute to the phenotype. There is increased urinary excretion of dermatan sulfate (a substrate for arylsulfatase B and iduronate sulfatase) and heparan sulfate (a substrate for heparan sulfamidase and iduronate sulfatase) and glycopeptides. Confusion with patients with a mucopolysaccharidosis is possible, especially in young patients.258 Within the first 2 years of life, patients demonstrate slow develop-

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ment, skeletal changes, coarse facial features, hepatosplenomegaly and ichthyosis (due to the deficiency of arylsulfatase C). Vacuolated lymphocytes and Alder–Reilly bodies are found. Death usually occurs within a few years of onset of symptoms after rapid neurodegeneration. Thirty mutations have been found in the SUMF1 gene259 but the complexity of the biochemical and clinical phenotype due to deficiencies of several enzymes has made genotype/phenotype correlation difficult.260,261 The functional characterization of mutations in patients has shown that both the residual enzyme activity and the stability of the mutant FGE protein contribute to the clinical phenotype.259 The application of these functional assays of FGE should be of prognostic value. Diagnosis is made by demonstrating deficiencies of several sulfatases in plasma, leukocytes or fibroblasts.258,262 The pattern of sulfatase deficiencies varies, reflecting the clinical and biochemical heterogeneity of MSD. In general, the greater the decrease in the activities, the more severe is the phenotype. Parents of affected children do not have intermediate levels of sulfatases because the primary defect is not being measured. This has also prevented carrier detection in other members of an affected family. Now that the gene has been cloned and mutations identified in individual patients,251,259 carrier detection will be feasible. Prenatal diagnosis has been made by assaying sulfatases in AFC and CV.263 The availability of DNA analysis will greatly improve the reliability of prenatal diagnosis for families in which the mutations are known. No therapy is currently available for these children. Krabbe disease (globoid cell leukodystrophy) Krabbe disease results from a deficiency of galactocerebrosidase (EC 3.2.1.46) (GALC), which catalyzes the hydrolysis of the β-galactosidic linkages in various galactolipids, such as galactosylceramide, galactosylsphingosine, monogalactosyldiglyceride and possibly lactosylceramide (see Figure 12.1).264–266 Galactocerebrosidase is genetically distinct from the GM1-ganglioside β-galactosidase that is deficient in GM1-gangliosidosis. Galactocerebrosidase is a very hydrophobic protein and its activity towards galactosylceramide is stimulated by phosphatidylserine and saposins A and C. A

deficiency of galactocerebrosidase activity towards galactosyl ceramide due to a mutation in the saposin A coding region of the prosaposin gene has been found in an infant presenting as Krabbe disease.267 Galactosylceramide and its sulfated derivative, sulfatide, are found almost exclusively in myelin. Therefore, a deficiency of galactocerebrosidase activity leads to a progressive, cerebral degenerative disease affecting the white matter of the central and peripheral nervous systems.264–266 Pathologic examination of the brain268,269 shows that most but not all patients have characteristic, multinucleated globoid cells, containing undigested galactosylceramide. There is extensive depletion of glycolipids in the white matter but the total concentration of galactosylceramide in the brain does not increase because of the elimination of the cells synthesizing myelin during the course of the disease. The toxic metabolite galactosylsphingosine (psychosine) is also a substrate for galactocerebrosidase and it has been postulated that its accumulation is responsible for the early destruction of the oligodendroglia.266,270,271 The majority of patients (∼90%) have a severe infantile disease but patients with a later onset, even in adulthood, have been described.266,267,272–274 The onset in infancy usually occurs before 6 months of age, with irritability, hypertonicity, bouts of hypothermia, mental regression, and possibly optic atrophy and seizures.275 This can be followed by increased hypertonicity, opisthotonos, hyperpyrexia and blindness. Most patients die before 2 years of age.265,266,272–274 Cerebrospinal fluid protein is highly elevated (values of 100–500 mg/ dL are not unusual) and nerve conduction velocities are decreased. The age of onset and progress of the disease are highly variable even in patients with the same genotype. Neuroimaging can be a useful aid to diagnosis but it must be carried out in conjunction with biochemical and genetic testing to avoid misdiagnosis.276 BMT has provided alleviation of symptoms in some patients with the late-onset and more slowly progressing Krabbe disease.220,229,277 but others have died from complications of the procedure. Typical infantile patients are not considered good candidates for BMT because of the rapid course of their disease. However, recently transplantation of umbilical cord blood from unrelated donors has

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been carried out in babies with infantile Krabbe’s disease. 278 Most of those who underwent transplantation before the development of symptoms showed continuing myelination and neurologic improvement whereas those who underwent transplantation after the onset of symptoms had minimal neurologic improvement. A staging system has been set up to predict outcome after unrelated umbilical cord blood transplantation for infantile Krabbe disease.279 The GALC gene has been cloned280,281 and more than 70 mutations have been reported.264,265 The majority of patients are compound heterozygotes but several missense mutations have been found in homozygous form, permitting their designation as null or mild alleles, with the caveat of marked variability of phenotype. A 30 kb deletion accounts for 40–50 percent of the alleles in infantile patients of European ancestry and 35 percent in infantile Mexican patients.282 Some mutations, which presumably produce enzyme with residual activity, are homoallelic in juvenile/adult or adult patients. Patients who are compound heterozygotes for one of these mutations and the large deletion have a juvenile or adult phenotype, but with tremendous variation in severity. The GALC gene is highly polymorphic and about 80 percent of diseasecausing mutations occur on alleles with at least one polymorphism. These polymorphisms affect the activity in normal and mutant alleles. The most common polymorphism (I546T) has a frequency of 40–50 percent in the general population and decreases activity by up to 70 percent. The common deletion is always found in association with another polymorphism (502T). These polymorphisms are responsible for the wide reference ranges of activities in carriers and normal individuals and for some, but certainly not all, of the variation within a disease genotype. Diagnosis is based on demonstrating a marked deficiency of galactocerebrosidase (GALC) activity in leukocytes or cultured fibroblasts283–285 using the radiolabeled, natural substrate, galactosylceramide.286 A number of nonradioactive substrates have also been developed for the diagnosis of Krabbe disease.287–289 Carrier detection is by DNA testing because of the wide range of galactocerebrosidase activity in normal individuals due to polymorphisms in the gene. Healthy people with

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enzyme values almost as low as those measured in affected children285,290,291 occur, as well as obligate carriers with values clearly in the normal range. Prenatal diagnosis for Krabbe disease has been performed for more than 1,000 pregnancies at risk worldwide.265 Galactocerebrosidase can be assayed in CV directly292–293 and in CCV and cultured AFC.287,289 Knowledge of the levels of activity in the index case and in the obligate heterozygote parents is essential for interpretation of the results. A method based on the uptake and use of 14C fatty acid-labeled sulfatide in AFC has also been used to accurately identify fetuses affected with Krabbe disease.294 However, in families in whom the genotype is known, the enzyme assay can now be combined with detection of specific mutations. It is anticipated that the selective introduction of newborn screening for Krabbe disease46 in New York State and the transplantation of umbilical cord blood from unrelated donors278 will have an impact on the management of Krabbe disease and the demand for prenatal diagnosis. Niemann–Pick disease Niemann–Pick disease (NPD) consists of a group of autosomal recessive, lysosomal, lipid storage diseases, which have in common the storage of sphingomyelin, cholesterol and possibly other lipids in many tissues of the body.295–298 In Niemann–Pick disease types A and B, a primary deficiency of acidic sphingomyelinase (E.C. 3.1.4.12) due to mutations in the ASM gene leads to the lysosomal accumulation of sphingomyelin (see Figure 12.1).299–301 In contrast, in Niemann– Pick disease type C, mutations in two genes, NPC1 and NPC2/HEI, lead to altered trafficking of endocytosed cholesterol.302,303

Niemann–Pick disease types A and B (acid sphingomyelinase deficiency) There is massive accumulation of sphingomyelin in the liver and spleen of all patients with a deficiency of sphingomyelinase, but types A and B differ in their severity and neurologic involvement.299–301 NPD A is a severe neurovisceral disease, whereas there is only visceral involvement in NPD B with a chronic course.304 Patients with NPD type A usually present before 6 months of age with hepatomegaly and a slowing of motor and mental

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progress. This is followed by a general deterioration of neurologic function and health. About half of the children have a macular cherry-red spot, similar to that seen in Tay–Sachs disease. Death from respiratory infections usually occurs by 4 years of age. A higher incidence of NPD type A is found in children of Ashkenazi Jewish ancestry, in which the carrier frequency is about 1 in 90. Patients with NPD type B can present with hepatomegaly within the first few years of life, but adults can also be diagnosed because of their hepatomegaly. Continued storage of sphingomyelin and other lipids, especially cholesterol, in liver, spleen and lungs, causes many health problems. There is no obvious mental deterioration or retardation. Some have been found to have a cherry-red spot in the macular region.305,306 Complementation and molecular genetic studies have shown that NPD types A and B are allelic variants within the ASM gene. Patients with an intermediate phenotype, a protracted neuronopathic variant with overt, borderline or subclinical neurology, have been described indicating a wide clinical spectrum.307 The acid sphingomyelinase gene (ASM) has been characterized308 and more than 100 different mutations have been identified in Niemann–Pick A and B patients.299–301 Patients with NPD A have two null alleles and three mutations account for 92 percent of those found in Ashkenazi Jewish patients with NPD type A.309 A common mild mutation found in patients with type B has sufficient residual activity to prevent neurologic symptoms. Combinations of a milder allele and a null allele or two mild mutations are found in patients with NPD B. Several common mutations have been found in specific populations which might help screeening for NPD B in these groups. Patients with the more protracted neuropathic form of the disease than NPD A have a combination of mutations that produce less sphingomyelinase activity than typical NPD B mutations307 but this particular genotype does not always give an identical clinical course.310 As there is no significant involvement of the CNS in NPD B, the disorder is a potential candidate for enzyme replacement therapy by direct administration of recombinant enzyme, hematopoietic stem cell therapy or gene therapy. Liver transplantation was tried in one 4-month-old

patient with NPD A, but the results did not demonstrate a clear benefit from this drastic procedure.311 Some patients with NPD B were given amniotic membrane implants and a significant improvement in some clinical parameters was obtained.312 There have been three attempts at bone marrow transplantation313–315 with mixed outcome. Based on successful treament of the mouse model of NPD A/B, a phase I trial of enzyme replacement therapy using recombinant human acid sphingomyelinase is in progress for NPD B. Diagnosis of NPD A and B can be made by assaying acid sphingomyelinase in leukocytes or cultured cells using sphingomyelin radiolabeled in the choline moiety as the substrate. Several synthetic substrates have also been developed for the diagnosis of NPD297,316,317 but 3H choline-labeled sphingomyelin is still widely used because of its sensitivity, ease of assay and specificity.318 NPD types A and B cannot be distinguished by measuring the amount of residual acid sphingomyelinase in a conventional assay in vitro, but more residual activity is found in NPD type B than in type A cells when the activity is measured by loading cells with labeled sphingomyelin and measuring the rate of hydrolysis.319 This is consistent with the less severe phenotype of NPD type B. Heterozygote detection is unreliable by enzyme assay and should be based on DNA analysis. Prenatal diagnosis of NPD types A and B can be made by assaying acid sphingomyelinase in CV samples directly.318 Higher specific activities are obtained in CCV and AFC, but this delays the result. If the mutations are known in the index case and/or in the parents, mutation analysis on the CV sample is effective.

Niemann–Pick disease type C The genetic and metabolic basis of NPD type C is quite distinct from that of NPD types A and B. The lysosomal accumulation of unesterified cholesterol results from a defect in the processing and intracellular transport of endocytosed LDL-derived cholesterol and not from a primary defect in acid sphingomyelinase.302,303,320 NPD type C is more common than NPD types A and B combined and is panethnic. NPD type C is extremely heterogeneous clinically. Most patients have progressive neurologic disease with mild but variable visceral

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enlargement. The classic phenotype presents in childhood with ataxia, vertical supranuclear palsy, variable hepatosplenomegaly, dysarthria, dystonia and psychomotor regression. Death occurs in the second or third decade. Variants include an acute form with hydrops, an early form with fatal neonatal liver disease, an early-onset form with hypotonia and delayed motor development and adult variants.321 A small group of patients with severe pulmonary involvement and early death322 were shown by complementation studies to be genetically distinct.333 The two groups have been called NPC1 and NPC2, with about 95 percent of cases belonging to the NPC1 group.320 The two genes affected in these groups have been identified, NPC1324 and NPC2/HEI.316,325 Although mutation analysis is difficult with the NPC1 gene because of its size (56 kb and 25 exons), the presence of more than 50 polymorphisms and some unstable mutant transcripts,326 more than 140 disease-causing mutations have been identified.318,320 There are some common mutations and there is some correlation between genotype and residual NPC1 protein and clinical phenotype.320,327,330 However, the genotype of many patients is incomplete, despite complete sequencing of the gene.318 Several mutations have also been identified in the much smaller gene, NPC2/ HEI320,325,331–333 which encodes a small soluble protein that is secreted and recaptured and delivered to the lysosome by the mannose-6-phosphate pathway. There is a good correlation between the genotype and phenotype in the small number of families investigated.333 A group of patients concentrated in Nova Scotia have a homogeneous subacute phenotype and were originally designated as NPD type D.334 However, complementation studies335 and subsequently the discovery of a point mutation in the NPC1 gene336 showed that they are an allelic variant of NPC1, and the term NPD type D has been discontinued. A database has been established to catalogue mutations in the NPC1 and NPC2 genes to help understand genotype / phenotype correlation.337 The proteins encoded by NPC1 and NPC2 are presumed to act closely to one another in the intracellular pathway for endocytosed cholesterol but their precise functions are not known, although it is an area of very active research.338–340

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The diagnosis of NPC is complicated because there are two genetic defects, neither of which is a simple enzyme deficiency.318 If the genetic defect (i.e. NPC1 or NPC2) has been established in the index case and the mutations are known, diagnosis of other patients within the family and detection of carriers can be made by mutation analysis. Otherwise, a defect in the trafficking of cholesterol has to be demonstrated using cultured fibroblasts. The “filipin” test detects the accumulation of unesterified cholesterol in perinuclear vesicles in fixed cells stained with filipin by fluorescence microscopy. Alternatively, the kinetics of LDL-induced cholesterol ester formation can be measured using labeled oleate.327 Although these tests can detect both NPC1 and NPC2 with marked clinical defects, they may have to be modified to detect the less typical clinical variants, which account for 20 percent of the NPC1 cases.341 These cell-based assays can be applied successfully to the prenatal diagnosis of NPC1 and NPC2 in CCV for families with the classic, marked phenotype but not to the variant cases. Cultured AFC can also be used, but the result will be obtained much later in the pregnancy and there is a risk of a false-negative result with epithelial-like AFC.318 Because heterozygotes can show abnormal filipin staining comparable to that seen in the variants, it is advisable to examine the parents’ cells at the same time. If the genetic defect has been established in the index case and the mutations are known, then prenatal diagnosis by mutation analysis on CV samples is fast and reliable for both NPC1 and NPC2.302,303,318,331,342 Unfortunately, determining the full genotype of many patients with NPC1 has proved difficult because of the size and complexity of the gene. The problem with NPC2 is knowing that it is NPC2, because very few laboratories can carry out the complementation test. For these reasons, the prenatal diagnosis of NPC2 remains a complex procedure that should be undertaken only in very experienced laboratories. Testing by mutation analysis is reliable if the NPC2 family mutations are known. Several therapeutic strategies have been considered for NPC1. Some patients with NPD C have been placed on low-cholesterol diets and cholesterol-lowering drugs, but the results have not been encouraging.343 Another patient with NPD C

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underwent orthotopic liver transplantation with no evidence of improvement.344 There is secondary accumulation of GM2 and GM3-gangliosides in neurons, which is associated with neuron-specific dendritogenesis, in NPC.345 Miglustat, an inhibitor of glycolipid synthesis, was shown to decrease this accumulation of gangliosides in NPC mice and cats and to reverse the lipid-trafficking defect in blood lymphocytes in a human patient.346 On the basis of these results a trial of substrate deprivation using Miglustat was carried out with NPC patients over 12 years of age. After 12 months horizontal saccadic eye movement velocity and other neurologic parameters had improved in the treated patients.39 This is the first treatment for NPC to show benefit. Farber disease Farber disease is a rare, autosomal recessive, lysosomal sphingolipid storage disorder caused by a deficiency of acid ceramidase, also called N-acylsphingosine amidohydrolase (EC 3.5.1.23).347,348 The alkaline ceramidase present in most cells is not affected in this disorder. Ceramide is formed during the catabolism of all sphingolipids within the lysosomes349 (see Figure 12.1) and the deficiency of acid ceramidase leads to the intralysosomal accumulation of ceramide in most tissues, including heart, liver, lung and spleen. Extremely high levels of ceramide have been observed in the urine350 but it is not increased in the plasma of patients.348 Farber disease is also called Farber lipogranulomatosis because of the formation of the subcutaneous nodules near joints and other pressure points.348,351 The characteristic features include progressive hoarseness due to laryngeal involvement, painful swollen joints, subcutaneous nodules and pulmonary infiltrations. Initial signs appear between 2 and 4 months of age and death usually occurs before 2 years of age, but survival to the age of 16 years is known. Psychomotor development in the few patients described so far has been mostly normal, although deterioration has been observed in the later phases of this disorder.348,352 Conversely, very severe forms, with corneal clouding, hepatosplenomegaly, marked histiocytosis and death before 6 months of age348,353,354 or death in utero355 have been reported.Variable severity probably indicates the existence of juvenile and perhaps even

adult forms of this disorder, but too few patients have been described to define the clinical spectrum of this disease. Acid ceramidase is activated by saposin D in vitro356 and a deficiency of acid ceramidase has been found in the three patients identified with a prosaposin defect. These patients present neonatally with a rapidly progressing neurovisceral lipd storage disease.357 The acid ceramidase gene has been cloned358 and mutations identified in patients.359–362 It is not possible to make any deductions about a genotype– phenotype correlation because of the small number of patients analyzed.361 The clinical severity does not correlate with the residual activity measured under nonphysiologic conditions363 but there is a good correlation with the level of lysosomal storage of ceramide.364 A few patients have undergone bone marrow transplantation. There was improvement in the peripheral manifestations of infantile Farber disease, but neurologic deterioration continued even in mildly symptomatic patients.365 However, in patients without neurologic involvement, allogeneic stem cell transplantation resulted in almost complete resolution of granulomas and joint contractures, and considerable improvement in mobility and joint motility.366,367 Patients can be diagnosed by measuring the accumulation of ceramide in cultured fibroblasts either by including 14C stearic acid-labeled sulfatide in the medium for 1–3 days368 or by the enzymic determination of extracted, unlabeled ceramide.369 The residual acid ceramidase can also be measured using synthetic substrates in the presence of added detergent.370,371 A novel mass spectrometric method for measuring glycosphingolipids in extracts of cultured fibroblasts may also be applicable to the diagnosis of Farber disease.372 Reliable carrier detection should be based on mutation analysis. Prenatal diagnosis has been carried out by measuring the ceramidase activity in CV373 and AFC374 or by lipid-loading tests in AFC.375 Wolman disease and cholesteryl ester storage disease A deficiency of the lysosomal enzyme acid lipase (EC 3.1.1.13) leads to two main phenotypes, Wolman disease and cholesteryl ester storage

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disease (CESD). Wolman disease, or primary familial xanthomatosis with involvement and calcification of the adrenals, is due to a complete absence of acid lipase activity. It is an autosomal recessive disease marked by severe failure to thrive, diarrhea, vomiting and hepatosplenomegaly evident in the first few weeks of life.376–380 Death usually occurs within 6 months from cachexia complicated by peripheral edema. Although most patients have calcification of the adrenals, some severely affected patients do not.381 Foam cells are found in the bone marrow and other organs. The organs contain cells loaded with neutral lipids, especially cholesterol esters and triglycerides. Cholesteryl ester storage disease can be a relatively mild disorder due to the presence of some residual acid lipase activity. It is characterized by liver enlargement, short stature, chronic gastrointestinal bleeding, chronic anemia, headaches and abdominal pain.380,382–385 Patients usually have no calcification of the adrenals, but they may have sea-blue histiocytosis.386 Some die in their juvenile years but others live to adulthood with unpredictable presentation.387–389 Levels of cholesterol esters are markedly elevated in the liver; levels of triglycerides are only moderately elevated. There are several reports of successful stem cell transplantation in cases of Wolman disease by BMT390,391 or unrelated umbilical cord blood transplantation.392 The gene for acid lipase has been cloned393 and a number of mutations have been identified.392,394–403 The mutations in patients with Wolman disease result in no residual enzyme activity or no enzymic protein, whereas those found in patients with CESD produce an enzyme with some residual activity.394,396,398 Wolman disease appears to be more heterogeneous genetically than CESD.398,399 In contrast, most CESD patients carry a common splice site mutation, which produces a small amount of normal enzyme.402 Patients with both Wolman disease and CESD have a marked deficiency of acid lipase (EC 3.1.1.13) activity in all tissues examined, including liver, spleen, leukocytes, lymphocytes and cultured skin fibroblasts.383,404–406 A variety of substrates have been used in the in vitro assays. These include radiolabeled triglycerides and cholesterol esters as well as fatty acid esters of 4-methylumbelliferone and p-nitrophenol. In common with many of the other

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lysosomal storage disorders, mutation analysis is the preferred approach to accurate carrier detection in family members even though some heterozygotes can be identified by enzyme analysis. Prenatal diagnosis is possible by direct enzyme assay of CV and CCV and AFC using synthetic substrates407–409 and radiolabeled cholesterol oleate.410

Lysosomal storage diseases: the neuronal ceroid lipofuscinoses The neuronal ceroid lipofuscinoses (NCL), also collectively known as Batten disease, encompass a group of at least 11 genetically distinct, severe, progressive, degenerative disorders characterized by the accumulation of autofluorescent ceroid lipopigments in neural and peripheral tissues.411–413 Clinically, the patients show progressive visual failure, neurodegeneration, epilepsy and premature death. Historically, the NCLs were classified as infantile (INCL), late infantile (LINCL), juvenile (JNCL) and adult (ANCL) on the basis of the age at onset of symptoms and the ultrastructural morphology of the storage material (Table 12.2). Genetic variants have been found in sub-types in populations e.g. late infantile variants CLN5, 6 and 7 and phenotypic variants that differ from the classic presentation in age of onset or severity e.g. in INCL. The NCLs can be regarded as lysosomal storage diseases because the autofluorescent ceroid lipopigment accumulates in lysosomes but it is not diseasespecific i.e. it is not the substrate of a defective enzyme in each disease. Its main components are sub-unit c of mitochondrial ATP synthase, which is predominant in LINCL and JNCL 414 and sphingolipid activator proteins A and D (saposins A and D), particularly in INCL415 (see Table 12.2). Other proteins are also present.416 Historically classification of the NCL subtype was based on clinical symptoms, age of onset and the ultrastructural morphology and composition of the storage material in a skin biopsy or whitecell buffy coat (see Table 12.2). In patients classified as having INCL, with onset of symptoms in the first or second year of life, granular osmiophilic deposits (GROD) were observed by electron microscopy (EM). Curvilinear bodies (CL) were

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found in biopsies from patients with LINCL and in patients with JNCL vacuolated lymphocytes were seen under light microscopy and fingerprint profiles (FP) under EM. In other patients with a late infantile presentation, the storage material was shown to be a mixture of FP and CL, and in the northern Finnish patients with epilepsy who had mutations in the CLN8 gene, rectilinear profiles (RL) were evident. Adult patients have also been found in whom the storage material is usually a mixture of FP and CL, but in some patients only GROD is evident. Very little progress has been made in identifying the gene, CLN4, underlying this subtype (called Kuf disease), but it is now clear that adult patients with GROD have mutations in the CLN1 gene and a palmitoyl protein thioesterase deficiency. The relationships between the storage material, genetic defects and clinical symptoms are poorly understood but under active investigation.417–419 Great progress has been made in understanding the genetic basis of the different forms of NCL (see Table 12.2).412,413,417,419,420 Currently over 225 disease-causing mutations have been reported in nine human genes (www.ucl.ac.uk/ncl/). The NCLs are autosomal recessive disorders. Classic infantile ceroid lipofuscinosis results from mutations in the the CLN1gene, which encodes a typical lysosomal enzyme, palmitoyl protein thioesterase.421 Most mutations in this gene lead to the classic severe infantile disease but patients with late infantile, juvenile or adult-onset NCL have been identified with mutations in the CLN1 gene.417 A common mutation is found in the Finnish population. The CLN2 gene also encodes a lysosomal enzyme, tripeptidyl peptidase I,422,423 a deficiency of which leads to cases of classic late infantile NCL (LINCL, NCL2). Variants with a later onset of symptoms occur. There are two common mutations. Mutations in the CLN3 gene are associated with juvenile NCL (JNCL, NCL3)424 and a 1 kb deletion accounts for approximately 90 percent of the affected alleles.425 Some mutations cause a less severe phenotype. Various functions have been ascribed to the CLN3 protein but its precise role on the endosomal/lysosomal sytem remains to be established.413 The gene for the rare adult-onset form, CLN4, has not been isolated. Several genetic variants of late infantile NCL have been characterized (see Table

12.2). Mutations in the CLN5 gene are found in a late infantile-onset variant, CLN5, particularly prevalent in Finnish patients, in whom a 2 bp deletion accounts for 94% of the mutant alleles.426 In another group of patients also presenting in the late infancy (NCL6), mutations have been found in the CLN6 gene.427 It is not clear how the ubiquitous CLN6 protein, which is located in the endoplasmic reticulum, leads to dysfunction of the lysosomal system. A group of patients, from Turkey, with a late infantile presentation have mutations in the MFSD8 (lysosomal transmembrane protein of MFS facilitator family) or NCL7 gene.428 Another variant form of LINCL arises from mutations in the CLN8 gene.429 A common mutation in predominantly Finnish patients produces progressive epilepsy with mental retardation (Northern epilepsy) whereas other mutations in the CLN8 gene produce a more severe and typical form of LINCL.430 A small number of patients has been described with a clinical presentation very similar to that in JNCL but without mutations in the CLN3 gene.431 The underlying defective gene is unknown but it has been designated CLN9. Patients with a very severe congenital form of NCL have mutations in the gene encoding cathepsin D (CLN10).432 Defects in members of the family of chloride channel proteins (ClCN6 and 7) may contribute to lysosomal dysfunction because of their location and function in the endosomal membranes giving rise to some clinical featurs of NCL, as well as disrupting other cellular processes.433,434 The diagnosis of most cases of neuronal ceroid lipofuscinosis can be achieved by a combination of biochemeical and genetic techniques.435 CLN1 and CLN2 can be diagnosed reliably by assaying the activity of PPT1 and TPP1 in white blood cells,436,437 dried blood spots,438 saliva439 or cultured fibroblasts. For all the other subtypes definitive diagnosis is based on DNA analysis. However, it has become evident that mutations in different NCL genes can give rise to a similar phenotype and that diffferent mutations in the same gene can give rise to very different phenotypes. Therefore, PPT1 and TPP1 should always be assayed in cases with an unusual presentation or later onset and all diagnoses should be supported by mutation analysis if possible. The characteristic ultrastructural morphology of buffy coat leukocytes is still a very useful adjunct to diag-

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nosis and can sometimes help to resolve an atypical case. The presence of GROD indicates a deficiency of palmitoyl protein thioesterase or perhaps cathepsin D, whereas the presence of only CL would indicate a tripeptidyl peptidase I deficiency (classic NCL2) or perhaps CLN8. Vacuolated lymphocytes and only FP in the buffy coat would be consistent with NCL3. The presence of a mixed FP/CL profile would indicate NCL5, NCL6 or NCL7. There are no biochemical tests available for NCL 3 or NCLs 5–9. Carrier detection is not possible by histology and unreliable by enzyme assay for CLN1 and CLN2 and should always be based on mutation analysis. To offer reliable prenatal diagnosis for the NCLs it is essential to have studied the index case and to define the subtype of NCL precisely. Postnatal diagnosis should include histology, enzymology and mutation analysis. All these approaches can be used in combination in prenatal diagnosis, depending on the CNL sub-type.52,440 The first prenatal diagnosis of NCL was made for NCL2 on noncultivated AFC.441,442 EM of these cells showed the characteristic CL profile in both pregnancies. The first pregnancy reported was not terminated and the diagnosis was confirmed postnatally by EM of a skin biopsy and lymphocytes.443 In the second pregnancy the prenatal diagnosis was confirmed by EM studies in the aborted fetus.442 Skin, amnion, umbilical vessels, blood, liver and brain showed the classic CL profile. Subsequently, inclusions have been found in CV from two fetuses affected with NCL1.444,445 Currently, prenatal diagnosis is available by analysis of CV directly, CCV, AFC and, as reported in the cases of LINCL, noncultivated AFC. For NCL1 and NCL2, assay of the palmitoyl protein thioesterase436,446 or the tripeptidyl peptidase447 in CV directly is very fast and reliable. However, it is reassuring to confirm this result by either histologic analysis and/or mutation analysis if the mutation is known and sufficient material is available.52,448–451 A combination of histology and mutation analysis (if the mutation is known) of CV is the preferred approach to prenatal diagnosis of NCL3,452 NCL5,453 and NCL6–10.454 This allows a diagnosis to be made in the first trimester. Abnormal histology has been found in CV, in a pregnancy at risk for NCL6; the pregnancy was

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terminated. The diagnosis was confirmed histologically on the termination products (Anderson G, Histopathology, Great Ormond Street Hospital, personal communication). This pregnancy was monitored before mutation analysis of the CLN6 gene was available. Prenatal genetic testing of couples at low risk for infantile neuronal ceroid lipofuscinosis has been carried out in Finland.455 Currently there is no effective treatment for these disorders but various therapeutic strategies have been tried or are in preclinical development.456–458 BMT has been performed in two patients, one with LINCL and one with JNCL.459 Although the patient with LINCL was less severely affected than his older sister was at 5 years of age, the BMT did not prolong his life and he died before his older sister, who had not been treated (Lake B, personal communication). There have been no follow-up reports on the patient with JNCL. Subsequently patients with INCL460 and LINCl461 were transplanted but without any improvement in the pathoneurology. On the basis of success in animal models of other lysosomal storage diseases, human neural stem cells have been transplanted into brains of children with Batten disease in a phase I trial. 462 Ten children with LINCL have undergone a trial of gene therapy by direct injection into the CNS of adeno-associated virus (AAV) serotype 2 vector expressing human CLN2 cDNA without serious adverse effects and with a significantly reduced rate of neurologic decline compared with control subjects.14 Small molecular weight molecules are potentially attractive therapeutics for NCL because they may cross the blood–brain barrier and chaperone therapy may be applicable to both the soluble proteins and membrane-bound proteins. Phosphocysteamine may be helpful in patients with INCL463 and a phase II trial of a combination of cysteamine and N-acetylcysteine for children with INCL is in progress

Lipoprotein-associated disorders This group of genetic disorders is exemplified by changes in plasma lipids due to defects in the protein lipid-carriers (lipoproteins), lipoprotein receptors or enzymes responsible for the hydrolysis and clearance of lipoprotein–lipid complexes (see Table 12.3).6 The proteins responsible for the

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maintenance of normal plasma and tissue lipids, which are primarily triglycerides and free and esterified cholesterol, include the apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III, D, E, and LP(a) as well as lipoprotein lipase, hepatic triglyceride lipase, lecithin cholesterol acyltransferase, cholesterol ester transfer protein, low-density lipoprotein receptor (LDLR), chylomicron remnant receptor and scavenger receptor (see Table 12.3). The normal structure and metabolism of plasma lipoproteins have been reviewed.464 Defects in these proteins are rare except for defects in the LDLR, which lead to autosomal dominant familial hypercholesterolemia.465,466 Autosomal dominant familial hypercholesterolemia can also arise from defects in apolipoprotein (apo) B and proprotein convertase subtilisin/kexin 9 and autosomal recessive hypercholesterolemia (ARH) from defects in the adaptor protein.465–467 Most of the disorders can be managed by a combination of dietary control, cholesterol-lowering drugs and, in some cases, vitamin supplementation. Homozygotes for familial hypercholesterolemia, who frequently die by the age of 20 from myocardial infarction after coronary heart disease from childhood, are an exception.465,466 Long-term LDL plasmapheresis (together with statins and ezetimibe) can delay the onset and complications of severe CAD in homozygotes for familial hypercholesterolemia and can be continued during pregnancy to prevent superimposed hyperlipidemia and placental insufficiency.468,469 Many of the disorders do not affect children, although early diagnosis through screening programs and family histories could result in dietary management to prevent the onset of serious, lifethreatening coronary heart disease later in life. Most of the genes encoding these proteins have been cloned and mutations and informative polymorphisms have been identified.470–475 Over 750 mutations have been discovered in the LDLR and founder effects shown in genetically isolated communities. This information can be used to identify carriers of autosomal recessive disorders or asymptomatic or mildly affected members of families with autosomal dominant disorders. It can also be used to improve screening for people at risk in the general population and for identifying patients with known genetic defects from those with familial combined hyperlipidemia.476,477

Prenatal diagnosis by DNA analysis of fetal samples from pregnancies at risk is possible if a reliable DNA test is available. Although this would offer the advantage of dietary intervention soon after birth,478 not many prenatal tests are carried out for these disorders because of the possibility of testing and treating babies at risk soon after birth. Again the exception is prenatal diagnosis for homozygosity for familial hypercholesterolemia, which has been performed by a functional assay in AFC479 or by measurement of the cholesterol in fetal blood.480 If the mutation(s) in the LDL receptor or the haplotype of the mutated chromosome is known, prenatal diagnosis can be carried out reliably by DNA analysis.481,482 The concentrations of apolipoproteins (apo) A-I, A-II, B and E can be measured in human fetal blood sampled via fetoscopy as a potential method for the prenatal diagnosis of other congenital apolipoprotein deficiencies483 but DNA analysis is the preferred method. Quantitative profiling of high-density lipoproteins by proteomics may offer an alternative or complementary approach to DNA analysis and serum lipid profiling for the detection of individuals with different underlying causes of hypercholesterolemia.484 The investigation of these disorders is yielding a better understanding of the delicate balance between diet and de novo lipid synthesis and of the functions of the many proteins responsible for transporting and processing cholesterol and triglycerides.466,467,485

Acknowledgments I acknowledge the dedication, experience and friendship of my colleagues in the Enzyme Diagnostic Laboratory at Great Ormond Street Hospital, London, the Supraregional Laboratory for Genetic Enzyme Defects, Guys & St Thomas’ NHS Foundation Trust, London, and the Willink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, particularly Elisabeth Young, Marie Jackson and Alan Cooper, respectively, with whom I have worked for many years and without whom this chapter could not have been written.

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novel predicted transmembrane protein. Am J Hum Genet 2002;70:537. Siintola E, Topcu M, Aula N et al. The novel neuronal ceroid lipofuscinosis gene MFSD8 encodes a putative lysosomal transporter. Am J Hum Genet 2007;81:136. Ranta S, Zhang Y, Ross B, et al. The neuronal ceroid lipofuscinosis in human EPMR and mnd mutant mice are associated with mutations in CLN8. Nat Genet 1999;23:233. Topcu M, Tan H, Yalnizoglu D, et al. Evaluation of 36 patients from Turkey with neuronal ceroid lipofuscinosis: clinical, neurophysiological, neuroradiological and histopathologic studies, Turk J Pediatr 2004;46:1. Schulz A, Dhar S, Rylova G, et al. Impaired cell adhesion and apoptosis in a novel CLN9 Batten disease variant. Ann Neurol 2004;56:342. Steinfeld R, Reinhardt K, Schreiber K, et al. Cathepsin D deficiency is associated with a human neurodegenerative disorder. Am J Hum Genet 2006;78:988. Kasper D, Planells-Cases R, Fuhrmann JC, et al. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J 2005; 24:1079. Poët M, Kornak U, Schweizer M, et al. Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. Proc Natl Acad Sci USA 2006;103:13854. Williams RE, Åberg L, Autti T, et al. Diagnosis of the neuronal ceroid lipofuscinoses: an update. Biochim Biophys Acta 2006;1762:865. van Diggelen OP, Keulemans JLM, Winchester B, et al. A rapid fluorogenic palmitoyl-protein thioesterase assay: pre- and postnatal diagnosis in INCL. Mol Genet Metab 1999;66:240. Junaid MA, Brooks SS, Pullarkat RK. Specific substrate for CLN2 protease/tripeptidyl-peptidase I assay. Eur J Paediatr Neurol 2001;5(suppl A):63. Lukacs Z, Santavouri P, Keil A, et al. A rapid and simple assay for the determinatin of tripeptidyl peptidase (TPP) and palmitoyl protein thioesterase(PPT) in dried blood spots. Clin Chem 2003;49:509. Kohan R, de Hala IN, Anzolini VT, et al. Palmitoyl protein thioesterase1 (PPT1) and of tripeptidyl peptidase-1 (TPP1) are expressed in human saliva. A reliable and non-invasive source for the diagnosis of infantile (CLN1) and CLN2 neuronal ceroid lipofuscinosis. Clin Biochem 2005;38:492. Kleijer WJ, van Diggelen OP. Prenatal diagnosis of the neuronal ceroid lipofuscinoses. Prenat Diagn 2000; 20:819. Macleod PM, Dolman CL, Nickel RE, et al. Prenatal diagnosis of neuronal ceroid lipofuscinosis. N Engl J Med 1984;310:595.

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442. Chow CW, Borg J, Billson VR, et al. Fetal tissue involvement in the late infantile type of neuronal ceroid lipofuscinosis. Prenat Diagn 1993;13:833. 443. Macleod PM, Dolman CL, Nickel RE, et al. Prenatal diagnosis of neuronal ceroid lipofuscinoses. Am J Hum Genet 1985;22:781. 444. Rapola J, Salonen R, Ammala P, et al. Prenatal diagnosis of infantile neuronal ceroid-lipofuscinosis, INCL: morphological aspects. J Inherit Metab Dis 1993;16:349. 445. Munroe PB, Rapola J, Mitchison HM, et al. Prenatal diagnosis of Batten’s disease. Lancet 1996;347:1014. 446. Voznyi YV, Keulemans JLM, Mancini GMS, et al. A new simple enzyme assay for pre- and postnatal diagnosis of infantile neuronal ceroid lipofuscinosis (INCL) and its variants. J Med Genet 1999;36:471. 447. Young EP, Winchester BG, Logan WP, et al. Exclusion of late infantile neuronal ceroid lipofuscinosis (LINCL) in a fetus by assay of tripeptidyl peptidase I in chorionic villi. Prenat Diagn 2000;20:337. 448. Goebel HH, Vesa J, Reitter B, et al. Prenatal diagnosis of infantile neuronal ceroid-lipofuscinosis: a combined electron microscopic and molecular genetic approach. Brain Dev 1995;17:83. 449. Berry-Kravis E, Sleat DE, Sohar I, et al. Prenatal testing for late infantile neuronal ceroid lipofuscinosis. Ann Neurol 2000;47:254. 450. Kleijer WJ, van Diggelen OP, Keulemans JL, et al. Firsttrimester diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) by tripeptidyl peptidase I assay and CLN2 mutation analysis. Prenat Diagn 2001;21:99. 451. Zhong N, Ju W, Moroziewicz D, et al. Prenatal diagnostic testing for infantile and late-infantile neuronal ceroid lipofusinoses (NCL) using allele specific primer extension (ASPE). Beijing Da Xue Xue Bao 2005;37:20. 452. Uvebrant P, Björck E, Conradi N, et al. Successful DNA-based prenatal exclusion of juvenile neuronal ceroid lipofuscinosis. Prenat Diagn 1993;13:651. 453. Rapola J, Lähdetie J, Isosomppi J, et al. Prenatal diagnosis of variant late infantile neuronal ceroid lipofuscinosis (vLINCL[Finnish]; CLN5). Prenat Diagn 1999;19:685. 454. Fritchie K, Siintola E, Armao D, et al. Novel mutation and the first prenatal screening of cathepsin D deficiency (CLN10). Acta NeuroPathol 2008 Sept 2 (ePub ahead of print). 455. Kallinen J, Heinonen S, Palotie A, Antenatal gene tests in low-risk pregnancies: molecular screening for aspartylglucosaminuria (AGU) and infantile neuronal ceroid lipofuscinosis (INCL) in Finland. Prenat Diagn 2001;21:409. 456. Hobert JA, Dawson G. Neuronal ceroid lipofuscinoses therapeutic strategies: past, present and future. Biochim Biophys Acta 2006;1762:945.

457. Pieret C, Morrison JA, Kirk MD. Treatment of lysosomal storage diseases: focus on the neuronal ceroidlipofuscinoses. Acta Neurobiol Exp 2008;68:429. 458. Cooper JD. Moving towards therapies for juvenile Batten disease? Exp Neurol 2008;211:329. 459. Lake BD, Steward CG, Oakhill A, et al. Bone marrow transplantation in late infantile Batten disease and juvenile Batten disease. Neuropaediatrics 1997;28:80. 460. Lonnqvist T, Vanhanen SL,Vettenranta T, et al. Hematopoietic stem cell transplantation in infantile neuronal ceroid lipofuscinosis. Neurology 2001;57: 1411. 461. Yuza Y, Yokoi K, Sakurai K, et al. Allogenic bone marrow transplantation for late-infantile neuronal ceroid lipofuscinosis, Pediatr Int 2005;47:681. 462. Taupin P. HuCNS-SC (stem cells). Curr Opin Mol Ther 2006;8:156. 463. Zhang Z, Butler JDeB, Levin S, et al. Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood. Nat Med 2001;7:478. 464. Havel RJ, Kane JP. Introduction: structure and metabolism of plasma lipoproteins. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGrawHill, 2001:2705. 465. Goldstein JL, Hobbs HL, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw-Hill, 2001:2863. 466. Marais AD. Familial hypercholesterolaemia. Clin Biochem Rev 25;2004:49. 467. Goldstein JL, Brown MS. Molecular medicine. The cholesterol quartet. Science 2001;292:1310. 468. Kroon AA, Swinkels DW, van Dongen PW, et al. Pregnancy in a patient with homozygous familial hypercholesterolemia treated with long-term low-density lipoprotein apheresis. Metabolism 1994;43:1164. 469. Teruel JL, Lasunción MA, Navarro JF, et al. Pregnancy in a patient with homozygous familial hypercholesterolemia undergoing low-density lipoprotein apheresis by dextran sulfate adsorption. Metabolism 1995;44:929. 470. Kim J-H, Choi H-K, Lee H, et al. Novel and recurrent mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia. Mol Cells 2004; 18:63. 471. Humphries SE, Cranston T, Allen M, Mutational analysis in UK patients with a clinical diagnosis of familial hypercholesterolaemia: relationship with plasma lipid traits, heart disease risk and utility in relative tracing. J Mol Med 2006;84:203. 472. Widhalm K, Dirisamer A, Lindemayr A, et al. Diagnosis of families with familial hypercholesterolaemia and/

CHAP T E R 1 2

473.

474.

475.

476.

477.

478.

479.

480.

481.

482.

483.

484.

Prenatal Diagnosis of Disorders of Lipid Metabolism

or Apo B-100 defect by means of DNA analysis of LDLreceptor gene mutations. J Inherit Metab Dis 2007; 30:239. Tosi I, Toledo-Leiva P, Neuwirth C, et al. Genetic defects causing familial hypercholesterolaemia: identification of deletions and duplications in the LDLreceptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic. Atherosclerosis 2007;194:102. Zeng Y, Miao F, Li L, et al. A rapid and accurate DHPLC assay for determination of apolipoprotein E genotypes. J Alzheimers Dis 2007;12:357. Wright WT, Heggarty SV, Young IS, et al. Multiplex MassARRAY spectrometry (iPLEX) produces a fast and economical test for 56 familial hypercholesterolaemia-causing mutations. Clin Genet 2008;74:463. Gaddi A, Cicero AFG, Odoo FO, et al. Practical guidelines for familial combined hyperlipidemia diagnosis: an up-date. Vasc Health Risk Manag 2007;3:877. Civeira F, Jarauta E, Cenarro A, et al. Frequency of low-density lipoprotein receptor gene mutations in patients with a clinical diagnosis of familial combined hyperlipidemia in a clinical setting. J Am Coll Cardiol 2008;52:1546. Heath KE, Luong L-A, Leonard JV, et al. The use of a highly informative CA repeat polymorphism within the abetalipoproteinaemia locus (4q22–24). Prenat Diagn 1997;17:1181. Brown MS, Kovanen PT, Goldstein JL, et al. Prenatal diagnosis of homozygous familial hypercholesterolaemia: expression of a genetic receptor disease in utero. Lancet 1978;i:526. de Gennes JL, Daffos F, Dairou F, et al. Direct fetal blood examination for prenatal diagnosis of homozygous familial hypercholesterolaemia. Arteriosclerosis 1985;5:440. Reshef A, Meiner V, Dann EJ, et al. Prenatal diagnosis of familial hypercholesterolaemia caused by the “Lebanese” mutation at the low density lipoprotein receptor locus. Hum Genet 1992;89:237. de Oliveira e Silva ER, Haddad L, Kwiterovich PO Jr, et al. Applicability of LDLR flanking microsatellite polymorphisms for prenatal diagnosis of homozygous state for familial hypercholesterolaemia. Clin Genet 1998;53:375. Fainaru M, Deckelbaum R, Golbus MS. Apolipoproteins in human fetal blood and amniotic fluid in midtrimester pregnancy. Prenat Diagn 1981;1:125. Heller M, Schlappritzi E, Stalder D, et al. Compositional protein analysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS and probabilistic peptide scoring. Mol Cell Proteom 2007; 6:1059.

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485. Vaughan AM, Tang C, Oram JF. ABCA1 mutants reveal an interdependency between lipid export function, apoA-I binding activity, and Janus kinase 2 activation. J Lipid Res 2009;50:285. 486. Norum RA, Lakier JB, Goldstein S, et al. Familial deficiency of apolipoproteins A-I and C-III and precocious coronary-artery disease. N Engl J Med 1982;306:1513. 487. Ordovas JM, Cassidy DK, Civeira F, et al. Familial apolipoprotein A-I, C-III and A-IV deficiency and premature atherosclerosis due to deletion of a gene complex on chromosome 11. J Biol Chem 1989;264:16339. 488. Tall AR, Breslow JL, Rubin EM. Genetic disorders affecting high plasma high-density lipoproteins. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw-Hill, 2001:2915. 489. Esperón P, Raggio V, Stol M, et al. A new APOA1 mutation with severe HDL-cholesterol deficiency and premature coronary artery disease. Clin Chim Acta 2008;388:222. 490. Pennacchio LA, Olivier M, Hubacek JA, et al. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing. Science 2001;294:169. 491. Weinberg RB, Cook VR, Beckstead JA, et al. Structure and interfacial properties of human apolipoprotein A-V. J Biol Chem 2003;278:34438. 492. Brunzell JD, Deeb SS. Familial lipoprotein lipase deficiency, apoC-II deficiency, and hepatic lipase deficiency. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw-Hill, 2001:2789. 493. Talmud PJ. Genetic determinants of plasma triglycerides: impact of rare and common mutations. Curr Atheroscler Rep 2001;3(3):191–9. 494. Mahley RW, Rall SC Jr. Type III hyperlipoproteinaemia (dysbetalipoproteinaemia). The role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw-Hill, 2001:2835. 495. Santamarina-Fojo S, Hoeg JM, Assmann G, et al. Lecithin cholesterol acyltransferase deficiency and fish-eye disease In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw-Hill, 2001:2817. 496. Wetterau JR, Aggerbeck LP, Bouma ME, et al. Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia. Science 1992;258: 999. 497. Zamel R, Khan RL, Pollex R, et al. Abetalipoproteinemia: two case reports and literature review. Orphanet J Rare Dis 2008;3:19.

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498. Assmann G, von Eckardstein A, Brewer BH. Familial analphalipoproteinemia disease. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw-Hill, 2001:2937. 499. Rust S, Rosier M, Funke H, et al. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1. Nat Genet 1999;22:352.

500. Singaraja RR, Visscher H, James ER, et al. Specific mutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro. Circ Res 2006;99:389. 501. Alrasadi K, Ruel IL, Marcil M, et al. Functional mutations of the ABCA1 gene in subjects of FrenchCanadian descent with HDL deficiency. Atherosclerosis 2006;188:281.

13

Prenatal Diagnosis of the Peroxisomal and Mitochondrial Fatty Acid Oxidation Deficiencies Ronald J.A. Wanders Laboratory of Genetic Metabolic Diseases, University of Amsterdam, Amsterdam, The Netherlands

Introduction Fatty acid oxidation is an essential metabolic process which, if deficient, causes major clinical abnormalities with considerable mortality and morbidity in patients. In principle, there are three different mechanisms by which fatty acids (FAs) can be oxidized, including fatty acid β-oxidation, fatty acid α-oxidation, and fatty acid ω-oxidation. So far no specific defects in ω-oxidation have been described. In addition, only one disorder of fatty acid α-oxidation has been identified, which is Refsum disease1 for which no prenatal diagnosis has been reported, although this is technically feasible using either enzymatic or molecular analysis of phytanoyl-CoA hydroxylase in chorionic villous (CV) biopsy material. With respect to FA β-oxidation it is important to realize that it can take place in two different subcellular organelles, i.e. the mitochondrion2 and peroxisome.3 The two systems fulfill completely different roles in whole-cell FA oxidation. Indeed, the mitochondrial β-oxidation system is responsible for the oxidation of the bulk of FAs derived from our daily diet including the major long-chain FAs, such as palmitic, oleic, linolenic, and linoleic acid,2 whereas the peroxisomal β-oxidation system

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

is not as important for energy purposes but, instead, catalyzes the oxidation of a range of minor FAs, including very long-chain FAs (VLCFA), notably C26:0, pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid.4 The different role of the mitochondrial and peroxisomal FA oxidation systems is also reflected in the widely different clinical phenotypes, associated with defects in each of the two systems. Indeed, the mitochondrial FA oxidation deficiencies, especially those in which the oxidation of long-chain FAs is impaired, are characterized by hypoketotic hypoglycemia and, more importantly, life-threatening cardiomyopathy, but no neurologic involvement.2 In contrast, patients with a deficiency at the level of the peroxisomal β-oxidation system do not show hypoketotic hypoglycemia, nor cardiomyopathy, but do present with usually severe neurologic abnormalities, among other derangements.5

Prenatal diagnosis of peroxisomal fatty acid β-oxidation deficiencies The peroxisomal β-oxidation deficiencies represent a subgroup of disorders within the larger group of peroxisomal disorders (Table 13.1). Within this group of peroxisomal β-oxidation deficiencies, two different groups can be distinguished: • the single peroxisomal β-oxidation deficiencies, due to mutations in one of the genes coding for a peroxisomal β-oxidation enzyme

489

490

Disorder

Abbreviation

Deficient protein/enzyme

Gene

Chromosome

MIM

References

Zellweger syndrome

ZS

Different peroxins

Different PEX-genes

Multiple loci

214100

17, 18, 19

Neonatal adrenoleukodystrophy

NALD

Different peroxins

Different PEX-genes

Multiple loci

214110

–

Infantile Refsum disease

IRD

Different peroxins

Different PEX-genes

Multiple loci

202370

–

Rhizomelic chondrodysplasia punctata type 1

RCDP Type 1

Pex7p

PEX7

6q22-q24

215100

22

X-linked adrenoleukodystrophy

X-ALD

ALDP

ABCD1

Xq28

300100

20, 21

Acyl-CoA oxidase deficiency (pseudo-neonatal ALD)

ACOX1-deficiency

Straight-chain acyl-CoA oxidase (SCOX/ACOX1)

SCOX/ACOX1

17q25

264470

15

D-bifunctional protein deficiency

D-BP deficiency

D-BP

HSD17B4

5q2

261515

23, 24

2-Methylacyl-CoA racemase deficiency

Racemase deficiency

AMACR

AMACR

5q13.2-q11.1

604489

–

SCPx deficiency

SCPx deficiency

SCPx

SCP2

1p32

–

–

Rhizomelic chondrodysplasia punctata type 2 (DHAPAT deficiency)

RCDP Type 2

DHAPAT

GNPAT

1q42.1-42.3

222765

–

Rhizomelic chondrodysplasia punctata type 3 (alkylDHAP synthase deficiency)

RCDP Type 3

AlkylDHAP synthase

AGPS

2q33

600121

–

ARD

Phytanoyl-CoA hydroxylase (PhyH)

PHYH / PAHX

10pter-p11.2

266500

–

PH1

Alanine glyoxylate aminotransferase (AGT)

AGXT

2q37.3

259900

25, 26

Disorders of peroxisome biogenesis

Single peroxisomal enzyme/protein deficiencies Disorders of peroxisomal β -oxidation

Disorders of etherphospholipid biosynthesis

Disorders of fatty acid α-oxidation Refsum disease (phytanoyl-CoA hydroxylase deficiency) Disorders of glyoxylate metabolism Hyperoxaluria type 1

Genetic Disorders and the Fetus

Table 13.1 The peroxisomal disorders including the peroxisomal fatty acid β-oxidation deficiencies and prenatal diagnosis

C H A P T E R 13

Prenatal Diagnosis of the Peroxisomal and Mitochondrial Fatty Acid Oxidation Deficiencies 491

• the peroxisomal disorders, in which the deficiency of peroxisomal β-oxidation is the secondary consequence of a defect in peroxisome biogenesis. The latter group includes the Zellweger spectrum disorders, with Zellweger syndrome (ZS) as prototype and neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) as milder variants.5–7 The single peroxisomal β-oxidation deficiencies now include five different disorders8: X-linked adrenoleukodystrophy (X-ALD); acyl-CoA oxidase (ACOX) deficiency; D-bifunctional protein (DBP) deficiency; 2-methylacyl-CoA racemase (AMACR) deficiency; and SCPx deficiency.9–13 X-linked adrenoleukodystrophy is most frequent among these disorders and, in fact, among the whole group of peroxisomal disorders. Second in frequency is D-bifunctional protein deficiency, followed by acyl-CoA oxidase deficiency, AMACR deficiency and SCPx deficiency. The last two disorders are rare, with SCPx deficiency only described in a single case, whereas racemase deficiency has only been described in some five patients. The characteristics of these deficiencies are depicted in Table 13.1. The procedures used for the prenatal diagnosis of X-linked adrenoleukodystrophy, D-bifunctional protein deficiency and acylCoA oxidase deficiency follow. X-linked adrenoleukodystrophy In X-ALD β-oxidation of VLCFAs is deficient, due to mutations in the gene ABCD1, which codes for a peroxisomal half-ABC transporter, named ALDP.14 The preferred method for prenatal diagnosis of X-ALD is molecular analysis of the ABCD1 gene, which requires prior knowledge of the mutation in ABCD1 in the index case. In exceptional cases in which the proband has not been studied in detail and no molecular analysis has been performed, prenatal diagnosis can also be done biochemically by measuring the βoxidation of C26:0 in cultured CV cells and, in addition, determining the very long-chain FA profile using either gas chromatography-mass spectrometry [GC(MS)] or tandem MS. In our own laboratory molecular analysis of ABCD1 is the standard procedure.

Acyl-CoA oxidase deficiency In patients with acyl-CoA oxidase deficiency plasma very long-chain fatty acids are abnormally high with normal values for the other peroxisomal FAs, including pristanic acid and di- and trihydroxycholestanoic acid. This is due to a functional deficiency of acyl-CoA oxidase as encoded by ACOX1.8 Acyl-CoA oxidase deficiency is a very rare disease with so far only some 20–25 patients described.10 Through the years we have had several requests for prenatal diagnosis of ACOX deficiency and we reported the first prenatal diagnosis in 1990.15 In principle, prenatal diagnosis can be done using biochemical and molecular methods. Since acyl-CoA oxidase is well expressed in CV biopsy material, direct enzymatic analysis of acyl-CoA oxidase can be done with this tissue. We always request CV fibroblasts for confirmatory studies by determining the oxidation of C26:0 in these cultured CV cells plus VLCFA profiling. Since we have introduced molecular analysis of ACOX1 in our laboratory and now perform molecular analysis in any patient we identify with acylCoA oxidase deficiency, molecular analysis of ACOX1 in CV material is the method of choice. Using either of the two approaches, we have encountered no difficulties in establishing the correct diagnosis. D-bifunctional protein deficiency In patients with D-bifunctional protein deficiency plasma, VLCFA levels are abnormally high but pristanic acid and di- and trihydroxycholestanoic acid are also elevated. This is due to the fact that D-bifunctional protein is involved in the oxidation of all peroxisomal FAs identified so far. D-bifunctional protein deficiency has been described in some 100–150 patients.11In the past we have performed prenatal diagnosis for D-bifunctional protein deficiency using enzymatic and molecular methods. The procedure followed is basically identical to that used for acyl-CoA oxidase deficiency. We have solid and fully validated enzyme assays available for D-bifunctional protein, which allow us to measure the activity in CV biopsy material, backed up by subsequent studies in CV fibroblasts. However, now in our center molecular analysis of the D-bifunctional protein gene (HSD17B4) is solely done for prenatal diagnosis of

492

Genetic Disorders and the Fetus

D-bifunctional protein deficiency. Thus far in the long series of prenatal diagnostic tests we have performed, no errors have occurred.

Prenatal diagnosis of the mitochondrial fatty acid β-oxidation deficiencies The mitochondrial β-oxidation deficiencies known thus far are listed in Table 13.2. Carnitine acylcarnitine translocase (CACT) deficiency; carnitine palmitoyltransferase-2 (CPT2) deficiency; very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency; and long-chain 3-hydroxyacylCoA dehydrogenase/mitochondrial trifunctional protein (LCHAD/MTP) deficiency, at least in their severe forms, are characterized by severe abnormalities including hypoketotic hypoglycemia and especially cardiomyopathy, which usually leads to early death. For these disorders prenatal diagnosis is warranted. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is most frequent among the mitochondrial β-oxidation disorders, but is a treatable disease. The same is true for OCTN2 deficiency, which is due to a defect at the level of the plasma membrane transporter for carnitine. Sup-

plementation with high doses of carnitine can reverse the abnormalities, including the cardiomyopathy, in these patients. CPT1 deficiency is one of the most infrequent mitochondrial β-oxidation disorders and no prenatal diagnosis has been performed. Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a mitochondrial β-oxidation defect, in which clarity is lacking between the clinical signs and symptoms and the enzyme defect. Finally, short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficiency has been described in a few cases only and prenatal diagnosis for SCHAD deficiency has not been reported, although the enzyme is clearly expressed in CV biopsy material as well as in CV fibroblasts. In principle, both biochemical as well as molecular methods can be used for prenatal diagnosis of SCHAD deficiency. The procedures used for the prenatal diagnosis of CACT deficiency, CPT2 deficiency, VLCAD deficiency, and LCHAD/MTP deficiency follow. CACT deficiency Carnitine acylcarnitine translocase is a mitochondrial transport protein which exchanges carnitine for acylcarnitines. Its activity can be measured in intact cells only, so that for this type of analysis

Table 13.2 The mitochondrial fatty acid oxidation deficiencies and prenatal diagnosis Disorder

Abbreviation

Deficient

Gene

Chromosome

MIM

References

OCTN2

OCTN2

5q31

212140

–

CPT1

CPT-I

11q13

600528

–

CACT-deficiency

CACT

CACT

3p21.31

212138

27, 28

Carnitine palmitoyltransferase-2 CPT2-deficiency

CPT2

CPT-II

1p32

600650

29

VLCAD deficiency VLCAD

ACADVL

17p11.2-p11.1 201475

30

MCAD deficiency

MCAD

ACADM

1p31

201450

31, 32

SCAD deficiency

SCAD

ACADS

12q22-qter

201470

–

MTP deficiency

MTP

HADHA

2p23, 2p23

600890,

33

protein/enzyme OCTN2 deficiency (primary

–

carnitine deficiency) Carnitine palmitoyltransferase-1 CPT1-deficiency deficiency Carnitine acylcarnitine translocase deficiency deficiency Very long-chain acyl-CoA dehydrogenase deficiency Medium-chain acyl-CoA dehydrogenase deficiency Short-chain acyl-CoA dehydrogenase deficiency Mitochondrial trifunctional protein deficiency Short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency

HADHB SCHAD deficiency SCHAD

HADHSC

600890 4q22-25

201470

–

C H A P T E R 13

Prenatal Diagnosis of the Peroxisomal and Mitochondrial Fatty Acid Oxidation Deficiencies 493

cultured chorionic fibroblasts are needed for the prenatal diagnosis of CACT deficiency. We have performed prenatal diagnosis of CACT deficiency using direct enzymatic analysis of CACT but have abandoned this procedure since the activity of CACT can be artificially low if cells show an inappropriate growth. Molecular analysis of CACT, using genomic DNA isolated from CV material, is the method of choice for the prenatal diagnosis of CACT deficiency. CPT2 deficiency The enzyme carnitine palmitoyltranferase 2 (CPT2) is an intramitochondrial enzyme catalyzing the formation of acyl-CoA esters from the corresponding acylcarnitines after they have entered the mitochondrial space via CACT. CPT2 is generally expressed in virtually every cell, and is also highly active in CV biopsy material as well as CV fibroblasts. This implies that prenatal diagnosis of CPT2 deficiency can be done by direct measurement of CPT2 in CV material and/or CV fibroblasts. We have used this procedure in our long history of prenatal diagnosis of CPT2 deficiency without any problem, although, again, we have now introduced molecular analysis of the CPT2 gene, particularly in any case in whom CPT2 has been found to be deficient in the index case. VLCAD deficiency As for CPT2, VLCAD is highly expressed in both CV biopsy material and CV fibroblasts. This implies that prenatal diagnosis can be done by enzymatic analysis of VLCAD in CV biopsy material, followed by confirmatory testing in cultured villi. We have introduced molecular analysis of the VLCAD gene as a standard procedure in the diagnostic work-up of patients, and molecular analysis of VLCAD is the method of choice for prenatal diagnosis. LCHAD/MTP The enzyme mitochondrial trifunctional protein is a trifunctional enzyme which catalyzes the second, third and fourth steps of mitochondrial β-oxidation of long-chain FAs. In general, three different forms of MTP deficiency have been identified, including LCHAD deficiency, in which the MTP protein complex is normally present but the 3-hydroxyacyl-CoA dehydrogenase component of MTP is

functionally deficient due to single amino-acid substitution.16 The fact that LCHAD/MTP is highly expressed in CV biopsy material has allowed us to perform prenatal diagnosis of LCHAD/MTP deficiency, using biochemical methods. A drawback is that the substrate required for these studies (3-ketopalmitoyl-CoA) needs to be synthesized enzymatically since it cannot be obtained via commercial sources. As for the other deficiencies, we have now moved to molecular methods for the prenatal diagnosis of LCHAD/MTP deficiency. To summarize, in recent years prenatal diagnostic methods have become available for both the peroxisomal and mitochondrial β-oxidation deficiencies. In the Amsterdam laboratory we have been performing prenatal diagnosis of these deficiencies for some 20 years and have encountered minimal difficulties with only one or two mistakes made. Since the introduction of molecular methods, we have experienced no further discrepancies.

References 1. Wanders RJA, Jakobs C, Skjeldal OH. Refsum disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw-Hill; 2001:3303. 2. Rinaldo P, Matern D, Bennett MJ. Fatty acid oxidation disorders. Annu Rev Physiol 2002;64:477. 3. Wanders RJA. Peroxisomes, lipid metabolism, and peroxisomal disorders. Mol Genet Metab 2004;83:16. 4. Wanders RJA, Waterham HR. Biochemistry of mammalian peroxisomes revisited. Annu Rev Biochem 2006;75:295. 5. Wanders RJA. Metabolic and molecular basis of peroxisomal disorders: a review. Am J Med Genet 2004;126:355. 6. Wanders RJA, Waterham HR. Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders. Clin Genet 2005;67:107. 7. Weller S, Gould SJ, Valle D. Peroxisome biogenesis disorders. Annu Rev Genomics Hum Genet 2003;4:165. 8. Wanders RJA, Waterham HR. Peroxisomal disorders: the single peroxisomal enzyme deficiencies. Biochim Biophys Acta 2006;1763:1707. 9. Bezman L, Moser AB, Raymond GV, et al. Adrenoleukodystrophy: incidence, new mutation rate, and results of extended family screening. Ann Neurol 2001;49:512. 10. Ferdinandusse S, Denis S, Hogenhout EM, et al. Clinical, biochemical, and mutational spectrum of peroxisomal acyl-coenzyme A oxidase deficiency. Hum Mutat 2007;28:904.

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11. Ferdinandusse S, Denis S, Mooyer PA, et al. Clinical and biochemical spectrum of D-bifunctional protein deficiency. Ann Neurol 2006;59:92. 12. Ferdinandusse S, Denis S, Clayton PT, et al. Mutations in the gene encoding peroxisomal alpha-methylacylCoA racemase cause adult-onset sensory motor neuropathy. Nat Genet 2000;24:188. 13. Ferdinandusse S, Denis S, van Berkel E, et al. Peroxisomal fatty acid oxidation disorders and 58 kDa sterol carrier protein X (SCPx). Activity measurements in liver and fibroblasts using a newly developed method. J Lipid Res 2000;41:336. 14. Mosser J, Douar AM, Sarde CO, et al. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature 1993;361:726. 15. Wanders RJA, Schelen A, Feller N, et al. First prenatal diagnosis of acyl-CoA oxidase deficiency. J Inherit Metab Dis 1990;13:371. 16. IJlst L, Wanders RJA, Ushikubo S, et al. Molecular basis of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: identification of the major disease-causing mutation in the alpha-subunit of the mitochondrial trifunctional protein. Biochim Biophys Acta 1994;1215:347. 17. Steinberg SJ, Elçioglu N, Slade CM, et al. Peroxisomal disorders: clinical and biochemical studies in 15 children and prenatal diagnosis in 7 families. Am J Med Genet 1999;85:502. 18. Johnson JM, Babul-Hirji R, Chitayat D. First-trimester increased nuchal translucency and fetal hypokinesia associated with Zellweger syndrome. Ultrasound Obstet Gynecol 2001;17:344. 19. Mochel F, Grébille AG, Benachi A, et al. Contribution of fetal MR imaging in the prenatal diagnosis of Zellweger syndrome. Am J Neuroradiol 2006;27:333. 20. Ke LF, Wang ZH, Huang HJ, et al. Prenatal molecular diagnosis of four fetuses at high risk for X-linked adrenoleukodystrophy. Zhonghua Fu Chan Ke Za Zhi 2008;43:25. 21. Maier EM, Roscher AA, Kammerer S, et al. Prenatal diagnosis of x-linked adrenoleukodystrophy combining biochemical, immunocytochemical and DNA analyses. Prenat Diagn 1999;19:364. 22. Bas¸bug˘ M, Serin IS, Ozçelik B, et al. Prenatal ultrasonographic diagnosis of rhizomelic chondrodysplasia punc-

23.

24.

25. 26.

27.

28.

29.

30.

31.

32.

33.

tata by detection of rhizomelic shortening and bilateral cataracts. Fetal Diagn Ther 2005;20:171. Paton BC, Solly PB, Nelson PV, et al. Molecular analysis of genomic DNA allows rapid, and accurate, prenatal diagnosis of peroxisomal D-bifunctional protein deficiency. Prenat Diagn 2002;22:38. Suzuki Y, Zhang Z, Shimozawa N, et al. Prenatal diagnosis of peroxisomal D-3-hydroxyacyl-CoA dehydratase/ D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency. J Hum Genet 1999;44:143. Rumsby G. Experience in prenatal diagnosis of primary hyperoxaluria type 1. J Nephrol 1998;11:13. Danpure CJ, Rumsby G. Strategies for the prenatal diagnosis of primary hyperoxaluria type 1. Prenat Diagn 1996;16:587. Yang BZ, Mallory JM, Roe DS, et al. Carnitine/acylcarnitine translocase deficiency (neonatal phenotype): successful prenatal and postmortem diagnosis associated with a novel mutation in a single family. Mol Genet Metab 2001;73:64. Costa C, Costa JM, Siama A, et al. Mutational spectrum and DNA-based prenatal diagnosis in carnitine-acylcarnitine translocase deficiency. Mol Genet Metab 2003; 78:68. Vekemans BC, Bonnefont JP, Aupetit J, et al. Prenatal diagnosis of carnitine palmitoyltransferase 2 deficiency in chorionic villi: a novel approach. Prenat Diagn 2003; 23:884. Andresen BS, Olpin S, Kvittingen EA, et al. DNA-based prenatal diagnosis for very-long-chain acyl-CoA dehydrogenase deficiency. J Inherit Metab Dis 1999;22: 281. Nada MA, Vianey-Saban C, Roe CR, et al. Prenatal diagnosis of mitochondrial fatty acid oxidation defects. Prenat Diagn 1996;16:117. Gregersen N, Winter V, Jensen PK, et al. Prenatal diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in a family with a previous fatal case of sudden unexpected death in childhood. Prenat Diagn 1995;15:82. Ibdah JA, Zhao Y, Viola J, et al. Molecular prenatal diagnosis in families with fetal mitochondrial trifunctional protein mutations. J Pediatr 2001;138: 396.

14

Prenatal Diagnosis of the Mucopolysaccharidoses and Postnatal Enzyme Replacement Therapy John J. Hopwood Lysosomal Diseases Research Unit, Women’s and Children’s Hospital, Adelaide, Australia

The mucopolysaccharidoses (MPS) are a group of 11 genetically transmitted lysosomal storage diseases that are clinically progressive and involve multiple organs with devastating clinical outcomes. The MPS (Table 14.1) were initially characterized by the lysosomal storage of mucopolysaccharides – more accurately known as glycosaminoglycans (GAGs). These GAGs – dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), chondroitin sulfate (CS) and hyaluronan (HA) – are primary storage products of enzymes involved in the degradation of these GAGs in the lysosome (see Table 14.1). A deficiency in any one of these enzymes may lead to the storage of one or more GAG substrates. Research over the past three decades has led to promising outcomes for the diagnosis and treatment of the MPS, which affect at least 1 in 20,000 individuals.1,2 With the exception of the X-linked recessive MPS II, the mode of inheritance of all the known MPS is autosomal recessive. Gene structures for all 11 MPS have been characterized and enable causative mutations to be identified within most individual families. These mutations have facilitated diagnosis (particularly

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

prenatal diagnosis), assisted the detection of heterozygotes in high-risk family members and have in some instances assisted counseling by enabling a connection to be made between genotype and clinical phenotype. Importantly, the isolation of each MPS gene has also enabled the preparation of recombinant human enzyme proteins and subsequent demonstration of efficacy and safety of enzyme replacement therapies for most of the MPS. These developments have established enzyme replacement therapies for MPS with noncentral nervous system (CNS) involvement – MPS I, MPS II and MPS VI. In light of these advances the focus here is the complex molecular genetics and biochemistry of the MPS and the option to prenatally detect them all. Further, with the introduction of effective treatments for the MPS, there is increasing urgency for the development of methods and disease biomarkers to enable early detection (newborn screening), prognostics and monitoring of treatment in asymptomatic MPS patients.

Characteristics of the mucopolysaccharidoses The biochemical and molecular genetic characteristics of the 11 known MPS are shown in Table 14.1. Theoretically, there are at least two other MPS that may result from a deficiency of glucuronate-2-sulfatase or glucosamine-3-sulfatase and

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Table 14.1 The biochemical and molecular genetic characteristics of the mucopolysaccharidoses Type (syndrome)

MPS I (Hurler, Scheie)

OMIM

607014

Enzyme activity

Chromosomal

(Abbreviation)

location

α-L-iduronidase (IDUA)

4p16.3

Substrates

DS, HS

607015 607016 MPS II (Hunter)

309900

iduronat-2–sulfatase (IDS)

Xq28

DS, HS

MPS IIIA (Sanfilippo)

252900

N-sulfoglucosamine

17q25.3

HS

MPS IIIB (Sanfilippo)

252920

α-N-acetylglucosaminidase

17q21

HS

8p11.1

HS

12q14

HS

16q24.3

KS, CS

sulfohydrolase (SGSH) (NAGLU) MPS IIIC (Sanfilippo)

252930

acetyl CoA: α-glucosamine N-acetyl transferase (HGSNAT)

MPS IIID (Sanfilippo)

252940

glucosamine-6-sulfatase (GNS)

MPS IVA (Morquio)

253000

galactosamine-6-sulfatase (GAL6S)

MPS IVB (Morquio)

253010

β-D-galactosidase (GLB1)

3p21.33

KS, GM1

253200

galactosamine-4-

15q12

DS, CS

MPS V (vacant)a MPS VI (Maroteaux-Lamy)

sulfatase(GAL4S) MPS VII (Sly)

253220

β-D-glucuronidase (GUSB)

7q21.11

DS, HS, CS

610492

hyaluronidase (HYAL1)

3p21.3

HA

MPS VIII (vacant)b MPS IX (Natowicz disease)

CS, chondroitin sulfate; DS, dermatan sulfate; GM1, GM1-gangliosides; HA, hyaluronan; HS, heparan sulfate; KS, keratan sulfate. a

Previously Scheie syndrome, later shown to be an allelic variation of Hurler.

b

Initially a KS-specific glucosamine-6-sulfatase deficiency – not confirmed.

may lead to lysosomal storage of GAGs (possibly HS/DS or HS) and the development of clinical symptoms and signs.3,4 The clinical history of the MPS has been reviewed by McKusick and others5 and updated.6 Each of the MPS is characterized by progressive multisystem disease that may involve the skeleton, joints and most somatic tissues – particularly heart, lung and CNS. There is a broad spectrum of disease phenotypes and variation in the rate of clinical progression, which range from early onset and rapid progression to later onset or slower progression or attenuated forms. Earlyonset MPS usually leads to early death, whereas patients affected by later onset forms may have a relatively normal lifespan, albeit with many clinically serious problems. Thus MPS patients are

often classified within a disease spectrum from “severe rapidly progressing” to “attenuated slowly progressing” disorders. This disease spectrum is best illustrated by the MPS I and II patient groups. Severe or rapidly progressing MPS I or II individuals have progressive CNS and skeletal disease, considerably shortened lifespan and additional somatic features, whereas individuals with the slowly progressing or attenuating form of the disease have no significant clinical CNS pathology, a relatively normal lifespan but significant disease burden that includes skeletal, joint and cardiopulmonary complications.5,6 MPS patients at the attenuated end of the clinical spectrum may not be clinically recognized and may go through life with substantial clinical problems without a MPS diag-

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nosis. Also, the attenuated phenotype is not necessarily less clinically severe than the “classic” severe phenotypes (e.g. MPS I, MPS II, MPS III) but may present with and display a different balance of clinical characteristics. Examples of unusual clinical presentations for attenuated MPS IIIA7,8 and MPS VI9 have highlighted the need to remain aware of possible new adult presentations of these disorders. Population screening programs for the MPS are likely to identify their true incidence, with screening for MPS types I, II, IIIA, IIIB, IVA, VI and VII possible with established technology10 (and unpublished).

Disease heterogeneity of the mucopolysaccharidoses Disease heterogeneity is attributable to allele differences, the relative level of effective residual enzyme activity, and yet to be defined contributory influences from the patient’s genome that, for example, contribute to the efficiency of GAG synthesis or lysosome biogenesis. For most of the MPS the difference between rapidly and slowly progressing phenotypes may only represent a few percent of normal enzyme activity. For instance, the difference in residual α-L-iduronidase (IDUA) activity between the rapidly progressing form of MPS I (Hurler) and the slower progressing form (Scheie) (see Table 14.1) ranges from “none detected” to 1 percent of normal IDUA activity toward natural substrates derived from HS or DS.11–13 Further, the IDUA genotype of some MPS I patients contains multiple base changes that may influence the specific activity of each individual’s IDUA activity.13 Similar, very low differences in enzyme activity have been reported for patients with the rapidly advancing forms of MPS II, MPS IIIA and MPS VI.14–17 More recently, electrospray ionization-tandem mass spectrometry has been used to measure the relative amount of primary storage of DS and HS oligosaccharide exoenzyme substrates in cultured fibroblasts and urine in MPS I patients and their relationship to the rate of clinical onset and the presence or absence of CNS pathology.18 Characterization and measurement of the amount of these GAG fragments may contribute methods to enable the prediction of clinical progression in the MPS.

Pathogenesis and pathophysiology of the mucopolysaccharidoses Processes and relationships between the nature of the primary storage products in the lysosome and the clinical outcome are unknown. However, analysis of lysosomal disease pathogenesis provides a unique window through which to observe the importance of the greater lysosomal system for normal cell health.19 Activation of inflammatory responses has been implicated in the cascade of events leading to the onset of CNS clinical pathology in MPS I and MPS IIIB20; inflammatory events are also likely to contribute to the pathologic cascade in other MPS. Patients accumulate compounds which are normally degraded in the lysosome. In many diseases this accumulation affects various organs, leading to severe clinical symptoms and premature death. Identification of the mechanism by which stored compounds affect cellular function is the basis for understanding the pathophysiology underlying lysosomal storage disorders. The fact that storage compounds interfere with various cellular processes such as receptor activation and membrane responses that may impair autophagy has only recently been appreciated. Many of these processes are associated with accumulation of storage material in nonlysosomal compartments that go on and affect the formation of autophagosomes.21 Autophagy has important housekeeping and quality control functions that contribute to health and longevity, and is reported to play a role in programmed cell death and neurodegeneration. Impaired autophagy is speculated to contribute to the pathogenesis of lysosomal storage disorders.21,22 In summary, as salvage and autophagic processes are normally controlled by endosomal/lysosomal systems, lysosomal storage may interfere with key regulatory mechanisms involved in these processes. A further contribution and concept to consider in the development of pathology in the MPS – and other lysosomal storage disorders – comes from an observation that material stored in the lysosome is unlikely to recycle to maintain homeostasis; energy is therefore likely to be diverted to synthesis at the expense of typical energy storage depots.23 Importantly, these and other studies suggest that lysosomal diseases should also be considered as states

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Genetic Disorders and the Fetus

of deficiency rather than simply overabundance or storage.

Synthesis, structure and function of glycosaminoglycans GAGs are complex molecules composed of characteristic repeating disaccharide units that may have a single chain containing from 10 to 150 disaccharide units. The HS-repeating disaccharide contains a D-glucuronic acid glycosidically linked β 1-4 to N-acetylglucosamine that, in turn, is glycosidically linked α 1-4 to the next glucuronic acid residue. Further complexity comes when “in-chain” glucuronic acid residues may be epimerized to α-Liduronic acid and then 2-O-sulfated; or “in-chain” N-acetylglucosamine residues may be de-Nacetylated and then N-sulfated, and O-sulfated at the 3 and/or 6 positions. Similarly, DS contains β 1-3 glucuronic acid and α 1-4 N-acetylgalactosamine residues, where “in-chain” glucuronic acid residues may be epimerized to iduronic acid and then 2-O-sulfated; or the C4 and/or C6 positions of “in-chain” N-acetylgalactosamine residues are O-sulfated. These modifications to both HS and DS occur within various segments/blocks of the GAG chain, creating considerable molecular sequence diversity that provides the basis of the broad structure/function diversity shown by HS and DS. Except for HA, all reducing end GAG chains are covalently linked through a specific linkage region structure to serine or asparagine residues on specific protein cores to form a number of different proteoglycans that influence different physiologic processes through affecting the cell and organ distribution of these GAG chains. Depending on the type of proteoglycan, there may be from one to more than 30 GAG chains linked to each protein core. The GAG components of proteoglycans are synthesized from nucleotide sugars and are assembled in the Golgi complex. HS proteoglycans such as the glypicans, perlecans and syndecans play important and varied roles in cell signaling and/or adhesion processes.24–26 The glypicans and other HS proteoglycans, such as agrin, which functions as a cell receptor,27 have particular roles in the CNS. It is therefore possible, in a process where HS turnover is disrupted, to speculate that these important functions may be disturbed and

thus contribute to the development of CNS clinical pathology that is characteristic of all MPS that store HS fragments. Further, the involvement of DS proteoglycans such as decorin28 in cartilage and skin function, and epiphycan29 in growth plate function, may render the disruption of their turnover a major contributor to the characteristic pathology seen in bone, joints and skin of MPS I, II, VI and VII patients who store large amounts of DS. There is speculation that the rate of clinical progression in all the MPS is directly related to the amount, type and location of GAG stored and present in urine. This hypothesis has been supported with extensive natural history studies of more than 100 MPS VI patients, where a relationship was established between the extent of MPS-uria, genotype and clinical phenotype.17,30 Obviously, however, there are further complexities to the process of defining the characteristics of a MPS clinical phenotype than simply evaluating the amount and type of GAG present.. For example, although MPS I and MPS II both store DS and HS fragments and have a similar clinical phenotype, they do differ in key aspects, notably the degree of corneal involvement, the age of clinical onset and the rate of development of CNS pathology. Each MPS is characterized by progressive multisystem disease with considerable clinical heterogeneity. The clinical heterogeneity is thought to relate to the degree of the metabolic block in GAG degradation which, in turn, is related to the underlying mutation at the respective locus. Currently, other than longitudinal clinical observation or the detection of a recurrent genetic mutation, there are no accurate methods to predict the clinical course for an individual patient, particularly when diagnosed early. In addition, there are no specific disease biomarkers that reflect the total body burden of disease. The lack of specific biomarkers has made monitoring the response to treatment and predicting disease course difficult in these disorders. The recent introduction of enzyme replacement therapy for MPS I, II and VI highlights the need for objective measures of disease burden and disease responsiveness to therapy.

Catabolism of proteoglycans Catabolism of GAG proteoglycans begins with proteolysis of the protein core and endohydrolysis of

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GAG polysaccharides to oligosaccharides, initially in endosomal compartments; this is followed by the sequential action of an array of exoenzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate in lysosomal compartments for reuse in biosynthetic pathways within the same cell. In MPS I, which is caused by a deficiency of the exohydrolase IDUA (see Table 14.1), many odd and even length oligosaccharide fragments of two different GAGs (DS and HS) have been shown to accumulate and appear in urine; these GAG fragments have been identified and characterized using electrospray ionization-tandem mass spectrometry.18,31 All the accumulating oligosaccharides have nonreducing terminal α-L-iduronate residues, which are susceptible to digestion with IDUA. The presence of odd and even oligosaccharides suggested the action of endo-β-glucuronidase and endo-N-acetylhexosaminidase activities toward both HS and DS.32 Cultured skin fibroblasts from MPS I patients accumulate the same DS- and HSderived oligosaccharides as identified in MPS I urine. A total of at least 15 different oligosaccharide species derived from HS and DS were measured in fibroblast extracts using mass spectrometry and shown to discriminate MPS I from controls; of these, two sulfated trisaccharides allowed the grouping of patients based on the presence/absence of clinical CNS disease. Moreover, the ratio of IDUA activity to these sulfated trisaccharides clearly discriminated MPS I patients with and without CNS pathology and suggested that this type of analysis may be used to predict disease severity in MPS I patients.18 The type and size of the GAG storage products in the MPS are determined by the particular enzyme deficiency. Current dye binding or electrophoresis methods for the detection and classification of various GAGs are generally considered complex, insensitive and inaccurate. The measurement of GAGs in urine should only be considered as a screening tool to indicate further diagnostic testing. Usually, enzyme activities (see Table 14.1) are determined to identify the specific enzyme deficiency based upon the results of a urinary GAG screen. A single enzyme deficiency should be used to confirm the MPS type, followed by mutation analysis to determine options for therapies and future pregnancies. Fuller et al.33 applied electro-

spray ionization-tandem mass spectrometry to identify GAG-derived sulfated oligosaccharide profiles in urine from a large group of MPS patients and unaffected controls. These profiles enabled the identification of all MPS patients and, with the exception of MPS IIIB and IIIC, their subtypes. Thus, the identification and measurement of GAGderived oligosaccharides in urine by mass spectrometry provide a sensitive and specific screen for the early identification of individuals with MPS. The resulting oligosaccharide profiles not only characterize subtype but also provide a diseasespecific fingerprint by which to biochemically monitor current and proposed therapies.33 Glycosphingolipids such as GM2- and GM3gangliosides have been found to accumulate alongside GAGs in MPS I, II, IIIA, IIIB, IIID, VI and VII. These gangliosides appear early in the disease process and are considered causative factors in the clinical development of CNS pathology. Importantly, gangliosides are not primary substrates for the enzymes deficient in the MPS, suggesting that their accumulation in each MPS results from secondary unknown processes initiated from the storage of the primary GAG substrates. The type and size of GAG stored in lysosomes are determined by the particular enzyme deficiency. Elevation of GAG is subsequently observed in tissue, circulation and urine. Electrospray ionization-tandem mass spectrometric measurement of simple fragments, such as sulfated N-acetylhexosamines and disaccharides, have been shown to be elevated in some MPS34: most MPS had urinary increases in di- and monosulfated N-acetylhexosamines and monosulfated N-acetylhexosamineuronic acid.33 Analysis of plasma and dried blood spots on filter paper collected from MPS patients has also shown elevations in total monosulfated Nacetylhexosamines but less than that seen in urine. Urine samples from MPS IVA and MPS VI patients who had received bone marrow transplant showed post-transplant decreases in these sulfated monosaccharide biomarkers. This decrease correlated with clinical improvement. Therefore, these metabolic markers have potential application in diagnosis, phenotype prediction and monitoring of current and future therapies, particularly for MPS IIID, IVA and VI.

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Biomarkers for monitoring pathogenesis and treatment in the mucopolysaccharidoses As we enter an era of advancing treatment opportunities for the MPS, there are unprecedented demands being placed on clinicians for early diagnosis and prediction of clinical outcomes, particularly on the question of whether the patient will develop CNS pathology. Biochemical monitoring of any therapeutic avenue will also be necessary and will be needed to assist with the prediction of clinical phenotype, particularly as more asymptomatic patients are identified via newborn screening or improved clinical detection by more informed medical geneticists. As discussed above, a number of biochemical parameters (sulfated oligosaccharides, residual enzyme activity, gangliosides) assist the discrimination of patients of different genotype/phenotype. Measurement of heparin co-factor II–thrombin complex in serum is a reliable biomarker for the MPS35: in untreated patients, serum levels range from 3- to 112-fold above unaffected control values. In a series of patients with varying severity of MPS I, the serum complex concentration was reflective of disease severity. In addition, serum heparin co-factor II–thrombin levels appear to respond to various treatment regimens. Serum heparin co-factor II–thrombin complex may therefore provide an important assessment and monitoring tool for patients with MPS.35

Biochemical genetics of the mucopolysaccharidoses MPS I α-L-Iduronidase (IDUA; EC 3.2.1.76), originally defined as the “Hurler corrective factor”,36 is the enzyme deficiency that results in MPS I.37 IDUA hydrolyzes the nonreducing terminal α-L-iduronic acid residues of DS and HS.6 The IDUA cDNA coding sequence, the gene and its chromosomal location at 4p16.3 were determined and characterized by Scott et al.38–40 The gene has been shown to span approximately 19 kb and contain 14 exons40; the first two exons are separated by an intron of 566 bp, a large intron of approximately 13 kb follows, and the last 12 exons are clustered within 4.5 kb.

IDUA activity is readily measured using a fluorogenic 4-methylumbelliferyl α-L-iduronide substrate that is both sensitive and specific.41 Care with the preparation of this substrate is required to recognize the occasional presence of trace contaminating amounts of the β-glucuronide fluorogenic equivalent, particularly if the analytical intent is to measure residual IDUA activity and relate this to clinical phenotype. Recognition of the importance of the aglycone secondary structure and the synthesis of radiolabelled substrates that represent natural substrate structures enabled discrimination of residual activities present in fibroblasts from patients with the slowly progressing form of MPS I (Scheie) and the rapidly progressing form (Hurler).12,41 More than 100 disease-producing mutations have been reported in the IDUA gene. Most are missense, with premature stop, insertions and deletions reported.42–44 Two common premature stop mutations (p.Q70X and p.W402X) have been identified as common in European populations, with p.Q70X representing approximately 60 percent of alleles in Scandinavian and some eastern Russian populations45 and p.W402X representing similar frequencies in northern European populations – UK and Germany. Homozygous p.W402X and p.Q70X have been identified with the expression of a rapidly progressing MPS I phenotype. A wide variation in clinical severity is apparent between the rapidly progressing form of MPS I and the slower progressing form, despite deficiencies in IDUA activity of less than a few percent of unaffected. As a result of IDUA deficiency, HS and DS accumulate in MPS I patients and are elevated in urine.

Prenatal diagnosis of MPS I Diagnosis using chorionic villus (CV) or amniotic fluid (AF) cells is routine in many centers, which also provide analysis of IDUA activity or selected mutation analysis. MPS II Wilson et al.46 isolated and sequenced a 2.3 kb cDNA clone coding for the entire sequence of human iduronate-2-sulphatase (IDS; EC 3.1.6.13), the enzyme deficient in sulfate.37 A strong sequence homology was found with other human sulfatase

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genes.47 The IDS cDNA detected structural alterations or gross deletions of the IDS gene in many of the clinically severe MPS II syndrome patients studied. The IDS gene contains nine exons and spans approximately 24 kb.48,49 The IDS gene is located on Xq28, distal to the fragile X site, and also shown to span the X chromosome breakpoint in a female MPS II patient with an X;autosome translocation.50,51 The GAGs HS and DS accumulate in MPS II as a consequence of IDS deficiency and are elevated in urine. IDS activity is conveniently measured with a fluorigenic 4-methylumbelliferyl iduronate2-sulfate substrate52; alternatively, IDS is also measured with a radiolabelled disaccharide substrate derived from heparin.53 An IDS pseudogene located 90 kb telomeric from the IDS gene was probably a result of an inversion of the intervening IDS sequence to disrupt intron 7 of the IDS gene. This pseudogene was found in a minority of patients with MPS II and was shown to contain sequence related to exons 2 and 3 as well as introns 2, 3 and 7 of the IDS gene. Nucleotide sequencing found that the inversion was caused by recombination between homologous sequences present in the IDS gene and the IDS pseudogene locus.54–56 Birot et al.57 described an MPS II patient in whom an exchange between the IDS gene and pseudogene through interchromosomal recombination had apparently caused internal deletion of exons 4, 5, 6 and 7. In the rearranged gene, the junction intron contained the pseudogene intron 3- and intron 7-related sequences. Some very large deletions of the IDS locus may extend to adjoining genes, resulting in a contiguous gene syndrome that displays a rapidly progressing MPS II phenotype with early onset of seizures.58 Although extremely rare, MPS II has been diagnosed in females.59,60 Missense, with premature stop, small and large insertions and deletions have been reported in the IDS gene to confirm the extreme heterogeneity of IDS gene alterations.61,62 Families in which the occurrence of MPS II was sporadic revealed mosaicism in the mothers of a small number of patients and a high frequency of de novo mutations occurring preferentially during male meiosis.62 The diagnosis of an MPS II female based on deficiency of IDS enzyme activity

should be checked for other sulfatase deficiencies to eliminate multiple sulfatase deficiency as an explanation.47

Prenatal diagnosis of MPS II This is best achieved by the assay of IDS activity or identification of an MPS II disease mutation in uncultured or cultured CV or AF cells. Assays using uncultured cells and CV cells allow for a rapid and reliable result. Measurement of IDS activity in AF supernatant is less reliable for the diagnosis of MPS II. Determination of fetal sex is helpful to exclude a female fetus with low IDS activity from being considered as an affected male fetus. Again, it is important to determine the contribution of maternal contamination to the IDS activity or mutation result. MPS IIIA The gene encoding N-sulfoglucosamine sulfohydrolase (SGSH), the enzyme deficient in MPS IIIA, is located on chromosome 17q25.3, spans 11 kb and includes eight exons.63,64 More than 70 diseasecausing mutations have been reported.65–67 Most are missense, with premature stop, insertions and deletions also reported. Founder effects for some common mutations have been proposed, with p. R245H most common in Dutch and German populations,68 p.R74C in Polish,69 p.S66W in Sardinian68,66 and 1091delC in Spanish populations.70 SGSH activity has been routinely measured with radiolabelled tetrasaccharides and fluorogenic substrates.71,72 Clinically, patients have progressive neurodegeneration, behavioral problems, mild skeletal changes, and shortened lifespan. SGSH deficiency results in the accumulation of HS, which is elevated in urine. A sensitive and specific immunoquantification SGSH protein assay in conjunction with measurement of SGSH activity in cultured skin fibroblasts – toward a natural tetrasaccharide substrate – was used to predict clinical severity in 35 MPS IIIA patients.16 It was further proposed that enzyme replacement to achieve a correction of approximately 10 percent of normal SGSH activity is required to avoid the onset of an MPS IIIA phenotype.16 Meyer et al.73 reported the natural history of a cohort of 54 MPS IIIA patients, based on a

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Genetic Disorders and the Fetus

detailed questionnaire and a four-point scoring system, and SGSH mutation analysis. They reported MPS IIIA patients with a broad clinical spectrum and identified patients with slowly progressing disease that appeared to be associated with the presence of p.S298P on one allele.

Prenatal diagnosis of MPS IIIA Diagnosis using CV or AF cells is routine in many centers where SGSH activity or selected mutations are directly measured.72 MPS IIIB Mucopolysaccharidosis IIIB is characterized by a deficiency of α-N-acetylglucosaminidase activity (NAGLU) that leads to the accumulation of HS.74 MPS IIIB patients are phenotypically similar to MPS IIIA patients, with progressive neurodegeneration, behavioral problems, mild skeletal changes and shortened lifespan; MPS IIIB patients also have HSuria. Clinical severity ranges from rapid to relatively slow progression. The NAGLU gene is located on chromosome 17q21, spans 8.5 kb and has six exons.75,76 NAGLU activity has been routinely measured using radiolabelled HS oligosaccharides and fluorogenic 4-methylumbelliferyl-2-acetamido-2deoxy-α-D-glucopyranoside substrates.77–79 Out of a total of 86 mutations in the NAGLU gene of MPS-IIIB patients, 58 missense/nonsense mutations, 27 insertions/deletions and one splice site mutation have been identified. Most of these are associated with severe clinical phenotypes. Many of the missense, nonsense and insertion/ deletion mutations have been expressed in mammalian cell lines to permit the characterization of their effects on NAGLU activity and intracellular processing and trafficking. The majority of the reported MPS IIIB mutations are unique, making mutation screening of the general population difficult.65,80 MPS IIIC Acetyl CoA:α-glucosamine N-acetyl transferase (HGSNAT) is a new structural class of protein involved in the transport of activated acetyl residues from cytoplasmic acetyl-CoA across the cell membrane to N-acetylate the nonreducing end glucosamine on HS in the lysosome.81 This is the

only bond-making activity known to function in the lysosome. A deficiency of HGSNAT activity leads to the accumulation of HS, HS-uria and a phenotype similar to MPS IIIA, MPS IIIB and MPS IIID. The HGSNAT gene is 62.4 kb in length, contains 18 exons and is located on 8p11.1.82–84 Diagnostic enzymology using radiolabelled glucosamine85 or fluorogenic 4-methylumbelliferyl β-D-glucosaminide86 substrates has been established. Four nonsense mutations, three frame-shift mutations due to deletions or duplication, six splice-site mutations, and 14 missense mutations have been identified among 30 probands with MPS IIIC.84 Fedele et al.87 reported splice-site mutations and frame-shift deletions resulting in premature stop codons, a nonsense mutation, and two missense mutations in a cohort of Italian MPS IIIC patients, whereas Ruijter et al.88 identified 14 different mutations in a cohort of 29 MPS IIIA Dutch patients that included splice-site mutations, a frame-shift mutation, nonsense mutations and missense mutations. Two mutations, p.R344C and p.S518F, were frequent among probands of Dutch origin, representing 22.0 percent and 29.3 percent of the mutant alleles, respectively. These MPS IIIC patients had a milder clinical course than previously reported MPS IIIC patients. More than 30 mutations have been identified. Specific assay of the transferase activity or the use of mutations identified in the proband will assist prenatal diagnosis of MPS IIIC. MPS IIID Kresse et al.89 identified a patient with a deficiency of glucosamine-6-sulfatase (GNS) and HS-uria. The GNS gene is located at chromosome 12q1490 and the cDNA has been isolated.91 The gene has 14 exons and spans approximately 43 kb; mutations have been identified.92,93 Diagnostic enzymology has been established using radiolabelled oligosaccharide sulfate substrates94,95 or a fluorogenic 4-methylumbelliferyl-α-N-acetylglucosamine-6sulfate substrate.96 Fibroblasts and leukocytes from three MPS IIID patients showed Stop at codon 1282

Severe

G551D

1.93

Gly>Asp at codon 551

Severe

621+1G>T

1.3

Splice mutation

Severe

N1303K

1.27

Asn>Lys at codon 1303

Severe

R553X

1.21

Arg>Stop at codon 553

Severe

∆I507

0.90

Deletion of Ile at codon 506 or 507

Severe

3120+1 G>A

0.86

Splice mutation

Severe

3849+10kbC>T

0.85

Aberrant splicing

Mild

R117H

0.54

Arg>His at codon 117

Mild

1717-1G>A

0.44

Splice mutation

Severe

2789+5G>A

0.38

Splice mutation

Mild

R334W

0.37

Arg>Trp at codon 334

Mild

R347P

0.36

Arg>Pro at codon 347

Variable

711+1G>T

0.35

Splice mutation

Severe

R560T

0.30

Arg>Thr at codon 560; splice mutation?

Severe

R1162X

0.30

Arg>Stop at codon 1162

Severe

3659delC

0.28

Frame-shift

Severe

A455E

0.26

Ala>Glu at codon 455

Mild

G85E

0.26

Gly>Glu at codon 85

Variable

2184delA

0.15

Frame-shift

Severe

1898+1G>A

0.13

Splice mutation

Severe

Reflex tests: 1. I506V, I507V, F508C when ∆F508 is detected. These variants may result in a false-positive homozygous ∆F508 status. 2. 5T/7T/9T when R117H is detected. 5T influences the severity of the R117H mutation.

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Genetic Disorders and the Fetus

R117H MSD 1

R347P R334W

MSD 2

G85E

NH2

A455E ∆I507 ∆F508 G542X

G551D R553X R560T

R1162X 3659delC NBD1

NBD2

R DOMAIN

COOH

2184delA

W1282X N1303K

Figure 17.1 Approximate position of ACMG-recommended panel of mutations occurring within the CFTR domain. NBD, nucleotide-binding domain; MSD, membrane-spanning domain.

The nonsense mutation W1282X is located in NBD2. Mutations within the first membranespanning domain are thought to reduce the ability of the CFTR to transport chloride anions. Mutations in this domain include R117H, R334W, R347P, and G85E. Mutations within the second transmembrane domain are hypothesized to affect chloride channel activity more mildly. Mutations in the regulatory domain are less common.53 To further understand how mutations affect CFTR function, mutations were divided into general classes according to the functional properties of the protein product with respect to chloride channel function in epithelial cells.52,54–57 Class I mutations result in defective protein synthesis due to premature termination or stop signals (e.g. the nonsense mutation G542X and the splice-junction mutation 1717–1G>A). Class II mutations (e.g. ∆F508) result in a protein that is not processed properly after translation, resulting in its inability to reach the cell surface and subsequent degradation. An example of a class III mutation is the missense mutation G551D. This type of mutation affects the ability of CFTR phosphorylation or ATP binding. Class IV mutations (e.g. R117H) produce

proteins that are oriented correctly in the apical membrane and bind and hydrolyze ATP but do not transport chloride normally. Class V mutations produce decreased amounts of normally functioning CFTR due to aberrant splicing at alternative sites (3849+10 kb C>T). This classification system for CF mutations may assist in improving therapeutic strategies for patients so that it may be possible to design a personalized treatment strategy based on the genotype of each CF patient.57 Ethnic variation Not only does the incidence of CF vary markedly among different populations, but the frequency of specific mutations also differs among ethnic groups. On average, ∆F508 occurs on 68 percent of all CF chromosomes worldwide14; however, the frequency of ∆F508 ranges from 20 percent in the Ashkenazi Jewish population in Israel58 and Northern Africa14 to approximately 100 percent in the Faroe Islands of Denmark.59 The worldwide frequency of most non-∆F508 mutations is low, although several occur in relatively high frequencies in specific ethnic groups. For example, between 48 and 60 percent of CF chromosomes in the

C H APTER 17

Ashkenazi Jewish population carry the nonsense mutation W1282X,58,60 whereas the worldwide frequency is 2 percent. In the US Ashkenazi Jewish population, screening for five mutations (∆F508, G542X, W1282X, N1303K, and 3849+10 kb C>T) will detect 92 percent of CF alleles.7,51 Screening for the American College of Medical Genetics (ACMG) core panel of 23 mutations will detect 94 percent of mutations among the Ashkenazi.7,51 In addition, the mutation, D1152H was reported to be present in high frequency among all Jewish ethnic groups, including the Ashkenazi,61 accounting for 12% of the mutations identified in an Ashkenazi Jewish carrier screening program.62 This mutation is associated with a broad phenotype ranging from CBAVD63 to pancreatic sufficient CF.64 Additional studies in CF patients are needed to determine the clinical significance of this mutation. The mutation detection rate for the 23-mutation core panel in various races/ethnic groups is shown in Table 17.1. Among non-Hispanic US Caucasians, ∆F508 accounts for 72% of the CF mutations. Mutations with a frequency >1% among Caucasian CF patients include G542X, W1282X, G551D, 621+1 G>T and N1303K. The ACMG 23-mutation panel permits the detection of greater than 88 percent of CF mutations in non-Hispanic Caucasians.51 A comprehensive analysis of CF mutations in African Americans showed that ∆F508 accounted for 48 percent of them.65 The second most common mutation among African Americans was 3120+1 G>A, which occurred with a frequency of 12 percent. The mutation detection rate among African Americans using the current ACOG/ ACMG panel is 64 percent. Other common mutations (not included in the current screening panel) identifed in this group include 2307insA, A559T and D1152H66 and G622D.67 Individuals of Mexican, Puerto Rican, Cuban, South or Central American or other Spanish culture (regardless of race) are considered Hispanic. The mutation detection rate using the ACMG 23-mutation panel among Hispanic Caucasian individuals is 72 percent.51 ∆F508 accounts for slightly greater than half of the mutations in this group. Other mutations with a >1% frequency among Hispanic American CF patients include G542X, R553X, R334W, 3849+10 kb C>T and N1303K. Other common mutations among His-

Prenatal Diagnosis of Cystic Fibrosis

583

panic Americans (not included in the common screening panel) include 3876delA, W1089X, R1066C, L206W and D1152H, the latter two being associated with a variable phenotype.66 The birth prevalence for CF among Asian Americans is very low, so there is limited information regarding the CF mutations common in this group. Among Asian Americans, the ACMG 23-mutation panel has a 49% detection rate, which is based on the analysis of only 86 CF chromosomes.6,51 Just five of the ACMG recommended 23 mutations have been detected in this group: ∆F508, G551D, N1303K, ∆I507, and 3849+10 kb C>T.6 Unfortunately, reports are limited regarding the population-specific mutation frequencies among individuals of other ethnicities. For example, based on several small studies, the ACMG core panel will detect less than 40 percent of Arab or Arab American CF mutations.11–13,68 The mutations 1548delG, I1234V, ∆F508, 3120+1 G>A, W1282X, N1303K, H139L, 4010del4, G115X, 711+1 G>A and S549R account for approximately 70% of the mutations in this population. Correlation between genotype and phenotype Although CF is a single gene disorder, genotype– phenotype comparisons in this condition are complicated and cannot be used to predict the clinical course of the disease. Analysis of relationships between genotype and phenotype shows a strong correlation between particular mutations and pancreatic function. Between 10 and 15 percent of patients with CF have pancreatic sufficiency (PS), low to normal sweat chloride levels, later age at diagnosis, better nutritional status, and slower decline in pulmonary function.69,70 Pancreatitis occurs in about 10% of patients with PS CF and 0.5% of patients with pancreatic insufficient (PI) CF.71 Pancreatitis may precede the diagnosis of CF, with an average age of onset of 20–22 years.70,71 No specific mutation is associated with this phenotype.70,71 In general, patients with PS have one or two mild mutations, whereas those with two severe mutations have PI and more severe clinical manifestations. Ten percent of CF mutations are mild alleles.72 The mild allele is presumed to confer a dominant phenotype over the severe allele,69 although the presence of one mild allele does not

584

Genetic Disorders and the Fetus

ensure pancreatic sufficiency.73 The status of each of the 23 ACOG/ACMG core panel mutations with respect to pancreatic function is included in Table 17.2 and is described as mild (PS) or severe (PI).

Table 17.3 R117H/polyT genotype-phenotype

R117H and the variable intron 8 polyT locus The CFTR intron 8/exon 9 acceptor-splice site contains a variable number of thymidines known as the polyT allele, with five, seven or nine thymidines present at the end of intron 8 (known as 5T, 7T and 9T).74 The presence of the 5T allele negatively affects RNA splicing, resulting in a CFTR transcript lacking exon 9. The 7T allele is associated with normal splicing and therefore normal levels of functional protein.75 The 5T allele is present in 5 percent of Caucasians and is associated with a wide range of clinical manifestations, including congenital bilateral absence of the vas deferens, mild CF, atypical CF symptoms, as well as in healthy, fertile men.76–78 The variant phenotype associated with the 5T allele correlates with the length of a polymorphic thymidine-guanine (TG) repetitive sequence adjacent to the polyT region. Longer TG tracts are associated with less efficient splicing of CFTR exon 9. The presence of 11 TG repeats is associated with a normal phenotype while the presence of 12 or 13 TG repeats is associated with an abnormal phenotype.79 The 5T allele can exist on the same chromosome as the R117H CF mutation, but has not been reported in cis with any other CF mutation.77 R117H has also been reported on a 7T and, rarely, a 9T background.77 A great deal of information regarding genotype–phenotype correlation between the R117H/poly T locus is known (Table 17.3). Homozygosity for R117H and the 7T allele is associated with CBAVD or a normal phenotype.81,84 It appears that although these individuals may have elevated or borderline sweat chloride levels, clinical CF is rare, but close follow-up should be considered.84 Homozygosity for R117H in cis with the 5T allele or R117H/5T in combination with another CF mutation on the opposite chromosome is associated with nonclassic CF or PS CF.20,75,84 R117H/7T in combination with another CF mutation on the opposite chromosome (such as ∆F508) is associated with a spectrum of phenotypes from normal to CBAVD to PS CF.80

R117H-5T/CF mutation

Nonclassic CF, PS CF

R117H-7T/CF mutation

Asymptomatic female, CBAVD,

5T/CF mutation

Asymptomatic, nonclassic CF,

7T or 9T/CF mutation

Asymptomatic

correlations20,75,77,80–83 Genotype

Phenotype includes (allele 1/ allele 2)

nonclassic CF, PS CF CBAVD

For individuals undergoing routine CF carrier screening, reflex testing to examine the polyT status is recommended when R117H is detected. This will determine which individuals are truly at risk for having CF or CBAVD in combination with a second disease-causing mutation on the opposite chromosome. PolyT testing in the absence of the R117H mutation is not recommended as part of routine screening for CF because this may create confusion in interpreting DNA testing results, especially as it relates to prenatal diagnosis.51 However, polyT testing is appropriate for males with CBAVD and patients with other nonclassic CF symptoms, such as pancreatitis. Congenital bilateral absence of the vas deferens Most males with CF are infertile, because of CBAVD, which is also a cause of infertility in otherwise healthy men. An association between male fertility and the presence of the 3849+10 kb C>T mutation has been reported, suggesting that this particular mutation may result in levels of normal CFTR expression necessary to avoid infertility.85 Initial studies reported that of men with CBAVD, approximately one-fourth were compound heterozygotes for two mutant alleles (at least one being a mild mutation), almost half of the men had one identifiable CF mutation, and the remainder had no detectable CF mutation.86 CFTR mutations are rarely detected in males with CBAVD and renal anomalies87; however, men with CBAVD, CFTR mutations, and renal agenesis have been reported.88 Although some cases of CBAVD may actually represent extremely mild forms of CF, it is recom-

C H APTER 17

Prenatal Diagnosis of Cystic Fibrosis

585

mended that men with CFTR mutations and CBAVD as the only clinical manifestation (even in the presence of positive sweat chloride tests) not be classified with CF.89,90 However, because the possibility of late-onset CF symptoms cannot be excluded, clinical follow-up of these men should be considered.90 Eighty-four percent of males with CBAVD who are heterozygous for a CF mutation and 25 percent with no detectable mutation carry the 5T allele.74 The 5T allele is present in men with CBAVD at a frequency four times higher than in the general population (20 versus 5 percent).76 Of males with CBAVD, 41–48 percent have either two CF mutations or one CF mutation and the 5T allele on the opposite chromosome. Twenty-five to 31 percent have either one mutation or the 5T allele, and approximately 25 percent have neither a CF mutation nor the 5T allele.81,82 The majority of males with CBAVD have a severe mutation on one chromosome and a mild/variable mutation on the other, with the most common genotype in CBAVD males being ∆F508/R117H (7T) followed by ∆F508/5T.63

lication studies have either not been done or produce conflicting results.94 Candidate modifiers of pulmonary function include glutathione-S-transferase (functions in the detoxification of aromatic compounds), tumor necrosis factor α (contributes to neutrophil-predominant inflammation), β2adrenergic receptor (influences airway reactivity), α1-antitrypsin (serine proteinase inhibitor), βdefensin (resistance of epithelial surfaces to microbial colonization), angiotensin-converting enzyme (proinflammatory molecule), α1-antichymotrypsin (inflammatory response), voltage-gated chloride channel 2 (epithelial ion transport), Fcγ receptor IIA (immune defense system), human leukocyte antigens (immune defense system), and nitric oxide synthase (host defense, inflammation and bronchomotor control).97,98,101–103 Candidate modifiers of liver disease include human leukocyte antigens (DQ6), angiotensin-converting enzyme, mannosebinding lectin, glutathione-S-transferase P1 and α1-antitrypsin.91,97 A modifier locus at 19q13 is associated with an increased risk of meconium ileus, though no candidate gene has yet been identified.104

Modifier genes Complications secondary to pulmonary dysfunction are the leading cause of death in patients with CF; however, the degree of respiratory complications in these individuals is highly variable.91 Twin studies92 and investigations involving patients with identical mutations do not demonstrate a strong correlation between pulmonary function and CFTR genotype.93 These results suggest that this aspect of CF is influenced by other factors.52,94 Secondhand smoke is adversely associated with lung disease in CF patients.95 Other environmental factors, including infection, nutrition, pollutants, age at diagnosis, and compliance and intensity of treatment are thought to influence lung function.96,97 Transforming growth factor β1(proinflammatory and anti-inflammatory properties) and mannose-binding lectin (component of the immune defense system) modify lung function in CF patients.94,98 Many other potential modifier genes have been studied for associations with pulmonary, liver and gastrointestinal complications.91,97,99,100 However, for many of the genes, rep-

Other conditions associated with CFTR mutations Although guidelines for making a clinical diagnosis of CF exist,26 the identification of other conditions associated with CFTR mutations, also referred to as nonclassic CF, further complicates our understanding of the CFTR genotype as it relates to clinical expression of disease.83 These conditions include sarcoidosis, asthma, disseminated bronchiectasis, chronic rhinosinusitis, pulmonary emphysema, and idiopathic pancreatitis and have been reported with a wide variety of CF mutations, some of which are unique to the specific conditions. Summary The genotype of the patient with CF is useful for predicting pancreatic function. The combination of many different CFTR mutations, absence of clinical CF in patients with two CFTR sequence changes originally thought to be disease-causing mutations, modifier genes, and the lack of a clear association between specific common mutations and symptoms and severity of disease complicates

586

Genetic Disorders and the Fetus

genotype–phenotype correlations. Prenatal counseling after the diagnosis of one or two mild mutations may include the likelihood of long-term pancreatic sufficiency, whereas two severe mutations would indicate early-onset PI. Currently, it is impossible to predict prenatally other manifestations of CF, especially the severity of pulmonary disease, based on genotype information.

Methods of detecting the mutation DNA can be extracted from leukocytes isolated from whole blood, dried blood or fetal cells (amniotic fluid, chorionic villus or fetal blood cells) but other specimens, such as buccal cells collected on cytology brushes, are also used.105 It is important to delineate the reason for molecular genetic testing for CF, since the methods may differ significantly. For example, in response to the recommended AMCG CF core mutation panel, commercial companies developed various mutation detection technologies (Table 17.4). These

include allele-specific oligonucleotides (ASOs), reverse dot blot hybridization, amplification refractory mutation system (ARMS), oligonucleotide ligation assay (OLA), liquid bead arrays, fluorescence resonance energy transfer (FRET) and microarray hybridization.7 For CF mutation analysis, laboratories currently choose between laboratory-developed methods or commercially available in vitro diagnostic (IVD) kits which are cleared and approved by the FDA. Laboratory-developed genetic tests for clinical use are regulated under the provisions of CLIA-88.7 Each method has its own inherent strengths and weaknesses, and an ordering physician should be aware of individual laboratory’s methods. Importantly, however, the ACMG core panel of mutations is recommended only for populationbased preconception and prenatal CF carrier screening.51 For diagnostic CF testing, a few laboratories offer larger mutation panels than the ACMG recommendations, but these extended panels may still fail to identify one or both of the CF mutations. Extensive sequencing of all exons, intron/exon

Table 17.4 Methods of mutation detection7,106 Method

Principle

Advantages

Disadvantages

Allele-specific

Individual wild-type or

Can be automated

Design and interpretation of

oligonucleotide

mutant probes hybridize to

hybridization (ASO)

target (patient) DNA

Multiplex possible

results can be complex Not commercially available

bound to membrane Capable of high throughput

Reverse dot blot

Probe pairs (wild type and

hybridization

mutant) are bound to

Multiplex possible

membrane and hybridized

Rapid and robust assay

Difficult to add new mutations

with target (patient) DNA Amplification

PCR primers designed to

refractory

amplify only a specific

mutation system

(typically mutant) sequence

Rapid and reliable

Absence of product implies negative result if there is no paired wild-type reaction Assays without paired

(ARMS), also called allele-specific

wild-type reactions cannot

amplification

differentiate between the heterozygous and homozygous mutant state

Oligonucleotide ligation assay

Allele-specific PCR followed by ligation with probes to

Capable of high throughput Software automatically

identify mutant and

analyzes data and creates

wild-type sequence

summary reports

Detection requires use of automated DNA sequencer

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Prenatal Diagnosis of Cystic Fibrosis

587

Table 17.4 Continued Method

Principle

Advantages

Disadvantages

Liquid bead arrays

Multiplex PCR followed by

Capable of high throughput

Detection requires the use of

hybridization to beads with

Software analyzes data

covalently bound universal

automatically and enables

tags or allele-specific

results for reflex

capture probes. A

polymorphisms to be

fluorochrome coupled to a

revealed only as

reporter molecule

appropriate or on demand

specialized equipment

quantifies hybridization Fluorescence

Hybridization of patient DNA

Capable of high throughput

resonance energy

to a normal or mutant

Rapid

transfer (FRET)

probe forms a structure

Software analyzes data

that is recognized and

automatically and enables

cleaved by a proprietary

results for reflex

enzyme. The released

polymorphisms to be

fragment hybridizes to a

revealed only as

cassette containing a

appropriate or on demand

Detection requires the use of specialized equipment

reporter and quencher molecule forming a second structure, which is enzymatically cleaved, generating fluorescence signal Microarray hybridization

Hybridization of patient DNA

Capable of high throughput

to probes on a microarray

Rapid

(or chip)

Relatively easy to add new

Detection requires the use of specialized equipment Expensive

mutations DNA sequencing

Sequence individual exons or complete CFTR gene

Can theoretically identify all mutations within the amplicons

Expensive Cannot identify large deletions Variants of uncertain clinical significance are difficult to interpret

Mutation scanning

Scanning to search for

Inexpensive

sequence alterations

Cannot identify specific mutations Must have alternative method to define specific variation identified Variants of uncertain clinical significance are difficult to interpret

Mass spectrometry time-of-flight

Primer extension to detect a specific mutation

Rapid

Cannot detect large deletions

High resolution

Only detects known mutations

Multiplex possible Robust Automated

588

Genetic Disorders and the Fetus

borders, promoter regions and specific intronic regions with a mutation detection rate as high as 98.7 percent has been described and is clinically available.107 Limitations of such technology include the identification of novel sequence changes in which the clinical significance is unknown. Once the mutations in a patient have been identified, specific mutation analysis for that family then becomes available. A reasonable approach for studying patients in whom CF is suspected is to first obtain mutation results for the ACMG core panel, because most patients with CF would be expected to have at least one of those mutations. If a mutation is not detected, the likelihood of CF is significantly reduced. However, if the diagnosis of CF remains strong, more extensive CF mutation studies could then be obtained. In selecting a reference laboratory for population-based preconception or CF carrier screening, it is essential to confirm that the laboratory is offering the current ACMG recommended mutation panel. Reference laboratories should participate in ACMG/College of American Pathologists or another comparable proficiency testing program and should be accredited through the CAP Molecular Pathology Accreditation process or another comparable agency. Laboratories should provide specific proficiency test results and CAP inspection information when requested. Although these processes do not guarantee accuracy, they will provide information that help determine a laboratory’s standards and commitment to quality.

Prenatal diagnosis of cystic fibrosis Assisted reproductive technology (ART) and preimplantation genetic diagnosis (PGD) All patients with CBAVD or atrophy of the vas deferens and their partners considering in vitro fertilization by sperm extraction procedure with intracytoplasmic sperm injection (ICSI)) should be offered genetic counseling to discuss testing for CF.108 ART can be used to achieve pregnancy in couples in whom the male is infertile as a result of CFTR gene mutations. Sperm can be aspirated by microscopic epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA)

or open testis biopsy followed by in vitro fertilization (IVF), usually by ICSI. The presence of CF mutations in the male with infertility does not negatively affect the rate of pregnancy or healthy livebirths compared with couples undergoing ART for other reasons.109,110 Because most men with CBAVD have at least one detectable CF mutation, screening for mutations, a detailed family and medical history, and genetic counseling should be offered to their partners when considering reproduction.108,111 Sequencing of the CFTR gene should be offered to men with CBAVD and only one recognized mutation if their partner has a known CF gene mutation. Couples at risk of having an affected child should then be offered PGD, CVS or amniocentesis.112 The first report of PGD for CF after IVF was in 1992.113 Three couples in which both parents carried the ∆F508 deletion were treated. One of the two women undergoing embryo transfer became pregnant and gave birth to a normal girl unaffected by CF and free of both parents’ ∆F508 alleles. The pregnancy rate for couples undergoing PGD is comparable to couples undergoing regular ICSI113,114 (see Chapter 29). During PGD, the correct genotype of the embryo is essential and misdiagnosis has been reported because of lack of amplification of a mutant allele (allele drop-out), resulting in a false-negative result. Initially, PGD for CF was performed only when both parents were ∆F508 carriers because the determination of compound heterozygous embryos is complicated and technically difficult. However, PGD is now available for mutations other than ∆F508. PGD has been improved with the use of whole-genome amplification prior to testing for the mutation of interest,115 biopsying two blastomeres when only one mutation is identified,116 analyzing informative, linked microsatellites instead of or in addition to the disease-causing mutations,117 and fluorescent-PCR technology (decreases the allele dropout rate and is more sensitive than conventional PCR). Prenatal diagnosis, through either CVS or amniocentesis, is still recommended to confirm the PGD results.118 Although PGD of CF has provided at-risk couples with an additional option, it currently is limited to couples with financial resources and selected centers. Alternative methods of concep-

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tion available to a known carrier couple to avoid the risk of CF in their offspring include artificial insemination with sperm from a screened male donor or with an ovum from a screened female donor, and adoption. Offering such alternatives may be an option for those who would have otherwise chosen to avoid the increased risk of having a child with CF. Significant advances in reproductive technologies in the past 20 years have greatly changed the nature of reproductive choices available to couples at risk of having a child with CF. For known carrier couples, prenatal diagnosis with the option of an elective pregnancy termination of an affected fetus remains the main option to avoid having a child with CF. For other couples in whom pregnancy termination is not an option, prenatal diagnosis is also utilized to prepare them for the birth of a child with CF. Genetic counseling for known CF carrier couples is essential. Various studies have explored the reproductive choices faced by at-risk couples. Parental attitudes towards prenatal diagnosis and pregnancy termination of an affected pregnancy have been studied.119–123 The results of some of those studies may be region specific.122 The availability of newborn screening for CF may also affect at-risk couples’ decisions regarding future prenatal diagnosis.123,124 There are few studies of reproductive decisions in screened populations, although one did demonstrate that 66% of Australian women who had a subsequent pregnancy used prenatal diagnosis, with 10/12 affected pregnancies resulting in pregnancy termination.123 Two studies have demonstrated a decreasing trend of the overall CF birth rate since the onset of genetic testing, demonstrating the effect that prenatal screening programs may have on the overall incidence of CF.124,125 The acceptance and uptake of populationbased carrier screening recommended by ACOG and ACMG will depend much on how it is presented to patients. Highly accurate prenatal diagnosis is available for families with a previously affected child with known mutations and for couples in which both members are known CF carriers. For couples in which both mutations are known, direct mutation analysis using fetal cells can be performed to determine the fetal genotype. The risk of maternal cell contamination of fetal DNA is a potential cause of

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erroneous test results, and laboratories should perform additional studies, such as analyzing polymorphic markers, to rule out maternal cell contamination.7 Because approximately 90 percent of non-Hispanic Caucasian CF mutations are routinely tested in most diagnostic laboratories, the necessity of linkage analysis for this disorder has been markedly reduced. Potential diagnostic pitfalls, such as nonpaternity and uniparental disomy, should always be investigated when unusual test results are reported.

Uniparental disomy Uniparental disomy (UPD), the inheritance of two homologous chromosomes from one parent in a diploid cell, arises from two abnormal cell divisions during meiosis and/or mitosis. Six cases of UPD causing CF (some with other associated disorders linked to chromosome 7) have been reported.126 UPD is estimated to account for only 1 in 10,000 CF cases127; thus, it is a rare cause of CF. A prenatal procedure generally is not recommended if only one partner is a carrier because the a priori risk of UPD is low.

Echogenic bowel and CF Fetal echogenic bowel is detected in 0.2–1.8 percent of all second-trimester pregnancies.128,129 In 25–35 percent of cases, this sign is associated with poor perinatal outcome, including chromosome abnormalities, congenital infections, intrauterine growth restriction, fetal death, intra-amniotic bleeds and CF, while in the other cases it is of no clinical significance.130,131 In a summary of previously reported studies, Carcopino et al. noted an incidence of CF of 2.4 percent in 1,682 fetuses with echogenic bowel, with the risk of CF ranging from 0 percent to 14 percent.132 DNA testing involved screening for various mutation panels, ranging from ∆F508 only to more extensive panels. That study utilized MRI to further evaluate fetuses with echogenic bowel, with improved resolution of dilated bowel. Using a Bayesian calculation, Hodge et al.133 and Bosco et al.134 determined the risk of CF in a fetus with echogenic bowel and one identifiable CF mutation as 1 percent. In a large series of patients, Strom et al. noted 100 cases referred for CF testing with an

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indication of echogenic bowel.135 They identified one fetus with two mutations and five additional fetuses with one mutation only. Monaghan and Feldman examined 159 cases of prenatally detected echogenic bowel in an ethnically and racially diverse population in the United States.136 The mutation detection rate in this study was 90 percent for Caucasians of European descent. Two affected fetuses were identified, and both had two severe mutations associated with PI CF. The overall frequency of CF associated with echogenic bowel in this diverse population was 1.3 percent. Using a Bayesian calculation, the remaining risk for CF in a Caucasian fetus with echogenic bowel decreased from 1.7 percent to 1 in 5,783 when no fetal mutation was identified and increased to 8 percent when one fetal mutation was identified. Ogino et al. provided Bayesian analysis for CF risks in a fetus with echogenic bowel.137 Various hypothetical scenarios were presented, allowing accurate genetic risk estimates for patients and their families. Based on these risks, parental or fetal CF mutation studies, along with a fetal karyotype and a search for a congenital infection, should be offered to all pregnant women with this fetal ultrasound finding. Direct fetal studies could be performed if a fetal sample is available; alternatively, both parents could be studied, although mistaken paternity could lead to a false-negative diagnosis if only the parents are studied. Fetal studies would be indicated if one or both of the parents is identified as a CF carrier. If two CF mutations are identified in a fetal sample, the diagnosis of CF is confirmed, whereas identifying only one CF mutation in the fetus creates a dilemma in which a definitive molecular diagnosis cannot be made. In this case DNA testing using an extended mutation panel or DNA sequencing may be useful and a Bayesian calculation can be made.

deferens is obtained, CF carrier testing should also be offered. Ideally, the affected individual is the person in whom testing should first be performed in order to know for certain whether a CF mutation is identifiable within that family. If a mutation is identified, a more accurate risk assessment can be calculated for other family members. Because population screening for CF is now recommended, some individuals may know about other CF carriers in their family even if there is no family history of CF. Individuals with a positive family history of CF and a negative mutation analysis can have a carrier risk calculated that takes into account available information regarding the known family history and the mutation information (see Table 17.1). For most cases in which one partner has a family history positive for CF, the family history in the other partner is usually negative. However, CF screening should still be offered to both individuals, so that a joint risk of having a child with CF can be calculated. For example, a Caucasian couple in which one partner is a known CF carrier and the other partner is not screened has a 1 in 100 chance of having an affected child ((1 × 1/2) (1/25 × 1/2)). If that partner is screened and found to be a carrier, their joint risk increases to 1 in 4 ((1 × 1/2) (1 × 1/2)). However, if that partner is screened and has a negative mutation analysis, using a Bayesian calculation and assuming an 88 percent CF detection rate, that couple’s risk of having an affected child is reduced to 1 in 804 ((1 × 1/2)(1/201 × 1/2)). Similar calculations with risk tables are available for a variety of races/ethnicities, CF carrier screening results and CF family histories.138 For most couples, this is reassuring information and prenatal diagnosis generally is not indicated under these circumstances. Although initially there was concern regarding pregnancy termination in situations in which a fetus had inherited one CF mutation, this has been shown to be exceedingly rare.139,140

Carrier detection

Family history of CF

Population screening

Cystic fibrosis carrier detection studies should be offered to relatives of patients with CF who are interested or who are considering a pregnancy. A family history obtained as part of routine primary or obstetric care should identify individuals with a positive family history of CF. Similarly, if a family history of unilateral or bilateral absence of the vas

The identification of the CF gene and its most common mutations introduced the possibility of widespread population screening for CF. Several reservations, however, were raised about the prospect of population-based carrier screening for CF. For some, acting to avoid the birth of children with CF seemed an inappropriate goal, in part because

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of the improved prognosis of individuals affected with CF and in part because of the belief that a cure for this disease would be developed in the near future.141 Other concerns were raised about the limitations of the test itself. Even when multiple mutations are screened for, the carrier detection rate in CF population screening remains, at best, 88–90 percent among Caucasians and is lower in other populations.14 Thus, some wondered whether this level of sensitivity was sufficient to warrant devoting the resources needed for populationbased screening.142 Others were concerned about the uncertainty that would be faced by couples when only one partner was shown to be a carrier.143 The projected demand and the perceived difficulties of informing people about CF carrier screening, particularly with regard to the possibility of false-negative results, led some to speculate that geneticists would be overwhelmed by the introduction of CF carrier screening.144 Additional concerns include the large size of the target population, the relatively high cost of carrier testing, and concerns that misunderstanding test results would lead to unwarranted prenatal procedures and pregnancy terminations. Statements were issued by American professional genetic groups advising a moratorium on widespread carrier testing until pilot studies were completed.143,145–147 The National Institutes of Health (NIH) and others then funded a series of pilot studies in the United States to determine how to offer voluntary CF carrier screening; other countries instituted similar pilot studies. The results of CF screening trials were reviewed in 1998 and again in 2002.148,149 An NIH Consensus Development Conference on Genetic Testing for Cystic Fibrosis in 1997 recommended that genetic screening for CF in the United States be offered to adults with a family history of CF, to partners of people with CF, to couples currently planning a pregnancy and to couples seeking prenatal care.150 Recognizing that laboratory testing for CF was not standardized and that educational materials to support such a massive undertaking were not readily available, an NIH-sponsored conference was held shortly thereafter to discuss the implementation of the NIH Consensus Conference recommendations.151 In the meantime, the ACMG and American College of Obstetrics and Gynecology (ACOG)

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issued statements that routine prenatal screening for CF was not standard practice and would not be until the above requirements were met.152,153 Eventually, the ACMG identified and recommended a core panel of mutations for CF population-based CF carrier screening.154 These recommendations were based on the prevalence of these mutations in more than 20,000 patients with classic CF; any mutation representing 0.1 percent or more of CFTR alleles in this pan-ethnic population was included. The original panel included 25 mutations and four variants, one of which was known to modify the expression of one of the mutations. The recommended CF mutation screening panel was expected to be modified as new information was learned regarding the phenotype associated with specific mutations and allele frequencies in various populations. Subsequently, in 2004 two mutations were removed from the panel: 1078delT, due to a lower than expected frequency, and I148T, following the discovery that this sequence change was not associated with a classic CF phenotype.51,155 The current ACMG recommended CF carrier screening mutation panel is shown in Table 17.2. Because the mutation panel is pan-ethnic, the residual remaining carrier risk for an individual is based on their race/ethnic background and prior carrier risk (see Table 17.1). Expanded CF mutation panels should not be offered routinely, even in couples where one member is identified as a CF carrier and the other tests negative for the standard CF mutation panel.7,156 In a collaborative effort, the ACOG, ACMG, and the National Human Genome Research Institute’s Ethical, Legal and Social Implications program developed the materials and standards needed to ensure the appropriate implementation of these guidelines, which were further refined by these organizations.157 The original report, issued in 2001, noted that the counseling, offer of screening and the couple’s decision regarding screening should be discussed and documented in the medical record and that written, informed consent should be obtained only after the woman and her partner have an opportunity to review the educational material and receive pretest counseling. Documentation of the patient’s decision to accept or decline screening should be incorporated into the medical record. Two different screening

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strategies were discussed as appropriate: concurrent screening (both partners are tested simultaneously) or sequential screening (one person is tested, then the partner is tested only if the first person is identified as a carrier). The guidelines also stated that patients should receive information that includes a brief description of the following considerations: • the purpose of screening • the voluntary nature of the testing • the range of symptoms and severity of CF, the treatment of the disease, and life expectancy • the genetics of CF and population estimates of carrier risk in their ethnic or racial group • the meaning of positive and negative test results • factors to consider in deciding to have or not have screening. In 2005, the ACOG Committee on Genetics issued an updated CF carrier screening policy to include the following recommendations.112 • It is reasonable to offer CF carrier screening to all couples, regardless of race or ethnicity, as an alternative to selective screening. Information about CF carrier screening should be made available to all couples. • CF screening should be offered to all couples where both partners are Caucasian, European or Ashkenazi Jewish. Screening should be performed before conception or early in pregnancy and can be done by either the sequential or concurrent model. • For individuals with a family history of CF, documentation of the mutation known to be present in the affected family member should be obtained when possible. Genetic counseling may be beneficial in this situation. • Complete analysis of the CFTR gene by DNA sequencing is not appropriate for routine carrier screening. This testing may be appropriate for patients with CF, a family history of CF, males with congenital bilateral absence of the vas deferens, or a positive newborn screening test when mutation testing using a panel of mutations has a negative result. • When both partners are CF carriers, genetic counseling is recommended and prenatal diagnosis by CVS or amniocentesis should be offered. • When one member of the couple is identified as a CF carrier and the other partner is unavailable for testing, genetic counseling may be helpful to

review the chance of having an affected child and prenatal testing options. • In the event that two mutations are identified during carrier screening in an individual who does not have a diagnosis of CF, referral to a CF clinic is recommended and genetic counseling may be beneficial. Since the implementation of CF carrier screening, some information has become available with regard to uptake of testing. According to a survey of ACOG members 2 years after implementation of CF carrier screening (prior to the most recent ACOG CF carrier screening policy update in 2005), the majority of obstetrician-gynecologists reported being familiar with the ACOG-ACMG guidelines.158 The majority were offering CF carrier screening to their pregnant patients, asking about their family history of CF and providing pregnant patients with information on CF and CF screening. Two-thirds of obstetricians reported offering CF carrier screening to all pregnant patients, as opposed to selecting patients at high risk based on ethnicity, race or family history. However, fewer obstetrican-gynecologists offered nonpregnant patients carrier screening, unless there was a family history of CF, the patient requested testing or the patient (or her partner) was a member of a higher-risk racial/ ethnic group.112,159 Of concern to the physicians surveyed were liability for not offering testing should the patient have a child with CF, confidence in their ability to interpret positive results, and level of familiarity with CF and genetics in general.159 From a laboratory standpoint, CFTR testing increased significantly following the publication of the guidelines in 2001. DNA-based testing for CF is now one of the most frequently ordered molecular genetic tests.160 In some laboratories, carrier screening accounts for as much as 95 percent of CF test indications.161 A predicted, although difficult, situation is the identification of asymptomatic individuals in whom two CF mutations are detected. Many laboratories and physicians have identified such individuals.135 Referral of the patient to a CF center is strongly recommended when that occurs. Interpreting, reporting and communication of test results are among the most challenging parts of screening. Accurate interpretation requires that ethnic information and family history be obtained and provided to the laboratory. Risk calculations

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for an individual with a negative test are not accurate unless that information is available. A limited survey regarding practices of US laboratories revealed that in one-fourth of cases, data needed to accurately interpret the CF test results (indication for testing, race/ethnicity and family history) were not provided to the laboratory by the ordering physician.161 It is of concern that some laboratories have chosen to include testing for the intron 8 polyT variant (5T/7T/9T) in their initial reporting process,135,162 even though the ACMG recommendations clearly indicated that testing for this variant should be performed only as a reflex test when the R117H mutation was identified.154 The reason that some laboratories have chosen this approach is unclear, although it may be related to the fact that some of the commercially available methods include this variant in an all-or-none single test. Although there are clear indications for studying the status of the polyT variant (see earlier discussion), reporting of the 5T variant in prenatal carrier screening settings has led to many difficult counseling dilemmas, as the goal of the prenatal screening program was to identify individuals at risk for classic CF, not CBAVD. Thus, it is apparent that many more individuals in the US are choosing to have CF carrier screening performed. The majority of clinicians are routinely offering this as part of their routine prenatal obstetric care. Because the ACOG has officially recommended offering CF carrier screening to all couples regardless of race or ethnicity either prior to pregnancy or early in pregnnacy, this has become the standard of care in the United States. Obstetric care providers who are not offering CF carrier screening may be at risk of litigation. Neonatal screening for CF Reliable screening of neonates for CF by determination of dried blood-spot immunoreactive trypsin (IRT) levels was first described in 1979.163 During the subsequent 30 years, there has been much debate regarding whether early diagnosis of CF by newborn screening is beneficial in reducing longterm morbidity and mortality. The underlying premise is that diagnosis of CF in early infancy will allow for aggressive nutritional supplementation and an improvement in long-term pulmonary

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function. Furthermore, because CF is often difficult to identify, delays mean that children are often not diagnosed until significant signs are already apparent. The identification of the CF gene made possible a different approach to neonatal screening, in which screeners used a combination of IRT determination and direct gene analysis with the same dried blood sample.164–167 In the US, newborn screening for CF has been implemented in most of the states following the Secretary’s Advisory Committee on Heritable Disorders and Genetics Diseases in Newborns and Children voted to recommend a uniform newborn screening panel, which included CF.168 By November 2008, 45 states had implemented CF screening: two others will be implementing testing in the near future.169 Newborn screening for CF is used widely, with implementation in Europe, United Kingdom, Australia and New Zealand.170 The list of benefits from newborn screening for CF continues to expand, and includes the opportunity to reduce complications, earlier diagnosis, timely genetic counseling, consideration of future prenatal options, improved growth, decreased risk of infection and a better quality of life.171–173 For the parents of the affected child, additional benefits of newborn screening include the identification of previously undiagnosed siblings with CF, genetic counseling concerning future reproduction and identification of CF carriers in the extended family.

Future directions The past two decades since the CF gene was identified have brought new methods of treatment, improved methods of population and newborn screening, increased life expectancy and the promise of additional treatment options. As individuals with CF live longer and reach reproductive age, many of them are now choosing to have children. Understanding of the basic defect and pathophysiology has progressed greatly. CF newborn screening has been mandated in 47/50 U.S. states and in many other countries. Population-based carrier screening is now deemed standard of care. Reproductive options for couples include population screening, prenatal diagnosis of at-risk pregnancies and initiation of prompt treatment for those infants born with CF. Assisted reproductive

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technologies allow males with CF to have children and women with CF, under close monitoring, can have healthy children at minimal risk to themselves. The ACMG CF core mutation panel has been revised and continues to be evaluated as technologies improve and additional information regarding specific CF mutations becomes available. Improved treatments have considerably increased the life expectancy of a child born today with CF. On the other hand, gene therapy trials have been relatively unsuccessful so far, though it is hoped that continued research will result in future successes. More than two dozen therapies at various stages of clinical trials are listed on the website of the CFF (www.cff.org), many directed at the basic defect while others are utilizing small molecules to increase residual CF activity or correct the resultant ion transport defects. It is clear that in the future, earlier, improved CF diagnosis, treatment and perhaps a cure will become real options for patients.

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Genetic Disorders and the Fetus mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease. Am J Hum Genet 1992;50:222. Schwartz M, Sørensen N, Brandt NJ. High incidence of cystic fibrosis on the Faroe Islands: a molecular and genealogical study. Hum Genet 1995;6:703. Abeliovich D, Lavon IP, Lerer I, et al. Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and predicts a carrier frequency of 1:29 in the Jewish Ashkenazi population. Am J Hum Genet 1992; 51:951. Orgad S, Neumann S, Loewenthal R, et al. Prevalence of cystic fibrosis mutations in Israeli Jews. Genet Test 2001;5:47. Kornreich R, Ekstein J, Edelmann L, et al. Premarital and prenatal screening for cystic fibrosis: experience in the Ashkenazi Jewish population. Genet Med 2004; 6:415. Dörk T, Dworniczak B, Aulehla-Scholz C, et al. Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum Genet 1997;100:365. Mussaffi H, Prais D, Mei-Zahav M, et al. Cystic fibrosis mutations with widely variable phenotype: the D1152H example. Pediatr Pulmonol 2006;41:250. Macek M, Mackova A, Hamosh A, et al. Identification of common cystic fibrosis mutations in AfricanAmericans with cystic fibrosis increases the detection rate to 75%. Am J Hum Genet 1997;60:1122. Sugarman EA, Rohlfs EM, Silverman LM, et al. CFTR mutation distribution among US Hispanic and African American individuals: evaluation in cystic fibrosis patient and carrier screening program. Genet Med 2004;6:392. Monaghan KG, Bluhm D, Phillips M, et al. Preconception and prenatal cystic fibrosis carrier screening of African Americans reveals unanticipated frequencies for specific mutations. Genet Med 2004;6:141. Wei S, Feldman GL, Monaghan KG. Cystic fibrosis testing among Arab Americans. Genet Med 2006;8: 255. Kerem E, Corey M, Kerem B, et al. The relation between genotype and phenotype in cystic fibrosis: analysis of the most common mutation (DF508). N Engl J Med 1990;323:1517. Durno C, Corey M, Zielenski J, et al. Genotype and phenotype correlations in patients with cystic fibrosis and pancreatitis. Gastroenterology 2002;123:1857. DeBoeck K, Weren M, Proesmans M, et al. Pancreatitis among patients with cystic fibrosis: correlation with pancreatic status and genotype. Pediatrics 2005;115:463. Kristidis P, Bozon D, Corey M, et al. Genetic determination of exocrine pancreatic function in cystic fibrosis. Am J Hum Genet 1992;50:1178.

73. Walkowiak J, Herzig K-H, Witt M, et al. Analysis of exocrine pancreatic function in cystic fibrosis: one mild CFTR mutation does not exclude pancreatic insufficiency. Eur J Clin Invest 2001;31:796. 74. Costes B, Girodon E, Ghanem N, et al. Frequent occurrence of the CFTR intron 8 (TG)n 5T allele in men with congenital bilateral absence of the vas deferens. Eur J Hum Genet 1995;3:285. 75. Kiesewetter S, Macek M Jr, Davis C, et al. A mutation in the CFTR produces different phenotypes depending on chromosomal background. Nat Genet 1993;5:274. 76. Chillon M, Casals T, Mercier B, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N Engl J Med 1995;332:1475. 77. Friedman KJ, Heim RA, Knowles MR, et al. Rapid characterization of the variable length polythymidine tract in the cystic fibrosis (CFTR) gene: association of the 5T allele with selected CFTR mutations and its incidence in atypical sinopulmonary disease. Hum Mutat 1997;10:108. 78. Noone PG, Pue CA, Zhou Z, et al. Lung disease associated with the IVS8 5T allele of the CFTR gene. Am J Respir Crit Care Med 2000;162:1919. 79. Groman JD, Hefferon TW, Casals T, et al. Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign. Am J Hum Genet 2004;74:176. 80. Taylor CG, Dalton A, Pirzada O. Cystic fibrosis mutations and disease phenotype. Arch Dis Child 2000;83:185. 81. Mak V, Zielenski J, Tsui L-C, et al. Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia. JAMA 1999; 281:2217. 82. Claustres M, Guittard C, Bozon D, et al. Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France. Hum Mutat 2000;16:143. 83. Noone PG, Knowles MR. CFTR-opathies: disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations. Respir Res 2001;2:328. 84. Massie RJH, Poplawski N, Wilcken B, et al. Intron-8 polythymidine sequence in Australasian individuals with CF mutations R117H and R117C. Eur Respir J 2001;17:1195. 85. Mickle JE, Cutting GR. Genotype-phenotype relationships in cystic fibrosis. Med Clin North Am 2000;84: 597. 86. Mercier B, Verlingue C, Lissens W, et al. Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients. Am J Hum Genet 1995;56:272.

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87. Schlegel PN, Shin D, Goldstein M. Urogenital anomalies in men with congenital absence of the vas deferens. J Urol 1996;155:1644. 88. Daudin M, Bieth E, Bujan L, et al. Congenital bilateral absence of the vas deferens: clinical characteristics, biological parameters, cystic fibrosis transmembrane conductance regulator gene mutations, and implications for genetic counseling. Fertil Steril 2000;74:1164. 89. Anguiano A, Oates RD, Amos J, et al. Congenital bilateral absence of the vas deferens: a primarily genital form of cystic fibrosis. JAMA 1992;267:1794. 90. Colin AA, Sawyer SM, Mickle JE, et al. Pulmonary function and clinical observations in men with congenital bilateral absence of the vas deferens. Chest 1996;110:440. 91. Salvatore F, Scudiero O, Castaldo G. Genotypephenotype correlation in cystic fibrosis: the role of modifier genes. Am J Med Genet 2002;111:88. 92. Santis G, Osborne L, Knight R, et al. Genotypephenotype relationship in cystic fibrosis: results from the study of monozygotic and dizygotic twins with cystic fibrosis. Pediatr Pulmonol 1992;14:239. 93. Cystic Fibrosis Genotype-Phenotype Consortium. Correlation between genotype and phenotype in patients with cystic fibrosis. N Engl J Med 1993;329:1308. 94. Collaco JM, Vanscoy L, Bremer L, et al. Interactions between second-hand smoke and genes that affect cystic fibrosis lung disease. JAMA 2008;299:417. 95. Collaco JM, Cutting GR. Update on gene modifiers in cystic fibrosis. Curr Opin Pulm Med 2008;14:559. 96. Acton JD, Wilmott RW. Phenotype of CF and the effects of possible modifier genes. Paediatr Respir Rev 2001;2:332. 97. Cutting GR. Modifier genetics: cystic fibrosis. Annu Rev Genom Hum Genet 2005;6:237. 98. Drumm ML, Konstan MW, Schluchter MD, et al. Genetic modifiers of lung disease in cystic fibrosis. N Engl J Med 2005;353:1443. 99. Merlo CA, Boyle MP. Modifier genes in cystic fibrosis lung disease. J Lab Clin Med 2003;141:237. 100. Slieker MG, Sanders EAM, Rijkers GT, et al. Disease modifying genes in cystic fibrosis. J Cystic Fibrosis 2005;4:7. 101. Garred P, Pressler T, Madsen HO, et al. Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis. J Clin Invest 1999;104:431. 102. Arkwright PD, Laurie S, Super M, et al. TGF-b1 genotype and accelerated decline in lung function of patients with cystic fibrosis. Thorax 2002;55:459. 103. Hull J, Thomson AH. Contribution of genetic factors other than CFTR to disease severity in cystic fibrosis. Thorax 1998;53:1018.

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104. Zielenski J, Corey M, Rozmahel R, et al. Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13. Nat Genet 1999;22: 128. 105. Richards B, Skoletsky J, Shuber AP, et al. Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs. Hum Mol Genet 1993;2:159. 106. Johnson MA, Yoshitomi MJ, Richards CS. A comparative study of five technologically diverse CFTR platforms. J Mol Diagn 2007;9:401. 107. Strom SM, Huang D, Chen C, et al. Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test. Genet Med 2003;5:9. 108. Committee on Obstetric Practice, Committee on Gynecologic Practice, Committee on Genetics. American College of Obstetricians and Gynecologists. ACOG Committee Opinion No 324. Perinatal risks associated with assisted reproductive technology. Obstet Gynecol 2005;106:1143. 109. Hubert D, Patrat C, Guibert J, et al. Results of assisted reproductive technique in men with cystic fibrosis. Hum Reprod 2006; 21:1232. 110. McCallum TJ, Milunsky JM, Cunningham DL, et al. Fertility in men with cystic fibrosis: an update on current surgical practices and outcomes. Chest 2000; 118:1059. 111. Josserand RN, Bey-Omar F, Rollet J, et al. Cystic fibrosis phenotype evaluation and paternity outcome in 50 males with congenital bilateral absence of vas deferens. Hum Reprod 2003;16:2093. 112. American College of Obstetricians and Gynecologists. Update on carrier screening for cystic fibrosis. Obstet Gynecol 2005;106:1465. 113. Handyside AH, Lesko JG, Tarin JJ, et al. Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N Engl J Med 1992;327:905. 114. Keymolen K, Goossens V, De Rycke M, et al. Clinical outcome of preimplantation genetic diagnosis for cystic fibrosis: the Brussel’s experience. Eur J Hum Genet 2007;15:752. 115. Coskun S, Alsmadi O. Whole genome amplification from a single cell: a new era for preimplantation genetic diagnosis. Prenat Diagn 2007;27:297. 116. Goossens V, Sermon K, Lissens W, et al. Clinical application of preimplantation genetic diagnosis for cystic fibrosis. Prenat Diagn 2000;20:571. 117. Goossens V, Sermon K, Lissens W, et al. Improving clinical preimplantation genetic diagnosis for cystic fibrosis by duplex PCR using two polymorphic markers or one polymorphic marker in combination with the

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Genetic Disorders and the Fetus detection of the ∆F508 mutation. Molec Hum Reprod 2003;9:559. Gazvani R, Lewis-Jones I. Cystic fibrosis screening in assisted reproduction. Curr Opin Obstet Gynecol 2006;18:268. Decruyenaere M, Evers-Kiebooms G, Denayer L, et al. Update and impact of carrier testing for cystic fibrosis: a review and theoretical framework about the role of knowledge, health beliefs and coping. Community Genet 1998;1:23. Farrell PM, Fost N. Prenatal screening for cystic fibrosis: where are we now? J Pediatr 2002;141:758. Scotet V, Duguépéroux I, Audrézet M-P, et al. Prenatal diagnosis of cystic fibrosis: the 18 year experience of Brittany (western France). Prenat Diagn 2008;28: 197. Sawyer SM, Cerritelli B, Carter LS, et al. Changing their minds with time: a comparison of hypothetical and actual reproductive behaviors in parents of children with cystic fibrosis. Pediatrics 2006;18:649. Dudding T, Wilcken B, Burgess B, et al. Reprdoductive decisions after neonatal screening. Arch Dis Child Fetal Neonatal Ed 2000;82:124. Dupus A, Hamilton D, Cole DEC, et al. Cystic fibrosis birth rates in Canada: a decreasing trend since the onset of genetic testing. J Pediatr 2005;147:312. Scotet V, Assael BM, Duguépéroux I, et al. Time trends in birth incidence of cystic fibrosis in two European areas: data from newborn screening programs. J Pediatr 2008;152:25. Le Caignec C, Isidor B, de Pontbriand U, et al. Third case of paternal isodisomy for chromosome 7 with cystic fibrosis: a new patient presenting with normal growth. Am J Med Genet Part A 2007;143:2696. Warburton D. Editorial: Uniparental disomy: a rare consequence of the high rate of aneuploidy in human genes. Am J Hum Genet 1998;42:215. Dicke JM, Crane JP. Sonographically detected hyperechoic fetal bowel: significance and implications for pregnancy management. Obstet Gynecol 1992;80:778. Nyberg DA, Dubinsky T, Resta RG, et al. Echogenic fetal bowel during the second trimester: clinical importance. Radiology 1993;188:527. Al-Kouatly HB, Chasen ST, Streltzoff J, et al. The clinical significance of fetal echogenic bowel. Am J Obstet Gynecol 2001;185:1035. Simon-Bouy B, Satre V, Ferec C, et al and the French Collaborative Group. Hyperchogenic fetal bowel: a large French collaborative study of 682 cases. Am J Med Genet 2003;121:209. Carcopino X, Chaumoitre K, Shojai R, et al. Fetal magnetic resonance imaging and echogenic bowel. Prenat Diagn 2007;27:272.

133. Hodge SE, Lebo R, Yesley AR, et al. Calculating posterior risk with echogenic bowel and one characterized cystic fibrosis mutation. Am J Med Genet 1999;82:329. 134. Bosco AF, Norton ME, Lieberman E. Predicting the risk of cystic fibrosis with echogenic fetal bowel and one cystic fibrosis mutation. Obstet Gynecol 1999;94: 1020. 135. Strom CM, Crossley B, Redman JB, et al. Cystic fibrosis screening: lessons learned from the first 320,000 patients. Genet Med 2004;6:136. 136. Monaghan, KG, Feldman, GL. The risk of cystic fibrosis with prenatally detected echogenic bowel in an ethnically and racially diverse North American population. Prenat Diagn 1999;19:604. 137. Ogino S, Wilson RB, Gold B, et al. Bayesian analysis for cystic fibrosis risks in prenatal and carrier screening. Genet Med 2004;6:439. 138. Lebo RV, Grody WW. Testing and reporting ACMG cystic fibrosis mutation panel results. Genet Test 2007;11:11. 139. Brambati B, Tului L, Fattore S. First-trimester fetal screening of cystic fibrosis in low-risk population. Lancet 1993;342:624. 140. Black SH, Bick DP, Maddalena A, et al. Pregnancy screening for cystic fibrosis. Lancet 1993;342:1112. 141. Colten HR. Screening for cystic fibrosis: public policy and personal choices. N Engl J Med 1990;322:328. 142. Faden RR, Tambor ES, Chase GA, et al. Attitudes of physicians and genetics professionals toward cystic fibrosis carrier screening. Am J Med Genet 1994;50:1. 143. Workshop on Population Screening for the Cystic Fibrosis Gene. Statement from the National Institutes of Health workshop on population screening for the cystic fibrosis gene. N Engl J Med 1990;323:70. 144. Wilfond BS, Fost N. The cystic fibrosis gene: medical and social implications for heterozygote detection. JAMA 1990;263:2777. 145. Caskey CT, Kaback MM, Beaudet AL. The American Society of Human Genetics statement on cystic fibrosis screening. Am J Hum Genet 1990;46:393. 146. Statement of the American Society of Human Genetics on cystic fibrosis carrier screening. Am J Hum Genet 1992;51:1443. 147. Committee on Obstetrics, Maternal and Fetal Medicine, American College of Obstetricians and Gynecologists. American College of Obstetricians and Gynecologists committee opinion: current status of cystic fibrosis carrier screening. Washington, DC: American College of Obstetricians and Gynecologists. 1992. 148. Haddow JE, Bradley LA, Palomaki GE, et al. Issues in implementing prenatal screening for cystic fibrosis: results of a working conference. Genet Med 1999;1: 129.

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149. Henneman L, Poppelaars FAM, Kate LP. Evaluation of cystic fibrosis carrier screening programs according to genetic screening criteria. Genet Med 2002;4:241. 150. NIH Consensus Statement. Genetic Testing for Cystic Fibrosis. NIH Consensus Statement Online 1997 Apr 14–16; 15(4): 1–37. Available at: http://consensus.nih. gov/1997/1997GeneticTestCysticFibrosis106html.htm. 151. Mennuti MT, Thomson E, Press N. Screening for cystic fibrosis carrier state. Obstet Gynecol 1999;93:456. 152. Holmes LB, Pyeritz RE, for the Clinical Practice Committee of the American College of Medical Genetics. Screening for cystic fibrosis. JAMA 1998;279:1068. 153. American College of Obstetricians and Gynecologists Statement on Cystic Fibrosis Testing. Washington, DC: American College of Obstetricians and Gynecologists, 1998. 154. Grody WW, Cutting GR, Klinger KW, et al. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 2001;3:149. 155. Rohlfs EM, Zhou Z, Sugarman EA, et al. The I148T CFTR allele occurs on multiple haplotypes: a complex allele is associated with cystic fibrosis. Genet Med 2002;4:319. 156. Grody WW, Cutting GR, Watson MS. The cystic fibrosis “arms race”: when less is more. Genet Med 2007; 9:739. 157. American College of Obstetricians and Gynecologists and American College of Medical Genetics. Preconception and prenatal carrier screening for cystic fibrosis: clinical and laboratory guidelines. Washington, DC: American College of Obstetricians and Gynecologists, 2001. 158. Morgan MA, Driscoll DA, Mennuti MT et al. Practice patterns of obstetrician-gynecologists regarding preconception and prenatal screening for cystic fibrosis. Genet Med 2004;6:450. 159. Morgan MA, Driscoll DA, Zinberg S, et al. Impact of self-reported familiarity with guidelines for cystic fibrosis carrier screening. Obstet Gynecol 2005;105:1355. 160. Richards CS, Grody WW. Prenatal screening for cystic fibrosis: past, present and future. Expert Rev Mol Diagn 2004;4:49. 161. Lubin IM, Caggana M, Constantin C, et al. Ordering molecular genetic tests and reporting results, practices in laboratory and clinical settings. J Mol Diagn 2008;10:459.

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162. Strom CM, Huang D, Buller A, et al. Cystic fibrosis screening using the College panel: platform comparison and lessons learned from the first 20,000 samples. Genet Med 2002;4:289. 163. Crossley JR, Elliott RB, Smith PA. Dried blood spot screening for cystic fibrosis in the newborn. Lancet 1991;2:472. 164. Ranieri E, Ryall RG, Morris CP, et al. Neonatal screening strategy for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis. BMJ 1991;302: 1237. 165. Gregg RG, Wilfond BS, Farrell PM, et al. Application of DNA analysis in a population screening program for neonatal diagnosis of cystic fibrosis: comparison of screening protocols. Am J Hum Genet 1993;52:616. 166. Larsen J, Campbell S, Faragher EB, et al. Cystic fibrosis screening in neonates: measurement of immunoreactive trypsinogen and direct gene analysis for ∆F508. Eur J Pediatr 1994;153:569. 167. Gregg RG, Simantel A, Farrell PM, et al. Newborn screening for cystic fibrosis in Wisconsin: comparison of biochemical and molecular methods. Pediatrics 1997;99:819. 168. Newborn screening: toward a uniform screening panel and sytstem. Available at: http://mchb.hrsa.gov/ screening/. 169. March of Dimes. Newborn Screening (United States). Last updated: November 2008. Available at: www. marchofdimes.com/peristats/level1.aspx?reg=99&top =12&stop=239&lev=1&slev=1&obj=20. 170. Southern KW, Munck A, Pollitt R, et al. On behalf of the European Cystic Fibrosis Society CF Neonatal Screening Working Group: a survey of newborn screening for cystic fibrosis in Europe. J Cystic Fibrosis 2007;6:57. 171. Sims EJ, Clark A, McCormick MD, et al. On behalf of the United Kingdom Cystic Fibrosis Database Steering Committee. Cystic fibrosis diagnosed after two months of age leads to worse outcomes and requires more therapy. Pediatrics 2007;119:19. 172. Grosse SD, Rosenfeld M, Devine OJ, et al. Potential impact of newborn screening for cysic fibrosis on child survival: a systematic review and analysis. J Pediatr 2006;149:362. 173. Farrell PM. The meaning of an early diagnosis in a new era of cystic fibrosis care. Pediatrics 2007;119:157.

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Prenatal Diagnosis and Treatment of Congenital Adrenal Hyperplasia Phyllis W. Speiser Division of Pediatric Endocrinology, Schneider Children’s Hospital, New York, USA

Congenital adrenal hyperplasia (CAH) is a family of autosomal recessive disorders of adrenal steroidogenesis in which there is deficient activity of one of the enzymes necessary for cortisol synthesis. As a result of deficient cortisol synthesis, corticotropin-releasing hormone and adrenocorticotropic hormone (ACTH) secretion are stimulated via negative feedback, with resultant adrenal hyperplasia. There is overproduction of the adrenal steroids preceding the deficient enzymatic step, and these precursors are channeled into production of potentially potent sex steroids. A simplified scheme of adrenal steroidogenesis, showing the series of enzymatic steps required for adrenal steroidogenesis, is depicted in Figure 18.1. Deficiency of each of the enzymatic activities required for cortisol synthesis has been described, and results in one of several phenotypes.1 Clinical and genetic heterogeneity of these disorders is well recognized, and is related to the nature of the respective genetic mutations and the extent to which they alter the balance of adrenal cortical hormone production. Specifically, the signs and symptoms of each disorder depend on which steroids are lacking and which are produced in excess. Measurement of the serum and urinary steroids helps to determine which are overproduced and which are deficient, and the precursor/ product ratio helps to localize the site of the disor-

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dered enzymatic step. In the prototypical case of steroid 21-hydroxylase deficiency, administration of glucocorticoid results in suppression of the overproduced hormones, restoring proper hormone balance.2 This chapter focuses on the prenatal diagnosis and treatment of virilizing CAH due to 21hydroxylase (21-OH) deficiency and touches on rarer defects, namely 11β-hydroxylase (11β-OH) deficiency, congenital lipoid adrenal hyperplasia and P450 oxidoreductase deficiency.

21-Hydroxylase deficiency Deficiency of steroid 21-OH (also termed cytochrome P450c21 or 21-mono-oxygenase) activity is the most common cause of CAH (MIM +#201910), accounting for more than 90 percent of cases. Failure to adequately 21-hydroxylate 17-hydroxyprogesterone to 11-deoxycortisol (compound S) results in deficient cortisol, increased ACTH, adrenal hyperplasia, and increased adrenal androgen secretion. Adrenal hyperplasia seems to be mediated by ACTHstimulated production of several growth factors, including insulin-like growth factors I and II (IGF-I and II).3 Excessive adrenal androgen production, mostly androstenedione (see Figure 18.1), and by peripheral conversion the more potent testosterone and dihydrotestosterone, produces the virilization that is the hallmark of this disorder. Beginning in utero, the affected female fetus develops clitoromegaly, with or without partial fusion of the labioscrotal folds. In the most severe cases,

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Figure 18.1 Simplified scheme of adrenal steroidogenesis. Chemical names for enzymes are shown above or to the right of arrow; circled numbers refer to traditional names: 1 = 20, 22-desmolase; 2 = 3β-hydroxysteroid dehydrogenase/isomerase; 3 = 21-hydroxylase; 4 = 11βhydroxylase; 5 17 α-hydroxylase; 6 = 17,20-lyase;

7 = 18-hydroxylase; 8 = 18-oxidase. Two unnumbered reactions shown with dotted arrows occur primarily in gonads, not in the adrenal gland. DOC, 11-deoxycorticosterone. Source: Adapted from Miller WL and Levine LS. Molecular and clinical advances in congenital adrenal hyperplasia. J Pediatr 1987;111:1.

there is complete fusion of the labioscrotal folds, with the appearance of a penile urethra. In approximately three-fourths of infants with 21-OH deficiency, inadequate 21-hydroxylation of progesterone to 11-deoxycorticosterone (DOC) results in aldosterone deficiency (see Figure 18.1) and salt-wasting crisis may occur, usually during the first few weeks of life. In the US, universal newborn screening for CAH-21-OH deficiency has largely mitigated neonatal adrenal crises and the former problems of progressive postnatal virilization.4 Unrecognized or inadequately treated CAH typically leads to children with inappropriate penile and clitoral enlargement, excessive growth, acne, and/or early onset of pubic hair. Untimely advancement in bone age reduces height potential and historically, the average CAH patient has grown to about 1–2 SD below the population mean in stature.5,6 Disordered puberty and infertility in patients with CAH are more common than in the general population, but reproduction is possible with appropriate treatment.7

The diagnosis of 21-OH deficiency is based on elevated baseline and ACTH-stimulated levels of serum 17-hydroxyprogesterone (17-OHP) and adrenal androgens, particularly androstenedione, and their suppression with glucocorticoid treatment.8 Elevated plasma renin activity (PRA)/aldosterone ratio is present in salt wasting. Genetics Each of the enzyme deficiency disorders herein discussed is attributable to inactivating recessive mutations in genes encoding the respective proteins. In the case of 21-OH deficiency, conversion of progesterone to deoxycorticosterone and 17OHP to 11-deoxycortisol is mediated by a cytochrome P450 enzyme, P450c21, found in the endoplasmic reticulum and predominantly expressed in adrenal cortical cells. The enzyme is encoded by CYP21A2 (also termed CYP21) localized to 6p21.3 near the HLA complex. An adjacent highly homologous pseudogene, CYP21A1P (also termed CYP21P), is the source of most of

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Figure 18.2 A, Map of the genetic region around the 21-hydroxylase (CYP21) gene. Arrows denote direction of transcription. CYP21P, 21-hydroxylase pseudogene; C4A and C4B, genes encoding the fourth component of serum complement; RP1, gene encoding a putative nuclear protein of unknown function; RP2, truncated copy of this gene. TNXB, tenascin-X gene and TNXA, a truncated copy of this gene, are on the opposite chromosomal strand overlapping the 3’ end of each CYP21 gene. The 30-kb scale bar is positioned to show the region involved in the tandem duplication location of the CYP21 genes within the HLA major histocompatibility complex on chromosome 6p21.3. B, An extended view of the short arm of chromosome 6. Numbers denote distances

between genes in kilobase pairs (kb). The HLA-B and HLA-DR histocompatibility genes flank the CYP21 gene. The centromere is nearest HLA-DR. TNF, tumor necrosis factor (actually two genes, TNF A and B), is situated between the C4/CYP21 region and HLA-B. There are many other genes in this region with functions as yet unknown. C, Diagram showing the location and functional significance of the nine most common mutations (other than deletion) found in patients with CAH due to 21-hydroxylase deficiency. Numbered boxes represent exons. A detailed description of these and other mutations is given in the text. Source: Modified from White PC and Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21:245.

the common mutations via gene conversion (Figure 18.2). Genetic linkage disequilibrium is observed between specific HLA genotypes and 21-OH deficiency. HLA Bw47,DR7 is associated with deletion of CYP21A2 in salt-wasting CAH frequently observed in Northern European Cau-

casians, and HLA B14,DR1 is associated with duplication of CYP21A1P in nonclassic CAH, especially prominent among Ashkenazi Jews. Prenatal diagnosis of CAH was originally done by HLA serotyping, but more recently by direct CYP21A2 genotyping.2

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Prenatal Diagnosis and Treatment of Congenital Adrenal Hyperplasia

The two closely homologous 21-OH genes are located in tandem with two highly homologous genes for the fourth component of complement (C4A, C4B)9 and a gene for an extracellular matrix protein, tenascin-X, which is also duplicated (X, XA).10 Deficiency of tenascin X and deletion of the corresponding genes are associated with one of the rarer forms of the connective tissue disease Ehlers– Danlos syndrome (MIM #606408), most of which are caused by mutations in the type V collagen genes. At least one case has been reported of a contiguous gene deletion syndrome including CAH and Ehlers–Danlos syndrome.11 More than 100 mutations have been identified in CYP21A2 among patients with 21-OH deficiency; these are summarized in the Human Gene Mutation Database.12 Among the various deletions and point mutations reported, approximately 70–75 percent of CAH haplotypes contain sequences identical to those in the CYP21A1P pseudogene, suggesting that these mutations arose via small-scale gene conversion events.13 Rarer mutations that do not seem to have arisen by unequal meiotic recombination between CYP21A2 and the pseudogene have also been reported.14 Approximately 10 percent of alleles have large macroconversions that change a major portion of the active gene sequence into a pseudogene sequence,15 and approximately 25 percent of affected alleles carry a 30 kb deletion.16 Most patients of mixed ethnic background are compound heterozygotes, having inherited different genetic lesions from each parent. Those of homogeneous parentage or the product of consanguineous unions are often homozygous for either one of the two most common mutations found in classic CAH: the 30 kb deletion or the splice mutation in intron 2 (656G). These two alleles account for nearly 50 percent of mutations in most populations. The severity of disease expression in the compound heterozygote is most often determined by the activity of the less severely affected of the two alleles.15,17 The functional effects of mutations in CYP21A2 gene have been examined. Amino acid substitutions present in patients with late-onset or nonclassic 21-OH deficiency (e.g. P30L, V281L or P453S) result in an enzyme with 10–50 percent of normal activity18–21; the mutation characteristic of

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simple virilizing 21-OH deficiency (I172N) results in an enzyme with 2 percent of normal activity22; and a cluster of mutations in exon 6 found in saltwasting 21-OH deficiency results in an enzyme with no detectable activity (see Figure 18.2).19 In general, there is a close correlation between genotype and phenotype.15 However, patients have been reported who were more or less severely affected than would have been predicted by genotype.23,24 The mechanism of this is not completely clear but may be due to the unrecognized presence of more than one mutation in any single allele, differing definitions of clinical forms of CAH, variation in splicing with the intron 2 mutation, variability in gene copy number and sequence, extra-adrenal 21-OH activity, and/or the genetic background against which the CYP21A2 genotype is expressed. Newborn screening Screening for CAH due to 21-OH deficiency became possible with the development of an assay for 17-OHP, using a heel-stick capillary blood specimen impregnated on filter paper.25 Newborn screening programs have been established in the United States, Canada, Europe, Japan, New Zealand, and South America. Results of worldwide screening of several million newborns for CAH due to 21-OH deficiency have been reported. The average incidence of classic CAH in most locales is ∼1 in 15,000 livebirths.26 The world’s highest incidence of CAH due to 21-OH deficiency is among the Yupik Eskimos of southwestern Alaska (1 in 282) and the island people of La Reunion, France (1 in 2,141).27 Salt wasting is diagnosed in approximately 75 percent of affected newborns. Cost/benefit analysis indicates that newborn screening for classic 21-OH deficiency is largely accurate and cost effective.28 A detailed review of factors impacting the accuracy of CAH newborn screening is presented by Honour and Torresani.29 These include the timing and type of assays performed, the cut-off designations for affected infants depending on birthweight and gestational age, whether second-tier tests such as tandem mass spectrometry and/or genotyping are incorporated into the screening procedure, and systems in place to notify healthcare providers of results and evaluate infants.

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Nonclassic forms of congenital adrenal hyperplasia Nonclassic 21-OH deficiency is among the most frequent autosomal recessive disorders in humans, with an estimated prevalence in Ashkenazi Jews of 3.7 percent (1 in 27) and in a diverse Caucasian population of 0.1 percent (1 in 1,000),30 confirmed by limited population screening.31,32 Nonclassic 21-OH deficiency, rarely detectable in newborn screening owing to lower levels of marker hormones,33 presents in later childhood, at puberty or in adult life with signs of androgen excess – early appearance of pubic and axillary hair, inappropriately rapid statural growth, advanced bone age, acne, hirsutism, temporal hairline recession, amenorrhea or infertility.34–36 It is relatively rare for females who carry a nonclassic allele to present with clitoromegaly; however, this is more commonly found with the P30L mutation.18 In males the phenotype is more subtle, and the consequences of adrenal androgen excess are less severe. This condition may also be relatively asymptomatic. Moreover, individuals with the same genetic mutations and biochemical abnormalities may manifest different clinical findings, and androgen excess symptoms may vary in severity over time. The genotype of inviduals with nonclassic 21-OH deficiency is either compound heterozygote or homozygous mild. This means that the disorder results either from the combination of a classic, severe mutant allele on one parental haplotype and a mild mutant allele on the other, or from a mild CYP21A2 mutation on each haplotype. Molecular genetic studies in patients with nonclassic 21-OH deficiency (see Figure 18.2) have revealed that the most common mutation associated with this disorder is a point mutation at codon 281 resulting in a single conservative amino acid change from valine to leucine (V281L). This allele is found in up to 70 percent of patients with the typically associated HLA-B14,DR1 haplotype.37 Other less commonly found missense mutations are P30L,18 P453S,38 H62L,39 and K121Q.21 The pattern of hormonal abnormalities in nonclassic CAH due to 21-OH deficiency demonstrates a less marked elevation in 17-OHP, androstenendione, and testosterone.8 The hormone levels correlate with genotype.15 It is important to note that

there is no clinically significant deficit in cortisol production in response to stress among patients with nonclassic forms of CAH,40 nor is there a problem with aldosterone production. Thus, such individuals are not at risk for hypotensive adrenal crises typically associated with the most severe forms of classic CAH. Many affected with nonclassic CAH can avoid any glucocorticoid treatment. A more detailed discussion of nonclassic CAH is provided in a recent review.41 Prenatal diagnosis and treatment of congenital adrenal hyperplasia due to 21-OH deficiency Classic CAH due to 21-OH deficiency is the most common cause of ambiguous genitalia in the newborn female. Advances within the past two decades have made possible the prenatal diagnosis and treatment of this disorder, making it among the first diseases amenable to prenatal medical therapy. The objective of prenatal diagnosis and treatment of 21-OH deficiency is the prevention of ambiguous genitalia in the female fetus affected with the classic, severe salt-wasting or simple virilizing forms of CAH, thus avoiding the attendant psychologic stress to families and patients caused by the genital ambiguity and the potential need for corrective surgery. Moreover, prenatal diagnosis can help avoid possible erroneous male sex assignment in the severely virilized female, salt-wasting crisis and death of (mostly male) infants with the salt-wasting form, and progressive virilization in undiagnosed infants and children, mainly those with less severe forms of CAH. Newborn screening mitigates these problems, but is too late to prevent genital virilization in affected females.

Prenatal diagnosis of 21-OH deficiency Prenatal prediction of CAH due to classic 21-OH deficiency has been performed using a number of methods: amniotic fluid (AF) hormone levels, HLA typing of chorionic villus (CV) cells and AF cells, and molecular genetic studies of chorionic villus cells and AF cells. Prenatal diagnosis of CAH was first reported in 1965, based on elevation of AF 17-ketosteroids and pregnanetriol.42 Pang et al. reviewed subsequent reports that suggested that elevated 17-OHP con-

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Figure 18.3 Guidelines for prenatal maternal dexamethasone therapy for fetal virilizing CAH due to 21-hydroxylase deficiency CAH.

centration in AF was a more accurate test for saltwasting CAH.43 Elevated androstenedione (D4-A) concentration in AF provides another diagnostic measure. These hormone markers (17-OHP and D4-A) may be in the normal range in some milder forms of CAH due to 21-OH deficiency, and thus are consistently reliable for prenatal prediction only when the fetus is affected with classic saltwasting CAH. Elevated AF 21-deoxycortisol may provide a more useful prenatal hormonal measure in nonsalt-wasting disease.44

The demonstration of the genetic linkage between CAH due to 21-OH deficiency and HLA made possible the prenatal prediction of this disorder by HLA typing of cultured AF cells45 and cultured CV cells. Use of CV cells permits earlier identification of the affected fetus than was possible by amniocentesis (typically at 10 versus 14 weeks). Recent advances in cell sorting and PCR may allow even earlier detection of fetal sex, and potentially fetal disease status, from maternal blood during the first trimester (see Chapter 30).46

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Genotyping using DNA extracted from CV cells or amniocytes for analysis of CYP21A2 for prenatal diagnosis is now the most frequently performed diagnostic procedure. Causative mutations can now be identified on ∼95 percent of alleles using PCR-based genotyping. Mutations not detected by this approach can be characterized by direct sequencing of CYP21A2. De novo mutations, found in patients with CAH but not in both parents, are found in 1 percent of disease-causing CYP21B mutations.15 Pitfalls in the molecular genetic diagnosis include allele “drop-out,” sample contamination, and inability to detect “phase” (i.e. whether two mutations are situated on the same allele or on each of two parental alleles).47

Prenatal treatment of congenital adrenal hyperplasia due to 21-OH deficiency Virilizing CAH requires inheritance of a mutant allele from both parents. Thus, partners who are obligate heterozygotes for classic CYP21A2 mutations have a 1 : 4 risk of having a CAH-affected child, and a 1 : 8 risk of an affected girl. Females exposed in utero to elevated androgen levels develop some degree of ambiguity of the external genitalia and often masculine behavior. Ideally, one would want to minimize or eliminate these traits because they cause distress to the parents and the daughter. Prenatal treatment of CAH was first reported in 1969.48 Repeated injection of hydrocortisone into a male fetus diagnosed with CAH on the basis of AF pregnanetriol concentration was associated with decreased AF pregnanetriol level. Experience since the early 1980s, when prenatal treatment gained support,49,50 has shown that administration of dexamethasone to the pregnant woman at risk for carrying a CAH fetus ameliorates or prevents genital ambiguity in about 80 percent of cases. The protocol used presently consists of starting oral dexamethasone for the pregnant woman at 25 percent risk of carrying a virilized CAH fetus during the early first trimester. The arbitrarily chosen dose is 20 mg/kg/day (based on pre-pregnancy weight of the mother) divided in three equal doses (Figure 18.3). Affected females subjected to prenatal treatment show fewer genital anomalies compared to their older affected sisters who were not treated, provided dexamethasone is reliably and continuously administered before 9 weeks’

gestation.51 Prenatally treated females may avoid psychosexual difficulties associated with genital ambiguity and the possible need for surgical reconstruction. Controversies in prenatal CAH treatment Despite this sanguine description, dexamethasone treatment for at-risk pregnancies remains controversial; these concerns are discussed below.

Unnecessary treatment To prevent female genital virilization, treatment must be instituted early in the first trimester, before it is possible to determine karyotype and CYP21A2 genotype; therefore seven of eight pregnancies will be treated unnecessarily, albeit briefly, to prevent one case of ambiguous genitalia. Families must therefore be fully informed when they consent to the diagnostic and putative therapeutic interventions.52,53 To minimize the duration of dexamethasone treatment for male or unaffected female fetuses, prompt and accurate diagnostic studies are needed. Most often, CVS is performed at 10–12 weeks’ gestation. Finding an XY karyotype permits discontinuation of prenatal treatment because males with 21-OH deficiency do not suffer from genital ambiguity, and thus prenatal dexamethasone serves no therapeutic purpose. If the karyotype is XX, prenatal treatment continues until CYP21A2 genotyping can be performed to determine whether the female fetus is affected. In unaffected females, treatment may be stopped; however, by the time the genotype is known, the fetus has been exposed to dexamethasone for several weeks. For CAH-affected females, treatment usually continues to term, since cessation of therapy for even a few days may result in genital ambiguity. Future methods for obtaining fetal tissue for diagnosis may include analysis of fetal nucleic acids or cells culled from the maternal circulation in the first trimester (see Chapter 30), and preimplantation genetic diagnosis (PGD) (see Chapter 29). Theoretically, if done early enough, the former test could obviate prenatal treatment of male fetuses, regardless of CAH status. PGD can help couples select either male or female embryos unaffected by the disease in question.

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Box 18.1 Fetal side effects of prenatal treatment for congenital adrenal hyperplasia with dexamethasone

Fetal death Intrauterine growth restriction/failure to thrive Hydrocephalus Hydrometrocolpos Vaginal cyst Shyness +/− Memory and other cognitive changes +/− Note: These side effects may not be directly attributable to dexamethasone.

Teratogenicity and late effects (Box 18.1) To date, glucocorticoids have not been linked causally with any congenital malformations in humans.51,54 These drugs are frequently used in pregnancy to promote fetal lung maturation before impending premature delivery. An important caveat, however, is that betamethasone is given to promote rapid lung maturation for a few days in a relatively late phase of fetal development, rather than starting from the first trimester and continuing for weeks or months, as in CAH prenatal therapy. The dexamethasone dose used in prenatal treatment of CAH is clearly supraphysiologic, and comparable doses used in rats have caused hypertension in later life.55 Currently, the US Food and Drug Administration classifies corticosteroids as Category B, indicating that there are no controlled studies to indicate teratogenicity in human pregnancy. Nevertheless, there are data to suggest that glucocorticoids are toxic to the developing central nervous system in primates and rodents.56,57 Repeated prenatal exposure of the human fetus to betamethasone to induce pulmonary surfactant in threatened early delivery may induce hyperactivity and attention deficit disorder.58 Spontaneous abortion, late pregnancy, fetal death, and intrauterine growth restriction (IUGR) occasionally have occurred in short-term treated unaffected pregnancies or longer treated affected pregnancies (see Box 18.1); however, the frequency of such events is no different from those observed

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Box 18.2 Maternal side effects of prenatal treatment for congenital adrenal hyperplasia with dexamethasone from first trimester until birth

Excess weight gain Cushingoid facial features Striae (broad, pigmented with scarring) Gestational diabetes or abnormal glucose tolerance Facial hair Hypertension Emotional lability, irritability Abdominal pain Fatigue Pedal edema

in untreated pregnancies.59 An additional observation in a child after prenatal treatment was a single case of hydrometrocolpos,60 although it is unclear whether this defect could be directly attributed to prenatal exposure to dexamethasone. Limited data suggest mild adverse cognitive or behavioral effects in these children,61 yet larger studies have not confirmed such findings.62 A salutary effect in the prenatal treatment group, other than improved genital appearance in affected girls, was less prominent masculine behavior.63 It should be noted that recent refinements in surgical technique permit potentially better outcomes with respect to genital appearance and sexual function.64 Long-term results of newer operations remain unproven and surgeries performed within past generations were somewhat unsatisfactory.65

Maternal effects (Box 18.2) The incidence of maternal complications varies, but is estimated at about 10–20 percent. Overt Cushing syndrome and hypertension have been reported in approximately 1–2 percent of all treated pregnancies, usually with subjects treated throughout pregnancy.66 Excessive weight gain occurs during the first trimester, but generally stabilizes during continued treatment. No significant differences have been observed between treated and untreated pregnant mothers in blood pressure,

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proteinuria, gestational diabetes or placental weight.67 Monitoring of urinary estriol to assess compliance, dosing, and efficacy has been sporadic. Estriol reflects fetal adrenal and placental activity and, if low, could permit a decrease in dexamethasone dose in later pregnancy; this approach has not been systematically tested. Summary In summary, prenatal treatment of pregnancies at risk for classic, severe forms of virilizing CAH is quite effective in ameliorating or preventing genital ambiguity, but there are some potentially concerning issues. The long-term safety of gestational dexamethasone should be monitored prospectively, preferably in an international database comprising data from specialized centers with ethically approved protocols. Women must be fully informed of the potential risks for themselves and the fetus and the possible lack of benefit in an affected female. Couples should be informed that dexamethasone is a highly potent steroid that has caused long-term side effects in animals exposed to this drug during early gestation. Mothers with previous medical conditions that may be aggravated by dexamethasone, such as hypertension, overt diabetes, gestational diabetes or toxemia, probably should not be treated or should be treated only with extreme caution. Maternal monitoring for physical, hormonal, and metabolic changes should begin at the initiation of treatment and should be continued throughout the pregnancy. Given the aforementioned risks, prenatal therapy should not be used routinely in families with mild forms of CAH. Since the benefit of prenatal dexamethasone is restricted to reducing genital ambiguity in severely affected females, healthcare providers and families should seriously consider the risk/benefit ratio when contemplating this experimental treatment. New protocols should require early identification (from fetal cells in maternal blood) and exclusion of male fetuses from treatment.

11β-Hydroxylase deficiency Congenital adrenal hyperplasia due to 11βhydroxylase (11β-OH) deficiency (MIM #202010) accounts for 5–8 percent of reported cases of CAH. It occurs in approximately 1 in 100,000 births in

the general Caucasian population, but is more common among Jews of North African origin (1/5,000–7,000 births).68 A deficiency of 11β-OH results in a defect in the conversion of 11-deoxycortisol (also termed compound S) to cortisol and DOC to corticosterone. Similar to 21-OH deficiency, there is virilization secondary to the excessive secretion of the adrenal androgens, resulting in virilization of the female fetus and postnatal virilization of males and females. Hypertension is commonly observed in this disorder after infancy, caused by chronic increased mineralocorticoid (DOC) secretion leading to sodium and water retention and consequent volume expansion. Hypokalemia may also be present. Glucocorticoid administration suppresses the overproduced adrenal steroids (S, DOC, and androgens), preventing continued virilization and resulting in remission of the hypertension in most cases. The external genitalia may be more severely virilized in these females compared to those with 21-OH deficiency. Optimal medical treatment with low-dose glucocorticoids should permit normal growth and pubertal development and fertility.69 The 11β-OH deficiency is diagnosed by the presence of elevated baseline and ACTH-stimulated serum levels of S, DOC, and androgens and their suppression with glucocorticoid therapy. In the untreated state, plasma renin activity and aldosterone often are suppressed because of the sodiumand water-retaining effect of excessive DOC. Molecular genetics Humans have two mitochondrial 11β-OH isozymes: 11β-OH and aldosterone synthase encoded by CYP11B1 and CYP11B2, respectively, located on chromosome 8q21-q22. The genes are 95 percent identical in coding sequences and approximately 90 percent identical in introns.70 The 11βOH activity encoded by CYP11B1 and expressed in the zona fasciculata converts 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone. Aldosterone synthase encoded by CYP11B2 expressed at relatively low levels exclusively in the zona glomerulosa converts corticosterone to aldosterone, and has weak 11β-OH activity. CAH caused by 11β-OH deficiency results from carrying two recessive inactivating mutations in

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CYP11B1. White reviewed the numerous mutations described in patients with classic CAH caused by 11β-OH.71 In Moroccan Jews, in whom this condition is apparently more prevalent than 21-hydroxylase deficiency, almost all affected CYP11B1 alleles carry the same mutation, R448H in exon 8, which abolishes enzyme activity.72 Genotype–phenotype correlations have been sought in classic 11β-OH deficiency; however, no strict correlation exists between the phenotypic features of hypertension and virilization and the hormonal profile or specific mutations.73 Nonclassic 11β-OH deficiency Nonclassic 11β-OH deficiency may present in a way similar to that of nonclassic 21-OH deficiency, although precocious pubarche resulting from mild 11β-OH deficiency seems to be quite rare, since these alleles are much rarer in the general population. Hypertension is not a typical feature of mild 11β-OH deficiency. The principles of diagnosis are the same as for the classic form of 11β-OH deficiency, although the hormonal abnormalities are less marked. As expected, the mutations detected in milder forms of the disease are less deleterious than those found in classic 11β-OH deficiency.74,75 Prenatal diagnosis and treatment of CAH due to 11β-OH deficiency Prenatal diagnosis of CAH due to 11β-OH deficiency was first reported from Israel based on high level of urinary metabolites; the ratio of tetrahydro-11-deoxycortisol (THS) to tetrahydro-cortisol (THF) + tetrahydro-cortisone (THE) was the best discriminatory index for an affected fetus.76 Dexamethasone administration to the mother can greatly reduce maternal urinary THS excretion and can allow normal female genital development.77 Prenatal diagnosis by CYP11B1 DNA analysis of CV cells or amniocytes has also been reported.78

Congenital lipoid adrenal hyperplasia Congenital lipoid adrenal hyperplasia (CLAH, MIM #201710)) is an even rarer form of CAH that results in a deficiency of all adrenal and gonadal hormones caused by one of two possible defects in

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the early stages of steroid hormone synthesis, resulting in inability to convert cholesterol to pregnenolone. Patients with this condition have been found with inactivating recessive mutations in either CYP11A1 (P450SCC or cholesterol sidechain cleavage enzyme)79 or steroidogenic acute regulatory protein (STAR).80 Deficient adrenal steroidogenesis leads to salt-wasting crisis, hyponatremia, hypovolemia, hyperkalemia, and acidosis with death usually in early infancy unless prompt diagnosis and treatment occur. Because there is deficient fetal testicular steroidogenesis in 46,XY patients, males with this disorder have apparent sex reversal with female external genitalia, but they lack mullerian structures and have dysgenetic gonads. Some females may undergo female puberty, whereas others will show no evidence of gonadal steroidogenesis. One case has been reported of in vitro fertilization and delivery of a healthy infant to an affected woman.81 As with other forms of CAH, the severity of the phenotype usually depends on the severity of the underlying mutation.79 Molecular genetics Mutations in the gene for steroidogenic acute regulatory protein (STAR), a protein that promotes the movement of cholesterol across the mitochondrial membrane, have been reported in patients from various ethnic and genetic backgrounds. The mutations were primarily found in three exons, and two mutations accounted for 70–80 percent of the mutations in Japanese and Palestinian patients.80 A small handful of CLAH patients have been found with CYP11A1 mutations. Further investigation revealed that in nine 46,XY infants with adrenal failure and disordered sexual differentiation, only two had compound heterozygous mutations in CYP11A1,79 implicating genetic hetergeneity. Prenatal diagnosis of congenital lipoid hyperplasia Prenatal diagnosis in pregnancies at risk for lipoid adrenal hyperplasia has been reported using AF levels,82 and also in pregnancies with 46,XY fetuses, by ultrasonographic examination of the external genitalia showing absent phallic structure. Low levels of 17-OHP, 17-hydroxypregnenolone, cortisol, dehydroepiandrosterone, androstenedione,

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and estriol in AF and female-typical genitalia on ultrasonography in two pregnancies with affected male fetuses suggested the diagnosis that was confirmed after termination of pregnancy or birth.82,83

P450 oxidoreductase deficiency The unusual phenotype of apparent defects in two or more steroidogenic enzymes, P450c17 (17α-OH/17,20 lyase), P450c21 (21-OH) and/or P450arom (aromatase), without and with the associated Antley–Bixler syndrome of skeletal malformations (ABS; MIM #207410), remained puzzling until the recent discovery of a form of CAH due to mutations in P450 oxidoreductase (POR) (MIM #201750), the flavoprotein that donates electrons to all microsomal P450 enzymes.84 The phenotype in this disorder is quite variable. Since most POR mutations result in retained partial enzyme activity, the pattern of diagnostic hormone measurements is not necessarily consistent, making the diagnosis challenging. POR deficiency can lead to genital ambiguity in either XX or XY individuals. In cases with severe impairment of P450c17, affected males have incomplete masculinization. In contrast, if the main enzyme affected is either 21-OH or placental aromatase, the female fetus will be virilized.85 Some patients have shown low cortisol levels and manifest clinical signs of adrenal insufficiency. Glucocorticoid replacement therapy is required, especially during periods of major stress. Low maternal estriol at prenatal screening can serve as a marker steroid facilitating early diagnosis, and this can be confirmed by genotyping to distinguish this condition from Smith–Lemli– Opitz syndrome.86

Conclusion Prenatal diagnosis has been reported in several forms of the disease: CAH due to both 21-OH and 11β-OH deficiencies, P450 oxidoreductase deficiency, and in lipoid adrenal hyperplasia. Prenatal diagnosis may be coupled with prenatal treatment to prevent female virilization. Dexamethasone treatment of fetuses at risk for the virilizing disorders must be evaluated carefully to determine the risk/benefit ratio of prenatal treatment. Families should be fully informed as to the potential for as

yet unknown long-term complications. In view of the recent introduction of universal newborn screening for 21-OH deficiency CAH in the US, earlier diagnosis and improved surgical techniques for genital repair, clinicians and families should not assume that prenatal dexamethasone is mandatory for good outcomes.

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40. Feuillan P, Pang S, Schurmeyer T, et al. The hypothalamic-pituitary-adrenal axis in partial (late-onset) 21hydroxylase deficiency. J Clin Endocrinol Metab 1988; 67:154. 41. New MI. Extensive clinical experience: nonclassical 21-hydroxylase deficiency. J Clin Endocrinol Metab 2006;91:4205. 42. Jeffcoate TNA, Fliegner JRH, Russell SH, et al. Diagnosis of adrenogenital syndrome before birth. Lancet 1965;2:553. 43. Pang S, Pollack MS, Loo M, et al. Pitfalls of prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia. J Clin Endocrinol Metab 1985;6:89. 44. Gueux B, Fiet J, Couillin P, et al. Prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia by simultaneous radioimmunoassay of 21deoxycortisol and 17- hydroxyprogesterone in amniotic fluid. J Clin Endocrinol Metab 1988;66:534. 45. Pollack MS, Maurer D, Levine LS, et al. HLA typing of amniotic cells: the prenatal diagnosis of congenital adrenal hyperplasia (21-OH-deficiency type). Transplant Proc 1979;11:1726. 46. D’Souza E, Sawant PM, Nadkarni AH, et al. Evaluation of the use of monoclonal antibodies and nested PCR for noninvasive prenatal diagnosis of hemoglobinopathies in India. Am J Clin Pathol 2008;130:202. 47. Day DJ, Speiser PW, Schulze E, et al. Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees. Hum Mol Genet 1996;5:2039. 48. Nichols J. Antenatal diagnosis and treatment of the adrenogenital syndrome. Lancet 1970;1:83. 49. David M, Forest MG. Prenatal treatment of congenital adrenal hyperplasia resulting from 21- hydroxylase deficiency. J Pediatr 1984;105:799. 50. Evans MI, Chrousos GP, Mann DW, et al. Pharmacologic suppression of the fetal adrenal gland in utero. Attempted prevention of abnormal external genital masculinization in suspected congenital adrenal hyperplasia. JAMA 1985;253:1015. 51. Nimkarn S, New MI. Prenatal diagnosis and treatment of congenital adrenal hyperplasia. Pediatr Endocrinol Rev 2006;4:99. 52. European Society for Paediatric Endocrinology and Lawson Wilkins Pediatric Endocrine Society. Consensus statement on 21-hydroxylase deficiency. Horm Res 2002;58:188. 53. Clayton PE, Miller WL, Oberfield SE, et al. Consensus statement on 21-hydroxylase deficiency from the Lawson Wilkins Pediatric Endocrine Society and The European Society for Pediatric Endocrinology. J Clin Endocrinol Metab 2002;87:448.

54. Gluck PA, Gluck JC. A review of pregnancy outcomes after exposure to orally inhaled or intranasal budesonide. Curr Med Res Opin 2005;21:1075. 55. Seckl JR, Miller WL. How safe is long-term prenatal glucocorticoid treatment? JAMA 1997;277:1077. 56. Liu L, Li A, Matthews SG. Maternal glucocorticoid treatment programs HPA regulation in adult offspring: sexspecific effects. Am J Physiol Endocrinol Metab 2001; 280:E729. 57. Uno H, Eisele S, Sakai A, et al. Neurotoxicity of glucocorticoids in the primate brain. Horm Behav 1994;28:336. 58. French NP, Hagan R, Evans SF, et al. Repeated antenatal corticosteroids: effects on cerebral palsy and childhood behavior. Am J Obstet Gynecol 2004;190:588. 59. New MI, Carlson A, Obeid J, et al. Prenatal diagnosis for congenital adrenal hyperplasia in 532 pregnancies. J Clin Endocrinol Metab 2001;86:5651. 60. Couper JJ, Hutson JM, Warne GL. Hydrometrocolpos following prenatal dexamethasone treatment for congenital adrenal hyperplasia (21-hydroxylase deficiency). Eur J Pediatr 1993;152:9. 61. Hirvikoski T, Nordenstrom A, Lindholm T, et al. Cognitive functions in children at risk for congenital adrenal hyperplasia treated prenatally with dexamethasone. J Clin Endocrinol Metab 2007;92:542. 62. Meyer-Bahlburg HF, Dolezal C, Baker SW, et al. Cognitive and motor development of children with and without congenital adrenal hyperplasia after earlyprenatal dexamethasone. J Clin Endocrinol Metab 2004;89:610. 63. Meyer-Bahlburg H, Dolezal C, Baker S, et al. Diminished behavioral masculinization in girls with congenital adrenal hyperplasia after prenatal dexamethasone exposure. Horm Behav 2003;44:64. 64. Graziano K, Teitelbaum DH, Hirschl RB, et al. Vaginal reconstruction for ambiguous genitalia and congenital absence of the vagina: a 27-year experience. J Pediatr Surg 2002;37:955. 65. Crouch NS, Minto CL, Laio LM, et al. Genital sensation after feminizing genitoplasty for congenital adrenal hyperplasia: a pilot study. BJU Int 2004;93:135. 66. Forest MG, Dorr HG. Prenatal therapy in congenital adrenal hyperplasia due to 21-hydroxylase deficiency: retrospective follow-up study of 253 treated pregnancies in 215 families. Endocrinologist 2003;13:252. 67. Lajic S, Wedell A, Bui TH, et al. Long-term somatic follow-up of prenatally treated children with congenital adrenal hyperplasia. J Clin Endocrinol Metab 1998; 83:3872. 68. Rosler A, Leiberman E, Cohen T. High frequency of congenital adrenal hyperplasia (classic 11 betahydroxylase deficiency) among Jews from Morocco. Am J Med Genet 1992;42:827.

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69. Rosler A, Leiberman E, Sack J, et al. Clinical variability of congenital adrenal hyperplasia due to 11 betahydroxylase deficiency. Horm Res 1982;16:133. 70. Mornet E, Dupont J, Vitek A, White PC. Characterization of two genes encoding human steroid 11 betahydroxylase (P-450(11) beta). J Biol Chem 1989;264: 20961. 71. White PC. Steroid 11 beta-hydroxylase deficiency and related disorders. Endocrinol Metab Clin North Am 2001;30:61. 72. White PC, Dupont J, New MI, et al. A mutation in CYP11B1 (Arg-448—His) associated with steroid 11 beta-hydroxylase deficiency in Jews of Moroccan origin. J Clin Invest 1991;87:1664. 73. Zhu YS, Cordero JJ, Can S, et al. Mutations in CYP11B1 gene: phenotype-genotype correlations. Am J Med Genet A 2003;122:193. 74. Joehrer K, Geley S, Strasser-Wozak EM, et al. CYP11B1 mutations causing non-classic adrenal hyperplasia due to 11 beta-hydroxylase deficiency. Hum Mol Genet 1997;6:1829. 75. Peters CJ, Nugent T, Perry LA, et al. Cosegregation of a novel homozygous CYP11B1 mutation with the phenotype of non-classical congenital adrenal hyperplasia in a consanguineous family. Horm Res 2007;67:189. 76. Rosler A, Leiberman E, Rosenmann A, et al. Prenatal diagnosis of 11beta-hydroxylase deficiency congenital adrenal hyperplasia. J Clin Endocrinol Metab 1979; 49:546. 77. Rosler A, Weshler N, Leiberman E, et al. 11 Betahydroxylase deficiency congenital adrenal hyperplasia: update of prenatal diagnosis. J Clin Endocrinol Metab 1988;66:830. 78. Cerame BI, Newfield RS, Pascoe L, et al. Prenatal diagnosis and treatment of 11beta-hydroxylase deficiency

79.

80.

81.

82.

83.

84.

85.

86.

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congenital adrenal hyperplasia resulting in normal female genitalia. J Clin Endocrinol Metab 1999;84:3129. Kim CJ, Lin L, Huang N, et al. Severe combined adrenal and gonadal deficiency caused by novel mutations in the cholesterol side chain cleavage enzyme, P450scc. J Clin Endocrinol Metab 2008;93:696. Bose HS, Sugawara T, Strauss JF3, Miller WL. The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. International Congenital Lipoid Adrenal Hyperplasia Consortium. N Engl J Med 1996; 335:1870. Sertedaki A, Pantos K, Vrettou C, et al. Conception and pregnancy outcome in a patient with 11-bp deletion of the steroidogenic acute regulatory protein gene. Fertil Steril 2008 (EPub ahead of print). Jean A, Mansukhani M, Oberfield SE, et al. Prenatal diagnosis of congenital lipoid adrenal hyperplasia (CLAH) by estriol amniotic fluid analysis and molecular genetic testing. Prenat Diagn 2008;28:11. Izumi H, Saito N, Ichiki S, et al. Prenatal diagnosis of congenital lipoid adrenal hyperplasia. Obstet Gynecol 1993;81:839. Fluck CE, Tajima T, Pandey AV, et al. Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome. Nat Genet 2004; 36:228. Arlt W, Walker EA, Draper N, et al. Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study. Lancet 2004;363:2128. Shackleton C, Marcos J, Arlt W, et al. Prenatal diagnosis of P450 oxidoreductase deficiency (ORD): a disorder causing low pregnancy estriol, maternal and fetal virilization, and the Antley-Bixler syndrome phenotype. Am J Med Genet A 2004;129:105.

19

Prenatal Diagnosis of Miscellaneous Biochemical Disorders David S. Rosenblatt and David Watkins Department of Human Genetics, McGill University, Montreal, QC, Canada

Inborn errors of folate and cobalamin metabolism Folate and cobalamin (vitamin B12) are B group vitamins that play interacting roles in cellular metabolism. Reduced derivatives of folic acid are required for de novo synthesis of purines, synthesis of thymidylate, and conversion of homocysteine to methionine, transferring one-carbon units derived from glycine, serine and histidine. Derivatives of cobalamin are required for the catabolism of propionylCoA, derived from breakdown of branchedchain amino acids and odd-chain fatty acids, and for the conversion of homocysteine to methionine in a reaction using 5-methyltetrahydrofolate as methyl donor. Disorders of folate metabolism1–3 and those of cobalamin metabolism2–5 are listed in Table 19.1. Advances in the prenatal diagnosis of these disorders have been summarized recently.6,7 The genes for all of these disorders have been identified, and prenatal diagnosis by molecular genetic analysis is now possible in families in which mutations have been identified. In addition, for any disorder in which the gene is known, it may be possible to perform linkage analysis using single nucleotide Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

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polymorphisms (SNPs) at the relevant gene locus if the family is informative.6 This depends on accurate diagnosis of the disorder, preferably by complementation analysis, to ensure that the correct gene is analyzed.

Inborn errors of folate metabolism A number of genetic disorders affecting folate metabolism have been proposed, but not all of these have been confirmed. At the present time, three genetic disorders are generally recognized: hereditary folate malabsorption, methylenetetrahydrofolate reductase (MTHFR) deficiency, and glutamate formiminotransferase deficiency. The disorders that affect activity of methionine synthase (cblE and cblG), which uses 5methyltetrahydrofolate as a methyl donor, are considered in the section on inborn errors of cobalamin metabolism. Hereditary malabsorption of folate Hereditary folate malabsorption results in a specific defeciency of folate transport across the intestine and across the blood–brain barrier.8 It is the result of mutations affecting the SLC46A1 gene, which encodes the proton-coupled folate transporter (PCFT) responsible for folate transport at both sites.9–12 Patients with this disorder typically

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Table 19.1 Disorders of folate metabolism Disorder

Cause

Disorders of folate metabolism Hereditary folate malabsorption (229050)

Due to mutations in the SLC46A1 gene encoding the

Glutamate formiminotransferase deficiency (229100)

Due to mutations in the FTCD gene encoding Glutamate

proton-coupled folate transporter formiminotransferase (EC 2.1.2.5.) and formiminotetrahydrofolate cyclodeaminase (EC 4.3.1.4) Methylenetetrahydrofolate reductase deficiency (236250)

Due to mutations in the MTHFR gene encoding methylenetetrahydrofolate reductase (EC 1.5.1.20)

Disorders of cobalamin metabolism Intrinsic factor deficiency (261000)

Due to mutations at the GIF gene encoding intrinsic factor

Imerslund–Gräsbeck syndrome (261100)

Due to mutations in the CUBN and AMN genes encoding the

Transcobalamin deficiency (275350)

Due to mutations in the TCN2 gene encoding transcobalamin

cubilin and amnionless subunits of the cubam receptor

Isolated methylmalonic aciduria cblA (251100)

Due to mutations in the MMAA gene

cblB (251110)

Due to mutations in the MMAB gene, encoding

cblD variant 2 (277410)

Due to mutations in the MMADHC gene

ATP:cob(I)alamin adenosyltransferase (EC 2.5.1.17)

Isolated homocystinuria cblD variant 1 (277410)

Due to mutations in the MMADHC gene

cblE (236270)

Due to mutations in the MTRR gene encoding methionine

cblG (250940)

Due to mutations in the MTR gene encoding methionine

synthase reductase synthase (EC 2.1.1.13) Combined methylmalonic aciduria and homocystinuria cblC (277400)

Due to mutations at the MMACHC gene

“Classic”cblD (277410)

Due to mutations in the MMADHC gene

cblF (277380)

Due to mutations at the LMBRD1 gene

Note: The numbers in parentheses after the names of the disorders are the McKusick catalog numbers.

come to medical attention during the first year of life with megaloblastic anemia, failure to thrive and progressive neurologic deterioration. The gene is expressed in the placenta9 but it is not known whether its absence affects transplacental transfer of folate. The goal of therapy is to maintain blood and CSF folate at adequate levels; therapy with systemic folinic acid has been only partially successful in this disorder because of the difficulty in getting folate to the brain,13 although successful long-term therapy has been reported.12 With identification of the SLC46A1 gene, prenatal diagnosis by molecular genetic analysis is possible.

Glutamate formiminotransferase deficiency Glutamate formiminotransferase deficiency has a variable phenotype, ranging from severe neurologic disease14 to benign excretion of formiminoglutamate (FIGLU). The disorder is the result of mutations in the FTCD gene, which encodes enzymes that catalyze successive steps in the metabolism of formiminoglutamate generated during histidine catabolism: transfer of the formimino group to tetrahydrofolate followed by conversion of formininotetrahydrofolate to 10-formyltetrahydrofolate. Neither activity of the bifunctional enzyme is expressed in cultured

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fibroblasts.1 Mutations in the FTCD gene have been described in patients with glutamate formiminotransferase deficiency.15 This would allow for prenatal diagnosis in families in which the mutations are known, but because of the mild phenotype in most families, prenatal diagnosis is not usually a consideration. Methylenetetrahydrofolate reductase deficiency MTHFR is the most common and best characterized inborn error of folate metabolism.16 There is a wide range of phenotypes, from seizures, apnea, coma, and death in infancy17 to mild mental retardation and neurologic impairment in adolescence.18,19 Other phenotypes also have been reported.20–22 Megaloblastic anemia is not a feature of severe reductase deficiency, which is characterized by hyperhomocysteinemia and homocystinuria without hypermethioninemia. MTHFR is expressed in cultured fibroblasts23 and the levels of the different folate cofactors can be measured directly.24,25 There is a direct correlation between the residual proportion of methyltetrahydrofolate in cultured fibroblasts, the residual enzyme activity, and the clinical severity of the disease.25,26 Therapy with folate has been only partially successful.1,27 Betaine seems to be the single most beneficial agent, particularly if started early.28–32 The use of very high doses of folic acid has been reported from Japan.33 One infant showed a good response to multivitamin therapy along with methionine supplements34 but subsequently deteriorated when the family stopped therapy. MTHFR is expressed in amniocytes and chorionic villus cells35 and pregnancies at risk for severe MTHFR have been assessed by measurement of enzyme activity in extracts of both types of cells6,35–39 and by measurement of formation of labeled methionine from [14C]formate in amniocytes.40 Measurement of enzyme specific activity in extracts of amniocytes and chorionic villus cells has resulted in successful identification of affected individuals, unaffected carriers and unaffected noncarrier individuals.6 However, overlap in activity between affected individuals and carriers in some families has made interpretation of enzyme assay results difficult.37 In one study, enzyme activity was in the heterozygous range in the prenatal

studies, but the activity measured after birth was very low.38 The gene for MTHFR has been cloned and over 40 different mutations causing severe enzyme deficiency have been identified.41 Virtually all these mutations have been identified only in one or two families; an exception is a mutation that is present at a frequency of 30 percent among Old Order Amish.32 In cases in which the segregating mutations are not known, it is often possible to determine whether a fetus is affected by linkage analysis using common polymorphisms in the MTHFR gene, if the family is informative.6

Inborn errors of cobalamin metabolism Derivatives of cobalamin are required for the activity of two enzymes in human cells. Adenosylcobalamin (AdoCbl) is required for activity of the mitochondrial enzyme methylmalonylCoA mutase, which catalyzes the conversion of methylmalonylCoA to succinylCoA, and methylcobalamin (MeCbl) is required for activity of the cytosolic enzyme methionine synthase, which catalyzes the methylation of homocysteine to form methionine. Cellular deficiency of cobalamin therefore results in accumulation of the substrates methylmalonic acid and homocysteine in the blood and urine. Decreased methylmalonylCoA mutase activity is generally associated with increased susceptibility to lifethreatening acidotic crises, but neurologic damage can occur even in the absence of episodes of decompensation. Decreased activity of methionine synthase is associated with megaloblastic anemia and neurologic symptoms, including developmental delay, hypotonia, seizures and MRI abnormalities. Disorders of cobalamin uptake Intrinsic factor deficiency and Imerslund– Gräsbeck syndrome both affect intestinal absorption of ingested cobalamin. Patients typically present between 1 and 5 years of age with failure to thrive, megaloblastic anemia and usually mild neurologic problems. Intrinsic factor deficiency is the result of mutations affecting the GIF gene on chromosome 11q13, and results in deficiency of the protein that binds cobalamin in the intestine and facilitates its absorption in the distal ileum.42

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Imerslund–Gräsbeck syndrome is caused by mutations affecting the CUBN gene on chromosome 10p12.1 or the AMN gene on chromosome 14q3243–45; the genes encode the protein subunits of the intestinal receptor for the intrinsic factor– cobalmin complex, called cubam. Additional cases of Imerslund–Gräsbeck syndrome are linked to an as yet unidentified third gene. None of these genes is expressed in amniocytes or chorionic villus cells. However, with the identification of the underlying genes, molecular diagnosis is now an option.

Transcobalamin deficiency Transcobalamin (TC) deficiency is associated with megaloblastic anemia and pancytopenia, and with immunologic, gastrointestinal, and mental disorders.46–52 There may be decreased levels of immunoreactive TC (the most common form), presence of immunoreactive TC that cannot bind cobalamin or presence of immunoreactive TC that binds cobalamin but cannot support cobalamin uptake at the cellular level. Patients with TC deficiency are born healthy and without signs of cobalamin deficiency, which suggests that physiologic consequences of TC deficiency can be bypassed during fetal life. Treatment with pharmacologic doses of cobalamin results in normal hematologic development. Because cobalamin therapy will depress the unsaturated TC level, a patient’s serum must be taken before beginning therapy or after the cessation of therapy for several weeks to obtain relevant data with respect to TC deficiency. There are receptors for TC-cobalamin in the placenta, suggesting a role for maternal TC in transfer of cobalamin to the fetus53; however, a mother with TC deficiency has given birth to two normal children.50 Twins with TC deficiency had no measurable TC in cord blood,54 leading to suggestions that a fetal TC-like binder might be important for transfer of cobalamin across the placenta. Cord blood contains TC activity that corresponds to that of the fetus and not that of the mother,55,56 which does not explain why TCdeficient infants are healthy at birth. Because cultured amniotic cells have been shown to synthesize and secrete TC,57–59 prenatal diagnosis is possible. Three at-risk pregnancies have been investigated by measurement of TC production by cultured amniocytes.7,60 All three fetuses were

shown to be unaffected. The gene for human TC (TCN2 on chromosome 22q11.2-qter) has been cloned and mutations have been identified in several patients with TC deficiency.61–66 Disorders of cobalamin utilization A series of inborn errors of cobalamin (designated cblA-cblG) have been identified. These result in isolated methylmalonic aciduria (cblA, cblB, cblD variant 2), isolated homocystinuria (cblE, cblG, cblD variant 1) or combined methylmalonic aciduria and homocystinuria (cblC, cblF, classic cblD), depending on which step in cobalamin metabolism is affected.3 Analysis of the cobalamin pathway has involved measurement of incorporation of label from [14C]-labeled propionate into cellular macromolecules, a measure of the function of methylmalonylCoA mutase in intact cells67; measurement of incorporation of label from [14C]-labeled methyltetrahydrofolate into cellular macromolecules68 or incorporation of [14C]-labeled formate into methionine,40 a measure of the function of methionine synthase in intact cells; and measurement of conversion of exogenous [57Co]-cyanocobalamin (CNCbl) into AdoCbl and MeCbl.68 Assignment of patients to specific classes has depended on somatic cell complementation analysis.67,69–71

Isolated methylmalonic aciduria Methylmalonic aciduria and acidemia in the absence of homocystinuria is seen in patients with the cblA, cblB and cblD variant 2 disorders. The cblD disorder and its variants are described below. Cells in culture from these three groups are chracterized by decreased AdoCbl synthesis and decreased methylmalonylCoA mutase function in the presence of normal MeCbl synthesis and methionine synthase function. The cblB disorder is caused by mutations in the MMAB gene on chromosome 12q24, which encodes a cobalamin adenosyltransferase of the PduO family that catalyzes the final step in the synthesis of AdoCbl.72 The MMAB gene product may also act as a chaperone, delivering AdoCbl to methylmalonylCoA mutase in an activated base-off form.73 More than 20 mutations in the MMAB gene have been identified in cblB patients,72,74–79 almost all of which affect the active site of the enzyme as identified by X-ray crystallographic studies of human80,81 and bacterial

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PduO cobalamin transferases.82–84 The cblA disorder is caused by mutations of the MMAA gene on chromosome 4q31.1-2,85 which encodes a protein of poorly understood function. Studies of its bacterial homolog suggest that it acts as a chaperone, catalyzing and stabilizing the association of the AdoCbl cofactor with methylmalonylCoA mutase.86 Over 30 different mutations have been identified in the MMAA gene from cblA patients.75,78,85,87,88 Treatment of cblA and cblB patients consists of protein restriction and vitamin supplementation. Surveys of patients with methylmalonic aciduria have shown that over 90% of cblA patients responded clinically to therapy with decreased serum or urine methylmalonic acid, while fewer than half of cblB patients responded.89,90 It has been suggested that some patients who are unresponsive to hydroxycobalamin (OHCbl) might respond directly to AdoCbl. However, treatment with AdoCbl of a 30-month-old girl who was unresponsive to OHCbl failed to give a sustained clinically significant response, which suggests that the cofactor was unavailable to the mutase enzyme.91 Patients who live to adulthood often develop endstage renal failure that requires transplantation90; again, this occurs more frequently in patients with cblB disease compared to patients with the cblA disorder.

Isolated methylcobalamin deficiency Homocystinuria in the absence of methylmalonic aciduria occurs in patients with the cblE, cblG and cblD variant 1 disorders. Patient cells in culture are characterized by decreased synthesis of MeCbl and decreased function of methionine synthase, in the presence of normal AdoCbl synthesis and methylmalonylCoA mutase function.3 Decreased methylcobalamin synthesis in these disorders reflects decreased activity of methionine synthase; under normal circumstances, synthesis of MeCbl occurs during the catalytic cycle of methionine synthase.92 The cblG disorder is caused by mutations affecting the MTR gene on chromosome 1p43, which encodes the methionine synthase enzyme.93,94 Methionine synthase specific activity is decreased under all assay conditions in extracts of cblG cells.71,95 The cblE disorder is caused by mutations in the MTRR gene on chromosome 5p15.2-15.3,

which encodes methionine synthase reductase.96 This protein is required to maintain the cobalamin prosthetic group on methionine synthase in its active, fully reduced form. Methionine synthase specific activity in extracts of cblE cells is normal when the assay is performed in the presence of exogenous reducing agents, but is decreased when the amount of reducing agent is decreased.68 Twenty mutations have been identified in the MTR gene in patients with the cblG disorder.93,94,97,98 The most common of these, c.3518C→T (P1173L), is present only in the heterozygous state. Eighteen mutations in the MTRR gene have been identified in cblE patients.96,99,100 The most common of these, c.903+469T→C, results in presence of 140 bp of intronic sequence in mRNA. A c.1361C→T (S545L) mutation appears to be associated with a mild form of the cblE disorder that lacks neurologic involvement.

Combined methylmalonic aciduria and homocystinuria Combined methylmalonic aciduria and homocystinuria occurs in patients with mutations affecting early steps in cellular cobalamin metabolism common to synthesis of both cobalamin coenzymes. In the cblF disorder, total intracellular cobalamin is elevated, but virtually all of this cobalamin is unmetabolized vitamin trapped within the lysosome.101,102 Clinical findings have included anemia, failure to thrive, developmental delay, hypotonia, lethargy, hepatomegaly, encephalopathy, recurrent infections, rheumatoid arthritis, and a pigmentary skin abnormality. Both methylmalonic aciduria and homocystinuria are typically present, although levels may be low. In one case, no homocystinuria could be identified.101 Response to therapy with cobalamin has generally been good, although the second cblF patient identified had an unexpected sudden death.103 The disorder has recently been shown to be caused by mutations in the LMBRD1 gene on chromosome 6q13.104 The cblC disorder is the most common inborn error of cobalamin metabolism, with over 300 patients recognized. Synthesis of both AdoCbl and MeCbl is impaired in cells from cblC patients, with decreased activity of both cobalamin-dependent enzymes. Total intracellular cobalamin is decreased,

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apparently because cobalamin cannot become associated with cobalamin-dependent enzymes and is lost from cells.105 Clinically, patients with the cblC disorder usually present during the first months of life with developmental delay, feeding difficulties, hypotonia, seizures and microcephaly106–114 but some patients have presented later in childhood or as adults, with ataxia, subacute combined degeneration of the spinal cord, dementia or psychosis.115–119 The disorder is caused by mutations affecting the MMACHC gene on chromosome 1p34.1.120,121 The function of the gene product is unclear, although it has been suggested that it might act as a chaperone, activating internalized cobalamin and passing it on to subsequent steps of cobalamin metabolism.122 Over 50 mutations in the MMACHC gene have been identified in cblC patients, and genotype/phenotype associations have been described. The most common MMACHC mutation, c.271dupA, is associated with a severe early-onset phenotype. The phenotype of cells from the first two siblings with the cblD disorder was identical to that of cblC cells.67,123 However, it was subsequently shown that some cblD patients present with isolated homocystinuria (cblD variant 1) or methylmalonic aciduria (cblD variant 2).124 The cblD disorder is caused by mutations in the MMADHC gene on chromosome 2q32.2.125,126 Initial studies have suggested that mutations affecting the N-terminal domain of the MMADHC protein result in isolated methylmalonic aciduria, while mutations affecting the C-terminal domain result in homocystinuria.125 Classic cblD with decreased synthesis of both cobalamin coenzyme derivatives is associated with mutations that result in decreased expression of the gene product. Prenatal diagnosis Prenatal diagnosis of inborn errors of cobalamin has been achieved using a variety of techniques: measurement of methylmalonic acid in amniotic fluid (AF) or maternal blood by gas chromatography-mass spectrometry or tandem mass spectrometry; measurement of homocysteine in amniotic fluid by amino acid analysis, gas chromatographymass spectrometry or tandem mass spectrometry; measurement of biochemical parameters (incorporation of label from [14C]-propionate and

[14C]-methyltetrahydrofolate into cellular macromolecules, synthesis of cobalamin co-enzymes from exogenous [57Co]-cobalamin, enzyme assay) in chorionic villus samples, cultured chorionic villus cells or cultured amniocytes; and, as the genes for the various disorders have been identified, by molecular genetic testing.7,127–130 Successful prenatal diagnoses have been reported for cblA,7,131 cblB,7 cblC,7,132–136 cblE,7,137,138 cblF7 and cblG.7 In addition, prenatal diagnoses in several pregnancies at risk for unspecified cobalamin-responsive methylmalonic aciduria have been reported.139–149 Because both measurement of metabolites in AF and biochemical studies of amniocytes or chorionic villus cells have given rise to false-positive and false-negative results, it has been suggested that all diagnoses should make use of two independent methods.7 Chorionic villus samples may give inconsistent results7,132,145,146,150,151 and therefore cultured amniocytes should be used to assess biochemical parameters when possible. Prenatal therapy Prenatal therapy has been attempted for several fetuses with cobalamin-responsive methylmalonic aciduria using vitamin B12 (CNCbl) or OHCbl, administerd intramuscularly or orally to the mother.129,131,147,148,152,153 Therapy resulted in decreased maternal plasma and urine levels of methylmalonic acid in most cases, suggesting successful treatment of the fetus. In one case therapy was started too late in the pregnancy to have any effect.148 Levels of odd-chain fatty acids were near the control range in the cord blood and red blood cell lipids of treated fetuses but not in their adipose tissue,153 indicating some accumulation of propionylCoA in tissues despite treatment. In cases where prenatal therapy was carried out, infants were born healthy and appeared to develop normally.131,147,152 Prenatal therapy was also performed on a fetus with the cblE disorder, with 1 mg hydroxycobalamin administered intramuscularly weekly to the mother.154 The infant showed no signs of the disorder at birth and has continued to develop normally. Prenatal therapy has also been carried out on two fetuses with the cblC disorder. In one case, the mother was given cobalamin and folate supplements, leading to delivery of an apparently normal infant who was treated from birth with intramus-

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cular hydroxycobalamin and oral carnitine, folate and betaine, and was reported well with no effects of the disorder at 18 months of age.136 In the second case, the mother was treated with intramuscular hydroxycobalamin twice weekly from the 24th week of gestation. The child was born with no signs of the disorder, and was treated with intramuscular hydroxycobalamin and oral folate, carnitine and betaine. The child has had normal developmental milestones but has nystagmus, hyperpigmented retinopathy and hypotonia.135

Prenatal diagnosis of cystinosis Cystinosis is an autosomal recessive disorder characterized by the accumulation of free, nonprotein cystine within the lysosomes of most tissues. The cystine accumulates at 10 to 1,000 times the normal levels and forms crystals within the lysosomes. The primary defect in cystinosis is a defective lysosomal transport system for cystine.155–157 The gene for cystinosis, CTNS, codes for the protein cystinosin and is located on the short arm of chromosome 17.158–160 The major features of cystinosis have been reviewed extensively.161–163 Clinical findings Children with cystinosis are not symptomatic at birth, but signs of renal Fanconi syndrome develop between 1 and 12 months of life.160 These signs include failure to grow, dehydration, electrolyte imbalance, vomiting, acidosis, and hypophosphatemic rickets. Affected children have normal intelligence and their weight is appropriate for their height. They remain short and develop progressive glomerular insufficiency, leading to endstage renal disease by the end of the first decade. Additional findings in the classic form of cystinosis include photophobia, hypothyroidism, and abnormal sweating. Cystinosis can be diagnosed by examining the cystine content of cultured fibroblasts or leukocytes. After 1 year of age, the diagnosis can also be made by slit-lamp examination for corneal crystals. There is considerable clinical heterogeneity in cystinosis. Three different forms have been described (infantile nephropathic, late-onset nephropathic, and ocular), and the different forms seem to breed true in families. The clinical severity cor-

relates with the extent of the accumulation of cystine.162 Treatment in cystinosis includes the management of the renal disease and dialysis or transplantation after end-stage renal disease develops. Although storage of cystine does not occur in the transplanted organ, storage in other host tissues may result in retinal blindness, corneal erosions, diabetes mellitus, myopathy, swallowing difficulties, and neurologic disease.163 It has been shown that cysteamine, a cystine-depleting agent, can retard growth failure and renal deterioration if begun early in life.161,164–168 It can also prevent most, if not all, of the late complications of cystinosis.169 However, even if begun before 3 weeks of age, cysteamine does not necessarily prevent the development of the renal Fanconi syndrome.161,170 Heterozygote detection has relied on determining the content of free cystine in leukocytes or cultured fibroblasts. Prenatal diagnosis The first prenatal diagnosis of cystinosis was accomplished in 1974 by growing amniocytes for 48 hours in a cystine-free medium containing 10 percent dialyzed fetal bovine serum in the presence of [35S]-cystine. The cells were lyzed at physiologic pH in the presence of N-ethylmaleimide (NEM), which reacts with free sulfhydryl groups and forms derivatives that are stable at acid pH. Skin fibroblasts and control amniocytes contained most of the label in the form of glutathione-NEM and cysteineMEM and almost none in cystine. Amniocytes from the cystinotic fetus had much higher levels of nonprotein cystine.171 A modification of the preceding technique was reported,172 as was the use of cystine dimethyl ester.173 There have been other modifications, including the use of highperformance liquid chromatography.174 Chorionic villi have been used for direct cystine measurement at 9 weeks gestational age.175 The assay used a specific cystine-binding protein.176 Fresh tissue from the fetus at risk contained 34.7 nmol 1/2 cystine/mg protein, as compared with 0.09–0.13 nmol 1/2 cystine/mg protein in control samples. Cultured cells from the fetus at risk contained 9.7 nmol 1/2 cystine/mg protein, as compared with 0.11–0.18 nmol 1/2 cystine/mg protein in control cells.175 A similar technique was

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used to exclude the diagnosis of cystinosis in another fetus at risk.177 Prenatal diagnostic studies using chorionic villi and cultured cells showed that a quantitative cystine assay method gave results comparable to those using the [35S]-cystine incorporation method.178 There are currently more than 50 mutations known in CTNS, with the most common being a large deletion than eliminates the first 10 exons of the gene.160 If both causal mutations are known for a pregnancy at risk, prenatal diagnosis using a molecular approach is theoretically possible. Because of the therapeutic successes with early cysteamine treatment in cystinosis,165,166,170 most parents decline prenatal diagnosis, and rapid diagnosis after birth has become important. The measurement of the cystine content of fetal placental tissue175 and of leukocytes can be used to make the diagnosis.

Acknowledgments We thank W.A. Gahl for assistance in the preparation of the cystinosis section of this chapter.

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Genetic Disorders and the Fetus ylmalonic aciduria (MMA): titration of treatment dose to serum and urine MMA. Fetal Diagn Ther 1997;12:21. Zammarchi E, Lippi A, Falorni S, et al. cblC disease: case report and monitoring of a pregnancy at risk by chorionic villus sampling. Clin Invest Med 1990;13:139. Chadefaux-Vekemans B, Rolland MO, Lyonnet S, et al. Prenatal diagnosis of combined methylmalonic aciduria and homocystinuria (cobalamin cblC or cblD mutant). Prenat Diagn 1994;14:417. Merinero B, Pérez-Cerdá C, Garcia MJ, et al. Reliability of biochemical parameters used in prenatal diagnosis of combined methylmalonic aciduria and homocystinuria. Prenat Diagn 1998;18:947. Huemer M, Simma B, Fowler B, et al. Prenatal and postnatal treatment in cobalamin C defect. J Pediatr 2005;147:469. Zhang Y, Yang Y, Hasegawa Y, et al. Prenatal diagnosis of methylmalonic aciduria by analysis of organic acids and total homocysteine in amniotic fluid. Chin Med J 2008;121:216. Rosenblatt DS, Cooper BA. Selective deficiencies of methyl-B12 (cblE and cblG). Clin Invest Med 1989;12:270. Zavadakova P, Fowler B, Zeman J, et al. CblE type of homocystinuria due to methionine synthase reductase deficiency: clinical and molecular studies and prenatal diagnosis. J Inher Metab Dis 2002;25:461. Mahoney MJ, Rosenberg LE, Lindblad B, et al. Prenatal diagnosis of methylmalonic aciduria. Acta Paediatr Scand 1975;64:44. Nakamura E, Rosenberg LE, Tanaka K. Microdetermination of methylmalonic acid and other short chain dicarboxylic acids by gas chromatography: use in prenatal diagnosis of methylmalonic acidemia and in studies of isovaleric acidemia. Clin Chim Acta 1976; 68:127. Willard HF, Ambani LM, Hart AC, et al. Rapid prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism: a sensitive assay using cultured cells. Hum Genet 1976;32:277. Naylor G, Sweetman L, Nyhan WL, et al. Isotope dilution analysis of methylcitric acid in amniotic fluid for the prenatal diagnosis of propionic acid and methylmalonic acidemia. Clin Chim Acta 1980;107:175. Zinn AB, Hine DG, Mahoney MJ, Tanaka K. The stable isotope dilution method for measurement of methylmalonic acid: a highly accurate approach to the prenatal diagnosis of methylmalonic acidemia. Pediatr Res 1982;16:740. Kleijer WJ, Thoomes R, Galjaard H, et al. First-trimester (chorion biopsy) diagnosis of citrullinaemia and methylmalonicaciduria. Lancet 1984;324:1340.

145. Fowler B, Giles L, Sardharwalla IB, et al. First trimester diagnosis of methylmalonic aciduria. Prenat Diagn 1988;8:207. 146. Sachs ES, Jahoda MGJ, Kleijer WJ, et al. Impact of first-trimester chromosome, DNA, and metabolic studies on pregnancies at high genetic risk: experience with 1,000 cases. Am J Med Genet 1988;20:293. 147. van der Meer SB, Spaapen LJM, Fowler B, et al. Prenatal treatment of a patient with vitamin B12-responsive methylmalonic acidemia. J Pediatr 1990;117:923. 148. Soda H, Ohura T, Yoshida I, et al. Prenatal diagnosis and therapy for a patient with vitamin B12-responsive methylmalonic acidaemia. J Inher Metab Dis 1995;18:295. 149. Shigematsu Y, Hata I, Nakai A, et al. Prenatal diagnosis of organic acidemias based on amniotic fluid levels of acylcarnitines. Pediatr Res 1996;39:680. 150. Fowler B, Giles L, Cooper A, Sardharwalla IB. Chorionic villus sampling: diagnostic uses and limitations of enzyme assays. J Inher Metab Dis 1989;12(suppl 1): 105. 151. Coude M, Chadefaux B, Rabier D, Kamoun P. Early amniocentesis and amniotic fluid organic acid levels in the prenatal diagnosis of organic acidemias. Clin Chim Acta 1990;187:329. 152. Ampola MG, Mahoney MJ, Nakamura E, Tanaka K. Prenatal therapy of a patient with vitamin B12responsive methylmalonic acidemia. N Engl J Med 1975;293:313. 153. Zass R, Leupold D, Fernandez MA, Wendel U. Evaluation of prenatal treatment in newborns with cobalamin-responsive methylmalonic acidaemia. J Inher Metab Dis 1995;18:100. 154. Rosenblatt DS, Cooper BA, Schmutz SM, et al. Prenatal vitamin B12 therapy of a fetus with methylcobalamin deficiency (cobalamin E disease). Lancet 1985;325:1127. 155. Gahl WA, Tietze F, Bashan N, et al. Defective cystine exodus from isolated lysosome-rich fractions of cystinotic leucocytes. J Biol Chem 1982;257:9570. 156. Gahl WA, Bashan N, Tietze F, et al. Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis. Science 1982;217:1263. 157. Gahl WA, Tietze F, Bashan N, et al. Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions. Biochem J 1983;216:393. 158. Cystinosis Collaborative Research Group. Linkage of the gene for cystinosis to markers on the short arm of chromosome 17. Nat Genet 1995;10:246. 159. McDowell G, Isogai T, Tanigami A, et al. Fine mapping of the cystinosis gene using an integrated genetic and physical map of a region within human chromosome band 17p13. Biochem Mol Med 1996;58:135.

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160. Town M, Jean G, Cherqui S, et al. A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nat Genet 1998;18:319. 161. Gahl WA, Thoene JG, Schneider JA. Cystinosis. N Engl J Med 2002;347:111. 162. Gahl WA, Thoene JG, Aula PP. Lysosomal transport disorders: cystinosis and sialic acid storage disorders. In: Scriver CR, Beaudet AL, Sly WS, et al, eds. The metabolic and molecular bases of inherited disease, 7th edn. New York: McGraw-Hill, 1995:3763. 163. Gahl WA, Thoene JG, Schneider JA. Cystinosis: a disorder of lysosomal membrane transport. In: Scriver CR, Beaudet AL, Sly WS, et al, eds. The metabolic and molecular bases of inherited disease, 8th edn. New York: McGraw-Hill, 2001:5085. 164. da Silva V, Zurbrugg RP, Lavanchy P, et al. Long-term treatment of infantile nephropathic cystinosis with cysteamine. N Engl J Med 1985;313:1460. 165. Gahl WA, Reed GF, Thoene JG, et al. Cysteamine therapy for children with nephropathic cystinosis. N Engl J Med 1987;316:971. 166. Markello TC, Bernardini IM, Gahl WA. Improved renal function in children with cystinosis treated with cysteamine. N Engl J Med 1993;328:1157. 167. Broyer M, Tete MJ, Guest G, et al. Clinical polymorphism of cystinosis encephalopathy. Results of treatment with cysteamine. J Inherit Metab Dis 1996;19:65. 168. van’t Hoff WG, Gretz N. The treatment of cystinosis with cysteamine and phosphocysteamine in the United Kingdom and Eire. Pediatr Nephrol 1995;9:685. 169. Gahl WA, Balog JZ, Kleta R. Nephropathic cystinosis in adults: natural history and effects of oral cysteamine therapy. Ann Intern Med 2007;147:242.

170. Reznik VM, Adamson M, Adelman RD, et al. Treatment of cystinosis with cysteamine from early infancy. J Pediatr 1991;119:491. 171. Schneider JA, Verroust FM, Kroll WA, et al. Prenatal diagnosis of cystinosis. N Engl J Med 1974;290: 878. 172. States B, Blazer B, Harris D, Segal S. Prenatal diagnosis of cystinosis. J Pediatr 1975;87:558. 173. Steinherz R, Makov N, Narinsky R, et al. Prenatal diagnosis of cystinosis upon exposure of amniotic cells to cystine dimethyl ester. Isr J Med Sci 1985;21: 537. 174. Hall NA, Young EP. A high performance liquid chromatography method for the analysis of 35S-cystine: application to the diagnosis of cystinosis. Clin Chim Acta 1989;184:1. 175. Smith ML, Pellet OL, Cass MM. Prenatal diagnosis of cystinosis utilizing chorionic villus sampling. Prenat Diagn 1987;7:23. 176. Oshima RG, Willis RC, Furlong CE, Schneider JA. Binding assays for amino acids. The utilization of a cystine binding protein from Escherichia coli for the determination of acid-soluble cystine in small physiological samples. J Biol Chem 1974;249:6033. 177. Gahl WA, Dorfman A, Evans MI. Chorionic biopsy in the prenatal diagnosis of nephropathic cystinosis. In: Fraccaro M, Simmoni G, Brambti B, eds. First trimester fetal diagnosis. Berlin: Springer-Verlag, 1985: 260. 178. Jackson M, Young E. Prenatal diagnosis of cystinosis by quantitative measurement of cystine in chorionic villi and cultured cells. Prenat Diagn 2005;25:1045.

20

Prenatal Diagnosis of Primary Immunodeficiency Diseases Jennifer M. Puck Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, CA, USA

The immune system is a part of the general defense system that evolved to protect humans from harmful invasion by micro-organisms. The phagocytes and lymphocytes and their secreted products constitute a highly specialized and co-ordinated network responsible for selective recognition and elimination of micro-organisms that have passed through the body’s outer barriers. The most common causes of immunodeficiency worldwide are acquired. These are most often malnutrition and immunosuppression secondary to infection, not only by human immunodeficiency virus (HIV) but also by measles, tuberculosis, and other agents. In developed countries immunosuppression is caused by drugs used to combat malignancy or autoimmune or allergic diseases. Primary disorders of the immune system caused by heritable defects in specific genes are infrequent. Nonetheless, these diseases have been critical in demonstrating the roles played by specific genes and immune pathways in the development of normal immune responses. Moreover, the diagnosis, treatment, and genetic management of families with these diseases have undergone a fundamental shift in the past 15 years, with the identification and molecular cloning of more than 150 newly recognized host defense disease genes. Prenatal diagnosis of specific immunodeficiencies has now opened

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

628

up a broad range of choices for families who know they are at risk for having affected offspring, only one of which is the termination of an affected pregnancy. Neonatal treatments such as bone marrow transplantation for severe combined immunodeficiency are associated with improved outcome; in utero bone marrow transplantation has been achieved; and gene therapy has proven beneficial, although with an associated risk in some settings of leukemia due to insertional mutagenesis.

Family history There is a broad range of severity and age at presentation of inherited immunodeficiency disorders. The frequency of these disorders is largely unknown because they are rare and in some instances not recognized in infants or children who die of infections. Disorders limited to B lymphocytes are more common, but may present later in life than combined T and B lymphocyte disorders. Life-threatening immunodeficiencies such as severe combined immunodeficiency (SCID) are estimated to occur in around 1 in 50,000–100,000 births. Therefore, prenatal evaluation is generally requested in the context of an affected relative. A definitive evaluation of an affected proband in the kindred is a tremendous aid in directing fetal diagnosis. On the other hand, immunodiagnostics have become much more precise in recent years; the significance of a family history of early deaths due to infection must be appreciated and investigated, and review of an affected relative’s medical

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Prenatal Diagnosis of Primary Immunodeficiency Diseases 629

records or an autopsy report can provide important clues. When encountering a family history of individuals with recurrent infections, it is helpful to know that children with normal immune systems have an average of 6–8 respiratory infections per year for the first 10 years of life. Healthy children generally handle these infections well. In contrast, children with impaired host defenses have more severe or even fatal infections, persistent infections, and recurrences despite standard therapy. A very significant indicator of the seriousness of infections is failure to thrive. The timing of infections is also important; infants with immunodeficiency may be protected by transplacentally acquired maternal immunoglobulin G (IgG) for the first 3–6 months of life. Many children with immunodeficiency have chronic skin rashes. A number of primary immunodeficiencies occur in infants with other congeni-

tal disorders, such as developmental anomalies of the face, skeleton, heart, dentition or intestine or disorders of pigmentation and hair. The nature of the pathogens causing infections not only can strongly suggest immunodeficiency, as when an opportunistic pathogen such as Pneumocystis jirovecii is found, but also can point to the specific nature of the immune defect. The infectious agents commonly found in disorders of the various compartments of the immune system are summarized in Table 20.1. Although T cells are essential for controlling viral and fungal diseases, they also provide helper functions to B cells for effective antibody responses and macrophages for activation to kill ingested organisms. Thus, T cell disorders present as combined T and B cell immunodeficiency, with increased susceptibility to all types of bacterial infections as well as infections with viruses and fungi. Pure B cell defects produce

Table 20.1 Pathogens that cause disease when particular immune system compartments are defective Pathogen type

T cell defect

B cell defect

Macrophage

Activation defect

Complement defect

–

Neisseria infections,

granulocyte defect Bacteria

Bacterial sepsis

Streptococcus,

Staphylococcus, Pseudomonas

Staphylococcus,

other pyogenic bacterial

Haemophilus

infections Viruses

Cytomegalovirus,

Enteroviral

–

–

–

–

–

encephalitis

Epstein–Barr virus, severe varicella, chronic infections with respiratory and intestinal viruses Fungi and parasites

Candida,

Severe intestinal

Pneumocystis

Candida,

giardiasis

Nocardia, Aspergillus

Mycobacteria

Disseminated BCGosis

–

–

Disseminated

–

and severe typical and atypical mycobacterial disease Special features

Aggressive disease

Recurrent

with opportunistic

sinopulmonary

pathogens; failure

infections;

to clear infections

sepsis chronic meningitis

–

–

Autoimmunity

630

Genetic Disorders and the Fetus

recurrent sinopulmonary infections, often accompanied by bacterial septicemia and inability to mount lasting, or memory, responses that would prevent repeated illness with the same pathogen. Patients lacking mucosal antibody defenses are also particularly susceptible to invasive disease with enteroviruses, leading to chronic viral meningitis and severe gastroenteritis. Granulocyte disorders predispose to invasive staphylococcal infections because this organism is normally controlled by phagocytosis and superoxide-mediated killing in granulocytes. Macrophage defects lead to susceptibility to atypical mycobacterial infections. Finally, complement fixation has recently been recognized as an important mechanism for controlling neisserial species of bacteria, and patients with late complement component deficiencies are prone to septic arthritis, meningitis, and overwhelming sepsis with these organisms. Immunologic tests to review from probands are listed in Table 20.2. Autosomal recessive disorders affect both males and females, but low carrier frequencies make it unlikely to find affected relatives other than siblings. Important exceptions occur in cases of consanguineous matings and in population groups

that are closely inter-related or are descended from a limited ancestor pool. There are at least 11 X-linked immunodeficiencies, including Wiskott– Aldrich syndrome, X-linked chronic granulomatous disease, SCID, agammaglobulinemia, hyper-IgM syndrome, properdin deficiency, and X-linked lymphoproliferative disease. Because the ability to diagnose specific immunodeficiencies has been limited until recently, the family history may be ambiguous. An astute questioner can sometimes elicit a history of maternal male relatives who died at a young age with poor weight gain, diarrhea or pneumonia. Such patients were not infrequently empirically misdiagnosed as having cystic fibrosis instead of X-linked SCID. Furthermore, the rate of cases caused by new mutations, especially for X-linked disorders, is so significant that the majority of probands with proven X-linked immunodeficiency mutations have no history of affected male relatives. Autosomal dominant diseases of the immune system are a minority, but two important disorders are autoimmune lymphoproliferative syndrome (ALPS) and hyper-IgE syndrome, or Job’s syndrome.

Table 20.2 Immunologic tests for patients with suspected immunodeficiency Type of defect

Test

Specific aspects to note

Any immunodeficiency

Complete blood count; differential count;

Lymphocyte, neutrophil and eosinophil

platelet count

numbers, granule morphology; lymphocyte number; platelet size

Antibody deficiency

Quantitative immunoglobulins, B cell

Poor specific antibody responses to antigens such as vaccinations

number; antitetanus antibody, etc. before and after booster immunization T cell deficiency

Skin tests of delayed type hypersensitivity;

Skin test anergy cannot be diagnosed

T cell surface marker subsets

before 2 years of age; use agematched normal values for lymphocyte subsets

CD3, CD4, CD8; in vitro responses to

–

mitogens and antigens Phagocyte deficiency

Neutrophil count; neutrophil oxidative

–

function (neutrophil oxidative index or NOI) Complement deficiency

CH50 assay

–

Molecular diagnosis

Gene sequenceing

When a specific gene defect is suspected; gene sequencing is increasingly available in clinical laboratories

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Prenatal Diagnosis of Primary Immunodeficiency Diseases 631

Specific immune defects A classification of selected primary immune disorders is presented in Table 20.3. The disease classification1,2 is partially based on the reports of the International Union of Immunological Societies Committee on Primary Immunodeficiencies.3 Currently, several molecular diagnostic laboratories perform clinical diagnostic testing, including prenatal testing for several of the more common of these disorders. However, testing is costly and lags behind the number of diseases for which such tests are possible. The translation of basic discoveries into clinically available services for families depends on availability, cost, evolving diagnostic methods, laboratory regulation and certification, and ability to provide appropriate counseling before and after testing. None of the genetic immunodeficiencies discovered thus far demonstrate a single major or common mutation, such as the ∆F508 mutation in cystic fibrosis. Rather, as a rule, a great variety of mutations are observed, primarily changes of one or a few nucleotides, throughout the length of the genes and regulatory and splice sequences. Mutational hot spots at CpG dinucleotides have emerged for many of the genes, but these have not been sufficiently frequent to make single-mutation screening worthwhile. The great variety of mutations, combined with the overall rarity and broad spectrum of genes responsible for immune disorders, has meant that most prenatal diagnoses for these conditions have been conducted in a research setting. For further information about specific diseases, there are Internet resources including mutation databases for an increasing number of the diseases4–8 and the GeneTests database,9 which lists molecular diagnostic laboratories performing specialized tests. The Immune Deficiency Foundation10 and the Modell Foundation11 also provide information for physicians and families about the diagnosis and treatment of primary immunodeficiencies. Lymphocyte deficiencies

T cell and combined deficiencies Combined lymphocyte deficiencies include those with primary abnormalities in both T and B cells as well as those in which T cell defects prevent normal T cell/B cell co-operation. Infections in the

presence of these disorders do not respond to conventional treatment, and in the most severe forms (SCID), survival beyond the first year of life is rare unless the immune system can be reconstituted, such as by bone marrow transplantation. The most common form of SCID is the X-linked form, and more than 80% of SCID cases in some series are male.12 In 1993, X-linked SCID was found to be due to defects in IL2RG, the gene encoding the γ-chain of the interleukin-2 (IL-2) receptor.13,14 This transmembrane cytokine receptor protein is also part of the receptor complexes for IL-4, IL-7, IL-9, and IL-15; for this reason it is called the common γ-chain (γ-c). In X-linked SCID, B cells are usually present, but B cell function is intrinsically abnormal and specific antibody responses do not occur. Healthy carrier females can be identified by nonrandom X chromosome inactivation in their lymphocytes, but not in their granulocytes or nonlymphoid cells.15,16 This skewed X inactivation is a result of the selective disadvantage of lymphocyte precursors that have inactivated the X chromosome with an intact IL2RG gene. However, as expected with X-linked lethal disorders, new mutations are relatively common and can make predictions based on maternal X inactivation testing inaccurate; female germline mosaicism has been documented17 and women have been identified whose lymphocytes had no mutation and random X inactivation, but who passed on a germline IL2RG mutation to multiple affected offspring.17,18 As is typical of all the X-linked immunodeficiency disease genes recently identified, individual patient mutations are extremely diverse and consist primarily of changes of one to a few nucleotides; 153 different mutations were identified in a series of 240 patients in one cohort, and new mutations continue to be found.6,19,20 The best current treatment is human lymphocyte antigen (HLA)-matched bone marrow transplantation from a sibling or other relative, but most patients lack a matched related donor. Haploidentical, T cell-depleted bone marrow transplantation from a parent has been quite successful.2 Recent alternative sources of donor hematopoietic stem cells, banked umbilical cord blood and unrelated donor registries, have grown to sufficient size to make unrelated matched transplants possible for many patients. Nevertheless,

Category

Designation

Immunologic abnormalitya

632

Table 20.3 Classification of selected primary immunodeficiency diseases Gene defect; pathogenesis

Genetic locus

Prenatal diagnosis

Combined lymphocyte

X-linked severe combined immune deficiency (XSCID)

Low T cells; abnormal B cells; low Ig

IL2R (SCIDX1), Xq13.1

CVS, amnio: L, G; FB: CP

JAK3 intracellular signaling kinase defect

JAK3, 19p13.1

CVS, amnio: L, G; FB: CP

IL-7 α-chain defect

IL7RA, 5p13

CVS, amnio: L, G; FB: CP

Selective lymphocyte toxicity of purine

ADA, 20q13.11

CVS, amnio: E, L, G; FB: E

RAG1, RAG2, 11p13

CVS, amnio: L, G; FB: CP

DCLRE1C, 10p

CVS, amnio: L, G, FB: CP

Defect of γ-chain of IL-2 receptor and receptors for other cytokines, IL-4, 7, 9, 15, and 21

defects JAK3 deficiency (JAK3 SCID)

Low T cells; abnormal B cells; low Ig levels

IL-7 receptor deficiency (IL7RA SCID) Adenosine deaminase (ADA) deficiency Recombinase activating gene (RAG-1, RAG-2) deficiency Artemis recombination protein deficiency (Navajo

Low T cells; abnormal B cells; low Ig; NK cells present Low T cells and B cells; low Ig Low T cells; absent B cells; absent Ig Absent T, B cells; absent Ig; radiation sensitivity

pathway intermediates No T or B cell receptor rearrangement; blocked lymphocyte development No B or T cell rearrangement; blocked lymphocyte development

Indian SCID) SCID, autosomal recessive,

Low T cells

Unknown and rare; multiple defects

Low T cells, abnormal B cells;

Lymphocyte toxicity of purine pathway

FB: CP, CVS, amnio: G if

unknown genotype Purine nucleoside phosphorylase (PNP)

mutation known low Ig

PNP, 14q13

CVS, amnio: E, FB: E

Mutation in factors controlling RFX5, 1q

CIITA, 16p13, FB: CP

CVS, amnio: L, G

Thymocyte intracellular kinase defect;

ZAP-70, 2q12

CVS, amnio: L, G, FB: CP

intermediates

deficiency MHC class II deficiency

Low CD4 T cells, MHC II gene expression

ZAP-70 kinase deficiency

Low CD8 T cells

blocked maturation of T cells Reticular dysgenesis

Low T, B cells; low Ig

Unknown bone marrow stem cell defect

AR

FB: CP

Omenn syndrome

Low T, B cells; low Ig

RAG-1/RAG-2 or other SCID gene defects

AR, X

CVS, amnio: L, G

HIGMX, Xq25-q26

CVS, amnio: L, G

No IgG, IgA, IgE B cells

CD40L, Xq26

Potentially CVS, amnio: G

Embryologic defect of thymic

22q11.2 and rarely

CVS, amnio: L, FISH,

with residual activity X-linked hyper-IgM syndrome

Normal to high IgM; low IgA, IgG

CD40 ligand deficiency

Normal or high IgM; other

Defect of CD40 ligand, expressed on T cells; block in B cell isotype switch

antibody isotypes low DiGeorge syndrome

Normal to low T, B cells; normal to low Ig

development; variable defects (e.g. heart, parathyroid, face)

other loci

microarray

Genetic Disorders and the Fetus

optionsb

Category

Designation

Immunologic abnormalitya

Gene defect; pathogenesis

Genetic locus

Prenatal diagnosis optionsb

Antibody

X-linked

Low B cells; low to absent Ig

agamma-globulinemia

Defect of B cell-specific Bruton tyrosine

XLA, Xq22

CVS, amnio: L, G, FB: C

kinase Low B cells; low to absent Ig

Defect of cell surface µ-chain expression

IGM, 14q32.3

CVS, amnio: L, G, FB: CP

Low to absent B cells and Ig

Defects of m heavy- chain gene; l5

AR

Potentially CVS, amnio: G

AICDA, 12p13, AR

Potentially CVS, amnio G

Complex

Unknown

Complex; rare

Unknown

Vpreb gene; BLNK gene; syk gene

globulinemias, autosomal recessive Autosomal deficiency in IgG,

Selected isotype deficiency

IgA, IgE Immunoglobulin subclass

Activation-induced cytidine deaminase deficiency

One or more Ig subtypes low

Unknown defects in B cell isotype

deficiency, most commonly

expression; IgG subclass deficiencies

IgA

associated with Ig heavy- or lightchain gene deletions

Common variable immunodeficiency

Normal to low B cells; one or more Ig subtypes low

Unknown late-onset variable defects in B and T cell function and regulation

families with dominant defect in ICOS, 2q33

Hyper-IgE syndrome

High IgE, boils, pneumonia with lung cysts

STAT3 signaling defect

Dominant STAT3 defects, 171q21

CVS, amnio: L, G

Prenatal Diagnosis of Primary Immunodeficiency Diseases 633

µ-heavy-chain deficiency Other agamma-

C H A PTER 20

deficiencies

634

Category

Designation

Immunologic abnormalitya

Gene defect; pathogenesis

Genetic locus

Prenatal diagnosis optionsb

Other distinctive

Wiskott–Aldrich syndrome

Variable T, B, and Ig defects

syndromes

Defect of WASP gene involved in

WASP, Xp11.23

CVS, amnio: L, G, FB: CP

ATM, 11q22-q23

CVS, amnio: L, G, FB: CP

BLM, 15q26.1

CVS, amnio: L, G, FB: CP

XLP, Xq24-q26

CVS, amnio: L, G

FAS, 10q24; complex

CVS, amnio: G

cytoskeleton; sparse, small platelets; eczema Ataxia–telangiectasia

Variable

DNA repair defect in ATM gene; ataxia, progressive neurodegeneration; cancer; radiation sensitivity

Bloom syndrome

Normal

DNA repair defect in BLM gene; progressive neurodegeneration; cancer; radiation sensitivity

X-linked lympho-proliferative

Normal

disease Autoimmune lympho-

on Epstein–Barr virus encounter Elevated CD4−/CD8−

proliferative syndrome Immune dysregulation,

Fatal infection or immunocompromise Impaired Fas-mediated apoptosis of B and T cells; lymphadenopathy

T cells; high Ig

autoimmunity

Regulatory T cells

Defect of immune regulation

FOXP3, Xp11.23

CVS, amnio: L, G

Autoimmune poly-

Defect of immune tolerance

AIRE, 21q22.3, AR

Potentially CVS, amnio: G

polyendocrinopathy, enteropathy, X-linked IPEX Autoimmune poly-endocrinopathy

endocrinopathy, candidiasis, and ectodermal dystrophy

and AD forms

Genetic Disorders and the Fetus

Table 20.3 Continued

Category

Designation

Immunologic abnormalitya

Gene defect; pathogenesis

Genetic locus

Prenatal diagnosis optionsb

Phagocyte disorders

Chronic granulomatous

Normal

disease (CGD)

Impaired killing of ingested organisms

CYBB (gp91phox),

due to defects in four genes encoding

Xp21.1, CYBA

enzymes of cytochrome oxidase system

(p22phox),

CVS, amnio: L, G

16q24.1, NCF1 7q11.23, NCF2 (p67phox), 1q25 Leukocyte adhesion type 1

Normal

deficiency (LAD1)

Defects of CD18 or other leukocyte

CD18, 21q22.3

CVS, amnio: L, G, FB: CP

surface proteins required for motility, Normal

Defects of fucose glycosylation

FUCT1, 11p11.2

CVS, amnio: L, G

Normal

Defect of CHM gene causing faulty

CHS, 1q42-q44

CVS, amnio: L, G, ?CP,

deficiency (LAD2) Chediak–Higashi syndrome

FB: CP

lysosomal assembly, giant cytoplasmic granules Complement disorders

Individual component deficiencies

Normal

C1, C2, C3, C4 deficiencies, autoimmunity

AR: chromososmes6p,

and pyogenic infections; C3, C5–9 and

1q, etc.; X:

properdin deficiencies: Neisseria

properdin

Unknown

infections a

Ig, Immunoglobulin levels.

b

Prenatal diagnosis options.

amnio, amniocyte sample; CP, cellular phenotyping (leukocyte numbers, cell surface characteristics or in vitro function); CVS, chorionic villus sample; E, enzyme or biochemical assay; FB, fetal blood sample; FISH, fluorescence in situ hybridization; G, genotyping (i.e. specific mutation detection): L, linked polymorphic marker analysis.

Prenatal Diagnosis of Primary Immunodeficiency Diseases 635

adherence, and endocytosis Leukocyte adhesion type 2

C H A PTER 20

(p47phox),

636

Genetic Disorders and the Fetus

post-transplant complications include graftversus-host disease, failure to make adequate antibodies and necessity for long-term immunoglobulin replacement. Some late post-transplant patients have autoimmune diseases due to lymphocyte dysregulation, poor growth, chronic lung disease and gastrointestinal disease, but for the most part SCID survivors of transplantation are healthy and some are now having their own children. Gene therapy has also been piloted in X-linked SCID and adenosine deaminase deficient (ADA) SCID with success, making this the first human disease to be cured with gene therapy as the sole treatment.21,22 The group of Alain Fischer in Paris treated 10 infants with mutation-proven X-linked SCID by aspirating their bone marrow, enriching for stem cells, culturing the cells with activating cytokines and a retrovirus vector carrying a correct copy of the IL2RG cDNA, and then reinfusing the autologous cells into the infants. In all but one case, the cells found their way to the bone marrow and grew and differentiated into normal, functional T lymphocytes; other hematopoietic cell lineages were also generated containing the retroviral provirus. Both T and B cell function improved. However, 2 years or more after treatment, leukemia developed in four recipients of XSCID gene therapy in France and an additional child similarly treated in England, and one child died. Their leukemias were found to have been caused by insertional mutagenesis.23 The IL2RG retroviral vector, when integrated into genomic DNA near a gene encoding a growth factor such as LMO-2, caused inappropriate expression and unrestrained clonal expansion of cells.24 No leukemias have yet been reported in over a dozen patients who have received ADA gene therapy.24 Prenatal diagnosis can be performed by linkage analysis or by specific mutation detection on chorionic villus samples (CVS) or amniocyte DNA.16,25 Fetal blood sampling has also been used, as lymphocytopenia, low numbers of cells bearing the T cell marker CD3, and poor T cell blastogenic responses to mitogens can be definitively demonstrated in affected fetuses by week 17 of gestation.26 These options should be weighed against testing at birth for families who would not terminate an affected pregnancy. Regardless of whether prenatal testing is undertaken, education and counseling

should emphasize early definitive diagnosis and transplantation for affected infants. Infants treated immediately after birth have more rapid engraftment, fewer serious infections, less serious graftversus-host disease, and shorter hospitalizations than those whose transplants are delayed.27 The better outcomes of patients diagnosed perinatally argue in favor of universal newborn screening for SCID, which is now possible using dried blood samples and an assay for T cell receptor excision circles developed by Chan and Puck.28 Absence of these circular DNA by-products of T cell receptor rearrangement, detectable by PCR, identify SCID regardless of genotype. SCID screening is now being piloted in several states. The use of prenatal diagnosis for X-linked SCID was studied by Puck et al.,25 who found that the great majority of families at risk for an affected pregnancy desired prenatal testing, whether or not termination of pregnancy was a consideration. In fact, parents chose to terminate the pregnancy in only two of 13 instances of a predicted affected male fetus. To prepare for optimal treatment of an affected newborn, families and their medical providers selected bone marrow transplant centers, had HLA testing of family members, and even began a search for a matched, unrelated bone marrow donor. One family chose an experimental in utero bone marrow transplant, which was successful29 (see below). The concept of prenatal treatment for SCID is controversial. Theoretical advantages of in utero treatment include early reconstitution, an intrauterine environment protected from infections, and the possibility of introducing normal bone marrow stem cells at the gestational age when fetal hematopoiesis is shifting from fetal liver to bone marrow. Previous attempts at human in utero bone marrow transplantation were severely compromised by technologic limitations, septic complications, and the lack of methods to remove from the graft population the mature T cells capable of reacting to fetal tissues and causing graft-versus-host reactions. In at least three patients, these difficulties have been overcome.29–31Fetuses affected with X-linked SCID have been infused intraperitoneally with haploidentical T cell-depleted CD34+ parental bone marrow cells between 17 and 20 weeks gestation. Infants have been born with engrafted,

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functional T cells and have, to date, done at least as well as postnatally transplanted patients. However, the risks of in utero treatment must be weighed against the excellent results of hematopoietic cell transplantation in SCID newborns who are diagnosed prior to developing infectious complications. Over 16 genes are now known to be defective in SCID, including intracellular kinase JAK3, a downstream mediator of signals from γ-c.32 Males and females may have autosomal recessive SCID immunologically identical to XSCID but caused by JAK3 protein defects. Prenatal diagnosis ruling out JAK3 SCID by mutation analysis has been performed on DNA from a CVS.33 The IL-7 receptor α-chain gene is also a SCID disease gene.34 Adenosine deaminase (ADA) deficiency, the first genetic defect associated with SCID, is less than half as common as X-linked SCID. ADA is found in all tissues and is important in purine metabolism. The lack of this enzyme, which is most abundant in lymphocytes, causes intracellular accumulation of toxic levels of purine intermediates, particularly deoxyadenosine.35,36 Characteristic skeletal abnormalities of the ribs and hips are seen, along with extremely low numbers of T and B cells. Deafness and cognitive impairments are more frequent than in other SCID genotypes in which lymphocytelimited genes are mutated. Partial deficiency of ADA due to mutations that preserve some enzyme activity can cause milder forms of combined immunodeficiency presenting in childhood or even adulthood with declining T cell numbers. Diagnosis at any age depends on the measurement of low ADA enzyme activity and high levels of circulating deoxyadenosine. Although HLA-matched bone marrow transplantation is the treatment of choice for severe ADA deficiency, haploidentical T celldepleted transplants and enzyme replacement with ADA coupled to polyethylene glycol (PEG-ADA) are used for patients without a matched sibling donor, and experimental gene therapy has also been successful in a protocol similar to that described above for X-linked SCID, except that the patients also received cytoreductive chemotherapy before reinfusion of their autologous gene-corrected cells.37 Aiuti et al. recently reported on the successful long-term outcome of gene therapy for ADA SCID in 10 children.37a

Prenatal diagnosis of ADA deficiency is facilitated by the ubiquitous expression of the enzyme; CVS and amniocyte samples have successfully yielded prenatal determinations.38–40 However, as discussed by Hirschhorn,38 the variable enzyme activities in carrier parents make it important to relate fetal enzyme activities to those of all available family members. ADA activity may also vary in cultured cells. Ambiguous results from amniocyte testing could be clarified with a subsequent fetal blood sample in which red cell and lymphocyte enzyme levels can be measured, in addition to determining lymphocyte number. DNA-based prenatal diagnosis, by either linked markers or specific mutation detection, has also been accomplished. With the discovery of disease genes for X-linked immunodeficiencies and an increasing number of autosomal forms of SCID, it is now possible to define the genetic defect at the molecular level in around 90% of affected patients. This is a marked contrast to the situation two decades ago, when 80% of patients with SCID had undetermined genetic lesions. Disease genes for autosomal recessive SCID include recombinase-activating genes RAG1 and RAG2 that are required for the DNA rearrangements of variable (V), diversity (D), and joining (J) domains of the T cell receptor and immunoglobulin genes. RAG1 and RAG2 are adjacent to each other on chromosome 11p13.41 Some ethnic groups have an increased incidence of autosomal recessive SCID, such as the Amish, who have both ADA and RAG1 mutations,42 and Navajo Native Americans of Athabascan origin.43 The gene for Athabascan SCID has now been identified to be another protein involved T and B cell antigen receptor gene recombination as well as DNA repair.44 Prenatal diagnosis of the Athabascan SCID mutation can be readily performed.45 The recurrence risk for couples who have had an infant with non-X SCID is 25%. In the absence of specific molecular diagnostic studies, the prenatal diagnosis of SCID of unknown genotype is possible through fetal blood sampling after 17 weeks’ gestation. There are available data on normal fetal blood leukocytes.26,46–48 Potential abnormalities that can be expected in a fetus at risk can be predicted from careful analysis of the immunologic profile of the affected proband.

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Purine nucleoside phosphorylase (PNP) deficiency, an extremely rare disorder, is also associated with immunodeficiency involving both T and B cells. Although severe cases may present in infancy, PNP immunodeficiency is usually more mild than SCID, coming to medical attention later in childhood. Neurologic abnormalities, including spasticity, hypotonia, and developmental delay, are prominent in PNP deficiency and may be recognized first. As with ADA, PNP is found in all tissues, including CVS cells and amniocytes. Diagnosis can be made by assay of levels of the enzyme.49 Another very rare human SCID disease first recognized in Mennonites in 1994 is caused by lack of a T cell-specific signaling kinase called ZAP-70 kinase, or ζ-chain (a T cell-receptor component) associated protein kinase. These patients have natural killer (NK) cells but no functional T cells, even though T cells with surface expression of CD4 are present.50,51 Autosomal recessive mutations in the ZAP-70 gene, resulting in deficient expression of ZAP-70 protein, interfere with the thymic development of CD8 T cells and antigen activation in CD4 T cells. Despite the presence of B cells and serum immunoglobulins, specific antibody responses are impaired. Major histocompatibility complex class II (MHC-II) nonexpression has also been associated with moderate to severe immunodeficiency, originally classified as a form of “bare lymphocyte syndrome.”52,53 Patients present from early infancy to childhood with normal numbers of T and B cells but a preponderance of CD8 T cells, as opposed to the normal CD4/CD8 ratio of 2/1. Immunoglobulins are decreased, specific antibody production is poor, and a variety of severe bacterial and opportunistic infections can occur. Although no abnormalities of the MHC-II genes themselves have been found, three different genes regulating MHC-II expression, CIITA and RFX5 can be defective in some of the patients with this form of immunodeficiency,54–56 and other transcription factors involved in this regulatory process are likely to be mutated in additional patients. Although fetal blood sampling to assess fetal lymphocyte expression of MHC-II has been used for prenatal diagnosis,57 diagnosis of specific gene defects by linkage or specific mutation detection would be definitive

and could be performed on CVS or amniocyte DNA. Hyper-IgM syndrome was originally thought to be a disorder of B cells because in affected patients isotypes fail to switch from IgM to IgG, IgA or IgE. Affected patients sometimes, but not always, have very high levels of IgM, which gave the disease its name. Secondary or booster B cell-antibody responses are absent. However, Candida and Pneumocystis infections in these patients suggested a T cell component to the disease now known to be caused by deficient CD40 ligand (CD40L), a receptor expressed on activated T cells that is critical for stabilizing T–B interactions and activating B cell isotype switching.58,59 Prenatal diagnosis has been performed on CVS DNA by means of a highly informative dinucleotide repeat polymorphism in the 3’ untranslated region of the CD40L gene, confirmed by specific mutation detection.60 A spectrum of X-linked and autosomal recessive diseases similar to that seen in CD40L deficiency is caused by defects in additional genes governing T cell–B cell interactions. These genes include CD40, NEMO, and a gene encoding an activation-induced cytidine deaminase (AID), but additional cases exist without mutations in these genes.61 Patients are effectively treated with antibiotics and γglobulin replacement therapy but do experience morbidity and are at risk for premature death. Finally, additional well-recognized immunodeficiency syndromes exist for which the specific genetic causes continue to be found. DiGeorge syndrome, the most common interstitial deletion or copy number variant, is associated with variable occurrence of multiple anomalies of the fetal third and fourth pharyngeal pouch structures, including thymic dysplasia or aplasia.62 Although facial clefts, hypocalcemia due to hypoparathyroid maldevelopment, and cardiac defects may be more striking early in life, variable degrees of immunodeficiency, from mild T and B cell defects to autoimmune phenomena to SCID, are commonly seen. Cytogenetics with fluorescence in situ hybridization (FISH) can identify the common deletion at chromosome 22q11.2 found in 90% of affected patients (see Chapter 8).63 The transcription factor TBX1 located in the DiGeorge critical region has been found to harbor intragenic mutations that cause DiGeorge syndrome in humans.64 Fetal sonogra-

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phy and echocardiography have been useful in evaluating the nonimmune aspects of this syndrome. The chromosome 22 microdeletions can be evaluated by FISH or microarray (see Chapter 10) in CVS cells or amniocytes for prenatal diagnosis. A minority of patients with DiGeorge sequence do not have chromosome 22 deletions but instead may have microdeletions in chromosome 10p13p14.65 The genetic heterogeneity of this syndrome makes study of family members a necessary part of any prenatal evaluation. Moreover, highly variable expressivity makes the interpretation of a prenatally diagnosed abnormality complex, particularly in families with both severely affected and mildly affected members. Antibody deficiencies The most common complications of antibody deficiencies are recurrent sinopulmonary infections and septicemias with encapsulated bacteria. The most severe defect in this category is agammaglobulinemia, which is much more often seen in males, frequently with an X-linked inheritance pattern. The disease gene for X-linked agammaglobulinemia (XLA) was identified in 1993 as encoding Btk,66,67 for Bruton tyrosine kinase, named after the discoverer of human immunodeficiency due to agammaglobulinemia. B cells lacking Btk fail to develop from pre-B cells in the bone marrow. Diagnosis, including prenatal diagnosis,68 is made by finding extremely low or absent immunoglobulins and few to no B cells; specific mutation detection or measurement of Btk kinase activity can confirm the genetic cause in patients without an X-linked family history. Lifelong γ-globulin replacement keeps many patients free of infection when instituted early. However, pulmonary insufficiency due to recurrent pneumonias and bronchiectasis limits lifespan, and a particularly difficult complication is the development of chronic enteroviral meningitis. Prenatal detection of XLA was first accomplished by fetal blood enumeration of B cells,68 but now specific mutation detection or linked markers are used to make a fetal DNA diagnosis. Additional gene defects that cause agammaglobulinemia have been found in males and females without Btk mutations. These include defects in the autosomal immunoglobulin m heavy chain

locus itself.69 Patients have a clinical picture very similar to individuals with XLA, with complete absence of B cells, indicating that intact membrane-bound µ-chain expression is essential for B cell maturation. Prenatal diagnosis is possible by DNA-based methods or fetal blood phenotype. Other diseases characterized by antibody defects are IgA deficiency, other immunoglobulin subclass deficiencies, and common variable immunodeficiency (CVID). Although relatively common, IgA deficiency is complex in its inheritance pattern, and some relatives of patients with IgA deficiency have CVID. Some patients with no IgA are entirely without symptoms. CVID, often presenting in late childhood to adulthood, can present in many forms: hypoglobulinemia or agammaglobulinemia, subclass deficiency or dysregulation of the immune system with autoantibodies, lymphadenopathy, splenomegaly and/or hemolytic anemia, pernicious anemia, and other autoimmune diseases. Patients with CVID, especially women, are at increased risk for neoplasms, particularly lymphomas. In rare families, mutations in ICOS and TACI genes have been found, but CVID is genetically heterogeneous70 and the great majority of cases are not currently attributable to particular gene defects, making prenatal diagnosis impossible. Phagocyte deficiencies Patients with chronic granulomatous disease (CGD) present with lymphadenopathy, high lymphocyte counts, and recurrent infections, as listed in Table 20.1. The X-linked form of CGD, accounting for two-thirds of cases, was one of the first human diseases for which the disease gene was found by positional cloning.71 Three autosomal recessive gene defects are also recognized to cause CGD. All four genes encode proteins that are part of the oxidative killing pathway for ingested microorganisms. The disease is diagnosed by demonstration of failure of the normal respiratory burst upon activation of neutrophils (see Table 20.2). There is no specific therapy for CGD other than bone marrow transplantation, which can be curative if a matched donor is available, and stem cell gene therapy which has been tried experimentally. Continuously administered antibiotics have greatly reduced the frequency of severe infections, but

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autoimmune disease is a frequent late complication. Linkage and specific mutation diagnosis of fetal samples are used for prenatal diagnosis. Leukocyte adhesion deficiency (LAD) is a rare phagocyte defect of patients whose neutrophils fail to mobilize and migrate to sites of tissue injury. Delayed separation of the umbilical cord in infancy is a clue. Severe scarring infections of skin and soft tissue, gingivitis, and systemic bacterial infections occur. The original gene defect associated with the majority of cases is in the gene encoding CD18, the β-chain common to several leukocyte surface integrin complexes. Additional very rare defects have been associated with the LAD, including defects in the fucosylation of selectin ligands in conjunction with developmental and growth retardation.72 DNA-based prenatal diagnosis is possible for families with known genotypes. Chediak– Higashi syndrome (CHS) is an autosomal recessive disorder characterized by giant lysosomal granules in phagocytes, melanocytes, and other cells, including amniotic cells and chorionic villus cells. Patients have hypopigmentation and recurrent pyogenic infections that do not respond well to conventional therapy. One genetic locus for CHS is the LYST gene that encodes a large protein involved in either vesicle fusion or fission.73,73a However, genetic heterogeneity has been demonstrated and thus, prenatal diagnosis by linkage to chromosome 1q markers is not possible without having confirmed that CHS in the family is due to a chromosome 1q mutation. Prenatal diagnosis has been performed by demonstrating the abnormal granulocytes in fetal blood and by DNA analysis.74,75 Complement deficiencies The complement system involves more than 30 proteins encoded throughout the genome, with important clusters on chromosome 1q and within the MHC region on 6p. Because deficiencies of nearly all these proteins have been described,76 the topic is beyond the range of this chapter. Complement deficiencies can cause increased susceptibility to infection, rheumatic disorders or angio-edema. Defects in the terminal lytic components of complement, C5 through C9, and alternative pathway components predispose patients to invasive neisserial infections. In early component defects, C1, C4,

and C2, recurrent bacterial infections are seen. Prenatal testing for complement deficiencies has not been reported. Unclassified deficiencies Wiskott–Aldrich syndrome (WAS) is characterized by thrombocytopenia with small dysfunctional platelets, eczema, and variable immunodeficiency in males. Its inheritance is X-linked recessive. Infants present with petechiae or bleeding; rashes develop in the first 1–2 years of life; increased susceptibility to pneumonias, sepsis, and chronic viral infections as well as autoimmune disease are typically seen in childhood; and survivors to young adulthood have a high risk of lymphoma.77,78 In 1994, the disease gene was identified and named WASP for WAS protein.79 WASP can associate with actin in lymphoid cells and is involved in transmitting intracellular signals.80 Some patients have a mild phenotype, with thrombocytopenia and little or no immunodeficiency; part of this variability is due to the location and type of mutation within the WASP gene. The gene mutations are tracked on a centralized database.4 HLAmatched bone marrow transplantation is the treatment of choice; treatment decisions for affected boys without a matched donor are complicated by the variable expressivity of the disease. Prenatal diagnosis can be performed by linked marker analysis or mutation detection in fetal DNA.81 Ataxia–telangiectasia is characterized by progressive neurologic impairment with ataxia, variable immunodeficiency, and ocular and cutaneous telangiectasias. Increased frequency of solid tumors and lymphoreticular malignancies is well documented, and patients are hypersensitive to radiation. The disease gene ATM (“ataxia–telangiectasia mutated”) is a member of a family of phosphatidylinositol-3-kinase genes involved in cell cycle control.82 The important role of the ATM protein in DNA damage repair and lymphocyte DNA recombination helps explain the clinical features of this disease, and heterozygotes for ATM defects may also be at increased risk for cancer. Prenatal diagnosis was carried out in the past by analysis of new DNA synthesis in response to radiation of amniocytes,83 but specific mutation diagnosis can provide more definitive information. A related DNA breakage syndrome with cancer predisposi-

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tion and accompanying variable combined immunodeficiency is Bloom syndrome.84,85 Dominant interfering mutations in the intracellular signal transmitter STAT3 have been found to underlie hyper-IgE syndrome, also called Job syndrome for the recurrent boils noted in the patients first described.86,87 This is a multisystem disorder in which severe infections include pneumonia followed by pneumatocele formation. IgE antibody levels are extremely elevated. Non-immune features include distinctive facies with a wide nose and thickened skin, frequent bone fractures, hyperextensible joints and delayed shedding of primary dentition. Another X-linked immunodeficiency disease is X-linked lymphoproliferative syndrome (XLP). Affected males have no consistent immune dysfunction until they encounter Epstein–Barr virus (EBV). Most then die from severe mononucleosis, while a range of abnormalities subsequently develop in survivors, from aplastic anemia to B cell aplasia to B cell lymphomas. T and NK cell abnormalities as well as hypogammaglobulinemia have been noted after EBV infection. When the gene was mapped to Xq25q26, prenatal diagnosis by linkage could be performed.88,89 Now mutations can be sought in the SH2D1A gene, an SH2 domain-containing adaptor protein found in lymphocytes.90 Bone marrow transplantation has been performed presymptomatically on boys who have been determined to have inherited XLP because in retrospective series half of the initial EBV infections were fatal. Finally, gene defects can cause heritable disorders of regulation of immune responses or autoimmune disease. The first such genetic disorder, autoimmune lymphoproliferative syndrome (ALPS), is a consequence of defective apoptosis or programmed cell death.91–93 ALPS is most often caused by heterozygous dominant, interfering mutations in the cell surface receptor Fas, an important mediator of lymphocyte apoptosis. Children with ALPS have lymphadenopathy, autoimmunity, and expansion of a normally rare population of CD4−CD8− T cells. They also have impaired T–B cell apoptosis in vitro. Cellular apoptosis defects are inherited in families with ALPS as autosomal dominant traits, but the development of overt autoimmunity may depend on additional factors. Rare infants with homozygous Fas defects have developed hydrops due to severe

autoimmune hemolytic anemia in utero. Another inherited autoimmune condition is X-linked immunodysregulation, polyendocrinopathy and enteropathy (IPEX), in which defects of the transcriptional regulator FOXP3 lead to absence of regulatory T cells and in turn, neonatal diabetes, severe diarrhea, rashes and early death.94 A third well-recognized inherited autoimmune disorder is autoimmune polyendocrinopathy with candidiasis and ectodermal dysplasia (APECED or APS1), caused by autosomal recessive defects in the transcription factor AIRE, which regulates development of tolerance to self antigens.95–96 This disease is characterized by hypoparathyroidism, adrenal insufficiency or other endocrine deficiencies plus variable presence of alopecia, candidiasis and recurrent infections. Mutation diagnosis can be performed on prenatal DNA samples for each of these diseases. Notably, fetal-specific methylation of the AIRE gene, encoded on chromosome 21q22.3, has been claimed to constitute a marker that may assist diagnosis of Down syndrome using maternal peripheral blood (see Chapter 30).97

Conclusion Whenever an infant is born who is known or suspected to be at risk for an inherited host defense defect, immunologic evaluation should be performed, as outlined in Table 20.2. Until the immune status of the infant is clear, he or she should be protected from exposure to infection and iatrogenic administration of potentially lethal treatments. Live vaccines should not be given to such infants until diagnostic studies have ruled out immunodeficiency. Similarly, only irradiated blood products should be given, to avoid transfusion-mediated graft-versus-host disease from transfused lymphocytes, which cannot be eliminated when patients lack functional T cells of their own. Prophylactic measures including administration of intravenous gammaglobulin (IVIG) and antibiotics should be used until definitive diagnosis is made and treatment instituted.

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31. Touraine JL. Perinatal fetal-cell and gene therapy. Int J Immunopharmacol 2000;22:1033 32. Macchi P, Villa A, Giliani S, et al. Mutations of JAK3 gene in patients with autosomal severe combined immunodeficiency (SCID). Nature 1995;377:65. 33. Schumacher RF, Mella P, Lalatta F, et al. Prenatal diagnosis of JAK3 SCID. Prenat Diagn 1999;19:653. 34. Puel A, Ziegler SF, Buckley RH, Leonard WJ. Defective IL7R expression in T(−)B(+)NK(+) severe combined immunodeficineyc. Nat Genet 1998:20:394. 35. Hirschhorn R. Adenosine deaminase deficiency. Immunol Rev 1991;3:45. 36. Hershfield MS, Mitchell BS. Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The metabolic basis of inherited disease, 7th edn. New York: McGraw-Hill, 1995:1725. 37. Aiuti A, Cassani B, Andolfi G, et al. Multilineage hematopoitic reconstitution without clonal selection in ADASCID patients treated with stem cell gene therapy. J Clin Invest 2007;117:2233. 37a. Aiuti A, Cattaneo F, Galimberti S, et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N Engl J Med 2009;360:447. 38. Hirschhorn R. Prenatal diagnosis of adenosine deaminase deficiency and selected other immunodeficiencies. In: Milunsky A, ed. Genetic disorders and the fetus: diagnosis, prevention, and treatment, 3rd edn. Baltimore, MD: Johns Hopkins University Press, 1992:453. 39. Aitken DA, Gilmore DH, Frew CA, et al. Early prenatal investigation of a pregnancy at risk of adenosine deaminase deficiency using chorionic villi. J Med Genet 1986;23:52. 40. Perignon JL, Durandy A, Peter MO, et al. Prenatal diagnosis of inherited severe immunodeficiencies linked to enzyme deficiencies. J Pediatr 1987;111:595. 41. Schwarz K, Gauss G, Ludwig L, et al. RAG mutations in human B cell-negative SCID. Science 1996;274:97. 42. Strauss KA, Puffenberger EG, Bunin N, et al. Clinical application of DNA microarrays: molecular diagnosis and HLA matching of an Amish child with severe combined immune deficiency. Clin Immunol 2008;128: 31. 43. Jones JF, Ritenbaugh CK, Spence MA, et al. Severe combined immunodeficiency among the Navajo. I. Characterization of phenotypes, epidemiology, and population genetics. Hum Biol 1991;63:699. 44. Moshous D, Callebaut I, de Chasseval R, et al. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell 2001;105:177.

45. Li L, Zhou Y, Wang J, et al. Prenatal diagnosis and carrier detection for Athabascan severe combined immunodeficiency disease. Prenat Diagn 2002;22:763. 46. Rainaut M, Pagniez M, Hercent T, et al. Characterization of mononuclear cell subpopulations in normal fetal peripheral blood. Hum Immunol 1987;18:331. 47. Linch DC, Beverly PCL, Levinsky RJ, et al. Phenotypic analysis of fetal blood leukocytes: potential for prenatal diagnosis of immunodeficiency disorders. Prenat Diagn 1982;2:211. 48. Durandy A, Oury C, Griscelli C, et al. Prenatal testing for inherited immune deficiencies by fetal blood sampling. Prenat Diagn 1982;2:109. 49. Kleijer WJ, Hussaarts-odijk LM, Pijpers L, et al. Prenatal diagnosis of purine nucleoside phosphorylase deficiency in the first and second trimesters of pregnancy. Prenat Diagn 1989;9:401. 50. Elder ME, Lin D, Clever J, et al. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science 1994;264:1596. 51. Chan AC, Kadlecek TA, Elder ME, et al. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 1994;264:1599. 52. Steimle V, Reith W, Mach B. Major histocompatibility complex class II deficiency: a disease of gene regulation. Adv Immunol 1996;61:327. 53. Klein C, Lisowska-Grospierre B, Le Deist F, et al. Major histocompatibility complex class II deficiency: clinical manifestations, immunologic features, and outcome. J Pediatr 1993;123:921. 54. Steimle V, Otten LA, Zufferey M, et al. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome). Cell 1993;75:135. 55. Zhou H, Glimcher LH. Human MHC class II gene transcription directed by the carboxyl terminus of CIITA, one of the defective genes in type II MHC combined immune deficiency. Immunity 1995;2: 545. 56. Durand B, Sperisen P, Emery P, et al. RFXAP, a novel subunit of the RFX DNA binding complex is mutated in MHC class II deficiency. EMBO J 1997;16:1045. 57. Durandy A, Cerf-Bensussan N, Dumez Y, et al. Prenatal diagnosis of severe combined immunodeficiency with defective synthesis of HLA molecules. Prenat Diagn 1987;7:27. 58. Notarangelo LD, Duse M, Ugazio AG. Immunodeficiency with hyper-IgM (HIM). Immunodefic Rev 1992;3:101. 59. Conley ME, Larche M, Bonagura VR, et al. Hyper-IgM syndrome associated with defective CD40-mediated B cell activation. J Clin Invest 1994;94:1404.

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60. DiSanto JP, Markiewicz S, Gauchat J-F, et al. Prenatal diagnosis of X-linked hyper IgM syndrome. N Engl J Med 1994;330:969. 61. Imai K, Catalan N, Plebani A, et al. Hyper-IgM syndrome type 4 with a B lymphocyte-intrinsic selective deficiency in Ig class-switch recombination. J Clin Invest 2003;112:136. 62. Muller W, Peter HH, Kallfelz HC, et al. The DiGeorge sequence. II. Immunologic findings in partial and complete forms of the disorder. Eur J Pediatr 1989;149:96. 63. Gong W, Emanuel BS, Collins J, et al. A transcription map of the DiGeorge and velo-cardio-facial syndrome minimal critical region on 22q11. Hum Mol Genet 1996;5:789. 64. Yagi H, Furutani Y, Hamada H, et al. Role of TBX1 in human del22q11.2 syndrome. Lancet 2003;362:1366. 65. Daw SCM, Taylor C, Kraman M, et al. A common region of 10p deleted in DiGeorge and velocardiofacial syndromes. Nat Genet 1996;13:458. 66. Vetrie D, Vorechovsky I, Sideras P, et al. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature 1993;361:226. 67. Tsukada S, Saffran DC, Rawlings DJ, et al. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 1993;72: 279. 68. Durandy A, Griscelli C. Prenatal diagnosis of severe combined immunodeficiency and X-linked agammaglobulinemia. Birth Defects 1983;19:125. 69. Yel L, Minegishi Y, Coustan-Smith E, et al. Mutations in the m heavy-chain gene in patients with agammaglobulinemia. N Engl J Med 1996;335:1486. 70. Schäffer AA, Salzer U, Hammarström L, Grimbacher B. Deconstructing common variable immunodeficiency by genetic analysis. Curr Opin Genet Dev 2007;149:201. 71. Orkin SH. Molecular genetics of chronic granulomatous disease. Annu Rev Immunol 1989;7:277. 72. Etzioni A. Leucocyte adhesion molecular deficiencies: molecular basis, clinical findings, and therapeutic options. Adv Exp Med Biol 2007;601:51. 73. Nagle DL, Karim MA, Woolf EA, et al. Identification and mutation analysis of the complete gene for Chediak– Higashi syndrome. Nat Genet 1996;14:307. 73a. Kaplan J, de Domenico I, Ward DM. Chediak–Higashi syndrome. Curr Opin Hematol 2008;15:22. 74. Diukman R, Tanigawara S, Cowan MJ, et al. Prenatal diagnosis of Chediak–Higashi syndrome. Prenat Diagn 1992;12:877. 75. Durandy A, Breton-Gorius J, Guy-Grand D, et al. Prenatal diagnosis of syndromes associating albinism and immune deficiencies (Chediak–Higashi syndrome and variant). Prenat Diagn 1993;13:13.

76. Sullivan KE, Winkelstein JA. Genetically determined deficiencies of complement. In: Ochs HD, Smith CIE, Puck JM, eds. Primary immunodeficiency diseases: a molecular and genetic approach, 2nd edn. New York: Oxford University Press, 2007:589. 77. Sullivan KE, Mullen CA, Blaese RM, et al. A multi-institutional survey of the Wiskott–Aldrich syndrome. J Pediatr 1994;125:876. 78. Sullivan KE. Genetic and clinical advances in Wiskott– Aldrich syndrome. Curr Opin Pediatr 1995;7:683. 79. Derry JM, Ochs HD, Francke U. Isolation of a novel gene mutated in Wiskott–Aldrich syndrome. Cell 1994;78:635. 80. Nonoyama S, Ochs HD. Wiskott–Aldrich syndrome. Curr Allergy Asthma Rep 2001;1:430. 81. Wengler GS, Notarangelo LD, Giliani S, et al. Mutation analysis in Wiskott Aldrich syndrome on chorionic villus DNA. Lancet 1995;346:641. 82. Savitsky K, Bar-Shira A, Gilad S, et al. A single ataxia– telangiectasia gene with a product similar to PI-3 kinase. Science 1995;268:1749. 83. Jaspers NG, Scheres JM, Dewit J, et al. Rapid diagnostic test for ataxia–telangiectasia. Lancet 1961;2:473. 84. German J. Bloom syndrome: a mendelian prototype of somatic mutational disease. Medicine (Baltimore) 1993;72:393. 85. Ellis NA, Groden J, Ye TZ, et al. The Bloom’s syndrome gene product is homologous to RecQ helicases. Cell 1995;83:655. 86. Holland SM, DeLeo FR, Elloumi HZ, et al. STAT3 mutations in the hyper-IgE syndrome. N Engl J Med 2007;357:1608. 87. Minegishi Y, Saito M, Tsuchiya S, et al. Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome. Nature 2007;448:1058. 88. Skare J, Milunsky A, Byron K, et al. Mapping the X-linked lymphoproliferative syndrome. Proc Natl Acad Sci USA 1987;84:2015. 89. Skare J, Madan S, Glaser J, et al. First prenatal diagnosis of X-linked lymphoproliferative disease. Am J Med Genet 1992;44:79. 90. Schuster V, Kreth HW. X-linked lymphoproliferative disease is caused by deficiency of a novel SH2 domaincontaining signal transduction adaptor protein. Immunol Rev 2000;178:21. 91. Puck JM, Sneller MC. ALPS: an autoimmune human lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis. Semin Immunol 1997;9: 77. 92. Rieux-Laucat F, Le Deist F, Hivroz C, et al. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 1995;268:1347. 93. Fisher GH, Rosenberg FJ, Straus SE, et al. Dominant interfering Fas gene mutations impair apoptosis in a

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human autoimmune lymphoproliferative syndrome. Cell 1995;81:935. 94. Wilden RS, Ramsdell F, Peake J, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nature Genet 2001;27:18. 95. Nagamine K, Peterson P, Scott HS, et al. Positional cloning of the APECED gene. Nature Genet 1997;17:393.

96. DeVoss JJ, Anderson MS. Lessons on immune tolerance from the monogenic disease APS1. Curr Opin Genet Dev 2007;17:193. 97. Old RW, Crea F, Puszyk W, Hulten MA. Candidate epigenetic markers for non-invasive prenatal diagnosis of Down syndrome. Reprod Biomed Online 2007; 15:227.

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Prenatal Diagnosis of the Hemoglobinopathies John M. Old National Haemoglobinopathy Reference Laboratory, Oxford Haemophilia Centre, Churchill Hospital, Oxford, UK

The hemoglobinopathies are a diverse group of autosomal recessive disorders characterized by either the synthesis of a structurally abnormal globin (the hemoglobin variants) or the reduced synthesis of one or more of the globin chains (the thalassemias). Together, they form the most common single-gene disorder in the world population, and they are a serious public health problem in many countries. Although there is no definitive cure for the hemoglobinopathies, the methods of clinical management have improved considerably during the last few years and the life expectancy of affected individuals has been significantly increased. However, the treatment required is very expensive and is not a realistic means of controling the disorders for many developing countries. Individuals with the carrier state are easily identifiable, permitting the control of the clinically significant hemoglobinopathies by a program of carrier screening, counseling and prenatal diagnosis.1 Prenatal diagnosis (PND)was first achieved in 1974 by the study of globin synthesis in fetal blood, following the development of the technique of fetal blood sampling.2 This approach was applied for all of the hemoglobinopathies and proved very successful.3 However, it had the disadvantage of not being possible until the 18th week of pregnancy, which means a long wait for the mother and, if

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indicated, a relatively difficult elective abortion. Once the common thalassemia mutations were characterized, globin chain synthesis was universally replaced by fetal DNA analysis. Prenatal diagnosis by direct mutation identification was originally developed using amniotic fluid DNA to avoid the small risk of fetal loss associated with fetal blood sampling.4,5 However, amniocentesis is also a midtrimester procedure, and most diagnostic centers switched to the first-trimester procedure of chorionic villus sampling (CVS) soon after chorionic villus samples were shown to be a better source of fetal DNA for molecular analysis.6 Initially, the sampling procedure was observed to carry a high risk to the fetus, but as more studies were completed, the fetal loss rate decreased to the point at which it is lower than the risk from fetal blood sampling, but 0.6 percent higher than that from amniocentesis.7 Studies have shown that CVS performed before 10 weeks of pregnancy can cause limb reduction defects8; therefore, most centers currently perform CVS at 11 weeks. This usually allows just enough time for a first-trimester prenatal diagnosis, for which the target time for completing the polymerase chain reaction (PCR) tests in the UK is 3 working days. New developments in prenatal diagnosis are directed towards new approaches for noninvasive prenatal diagnosis. These include the analysis of fetal cells circulating in maternal blood, the analysis of free fetal DNA in maternal plasma, and preimplantation genetic diagnosis. The first two techniques, while safer for the fetus, still require

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the termination of pregnancy when indicated. Currently they are subject to technical problems and have a limited clinical application for the hemoglobinpathy prenatal diagnosis. The latter approach has the advantage that the need to terminate an affected ongoing pregnancy is eliminated and despite being a very difficult service to organize, has been proved feasible with the result of the birth of a small number of unaffected babies. However, it is a very technically challenging, multistep procedure with high costs and the approach is expected to supplement CVS diagnosis rather than to replace it. Almost 1,000 mutant alleles that result in a thalassemia phenotype or abnormal hemoglobin have been characterized at the molecular level. The mutations are regionally specific and in most cases, the geographic and ethnic distributions have been determined to provide the foundation of a control program by prenatal diagnosis. Numerous PCRbased techniques can be used to diagnose the globin gene mutations; the aim of this chapter is to compare and contrast these different approaches and then to describe the methods used in our laboratory in greater detail. The main requirements for methodologies providing molecular diagnosis of the hemoglobinopathies are speed, cost, convenience, and the ability to test for multiple mutations simultaneously. For β-thalassemia point mutations, the procedures that meet these requirements are the amplification refractory mutation system (ARMS) and the reverse dot-blot hybridization system. For β-thalassemia deletion mutations, some common ones can be diagnosed by gap-PCR, but multiplex ligation-dependent probe amplification (MLPA analysis) can now be used for both common and rare deletions. This technique is also used for the diagnosis of all α-thalassemia deletions, although the technique of gap-PCR is still useful to target the common deletion mutations. Finally, the nondeletion α-thalassemia mutations can be identified by a variety of PCR-based methods following selective amplification of the α-globin genes.

Clinical types The World Health Organization estimated in 1995 that there were more than 200 million carriers for

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the inherited disorders of hemoglobin (Hb) and that approximately 300,000 infants with severely affected homozygotes or compound heterozygotes would be born that year.9 Clinically, the most important of these conditions are α-thalassemia, β-thalassemia, sickle cell anemia, and the various compound heterozygous states which result in a clinically significant disease such as Hb E and βthalassemia.10 A brief account of the molecular pathology and phenotypic diversity of these disorders will be given here, but for more extensive coverage, the reader is referred to several review chapters in comprehensive textbooks on hemoglobin disorders.11–13 The hematologic features of the main types of hemoglobin disorders and the method of diagnosis by DNA analysis are summarized in Table 21.1.

The globin genes Hemoglobin is a tetrameric protein made up of two α-like (α or ζ) and two β-like (ε, γ, δ, or β) globin chains. Each globin chain is synthesized from its own globin gene located in two gene clusters, the α-like globin genes on chromosome 16 and the β-like genes on chromosome 11. The αglobin cluster includes an embryonic gene (ζ2), two fetal/adult genes (α1 and α2), several pseudogenes (ψζ1, ψα1, and ψα2), and a gene of undetermined function (θ1) arranged in the order ζ2–ψζ1–ψα2–ψα1–α2–α1–θ. The β-globin cluster includes an embryonic gene (ε), two fetal genes (Gγand Aγ), two adult genes (β and δ), and a pseudogene (ψβ) in the order ε-Gγ-Aγ–ψβ–δ–β. Throughout development, there is a series of coordinated switches of the production of one type of hemoglobin to another. Embryonic hemoglobin (α2γ2 – Hb Gower, ζ2γ2 – Hb Gower 1 and ζ2γ2 – Hb Portland) gives way to fetal hemoglobin (α2ζ2 – Hb F), which then switches to adult hemoglobin (α2β2 – Hb A and α2δ2 – Hb A2). The molecular mechanisms responsible for switching on and off the various globin genes have been the subject of intense research for many years. The phenotype of β-thalassemia or sickle cell disease in individuals with a naturally elevated level of Hb F is less severe, and thus the goal of this research is to understand and manipulate the switch in affected patients to ameliorate their disease. Several chemical agents,

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Table 21.1 Phenotypes of thalassemias, sickle cell disease and various thalassemia interactions Type

Phenotype

DNA diagnosis

1. Homozygous state α0-thalassemia (--/--)

Hb Bart’s hydrops fetalis

MLPA, Gap-PCR

α+-thalassemia (-α/-α)

No clinical problems

MLPA, Gap-PCR

α+-thalassemia (αTα/αTα)

Hb H disease

PCR, sequencing

β-thalassemia: β0 or severe β+ mutation

Thalassemia major

ASO, ARMS, sequencing

Mild β+ mutation

Thalassemia intermedia

ASO, ARMS, sequencing

δβ0-thalassemia

Thalassemia intermedia

MLPA, Gap-PCR

HPFH

No clinical problems

MLPA, Gap-PCR

Hb Lepore

Variable: intermedia to major

Gap-PCR

Hb S

Sickle cell disease

RE-PCR, ASO, ARMS

Hb C

No clinical problems

ASO, ARMS

Hb D

No clinical problems

RE-PCR, ASO or ARMS

Hb E

No clinical problems

RE-PCR, ASO or ARMS

2. Compound-heterozygous state α0-thal/α+-thal (–/-α)

Hb H disease

MLPA or Gap-PCR/PCR

α0-thal/α+-thal (-/αTα)

Hb H disease or Hb H hydrops

MLPA or Gap-PCR/PCR

β0/severe β+-thal

Thalassemia major

ASO or ARMS, sequencing

Mild β++/β0 or severe β+-thal

Variable: intermedia to major

ASO or ARMS, sequencing

δβ0/β0 or severe β+-thal

Variable: intermedia to major

MLPA or Gap-PCR/PCR

δβ0/mild β++-thal

Mild thalassemia intermedia

MLPA or Gap-PCR/PCR

δβ0/Hb Lepore

Thalassemia intermedia

MLPA or Gap-PCR

ααα/β0 or severe β+-thal

Mild thalassemia intermedia

Gap-PCR/ASO or ARMS

Hb Lepore/β0 or severe β+-thal

Thalassemia major

Gap-PCR/ASO or ARMS

Hb C/β0 or severe β+-thal

Variable: β-thal trait to intermedia

ASO, ARMS, sequencing

Hb C/mild β++-thal

No clinical problems

ASO, ARMS, sequencing

Hb D/β0 or severe β+-thal

No clinical problems

ASO, ARMS, sequencing

Hb E/β0 or severe β+-thal

Variable: intermedia to major

ASO, ARMS, sequencing

Hb O-Arab/β0-thal

Severe thalassemia intermedia

ASO, ARMS, sequencing

Hb S/β0 or severe β+-thal

Sickle cell disease

RE-PCR, ASO or ARMS

Hb S/mild β++-thal

Usually mild sickle cell disease

RE-PCR, ASO or ARMS

Hb S/δβ0-thal

Usually mild sickle cell disease

RE-PCR, MLPA/Gap-PCR

Hb S/Hb C

Sickle cell disease, variable severity

RE-PCR, ASO or ARMS

Hb S/Hb D-Punjab

Sickle cell disease

RE-PCR, ASO or ARMS

Hb S/Hb O-Arab

Sickle cell disease

RE-PCR, ASO or ARMS

Hb S/HPFH

Sickle cell trait

RE-PCR, MLPA/Gap-PCR

Hb E + α0-thal/α+-thal

Similar to Hb H disease

ASO or ARMS/ Gap-PCR

Hb EE + α0-thal/α+-thal

Severe thalassemia intermedia

ASO or ARMS/ Gap-PCR

Hb EE + αTα/αTα

Mild thalassemia intermedia

ASO, ARMS, sequencing

3. Hb E disorders

Hb, hemoglobin; PCR, polymerase chain reaction; RE, restriction enzyme, ASO, allele-specific oligonucleotide analysis; ARMS, amplification refractory mutation analysis; HPFH, hereditary persistence of fetal hemoglobin.

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such as hydroxyurea and butyrate, have been shown to induce the production of Hb F.14 Although the precise mechanism of action of these agents remains unknown, the most effective results obtained to date have been with hydroxyurea therapy in sickle cell and sickle-β thalassemia patients.15 This is in contrast to β-thalassemia patients, for whom the results have been disappointing, except in some patients with Hb Lepore or thalassemia intermedia who were not transfusion dependent.16

α-Thalassemia α-Thalassemia results from a deficiency of αglobin chain synthesis and can be divided into two forms: a severe form (called α-1 or α0-thalassemia), which produces a typical thalassemic blood picture in heterozygotes, and a mild form (α-2 or α+thalassemia), which is almost completely “silent” in heterozygotes. Although a few types of α+thalassemia have been shown to result from a nondeletion type of molecular defect, the most common cause of α-thalassemia is a series of gene deletions. α+-Thalassemia results from at least six different deletions, which effectively remove one of the two α-globin genes on chromosome 16.10 The genotype of the heterozygous state can be represented as -α/αα and that of the homozygous state as -α/–α. The clinical phenotype of the homozygous state is similar to that of α0thalassemia trait (--/αα genotype), and the two conditions are best differentiated by restriction enzyme mapping. α0-Thalassemia can result from 17 different gene deletions,11 all of which effectively delete both α-globin genes. Hb Bart’s hydrops fetalis syndrome The most severe form of α-thalassemia is the homozygous state for α0-thalassemia, known as Hb Bart’s hydrops fetalis syndrome. This condition results from a deletion of all four globin genes, and an affected fetus cannot synthesize any α-globin to make Hb F or Hb A. Fetal blood contains only the abnormal hemoglobin Bart’s (γ4) and a small amount of Hb Portland. The resulting severe fetal anemia leads to asphyxia, hydrops fetalis, and stillbirth or neonatal death, and prenatal diagnosis is

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always indicated to avoid the severe toxemic complications that occur frequently in pregnancy with hydropic fetuses. Hb H disease Hb H disease results from the compound heterozygous state for α0- and α1-thalassemia (--/-α) or, more rarely, from the homozygous state of nondeletion α+-thalassemia mutations affecting the dominant α2 gene (αTα/αTα). Individuals with Hb H disease have a moderately severe hypochromic microcytic anemia and produce large amounts of Hb H (β4) as a result of the excess β chains in the reticulocyte. Patients may suffer from fatigue, general discomfort, and splenomegaly, but they rarely require hospitalization and lead a relatively normal life. However, there also is a more severe form of Hb H disease arising from the compound heterozygous state of α0-thalassemia and nondeletion α+-thalassemia (--/αTα) or homozygous nondeletion α+-thalassemia (αTα/αTα). Such patients seem to exhibit more severe symptoms with a possible requirement of recurrent blood transfusions and splenectomy.10 In each case the α+-thalassemia resulted from a mutation in the α2 gene associated with a highly unstable α-globin variant17 or a polyadenylation signal.18 The unstable thalassemic αchain variants included Hb Adana, Hb Quong Sze, Hb Dartmouth, Hb Suan Dok and Hb Taybe. In a few cases, the severity of the phenotype has been sufficient to result in Hb H hydrops fetalis syndrome. This syndrome is different from Hb Bart’s hydrops caused by homozygous α0-thalassemia, as the fetus has around 35percent Hb Bart’s and 65 percent Hb F + Hb A. The hydropic changes of the fetus are likely due to a severe in utero anemia. Thus some couples at risk for this more severe form of Hb H disease have opted for prenatal diagnosis and termination of an affected fetus.19,20

β-Thalassemia The β-thalassemias are a heterogeneous group of disorders characterized by either an absence of βglobin chain synthesis (β0--type) or a much reduced rate of synthesis (β+ type). More than 200 different β-thalassemia mutations have been identified21

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and new ones are still being characterized by sequencing of unknown samples from screening programs (an updated list can be found at the website http://globin.cse.psu.edu). Only 17 mutations consist of large gene deletions, ranging from 25 bp to 67 kb; the remainder are single nucleotide changes or small deletions/insertions in the βglobin gene or its flanking sequences. The mutations cause defects in transcription, RNA splicing or modification, RNA translation through a frameshift effect or the presence of a new nonsense codon, and finally some create unstable β-globin chains. They can be classified into several groups according to their phenotypic effect. Most β0 and β+ type of mutations are called severe mutations because in either the homozygous or compound heterozygous state, they give rise to the phenotype of β-thalassemia major, a transfusion-dependent anemia from early in life. β-Thalassemia major At birth, infants with β-thalassemia major are asymptomatic because of the high production of Hb F but as this declines, affected infants present with severe anemia during the first or second year of life. Treatment is by frequent blood transfusion to maintain a hemoglobin level above 10 g/dL, coupled with iron chelation therapy to control iron overload, otherwise death results in the second or third decade from cardiac failure. This treatment does not cure β-thalassemia major, although many patients have now reached the age of 40 years in good health and have married and produced children. With the prospects for gene therapy remaining as distant as ever, the only cure for β-thalassemia for the foreseeable future is bone marrow transplantation. Although this form of treatment has proved successful when performed in young children, it is limited by the requirements of an HLAmatched sibling or relative. β-Thalassemia intermedia Some β-thalassemia mutations in the homozygous state are associated with a milder clinical condition called thalassemia intermedia. Patients with thalassemia intermedia present later in life relative to those with thalassemia major and are capable of maintaining a hemoglobin level higher than 6 g without transfusion. Thalassemia intermedia is

caused by a wide variety of genotypes, including β-thalassemia, δβ-thalassemia, and Hb Lepore, and covers a broad clinical spectrum. Patients with a severe condition present between 2 and 6 years of age, and, although they are capable of surviving with an Hb level of 5–7 g/dL, they will not develop normally and are treated with minimal blood transfusion. At the other end of the spectrum are patients who do not become symptomatic until they reach adult life and remain transfusion independent with Hb levels of 8–10 g/dL. However, even these milder patients tend to accumulate iron with age, and clinical problems relating to iron overload develop in many patients with thalassemia intermedia after the third decade. Prenatal diagnosis is often requested by couples at risk of having a child with thalasssemia intermedia due to the unpredictability of the phenotype. Thalassemia intermedia may result from a moderating effect due to the co-inheritance of two severe β-thalassemia mutations with either α0thalassemia trait or a hereditary persistence of fetal hemoglobin (HPFH) determinant such as the partial up promoter substitution (C → T) at −158 to the Gγ globin gene.22 For example the homozygous state for the β-thalassemia mutation IVSII-1 G → A results in thalassemia intermedia in some ethnic groups due to increased Hb F synthesis from the linked HPFH mutation. However, some individuals with β-thalassemia intermedia are simply homozygous for a mild type of β-thalassemia mutation. Specifically, these are IVSI-6 (T → C), CAP +1 (A → C), the transcription mutations occurring upstream of the β globin gene in the promoter sequences at approximately −30, −90, and −105 nucleotides, and the poly (A) AATAAA → AACAAA mutation.10 There is one exception, −29 (A → G), which has a mild phenotype in Africans but is severe in Chinese individuals, resulting in β-thalassemia major in the homozygous state.23 This is because the mutation is associated with the −158 Gγ globin HPFH mutation in African but not in Chinese individuals. Thus homozygosity for these mild β-thalassemia mutations usually results in a very mild disorder and prenatal diagnosis is not usually indicated. However, the situation for the compound heterozygous state when one of these mild mutations is coupled with a severe mutation is less clear.

CHAP T E R 21

Some of these individuals have a mild clinical picture, especially if it involves one of the very mild mutations such as the “silent β-thalassemia” mutations (those associated with a normal Hb A2 and mean corpuscular hemoglobin (MCH)). One of the most common, the mutation −101 (C → T) has been found to produce very mild clinical phenotypes in the homozygous state or in interaction with severe β-thalassemia mutations.24 Therefore prenatal diagnosis in at-risk couples where this silent allele is present should not be considered. However, the position for the other silent mutations (e.g. −92 C → T, the 5′ UTR mutations, IVSII-844 C → G, +1480 C → G, and the UTR mutations) and other normal Hb A2 mutations such as CAP+1 A → C is less clear. The unpredictability of the phenotype in compound heterozygotes for these mutations remains a diagnostic and counseling problem. Because the mutations are very uncommon, homozygotes do not exist and there is a general lack of data on cases with the co-inheritance of other β-thalassemia alleles. An excellent summary of what data there are on the interactions of silent and mild alleles can be found in the fourth edition of the book of Weatherall and Clegg.10 Another genotype associated with thalassemia intermedia is the homozygous state for the Hb Lepore deletion mutation (although some such individuals have been reported to have the more severe phenotype of thalassemia major) and from the homozygous state for a couple of the very rare large deletion mutations that cause β0-thalassemia.25 This group of deletion mutations (which does not include the 619 bp Asian Indian deletion gene) is characterized in the heterozygous state by an unusually high Hb A2 value. Finally, a third class of mutations forms the other end of the spectrum of severity. These mutations are more severe than the main group of severe β0 and β+ mutations and produce a thalassemia intermedia phenotype in the heterozygous state, the so-called dominantly inherited inclusion body β-thalassemia.26 The mutations all occur in exon 3 and are believed to produce a highly unstable β-globin chain, which is quickly broken down, causing overloading of the proteolytic system inside the red cell and the subsequent precipitation of free α-chains as inclusion bodies.

Prenatal Diagnosis of the Hemoglobinopathies

651

Hb E disorders Hb E (β26, Glu → Lys) is the most common abnormal hemoglobin in Southeast Asians, found at gene frequencies above 0.10 percent in some areas. Hb E heterozygotes and homozygotes are asymptomatic. Heterozygotes are clinically normal with 25–30 percent Hb E (lowered by the presence of thalassemia), and homozygotes may be mildly anemic but clinical symptoms are rare. The importance of Hb E is that it combines with different α and β-thalassemias to produce a range of symptomatic disorders.27 Hb E-β-thalassemia The compound heterozygous state of Hb E and β-thalassemia is a common disease in Thailand and parts of Southeast Asia. It results in a variable clinical picture similar to that of homozygous β-thalassemia, usually of intermediate severity. However, the clinical spectrum is heterogeneous, ranging from a condition indistinguishable from thalassemia major to a mild form of thalassemia intermedia because of the range of different βthalassemia genes. The most severe conditions are found in individuals with β0-thalassemia who usually have about 40–60 percent Hb F, the remainder being Hb E. Compound heterozygotes for Hb E and β+-thalassemia have a milder disorder and produce variable amounts of Hb A. Hb AE Bart’s disease Hb AE Bart’s disease results from the interaction of Hb H disease with heterozygous state for Hb E. The disorder is characterized by the presence of Hb A, Hb E (13–15 percent) and Hb Bart’s on hemoglobin analysis. Although Hb H inclusions may sometimes be observed, Hb Bart’s is usually found on electrophoresis in adults with this disorder. The clinical manifestations are similar to Hb H disease, with patients having a variable degree of anemia and splenomegaly. Two common subtypes of Hb AE Bart’s disease have been observed: α0-thalassemia/α+ thalassemia-βA/βE and α0-thalassemia/Hb Constant Spring-βA/βE. The latter disorder was found to have a more severe clinical syndrome. Hb EF Bart’s disease Hb EF Bart’s disease results from the interaction of Hb H disease with homozygous Hb E. The disorder

652

Genetic Disorders and the Fetus

is characterized by the presence of Hb E, Hb F and Hb Bart’s on hemoglobin analysis. Hb E constitutes approximately 80 percent, Hb F 10 percent and the Hb Bart’s 10 percent. Patients with this condition have severe thalassemia intermedia, with a Hb level ranging from 6 to 10 g/dL and markedly reduced mean cell volume (MCV) and MCH values and moderate to severe anemia. No inclusion bodies or Hb H are present, probably because the abnormal βE-globin chains cannot form tetramers. Four genotypes for Hb EF Bart’s disease have been identified: • Hb H disease, due to α0/α+ -thalassemia, with homozygous Hb E • Hb H disease, due to α0-thalassemia/Hb Constant Spring, in combination with homozygous Hb E • Hb H disease, due to α0/α+-thalassemia, with Hb E/β-thalassemia • Hb H disease, due to α0-thalassemia/Hb Constant Spring, in combination with Hb E/β-thalassemia. To differentiate among these genotypes, family studies and further investigation by DNA analysis are required. Hb E/E plus αCSα/αCSα Individuals homozygous for Hb E and homozygous Hb Constant Spring have been observed. They have mild thalassemia intermedia. Compared with homozygous Hb E alone, there were minimal red cell changes. This may be due to the interaction of α-thalassemia with the β-thalassemia-like reduced globin synthesis of Hb E.

Sickle cell disease Sickle cell disease is characterized by a lifelong hemolytic anemia, the occurrence of acute exacerbations called crises, and a variety of complications resulting from an increased propensity to infection and the deleterious effects of repeated vasoocclusive episodes. With active management, the proportion of patients expected to survive to 20 years of age is approximately 90 percent. The course of the illness is very variable, even within individual sibships let alone different racial groups. Sickle cell disease can result from a variety of different genotypes. These include the homozygous state for the sickle cell gene (sickle cell anemia),

plus the compound heterozygous genotypes of Hb S with β-thalassemia, δβ-thalassemia, Hb Lepore, Hb D-Punjab, Hb O-Arab, Hb C and few other rare abnormal hemoglobins, such as Hb CHarlem, one of six sickling variants with two amino acid substitutions.28 Sickle cell anemia The classic picture of the homozygous state of Hb S disease is a chronic anemia, childhood susceptibility to overwhelming infections, and periodic painful or hemolytic crises. The most common cause of death in early life is infection. The mortality in childhood is believed to be approximately 1–2 percent per year in the United States and the United Kingdom; in less developed countries, such as those in Africa, the rate of infant mortality is higher. However, the clinical picture of sickle anemia actually is heterogeneous, with a wide range of variability in the phenotypic expression of the disease. This is due, in part, to the fact that the sickle cell mutation has arisen independently at least four times in Africa and once in Asia, according to data provided by β globin haplotype analysis.29 The haplotypes have been assigned the name of the geographic area in which they are most frequently found. The four African haplotypes most frequently found are the Benin, Senegal, Cameroon and Central African Republic (CAR) or Bantu types. DNA studies have shown that the sickle gene found in Mediterranean individuals is of African origin – the Benin haplotype. The fifth haplotype is the Arab-Indian haplotype, found with the Hb S gene in Saudi Arabia, Iran and India. Different Hb F levels are associated with homozygotes for different β-globin gene haplotypes: Cameroon (5–6 percent), Benin and Bantu (6–7 percent), Senegal (7–10 percent) and ArabIndian (10–25 percent). Epidemiologic studies have shown that haplotypes associated with the lowest Hb F levels are associated with the most clinically severe condition, while the one with the highest, the Arab-Indian, is associated with the mildest course of the disease.30 The other factor known to modify the disease is the co-inheritance of α-thalassemia. In Africans and Indians, this is always the α+ type. Hb SS patients homozygous for α+-thalassemia have lower levels of Hb F, but reduced levels of hemolysis as judged by a higher

CHAP T E R 21

hemoglobin level. Some of the variability of the disease within families could be due to different inheritance patterns of α-thalassemia. Hb S/β-thalassemia In Hb S/β-thalassemia, the β-thalassemia gene interacts with the βS gene to increase the level of Hb S from above 50 percent to a level near that observed in Hb SS individuals. The clinical course of sickle cell β-thalassemia is very variable, ranging from a disorder identical with sickle cell anemia to a completely asymptomatic condition. The Hb concentration varies from 5 g/dL to within the normal range. The heterogeneity is mostly due to the type of β-thalassemia mutation that is coinherited. It tends to be very mild in Africans because of the likelihood of the co-inheritance of one of three mild β+ mutations commonly found in this racial group (−88, C → T; −29, A → G; C24, T → A). However, those patients who inherit a βo-thalassemia allele exhibit a clinical disorder very similar to sickle cell anemia. Hb S/β-thalassemia is characterized by microcytic red and target cells with occasionally sickled forms. Hemoglobin electrophoresis reveals 60–90 percent Hb S, 0–30 percent Hb A, 1–20 percent Hb F, and an increased Hb A2 level above normal. The percentages of Hb S and Hb A vary depending on whether the β-thalassemia gene is β+ or β0 type. Co-existing α-thalassemia increases the Hb concentration, the MCV and MCH. Hb S/δβ-thalassemia Hb S/δβ-thalassemia is a milder form of sickle cell disease than sickle cell anemia, because the high percentage of Hb F produced by δβ-thalassemia allele protects against sickling. Hb S/δβ-thalassemia has been characterized in Sicilian, Italian, Greek, Arab and Afro-American individuals. Patients have a mild anemia with a Hb concentration in the range of 10–12 g/dL, a significantly reduced MCH and MCV, Hb S, Hb F and a normal or low Hb A2 level. Hb S/C disease Hb S/C disease is a milder version of sickle cell disease with a variable course. Most of the complications occurr less frequently than in Hb SS disease. Hb C is found in parts of West Africa, where it

Prenatal Diagnosis of the Hemoglobinopathies

653

co-exists with Hb S at frequencies of up to 0.15 percent. The Hb C mutation, β6 Glu → Lys (GAG → AAG), causes a decrease in solubility of both the oxygenated and deoxygenated forms of Hb C, resulting in the formation of crystals. In individuals homozygous for Hb C, the red cells become dehydrated and rigid, causing a hemolytic anemia, but such patients do not develop any sickling symptoms. Hb S/D disease Hb S/Hb D-Punjab (β121, Glu → Gln) results in a moderately severe form of sickle cell disease. This compound heterozygous state has been observed in patients of African origin, from Central and South America, India, and in individuals with only Mediterranean or northern European ancestry. Patients have a mild to moderate hemolytic anemia (Hb of 5–10 g/dL) with sickling crises. Hb S/Hb O-Arab disease Hb S/Hb O-Arab (β121, Glu → Lys) results in a severe type of sickle cell disorder. HbS/Hb O-Arab has been observed in Arabs, Africans, AfroCaribbeans and Afro-Americans. The Hb concentration varies between 6 and 10 g/dL and the blood film is similar to sickle cell anemia. Other rare sickle cell genotypes Hb S/C-Harlem (β6 Glu → Val and β73 Asp → Asn) is a severe sickle cell disorder. Hb C-Harlem has two amino acid substitutions, the sickle cell substitution at codon 6 and one at codon 73 which makes the hemoglobin move like Hb C in electrophoresis at alkaline pH. In combination with Hb S, it causes severe sickle cell disease. Hb S-Antilles (β6 Glu → Val and β23 Val → Ile) has two amino acid substitutions, similar to Hb C-Harlem. It is more prone to sickling than Hb S itself and in the heterozygous state it results in a mild anemia and a moderate sickling disorder. In combination with Hb S, it is reported to produce a very severe form of sickle cell disease with a severe chronic hemolytic anemia. Compound heterozygosity for Hb C and Hb S-Antilles also produces a severe sickle cell disorder. Hb S-Oman (β6 Glu → Val and β121 Glu → Lys) has two different phenotypes in the heterozygous

654

Genetic Disorders and the Fetus

state, depending upon whether the patients have co-inherited heterozygous or homozygous for αthalassemia (all patients described with Hb S-Oman have α-thalassemia). Patients with α+-thalassemia trait have about 20 percent Hb S and a moderate sickling disorder. The blood film shows a unique form of an irreversibly sickled cell called a “Napoleon hat cell” or “yarn and knitting needle cell.” In contrast, patients with Hb S-Oman trait and homozygous α+-thalassemia have about 14 percent Hb S-Oman and are asymptomatic. The compound heterozygous state for Hb S and Hb S-Oman has been described in a few Omani patients. Patients have 25 percent Hb S, 11 percent Hb S-Oman and the blood film shows Napoleon hat cells. Patients have very severe disease, with a Hb level of 7 g/dL. The interaction of Hb S with unstable β-variants may result in a mild form of sickle cell disease. Three such variants have been observed, namely Hb Quebec-Chori, Hb Hofu and Hb I-Toulouse. Hb S in combination with mildly unstable βvariants such as Hb Hope and Hb Siiraj can cause mild hemolysis.

Carrier screening Community control of sickle cell anemia and thalassemia by fetal diagnosis depends on a successful population screening program.31 Screening using hematologic methods is the first step in genetic diagnosis and normally consists of measurement of the red cell indices, hemoglobin electrophoresis, quantitation of Hb A2, Hb F, Hb H, and the determination of iron status.32 Guidelines and a flow chart using cut-off points are followed to establish a diagnosis of a possible thalassemia phenotype.33 It is important to note that such a screening program is designed to lead to a reliable presumptive diagnosis. If an unequivocal diagnosis is required, characterization methods based on DNA analysis must be used. Screening will detect most cases of β-thalassemia trait; however, there is no specific screening test for α-thalassemia trait, and this diagnosis is usually made by exclusion of a raised Hb A2 level and iron deficiency. If an abnormal hemoglobin is found by electrophoresis, again the results will give only a presumptive diagnosis of the variant.

Methods Traditionally, a starch gel has been the medium for hemoglobin electrophoresis, but now this has been replaced by the more rapid methods of electrophoresis using cellulose acetate membrane, acid agarose or citrate agar gel. Detailed procedures for these techniques have been published by Weatherall and Clegg.10 However, isoelectric focusing using precast agarose gels is the method of choice, as it gives better separation of hemoglobin variants with sharper bands. It has proved useful for screening large numbers of samples, and provides better resolution and sharper bands than ordinary electrophoresis. The Hb A2 is estimated following its separation from Hb A using cellulose acetate electrophoresis and elution, column chromatography or highperformance liquid chromatography (HPLC). The latter allows the rapid direct measurement of both Hb A2 and abnormal hemoglobins on large numbers of samples. The MCH is determined together with the other red cell indices by a standard electronic cell counter in fresh blood samples. Evaluation of blood count in samples more than 24 hours old should be treated with caution, as the red cells increase in size, leading to a falsely raised MCV (thus the MCH is the more reliable parameter to use for diagnosis). Reduced red cell indices with a raised Hb A2 value The heterozygous states for β-thalassemia are usually associated with reduced MCH values, in the 18–25 pg range (normal range, 26–33 pg), and reduced MCV values, in the 60–70 fL range, and a raised Hb A2 level. The red cells also have reduced osmotic fragility, which is the basis for the singletube osmotic fragility test, which can be used as an alternative screening test if the electronic measurement of MCV is not available. Individuals found to have a low MCH (below 27 pg) are then investigated by estimating the Hb A2 level. If the Hb A2 level is elevated above the normal range (0–3.5 percent), then β-thalassemia trait is indicated. If the Hb A2 level is unusually high (6.5–9.0 percent), then β-thalassemia trait resulting from one of the large gene deletions should be suspected.12 The hematologic values for MCH, MCV and Hb A2 found in our laboratory for carriers

CHAP T E R 21

of different hemoglobinopathies are listed in Table 21.2. Reduced red cell indices with a normal Hb A2 value When reduced MCV and MCH levels and a normal Hb A2 level (below 3.5 percent) are observed, the diagnosis may be iron deficiency, α-thlassaemia, δβ-thalassemia trait (εγδβ)0 thlassemia trait, βthalassemia plus δ-thalassemia trait, Hb Lepore trait or normal Hb A2 β-thalassemia trait. A raised Table 21.2 Comparison of various heterozygous conditions Disorder/genotype

MCH (fl)

MCV (pg)

Hb A2 (%)

αα/αα

30

90

2.0

α α α /α α

29

85

2.2

Normal

α-thalassemia -α/αα

28

85

2.4

-/αα

22

70

3.0

β0-thalassemia CD39 C → T

20

66

4.7

β+-thalassemia

20

66

7.5

IVSI-110 G → A

21

68

4.5

IVSI-6 T → C

23

72

3.4

CAP+1 A → C

25

80

3.3

−101 C → T

28

85

3.3

β0 trait + α+ trait

22

70

5.7

β0 trait + α0 trait

26

78

6.0

MCH, mean cell hemoglobin; fl, femtoliters; MCV, mean corpuscular volume; pg, picograms; Hb, hemoglobin; CD, codon; IVS, intervening sequence; kb, kilobase.

Prenatal Diagnosis of the Hemoglobinopathies

655

Hb F level of 5–15 percent is indicative of δβthalassemia trait. Hb Lepore (8–20 percent) can be identified by gel electrophoresis or isoelectric focusing. Normal A2 β-thalassemia and αthalassemia can be identified only by molecular analysis. The condition of β-thalassemia trait with a normal Hb A2 level can be due to the co-inheritance of a standard β-thalassemia mutation with a δ-thalassemia mutation, or to the inheritance of a mild β-thalassemia allele associated with a normal or borderline Hb A2 level (3.3–3.8 percent). These are listed in Table 21.3. Some, such as IVSI-6 T → C and CAP+1 A → C, are associated with reduced red cell indices. However, some of the rarer alleles such as −92 C → T, IVSII-844 C → G, and −101 C → T are truly silent, being associated with normal red cell indices, and thus will not be detected by hematologic screening. The values for the MCH, MCV, and Hb A2 associated with these silent and normal Hb A2 β-thalassemia alleles are summarized in Table 21.3. Strategy for fetal diagnosis In summary, the screening program used in most countries is based on the following strategy. The MCV is measured first by an electronic cell counter and then a hemolysate is prepared and examined for hemoglobin variants by starch gel or cellulose acetate electrophoresis, HPLC or isoelectric focusing. For samples with a normal hemoglobin phenotype (AA) and a low MCV, Hb A2 quantification is performed. A normal Hb A2 level would usually indicate α-thalassemia unless anemia due to iron deficiency is identified. For the purpose of antena-

Table 21.3 Genotypes associated with borderline HbA2 levels – a guideline of related hematologic and biosynthetic characteristics Genotype

MCV fl

MCH pg

Hb A2

α/β ratio

β −101 (C → T)

88.5 ± 7.8

30.1 ± 1.0

3.1 ± 1.0

1.3 ± 0.4

β −92 (C → T)

83.0 ± 6.0

28.3 ± 2.0

3.5 ± 0.4

1.3 ± 0.8

β +33 (C → G)

82.0 ± 9.2

27.1 ± 3.4

2.5 ± 1.4

1.3 ± 0.6

Cap+1 (A → C)

23–26

75–80

3.4–3.8

–

β IVS1-6 T → C

71.0 ± 4.0

23.1 ± 2.2

3.4 ± 0.2

1.9 ± 1.0

βIVS2-844 C → G

96.0 ± 4.0

30.3 ± 1.8

3.2 ± 0.2

1.0 ± 0.6

β +1480 (C → G)

88.3 ± 9.5

27.9 ± 6.0

2.7 ± 0.8

1.6 ± 0.4

ααα/αα

85.5 ± 7.8

30.4 ± 5.0

2.8 ± 0.6

1.2 ± 0.4

δ + β thalassemia

67.6 ± 7.6

21.8 ± 3.6

3.3 ± 0.4

1.7 ± 0.6

656

Genetic Disorders and the Fetus

tal screening, laboratories may decide not to investigate further if the partner is found to have normal red cell indices or not to carry any hemoglobin variant. If both partners appear to have α0thalassemia, β-thalassemia or a combination that can result in a serious hemoglobinopathy, then DNA analysis is indicated. If one partner appears to have α0-thalassemia trait and the other βthalassemia trait, then DNA analysis should still be considered for both partners. The couple could be at risk for β-thalassemia if one has normal Hb A2 β-thalassemia instead of α-thalassemia trait. Alternatively, the couple, particularly those of Southeast Asian origin, could be at risk for Hb Bart’s hydrops fetalis syndrome because the β-thalassemia trait may be masking co-existing α0-thalassemia trait.

Approaches to prenatal diagnosis Prenatal diagnosis by fetal blood sampling Prenatal diagnosis of the hemoglobinopathies was first achieved by fetal blood sampling and the estimation of the relative rates of globin chain synthesis by radiolabeling. This method, which directly measures the product of the mutant globin genes, was initiated in 1974 after the development of safe techniques for sampling fetal blood at 18–20 weeks’ gestation. More than 20 centers performed prenatal diagnosis by this method, and more than 13,000 cases for hemoglobinopathies had been reported to a WHO Registry by December 1989. Overall, the program was remarkably successful, with approximately 25 percent of the fetuses being diagnosed as affected, a fetal loss rate of 3 percent, and a diagnostic error rate of 0.5 percent.3 Although prenatal diagnosis using fetal blood was a remarkable technical achievement, it had the disadvantage that fetal blood sampling is not possible until about the 18th week of pregnancy which, if indicated, implies a late elective abortion. Fetal blood sampling has now been almost entirely replaced in most diagnostic centers by fetal DNA analysis, once the common thalassemia mutations had been characterized. Amniotic fluid DNA As soon as development of techniques for the detection of hemoglobinopathies by gene analysis

began, several antenatal diagnosis centers began using fetal DNA from amniocytes.4 Most prenatal diagnosis centers quickly adopted the techniques of DNA analysis, and by 1982, 175 cases of amniocyte DNA diagnosis had been reported to the WHO Registry.34 During 1982, CVS tissue was shown to be an alternative source of fetal DNA for molecular analysis6 and early experience showed that chorionic villi provided relatively large amounts of DNA, allowing prenatal diagnosis in nearly all cases by the 12th week of pregnancy.35 CVS soon replaced amniocentesis as the source of fetal DNA for prenatal diagnosis, and by December 1989, a total of 4,581 CVS diagnoses had been recorded by the WHO Registry, in comparison to 1,222 amniocyte DNA diagnoses.3 The amniocyte DNA approach will never be entirely replaced, because it will still be necessary in cases in which a couple present themselves too late for CVS or, more rarely, where there is a failure to obtain a villus sample. DNA can be prepared from amniotic fluid cells directly or after culturing. It takes 2–3 weeks to grow amniocytes to confluence in a 25 mL flask, but culturing has the advantage that a large amount of DNA is obtained (in our experience, the yield from such a flask has varied from 15 to 45 µg, enough DNA for all types of analyses). However, not all laboratories have the facilities for cell culture, and diagnosis can be made using DNA from noncultivated cells in most cases. Approximately 5 µg of DNA is obtained from a 15 mL amniotic fluid sample, and this is sufficient for any PCR-based method of analysis. However, for genotype analysis by Southern blotting, it is only enough for one attempt; therefore, a small portion should be prudently set aside for culturing in case of failure. The method of DNA preparation for both cultured and noncultivated cells is essentially the same as that for chorionic villi.36 Chorionic villus DNA Both of the two main approaches to CVS, ultrasound-guided transcervical aspiration37,38 and ultrasound-guided transabdominal sampling,39,40 provide good-quality chorionic villus samples for fetal DNA diagnosis, and sufficient DNA normally is obtained for any method of analysis of the globin genes. For our first 200 CVS DNA diagnoses, the

CHAP T E R 21

average yield of DNA was 46 mg; in only one instance was less than 5 mg obtained.36 A problem with this approach is the risk of contamination of the chorionic villus DNA with maternal DNA, which arises from the maternal decidua sometimes obtained along with the chorionic villi. However, by careful dissection and removal of the maternal decidua with the aid of a phase-contrast microscope, one can obtain pure fetal DNA samples,41 as demonstrated by the lack of maternal DNA in chorionic villus samples by hybridization studies with an X chromosome-specific probe. Maternal contamination can be ruled out in most cases by the absence of a second informative maternal marker after the amplification of highly polymorphic repeat markers.42 A study of 161 CVS DNAs by Southern blot analysis using the hypervariable allele probes α-globin 3′ HVR and plg3 revealed that the level of contamination from experienced centers was less than 1 percent.43 Current practice is to analyze up to 12 short tandem repeat markers so that there is always more than one informative maternal marker to shown the presence of any maternal DNA contamination. Fetal cells in maternal blood Fetal cells have long been known to be present in the blood of pregnant women, and they provide an attractive noninvasive approach to prenatal diagnosis provided that the fetal cells are specific for the ongoing pregnancy and a pure population of cells can be isolated for analysis. However, attempts to isolate the fetal cells as a source of fetal DNA using immunologic methods and cell sorting have had only moderate success in providing a population of cells pure enough for fetal DNA analysis. The technique has been applied for the diagnosis of β-thalassemia in women whose partners carried a different thalassemia allele, as reported for the detection of a paternal Hb Lepore mutation.44 Another approach is to identify single nucleated fetal red blood cells and use single cell PCR to make the diagnosis. Following enrichment, nucleated fetal red blood cells have been identified by staining on a microscope slide with anti-ζ chain antibodies and collected by micromanipulation under microscopic observation.45 This approach was used successfully for prenatal diagnosis in two pregnan-

Prenatal Diagnosis of the Hemoglobinopathies

657

cies at risk for sickle cell anemia and β-thalassemia.46 However, studies have shown that embryonic and fetal globins may be expressed in adult erythroid progenitor cells and thus more specific fetal cell markers are required for the technique to become reliable.47 One successful application of this technique has been the prenatal detection of Hb Bart’s hydrops fetails syndrome by antibody staining of fetal erythrocytes in maternal blood for α- and ζglobins, as these cells from an affected fetus cannot express α-globin.48 Because the micromanipulation approach has proved to be subject to technical difficulties as well as being costly and time consuming, other approaches are being developed, such as noncontact laser capture microdissection to isolate single fetal nucleated cells from slides49 and enrichment of antibody-stained nucleated red blood cells by flow cytometric sorting.50 Fetal DNA in maternal plasma Maternal plasma contains a relatively low amount of cell-free DNA, and in 1997 it was discovered that some of this was fetal DNA originating from dying placental cells.51 The analysis of cell-free fetal DNA in maternal plasma is a simpler procedure than the analysis of DNA in fetal nucleated red cells in maternal blood as no complicated enrichment processes are involved. In early pregnancy, approximately 3 percent of the cell-free DNA in maternal plasma is fetal DNA, the rest being maternal DNA, and the fetal DNA is very fragmented (less than 300 bp) compared to the maternal cell-free DNA (of which only half is less than 300 bp). Thus simple size fractionation techniques may be employed for the selective enrichment of the fetal DNA.52 However, because of its low concentration and short size, the detection of cell-free fetal DNA against a background of a larger amount of maternal DNA is a significant technical challenge. Highly sensitive methods being developed with promising results include real-time quantitative PCR,53 different microarray platforms such as arrayed primer extension,54 and mass spectrometry (MALDITOF).55 Other approaches being tried are the use of enrichment protocols for the selective amplification of the fetal DNA sequences, for example, by clamping maternal mutation sequences with peptide nucleic acids.56

658

Genetic Disorders and the Fetus

The approach is being used routinely for the prenatal diagnosis of sex-linked genetic disorders and for fetal RhD blood group typing in the UK.57 For recessive disorders, this approach can be used to detect or exclude the paternally inherited mutation in cases in which the paternal mutation is different from the maternal mutation. The technique has been applied for the prenatal testing of eight fetuses at risk for β-thalassemia major using allele-specific primers for the detection of the CD 41/42 (-CTTT) mutation by real-time PCR58 and for the detection of homozygous α0-thalassemia.59 However, in cases where the maternal and paternal mutations are the same, the use of linked methods using informative DNA polymorphic markers will need to be developed.60 Preimplantation diagnosis Preimplantation genetic diagnosis (PGD) represents a “state-of-the-art” procedure that allows atrisk couples to have disease-free children without the need to terminate affected pregnancies. PCRbased diagnostic methods can be potentially applied for preimplantation genetic diagnosis using three types of cells: polar bodies from the oocyte/zygote stage, blastomeres from cleavagestage embryos and trophoectoderm cells from blastocysts.61 Although the technique requires a combined expertise in both reproductive medicine and molecular genetics, a small number of centers around the world are now set up to carry out this procedure. PGD has been used successfully for both α-thalassemia,62,63 and β-thalassemia.64,65 The approach is a useful alternative to PND for couples who have already had one or more therapeutic abortions and for whom religious or ethical beliefs will not permit the termination of pregnancy, although a study of the attitude of Muslim women to PGD demonstrated that parents’ concerns were complex and PGD was only acceptable to 27 percent of couples questioned.66 However, PGD is technically challenging, multistep and an expensive procedure. The PCR protocol must be able to diagnose the required genotype in single cells reliably and accurately and it also has to be optimized to minimize PCR failure and to avoid the problem of allele drop-out which could lead to a misdiagnosis. Protocols designed to

monitor the occurrence of allele drop-out include multiplex PCR to detect both alleles that contribute to the genotype, such as denaturing gradient gel electrophoresis (DGGE), single strand conformation analysis (SSCA) and real-time PCR.67 The birth of a healthy unaffected baby depends not only on an accurate diagnosis, but also on the success of each of the multiple stages of the assisted reproduction procedure. Overall, the success rate of the procedure is only 20–30 percent and thus this approach is not likely to be used routinely for the monitoring of pregnancies at risk for hemoglobin disorders. One specific use of this approach is to allow the birth of a normal child that is HLA identical to an affected sibling, thus permitting a possible cure by stem cell transplantation.65

DNA diagnosis of the hemoglobinopathies This section will review the various techniques of DNA analysis that are used to diagnose the hemoglobinopathies and present the methods that currently are in use in our laboratory. α-Thalassemia α+-thalassemia has been found to result from five different sizes of gene deletion, although only two are commonly encountered in practice. These are the 3.7 kb deletion (-α3.7), which has reached high frequencies in the populations of Africa, the Mediterranean area, the Middle East, the Indian subcontinent, and Melanesia, and the 4.2 kb deletion (-α4.2), which is commonly found in the Southeast Asian and Pacific populations.68 These deletions were created by unequal crossing over between homologous sequences in the α-globin gene cluster, resulting in a chromosome with only one α-gene (-α) and a chromosome with three α-genes (ααα). An additional recombination event between the resulting chromosomes has given rise to a quadruplicated α-gene allele (αααα). Various nondeletion defects also have been found to cause α+-thalassemia, and a total of 17 mutations have been described to date, mostly in populations from the Mediterranean area, Africa, and Southeast Asia.68 α0-Thalassemia results from deletions that involve both α-globin genes, and to date at least 14

CHAP T E R 21

different deletions have been described.11 The deletions that have attained high gene frequencies are found in individuals from Southeast Asia and South China (--SEA), the Philippine Islands (--FIL), Thailand (--THAI), and a few Mediterranean countries, such as Greece and Cyprus (--MED and -(α)20.5). Although one α0-thalassemia mutation (--SA) has been described in Asian Indians, it is extremely uncommon, and no α0-thalassemia deletions have been reported in individuals from sub-Saharan Africa. In Northern Europe, αthalassemia occurs sporadically because of the lack of natural selection, and several α0-thalassemia deletions have been reported in single British families, although one particular defect (--BRIT) has been observed in a number of unrelated individuals living in Cheshire and Lancashire.

Prenatal Diagnosis of the Hemoglobinopathies

Table 21.4 Globin gene deletion mutations diagnosable by gap-PCR Disorder

Deletion

Distribution

α0-thalassemia

--SEA

Southeast Asia

α+-thalassemia

β0-thalassemia

Gap-PCR diagnosis The technique of gap-PCR provides a quick, simple and cheap method for the diagnosis of the two most common α+-thalassemia deletion alleles, -α3.7 and -α4.2, and the five most common α0-thalassemia deletion mutations, the --MED, -(α)20.5, --SEA, --THAI and --FIL alleles (Table 21.4). However, it cannot be used for any of the rarer deletions because their breakpoint sequences have not been characterized, and these had to be diagnosed by Southern blot analysis until recently, as reviewed in the previous edition of this book.69 The technique of multiplex ligation-dependent probe amplification (MLPA) has now replaced Southern blotting for the diagnosis of all the rare α0-thalassemia deletions, and MLPA is also used in our laboratory as a second technique to confirm any prenatal diagnosis result involving the common α0-thalassemia deletions. Gap-PCR involves the use of two primers complementary to the sense and antisense strand in the DNA regions that flank the α-thalassaemia deletion.70–72 Figure 21.1 shows the diagnosis of the --MED allele by this technique. Amplified product is obtained from only the deletion allele, because the distance between the two primers is too great to amplify normal DNA. The normal allele (αα) is detected by amplifying DNA sequences spanning one of the breakpoints, using a primer complementary to the deleted sequence. The primers can be multiplexed to detect more than one type of

659

--MED

Mediterranean

-(α)20.5

Mediterranean

--FIL

Philippines

--THAI

Thailand

-α3.7

Worldwide

-α4.2

Worldwide

290 bp deletion

Turkey, Bulgaria

532 bp deletion

African

619 bp deletion

India, Pakistan

1393 bp deletion

African

1605 bp deletion

Croatia

3.5 kb deletion

Thailand

10.3 kb deletion

India

45 kb deletion

Philippines, Malaysia

0

(δβ) -thalassemia

Hb Lepore

Mediterranean,

Spanish

Spain

Brazil Sicilian

Mediterranean

Vietnamese

Vietnam

Macedonian/

Macedonia,

Turkish (Aγδβ)0-thalassemia Indian

HPFH

Turkey India, Bangladesh

Chinese

Southern China

HPFH1 (African)

African

HPFH2 (Ghanaian) Ghana, African HPFH3 (Indian)

India

deletion per amplification reaction. We currently use a screening strategy of three amplification reactions: one for the two α+-thalassemia alleles, one for the two Mediterranean α0-thalassemia alleles and one for the three Southeast Asian α0thalassemia alleles. Screening of carriers is targeted according to the ethnic origin of the individual, except for carriers of unknown origin in which case all three multiplex reactions are used. Gap-PCR provides a quick diagnostic test for α0-thalassemia but requires careful application for prenatal diagnosis, and ideally the result should be confirmed by a different technique such as MLPA.

660

Genetic Disorders and the Fetus

Figure 21.1 Prenatal diagnosis of α0-thalassemia using gap-PCR to detect the --MED allele. The amplification products after agarose gel electrophoresis and ethidium bromide staining are shown as follows: track 1, maternal DNA; track 2, paternal DNA; track 3, normal DNA; tracks 4 and 5, different concentrations of chorionic villus DNA.

A diagram shows the location of the --MED deletion with respect to the α-globin gene cluster, together with the positions of primers 1 and 3 which amplify --MED DNA to give a 650 bp product and primers 2 and 3 which amplify only the normal allele to give a 1000 bp product.

Amplification of sequences in the α-globin gene cluster is technically more difficult than that of the β-globin gene cluster, possibly due to the higher GC content. Experience in our laboratory has shown that the first primer pairs to be published were unreliable, resulting occasionally in unpredictable reaction failure due to allele drop-out. This is illustrated in Figure 21.1, in which the results for the chorionic villus DNA sample clearly give a homozygous genotype for α0-thalassemia (--MED/--MED). However, Southern blot analysis of the same DNA sample clearly showed a heterozygous genotype (--MED/αα) and a heterozygous phenotype was confirmed subsequently. However, the more recently published multiplex primers72,73 have been found to be more robust and give more reproducible results. As well as a redesign of the primer sequences, the addition of betaine to the reaction mixture and the use of a “hot start” ampli-

fication protocol seem to be key to the improved reliability.

MLPA analysis Other methods developed to diagnose deletion mutations include include the use of real-time quantitative PCR analysis for the Southeast Asian α0-thalassemia deletion in Taiwan,74 the use of denaturing HPLC to diagnose the 4.2 kb α+thalassemia deletion gene in Chinese individuals,75 real-time quantitative PCR to detect the Southeast Asian α0-thalassemia deletion59 and the use of an oligonucleotide microarray to detect the Southeast Asian α0-thalassemia deletion and the 3.7 kb and 4.2 kb α+-thalassemia deletions.76,77 However, the most useful recent development for the diagnosis of deletion mutations is the technique known as multiplex ligation-dependent probe amplification (MLPA).78

CHAP T E R 21

MLPA is a simple technique designed for the rapid quantitative analysis of DNA sequence copy number. The technique is based on the ligation and PCR amplification of two adjacently hybridizing oligonucleotides. Each oligonucleotide pair is designed to give a product of a unique length, and by using common ends, all probes can be amplified with one primer pair. Using a fluorescent label allows probe separation on a capillary sequencing system. Comparison of the product quantities against a normal control permits the mapping of gene deletions that result in α-thalassemia and β-thalassemia. For the α-globin gene cluster, two probe sets have been developed which number 35 probe pairs covering a genomic region of ∼700 kb with an average distance of ∼20 kb. The probes can detect both rare and novel forms of deletional αthalassemia that cannot be diagnosed by gap-PCR, and MLPA provides an excellent back-up diagnostic method for prenatal diagnosis of homozygous α-thalassemia. However, the method does not provide a definitive diagnosis for any particular deletion mutation, as it simply detects the deletion of DNA sequence between two probe locations, and thus only a presumed identification of a deletion mutation can be made.

PCR techniques for nondeletion mutations The nondeletion α+-thalassemia mutations can be identified by PCR techniques following the selective amplification of the α-globin genes,73 and then by analysis of the product for the appropriate mutation. This technique allows the amplified product from each α-globin gene to be analyzed for the expected known mutation according to the ethnic origin of the patient or the gene. In theory, any other technique for the direct detection of point mutations, such as allele-specific oligonucleotide hybridization or allele-specific priming, may be used for the diagnosis of nondeletion α+thalassemia. For example, several of the nondeletion mutations alter a restriction enzyme site and may be analyzed for by restriction enzyme analysis, in a manner similar to that reported for the diagnosis of Hb Constant Spring by Mse I digestion,19 as illustrated in Figure 21.2 However, no simple strategy to diagnose all the known nondeletion mutations for both α-globin genes has been

Prenatal Diagnosis of the Hemoglobinopathies

1

2

3

661

4

700 bp → 627 bp →

400 bp → 300 bp →

Figure 21.2 RE-PCR analysis using Mse I for the diagnosis of the α2 termination codon mutation for Hb Constant Spring. Tracks 1 and 2 show the digested amplification products for a carrier (genotype αCSα/αα) and tracks 3 and 4 show the results for a normal individual (genotype αα/αα). The mutation for Hb Constant Spring destroys the Mse I site and this creates a larger diagnostic fragment of 700 bp.

reported. The only published approach to date is a complex strategy involving the combined application of the indirect detection methods of denaturing gradient gel electrophoresis (DGGE) and single-strand conformation analysis (SSCA), followed by direct DNA sequencing.79 Therefore direct DNA sequencing of the whole of the selectively amplified gene product for the α1 and α2 genes is often used for identification of a suspected nondeletion α+-thalassemia mutation. β-Thalassemia Although more than 200 different β-thalassemia mutations have been characterized, only about 25 occur at a frequency of 1 percent or greater and thus account for most mutations worldwide.80 All the mutations are regionally specific, and the spectrum of mutations has been determined. Each population has been found to have just a few of the commonly found mutations, together with a larger and more variable number of rare ones. The mutations can be classified broadly as being of Mediterranean, Indian, Chinese or African origin; Table 21.5 lists the frequencies of the common mutations found in several countries from each of these four

662

Genetic Disorders and the Fetus

Table 21.5 The distribution of the common β-thalassemia mutations expressed as percentage gene frequencies of the total number of thalassemia chromosomes studied Mutation

−88(C → T) −87(C → G)

Mediterranean

India

Italy

Greece

Turkey

Pakistan

–

–

–

–

0.4

1.8

Chinese India 0.8

African China

Thailand

African-American

–

–

21.4

1.2

–

–

–

–

–

2.5

–

–

–

–

–

−30(T → A)

–

–

−29(A → G)

–

–

–

–

–

1.9

−28(A → G)

–

–

–

–

–

11.6

CAP+1(A → C)

–

–

–

–

CD5(−CT)

–

CD6(−A)

0.4

1.7

–

60.3 4.9

–

–

– –

1.2

0.8

–

–

–

–

–

2.9

0.6

–

–

–

–

–

CD8(−AA)

–

–

–

–

–

–

CD8/9(+G)

–

–

–

28.9

12.0

–

–

–

CD15(G → A)

–

–

–

3.5

0.8

–

–

CD16(−C)

–

–

–

1.3

1.7

–

–

–

CD17(A → T)

–

–

–

–

v

10.5

24.7

–

CD24(T → A)

–

–

–

–

–

–

–

CD39(C → T)

40.1

17.4

–

–

–

–

CD41/42(−TCTT)

–

–

–

13.7

38.6

46.4

–

CD71/72(+A)

–

–

–

–

12.4

2.3

–

IVSI-1(G → A)

0.6

4.3

13.6

7.4

3.5

7.9 – 2.5

–

–

0.8

7.9 –

–

–

–

–

–

IVSI-1(G → T)

–

–

–

8.2

6.6

IVSI-5(G → C)

–

–

–

26.4

48.5

IVSI-6(T → C)

16.3

7.4

17.4

–

–

–

–

–

IVSI-110(G → A)

29.8

43.7

41.9

–

–

–

–

–

1.1

2.1

9.7

–

–

–

–

–

–

15.7

–

–

–

–

23.3

13.3

–

–

0.5

0.9

IVSII-1(G → A) IVSII-654(C → T)

–

IVSII-745(C → G) 619bp deletion Others

– 3.5

–

– 7.1

– 4.1

2.7 –

2.2

9.7

2.5

6.8

– 4.9

–

– 8.9

– – –

7.9

10.6

CD, codon; IVS, intervening sequence; bp, base pairs.

major ethnic groups. Within each ethnic group, there is still much variation in the distribution of mutations. For example, in Sardinia, the most common mutation is CD39 (C → T), which occurs at a frequency of 95 percent, whereas in Cyprus CD39 accounts for only approximately 2 percent of the mutations and the mutation IVSI-110 (G → A) is most common, at a frequency of 80 percent.81 The strategy for identifying β-thalassemia mutations in most diagnostic laboratories depends on knowing the spectrum of the common and rare mutations that have been characterized in the ethnic group of the individual being screened. Usually, the common ones are screened for first,

using a PCR technique that allows the simultaneous detection of multiple mutations. This approach will identify the mutation in more than 90 percent of cases; an additional screening for the possible rare mutations will identify the defect in most of the remaining cases. Mutations remaining unknown after this second screening are characterized by direct DNA sequence analysis, usually after the localization of the site of the mutation by the application of a nonspecific detection method such as DGGE. Although a bewildering variety of PCR techniques have been described for the diagnosis of point mutations, most diagnostic laboratories are using one or more of the techniques described below.

CHAP T E R 21

Allele-specific oligonucleotides The use of allele-specific oligonucleotide probes (ASOs) to hybridize to amplified DNA bound to nylon membrane in the form of dots was the first diagnostic PCR-based method to be developed. Since then it has been applied with great success, especially in populations such as the one in Sardinia with just one common mutation and a small number of rare ones.82 The method is based on the use of two oligonucleotide probes for each mutation, one complementary to the mutant DNA sequence and the other complementary to the normal β-gene sequence at that position. The probes can be labeled with 32P-labeled deoxynucleoside triphosphates, biotin or horseradish peroxidase, but the method is limited by the need for separate hybridizations when screening for more than one mutation. To overcome this problem, the method of reverse dot blotting has been developed in which the roles of the oligonucleotide probe and amplified target DNA are reversed.83 Probe pairs, complementary to the mutant and normal DNA sequences, are bound to nylon membrane in the form of dots or slots and the amplified DNA, labeled by either the use of end-labeled primers or the internal incorporation of biotinylated dUTP, is then hybridized to the filter. This procedure allows multiple mutations to be tested for in one hybridization reaction. It has been applied recently to the diagnosis of β-thalassemia mutations in Mediterranean carriers,84 African Americans,85 and Thais,86 using a two-step procedure with one nylon strip for the common mutations and the other for the less common ones. The principle of reverse dot blotting has been brought up to date by the development of microarrays for the simultaneous detection of multiple βthalassemia mutations. Several groups have now published details of a DNA chip platform which has been used to genotype β-thalassemia carriers and patients.77,87 The approach of tagged singlebased extension and hybridization to glass or flowthrough arrays has been developed for the detection of 17 β-globin mutations88 and a similar approach of arrayed primer extension has been used to detect 23 mutations.89 However, it is not clear whether these state-of-the-art methods will prove cheap enough to replace conventional techniques in the

Prenatal Diagnosis of the Hemoglobinopathies

663

future and whether there will be a viable market for thalassemia mutation chips, especially for the diagnosis of mutations in populations with just one or two very common mutations which can be easily screened for by rapid low-tech methods and for which the additional screening capacity on the chip would be redundant.

Primer-specific amplification A number of different methods have been developed based on the principle of primer-specific amplification, which is that a perfectly matched PCR primer is much more efficient in annealing and directing primer extension than one containing one or two mismatched bases. The most widely used technique is the amplification refractory mutation system (ARMS) and its application is described in greater detail below. The technique has allowed the development of simple diagnostic strategies for the diagnosis of β-thalassemia mutations in individuals of many countries, including India, Pakistan, Thailand, Syria, Mauritius and Sri Lanka.90 This quick screening method does not require any form of labeling, as the amplified products are visualized simply by agarose gel electrophoresis and ethidium bromide staining. More than one mutation may be screened for at the same time in a single PCR reaction (multiplexing), if the ARMS primers are coupled with the same common primer.91 Fluorescent labeling of the common primer allows the sizing of the amplification products on an automated DNA fragment analyzer.92 Multiplex allele-specific PCR has also been used in combination with chip-based capillary electrophoresis for the prenatal diagnosis of β-thalassaemia mutations.93 If the normal and mutant ARMS primers for a specific mutation are co-amplified in the same reaction, they compete with each other to amplify the target sequence. This technique is called competitive oligonucleotide priming (COP) and requires that the two ARMS primers be labeled differently. Fluorescent labels permit a diagnosis by means of a color complementation assay.94 A variation of this method is simply to use ARMS primers that differ in length; therefore, a diagnosis can be made by analysis of the different product sizes. This technique, called mutagenetically separated polymerase chain reaction (MS-PCR), has

664

Genetic Disorders and the Fetus

been applied to the prenatal diagnosis of βthalassemia in Taiwan.95

Restriction enzyme analysis Approximately 40 β-thalassemia mutations are known to create or abolish a restriction endonuclease site.96 Most of these can be detected quickly by restriction endonuclease analysis of amplified DNA. The presence or absence of the enzyme site is determined from the pattern of digested fragments after agarose or polyacrylamide gel electrophoresis. As a screening method, this approach is limited by the small fraction of β-thalassemia mutations that affect a restriction enzyme site and because many of the restriction enzymes involved are very expensive. Mutations that do not naturally create or abolish restriction sites may be detected by the technique of amplification-created restriction sites (ACRS). This method uses primers that are designed to insert new bases adjacent to the mutation sequence and thus create a new restriction site, allowing known mutations to be detected by restriction enzyme digestion of the PCR product.97

Other methods for point mutations Many other techniques have now been developed for the diagnosis of known β-globin gene point mutations, including the use of denaturing highperformance liquid chromatography (DHPLC), the DNA ligase reaction, minisequencing, realtime PCR and multiplex primer extension technology. For example, DHPLC has been used for the analysis of polymorphic duplexes created by allelespecific priming,98 for the analysis of five common Southeast Asian mutations by multiplex minisequencing,99 for multiplex primer extension analysis for 10 Taiwanese mutations100 and the most common Chinese mutations,101 and for the screening of the 11 most common Greek mutations.102 Real-time PCR quantification and melting curve analysis have been used to provide rapid genotyping for a panel of the 10 most frequent Greek mutations67 and for detecting six Lebanese mutations.103 The DNA ligase method has been updated by the development of a novel piezoelectric method which has been used for the detection of a codon 17 point mutation using nano-gold-amplified DNA probes.104 All these methods are reported to

provide rapid and accurate genotyping of the common mutations and are worth considering as alternative diagnostic approaches for point mutations.

Gap-PCR and MLPA The β-globin gene deletion mutations are diagnosed by Gap-PCR and/or MLPA analysis. Table 21.4 lists the deletions diagnosable by gap-PCR. Small deletion mutations can be detected by PCR using two primers complementary to the sense and antisense strand in the DNA regions which flank the deletion.105 For large deletions, amplified product using flanking primers is obtained from only the deletion allele, because the distance between the two primers is too great to amplify normal DNA. In such cases, the normal allele may be detected by amplifying sequences spanning one of the breakpoints, using one primer complementary to the deleted sequence and one complementary to flanking DNA.106 As well as deletion β-thalassemia, Hb Lepore and a number of δβthalassemia and HPFH deletion mutations can be diagnosed by this method.107 However, all β-globin gene cluster deletions can be now detected by MLPA. For the β-cluster a total of three probe sets have been developed, consisting of 50 probe pairs covering a region of ∼500 kb and with an average distance of ∼10 kb. Thus all of the larger β-thalassemia deletions which cannot be diagnosed by gap-PCR because their breakpoint sequences are unknown can now be detected by this method, although as for the α-thalassemia deletions, the technique cannot provide a definitive diagnosis of any particular deletion, just a presumed diagnosis. This technique now also provides a second method for the prenatal diagnosis of βglobin gene deletion mutations.

Unknown mutations A number of techniques have been applied for the detection of β-thalassemia mutations without prior knowledge of the molecular defect. The first to be developed was DGGE, which allows the separation of DNA fragments differing by a single base change according to their melting properties.108 Another approach was heteroduplex analysis using nondenaturing gel electrophoresis. Unique heteroduplex patterns can be generated for each muta-

CHAP T E R 21

tion by annealing an amplified target DNA fragment with an amplified heteroduplex generator molecule, a synthetic oligonucleotide of about 130 bases in length containing deliberate sequence changes or identifiers at known mutation positions.109 The above techniques simply pinpoint the presence of a mutation or DNA polymorphism in the amplified target sequence. Sequencing of the amplified product is then required to identify the localized mutation. This can now be performed very efficiently using an automated DNA sequencing machine using fluorescence detection technology. For many laboratories, direct DNA sequencing of amplified product has now become the primary method of identifying rare or unknown βthalassemia mutations. In our laboratory we amplify the β-globin gene using three pairs of primers. Once a rare or novel mutation has been identified through DNA sequencing, the DNA sample can be used as a control for the development of ARMS primers to provide a more rapid and cheaper screening of further cases.

Direct detection: ARMS-PCR The allele-specific priming technique, known as the amplification refractory mutation system (ARMS), was developed for the prenatal diagnosis of β-thalassemia in our laboratory.110,111 For prenatal diagnosis, two primers must be designed that will generate specific amplification products: one with the mutant allele and the other with the normal sequence. The nucleotide at the 3′-terminus of each primer is complementary to the base in the respective target sequence at the site of the mutation. In addition, a deliberate mismatch to the target sequence is included at the second, third or fourth base from the 3′ end. The deliberate mismatch enhances the specificity of the primer, because all 3′ terminal mismatches on their own, except for C-C, G-A, and A-A mismatches, will allow some extension of the primer and thus generate nonspecific amplification product.112 The mutation-specific ARMS primers for the most common β-thalassemia mutations and β-globin variants are listed in Table 21.6. All are the same length (30 mers), so all can be used at one annealing temperature (65 °C), enabling screening for multiple mutations simultaneously. Primers for

Prenatal Diagnosis of the Hemoglobinopathies

665

the specific detection of the corresponding normal allele are listed in Table 21.7. These are required for prenatal diagnosis of cases in which both partners of a couple at risk for β-thalassemia carry the same mutation. Each normal ARMS primer must be tested to check that it is working correctly, using DNA from an individual homozygous for the particular mutation. The list of primers in Table 21.7 is shorter than that in Table 21.6 because of the lack of appropriate DNA controls in the laboratory. Each ARMS primer requires a second primer to generate the allele-specific product; in addition, two control primers must be included in the PCR reaction to generate an unrelated product that indicates that the reaction mixture was set up properly and everything is working correctly. The DNA sample is amplified with each mutant ARMS primer in a separate amplification reaction and the products visualized after electrophoresis. Figure 21.3 illustrates a screening of a sample for seven Mediterranean β-thalassemia mutations at one time. A control DNA known to carry each mutation was also amplified for comparison (evennumbered tracks). The unknown DNA sample produced an amplified product with only the IVSI110 mutant primer (track 1). For couples of Cypriot origin, this mutation usually is screened for first because it is so common; if it is not found, all the others are screened for simultaneously afterward. Similarly, for Asian Indian couples, the four most common mutations, IVSII-5, IVSI-1, Fr.8/9, and Fr.41/42,113 are screened for first. The 619 bp deletion gene also is screened for in the same reaction as the control pair of primers are designed to span the deletion, and instead of a normal 861 bp fragment, a characteristic 242 bp fragment is produced.110 Note that the DNA from an individual doubly heterozygous for the 619 bp deletion and, say, the IVSI-5 mutation will produce three bands: the 861 bp fragment from the IVSI-5 allele, an IVSI-5-specific fragment of 285 bp from the mutant IVSI-5 ARMS primer, and the 242 bp fragment. Figure 21.4 illustrates a prenatal diagnosis for a fetus at risk for two different mutations: codon 39 and IVSI-110. A normal ARMS primer is not required in this case because the fetal DNA has to be tested with the mutant ARMS primer for each mutation. The CVS DNA in Figure 21.4 was diag-

666

Genetic Disorders and the Fetus

Table 21.6 Primer sequences used for the detection of the common β-thalassemia mutations by the allele-specific priming technique Mutation

Oligonucleotide sequence

Second primer

Product size (bp)

−88(C → T)

TCACTTAGACCTCACCCTGTGGAGCCTCAT

A

684

−87(C → G)

CACTTAGACCTCACCCTGTGGAGCCACCCG

A

683

−30(T → A)

GCAGGGAGGGCAGGAGCCAGGGCTGGGGAA

A

626

−29(A → G)

CAGGGAGGGCAGGAGCCAGGGCTGGGTATG

A

625

−28(A → G)

AGGGAGGGCAGGAGCCAGGGCTGGGCTTAG

A

624

CAP+1(A → G)

ATAAGTCAGGGCAGAGCCATCTATTGGTTC

A

597

CD5(–CT)

TCAAACAGACACCATGGTGCACCTGAGTCG

A

528

CD6(–A)

CCCACAGGGCAGTAACGGCAGACTTCTGCC

B

207

CD8(–AA)

ACACCATGGTGCACCTGACTCCTGAGCAGG

A

520

CD8/9(+G)

CCTTGCCCCACAGGGCAGTAACGGCACACC

B

225

CD15(G → A)

TGAGGAGAAGTCTGCCGTTACTGCCCAGTA

A

500

CD16(–C)

TCACCACCAACTTCATCCACGTTCACGTTC

B

238

CD17(A → T)

CTCACCACCAACTTCAGCCACGTTCAGCTA

B

239

CD24(T → A)

CTTGATACCAACCTGCCCAGGGCCTCTCCT

B

262

CD39(C → T)

CAGATCCCCAAAGGACTCAAAGAACCTGTA

B

436

CD41/42(–TCTT)

GAGTGGACAGATCCCCAAAGGACTCAACCT

B

439

CD71-72(+A)

CATGGCAAGAAAGTGCTCGGTGCCTTTAAG

C

241

IVSI-1(G → A)

TTAAACCTGTCTTGTAACCTTGATACCGAT

B

281

IVSI-1(G → T)

TTAAACCTGTCTTGTAACCTTGATACCGAAA

B

281

IVSI-5(G → C)

CTCCTTAAACCTGTCTTGTAACCTTGTTAG

B

285

IVSI-6(T → C)

TCTCCTTAAACCTGTCTTGTAACCTTCATG

B

286

IVSI-110(G → A)

ACCAGCAGCCTAAGGGTGGGAAAATAGAGT

B

419

IVSII-1(G → A)

AAGAAAACATCAAGGGTCCCATAGACTGAT

B

634

IVSII-654(C → T)

GAATAACAGTGATAATTTCTGGGTTAACGT*

D

829

IVSII-745(C → G)

TCATATTGCTAATAGCAGCTACAATCGAGG*

D

738

βSCD6(A → T)

CCCACAGGGCAGTAACGGCAGACTTCTGCA

B

207

βCCD6(G → A)

CCACAGGGCAGTAACGGCAGACTTCTCGTT

B

206

βECD26(G → A)

TAACCTTGATACCAACCTGCCCAGGGCGTT

B

236

The above primers are coupled as indicated with primers A, B, C or D. A: CCCCTTCCTATGACATGAACTTAA B: ACCTCACCCTGTGGAGCCAC C: TTCGTCTGTTTCCCATTCTAAACT; or D: GAGTCAAGGCTGAGAGATGCAGGA. The control primers used were primers D plus E: CAATGTATCATGCCTCTTTGCACC for all the above mutation-specific ARMS primers except the two marked *, with which the Gγ-Hind |III RFLP primers (Table 21.7) were used.

nosed as β-thalassemia trait having inherited the codon 39 mutation from the father and a normal allele from the mother.

Haplotype analysis Linkage analysis of restriction fragment length polymorphisms (RFLPs) within the β-globin gene cluster often can be used for prenatal diagnosis of

β-thalassemia in the rare cases in which one or both of the mutations remain unidentified after screening using a direct detection method such as ARMS. The method is simple, quick and cheap, and provides an alternative method for diagnostic laboratories lacking DNA sequencing facilities in developing countries. The technique also can enable the prenatal diagnosis of β- and δβ-

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Table 21.7 Primer sequences used for the detection of the normal DNA sequence by the allele-specific priming technique Mutation

Oligonucleotide sequence

Second primer

Product size (bp)

−87(C → G)

CACTTAGACCTCACCCTGTGGAGCCACCCC

A

683

CD5(−CT)

CAAACAGACACCATGGTGCACCTGACTCCT

A

528

CD8(−AA)

ACACCATGGTGCACCTGACTCCTGAGCAGA

A

520

CD8/9(+G)

CCTTGCCCCACAGGGCAGTAACGGCACACT

B

225

CD15(G → A)

TGAGGAGAAGTCTGCCGTTACTGCCCAGTA

A

500

CD39(C → T)

TTAGGCTGCTGGTGGTCTACCCTTGGTCCC

A

299

CD41/42(–TCTT)

GAGTGGACAGATCCCCAAAGGACTCAAAGA

B

439

IVSI-1(G → A)

TTAAACCTGTCTTGTAACCTTGATACCCAC

B

281

IVSI-1(G → T)

GATGAAGTTGGTGGTGAGGCCCTGGGTAGG

A

455

IVSI-5(G → C)

CTCCTTAAACCTGTCTTGTAACCTTGTTAC

B

285

IVSI-6(T → C)

AGTTGGTGGTGAGGCCCTGGGCAGGTTGGT

A

449

IVSI-110(G → A)

ACCAGCAGCCTAAGGGTGGGAAAATACACC

B

419

IVSII-1(G → A)

AAGAAAACATCAAGGGTCCCATAGACTGAC

B

634

IVSII-654(C → T)

GAATAACAGTGATAATTTCTGGGTTAACGC

D

829

IVSII-745(C → G)

TCATATTGCTAATAGCAGCTACAATCGAGC

D

738

βSCD6(A → T)

AACAGACACCATGGTGCACCTGACTCGTGA

A

527

βECD26(G → A)

TAACCTTGATACCAACCTGCCCAGGGCGTC

B

236

See Table 21.6 footnote for details of primers A–D and control primers.

thalassemia deletion mutations through the apparent nonmendelian inheritance of the RFLP (due to the hemizygosity created by the inheritance of deleted sequences on one chromosome). Finally, haplotype analysis may provide an alternative approach for the confirmation of a prenatal diagnosis result obtained by a direct detection method such as ARMS. At least 18 RFLPs have been characterized within the β-globin gene cluster.114 However, most of these RFLP sites are nonrandomly associated with each other and thus they combine to produce just a handful of haplotypes.115 In particular, they form a 5′ cluster that is 5′ to the δ gene and a 3′ cluster that extends downstream from the β-globin gene. In between is a 9 kb stretch of DNA containing a relative hot spot for meiotic recombination. The recombination between the two clusters has been calculated to be approximately 1 in 350 meioses.116 Hybridization studies have shown that each βthalassemia mutation is strongly associated with just one or two haplotypes,117 probably because of their recent origin compared with the haplotypes, and thus haplotype analysis has been used to study

the origins of identical mutations found in different ethnic groups. The β-globin gene cluster haplotype normally consists of five RFLPs located in the 5′ cluster (Hind II/ε-gene; Hind III/Gγ-gene; Hind III/Aγ-gene; Hind II/3′ψβ; and Hind II/5′ψβ) and two RFLPs in the 3′ cluster (Ava II/β-gene; BamH I/β-gene).118 All of the seven RFLPs except BamH I can be analyzed by PCR very simply and quickly.119 Primers have been designed to span the RFLP site and produce easily identifiable fragments after electrophoresis of the digested products The primer sequences and sizes of the fragments generated are listed in Table 21.8. The BamH I RFLP is located within an L1 repetitive element creating amplification problems and a Hinf I RFLP located just 3′ to the β-globin gene is used instead, because these two RFLPs have been found to exist in linkage disequilibrium.120 Three other RFLPs are included in Table 21.9. An Ava II RFLP in the ψβ-gene is extremely useful in haplotype analysis of Mediterranean β-thalassemia heterozygotes. The (-) allele for this RFLP is frequently found on chromosomes carrying the IVSI-110 mutation, whereas it is very

668

Genetic Disorders and the Fetus

Figure 21.3 The screening of a DNA sample for seven common Mediterranean mutations by the allele-specific priming technique known as ARMS. The diagram shows the β-globin gene and the positions of the seven mutations: 1, IVSI-110; 2, IVSI-1; 3, IVSI-6; 4 codon 39; 5, codon 6; 6, IVSII-1; 7, IVSII-745. The gel shows the amplification products from DNA of a β-thalassemia heterozygote in the odd numbered lanes, and products generated by control DNAs in the even numbered lanes

for each mutation screened for: IVSI-110, G → A (lanes 1 and 2); IVSI-1, G → A (lanes 3 and 4); IVSI-6, T → C, (lanes 5 and 6); codon 39, C → T (lanes 7 and 8); codon 6, -A, (lanes 9 and 10), IVSII-1, G → A (lanes 10 and 11), IVSII-745, C → G (lanes 13 and 14). In lanes 1–12, the control primers D and E produced an 861 bp fragment, and in lanes 13 and 14, a different pair of control primers produced a 323 bp fragment. The primers used are listed in Tables 21.5 and 21.6.

rare on normal β-globin chromosomes121 and thus is a very useful informative marker for individuals heterozygous for this mutation. The Rsa I RFLP located just 5′ to the β-globin gene is useful for linkage analysis because it seems to be unlinked to either the 5′ cluster or the 3′ cluster RFLPs and thus may be informative when the 5′ haplotype and the 3′ haplotype are not. Finally, the Gγ-Xmn I RFLP, created by the nondeletion HPFH C→T mutation at position −158, is included because of its use in the analysis of sickle cell gene haplotypes and in individuals with thalassemia intermedia. To obtain the linkage phase of informative RFLPs, one requires DNA from either (1) a normal or an affected child; (2) both sets of grandparents

if no children are available; or (3) one set of grandparents if a child heterozygous for β-thalassemia is available. It is essential that one of the grandparents on each side of the family is normal with respect to β-thalassemia, otherwise the linkage phase cannot be determined. Informative RFLPs are found in more than 80 percent of the families studied and thus, haplotype analysis is a very useful alternative approach to provide confirmation of a diagnosis obtained by the direct detection of mutations.110 δβ-Thalassemia, Hb Lepore, and HPFH δβ-thalassemia and the deletion types of HPFH are characterized by the complete absence of Hb A and

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Figure 21.4 Prenatal diagnosis of β-thalassemia by the allele-specific priming technique known as ARMS. The diagram shows the positions of the β-thalassemia mutations IVSI-110, G → A (1) and codon 39, C → T (2), plus the locations of the primers used to diagnose these two mutations (as specified in Table 21.5). The gel shows the amplification products using the mutant

ARMS primer for codon 39 (lanes 1, 2 and 3) and for IVSI-110 (lanes 4, 5 and 6). The DNA samples were: lane 1, fetal DNA; lane 2, maternal DNA; lane 3, paternal DNA; lane 4, maternal DNA; lane 5, paternal DNA; lane 6, fetal DNA. The 436 bp product is diagnostic for the codon 39 mutation and the 419 bp product for IVSI-110.

Hb A2 in homozygotes and an elevated level of Hb F in heterozygotes. Both conditions are caused by large DNA deletions involving the β-globin gene cluster affecting the β and δ genes but leaving either one or both of the γ-globin genes intact. More than 50 different deletion mutations have been identified, and they can be classified into the

(δβ)0 and (Aγδβ)0 thalassemias, HPFH conditions, fusion chain variants, and (εγδβ)0 thalassemia.10

δβ-Thalassemia The (δβ)0 thalassemias are characterized by the Hb F consisting of both Gγ- and Aγ-globin chains, as both γ-globin genes remain intact in these condi-

670

Genetic Disorders and the Fetus

Table 21.8 Oligonucleotide primers used for analysis of β-globin gene cluster RFLPs RFLP

ε-Hind II

G

γ-Xmn I

G

γ-Hind III

A

γ-Hind III

5′ψβ-Hind II

5′ψβ-Ava II

3′ψβ-Hind II

β-Rsa I

Primer sequence 5′-3′

Annealing

Product

Absence

Presence

temperature, oC

size, bp

of site, bp

of site, bp

760

314

–

446

650

450

–

200

323

235

TCTCTGTTTGATGACAAATTC

55

AGTCATTGGTCAAGGCTGACC

55

760

AACTGTTGCTTTATAGGATTTT

55

AGGAGCTTATTGATAACTCAGAC

55

AGTGCTGCAAAGAAGAACAACTACC

65

CTCGCATCATGGGCCAGTGAGCCTC

65

ATGCTGCTAATGCTTCATTAC

55

TCATTGTGTGATCTCTCTCAGCAG

55

TCCTATCCATTACTGTTCCTTGAA

55

ATTGTCTTATTCTAGAGACGATTT

55

TCCTATCCATTACTGTTCCTTGAA

55

ATTGTCTTATTCTAGAGACGATTT

55

GTACTCATACTTTAAGTCCTAACT

55

TAAGCAAGATTATTTCTGGTCTCT

55

–

AGACATAATTTATTAGCATGCATG

55

CCCCTTCCTATGACATGAACTTAA

55

– 650 – 323 –

–

635

98

635

327

–

308

794

687

–

107

794

442

–

352

914

480

–

434

1200

692

692

–

413

331

–

100

100

– 794 – 794 – 914

82 β-Ava II

β-Hinf I

GTGGTCTACCCTTGGACCCAGAGG

65

TTCGTCTGTTTCCCATTCTAAACT

65

GGAGGTTAAAGTTTTGCTATGCTGTAT

55

GGGCCTATGATAGGGTAAT

55

tions. Heterozygotes have normal levels of Hb A2 and an Hb F level of 5–15 percent which, for most mutations, is heterogeneously distributed in the red cells. There is a reduction of the non-α-globin chains compared to α-globin and the red cells are hypochromic and microcytic. Homozygotes for this condition have thalassemia intermedia. The (Aγδβ)0 thalassemias are characterized by the Hb F containing only Gγ-globin chains, as the G γ-globin gene has been deleted in these conditions. Apart from this distinction, the phenotypes of the heterozygous and homozygous states are identical to those for (δβ)0-thalassemia. The (εγδβ)0 thalassemias are conditions that result from several different long deletions that start upstream of the ε-gene and remove all of the β-globin gene cluster or, in two cases, the deletion

328 – 475

328

227

–

101

320

219

–

155

155

–

–

108

ends between the δ- and β-genes, thus sparing the β-globin gene, but in both cases no β-globin synthesis occurs. This is because the deletions remove the β-globin gene cluster locus control region (LCR) located 50 kb upstream of the ε-gene. In adult life, heterozygotes for this condition have a hematologic picture similar to that of β-thalassemia trait, with a normal Hb A2 level. The homozygous condition is presumed to be incompatible with fetal survival.

Hb Lepore Two deletions in the β-globin gene cluster create an abnormal Hb chain as a result of unequal crossing over between globin genes. Hb Lepore is a hybrid globin chain composed of δ and β gene sequences, and Hb Kenya is composed of γand β

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Table 21.9 Oligonucleotide primers for the detection of βS, βE, βD Punjab, and βo Arab mutations as RFLPs Mutation and

Primer sequences 5′-3′

affected RE site

Annealing

Productsize,

Absence

Presence

temperature, oC

bp

of site, bp

of site, bp

386

201

67

175

βSCD6 (A → T)

ACCTCACCCTGTGGAGCCAC

65

443

(Loses Dde I site)

GAGTGGACAGATCCCCAAAGGACTCAAGGA

65

–

–

–

–

67

βECD26 (G → A)

ACCTCACCCTGTGGAGCCAC

65

443

231

171

(Loses Mnl I site)

GAGTGGACAGATCCCCAAAGGACTCAAGGA

–

–

89

89

–

–

56

60

–

–

35

35

–

–

33

33

CAATGTATCATGCCTCTTTGCACC

65

861

861

552

GAGTCAAGGCTGAGAGATGCAGGA

65

–

–

309

CAATGTATCATGCCTCTTTGCACC

65

861

861

552

GAGTCAAGGCTGAGAGATGCAGGA

65

–

–

309

βD-Punjab CD121 (G → C) (Loses Eco RI site) O-Arab

β

CD121

(G → A) (Loses Eco RI site)

gene sequences. Hb Lepore homozygotes have a phenotype similar to that of thalassemia major or severe thalassemia intermedia. Hb Kenya has been observed only in the heterozygous state and is similar to heterozygous HPFH, with individuals having 5–10 percent Hb F, normal red cell morphology, and balanced globin chain synthesis.

HPFH The deletional HPFH conditions can be regarded as a type of δβ-thalassemia in which the reduction in β-globin chain production is almost completely compensated for by the increased γ-globin chain production. Homozygous individuals have 100 percent F composed of both Aγ and Gγ globin chains but, in contrast to (δβ)0 thalassemia homozygotes, are clinically normal. Heterozygotes have an elevated Hb F level of 17–35 percent, higher than that found in δβ thalassemia heterozygotes, and the Hb F is distributed uniformly (pancellular) in red cells with near-normal MCH and MCV values. Finally, there is a group of conditions called nondeletion HPFH in which heterozygous individuals have normal red cells and no clinical abnormalities and an elevated Hb F level as a result of a point mutation in the promoter region of the A γ or Gγ globin gene in most cases. The percentage Hb F is variable, ranging from 1–3 in the Swiss type

to 10–20 in the Greek type. The only recorded homozygotes for nondeletion HPFH are for the British type described in a single family.

Molecular diagnosis Molecular diagnosis is by Gap-PCR and MLPA. Gap-PCR provides a definitive diagnosis for six δβ-thalassemia, three HPFH deletion mutations and for Hb Lepore,44 all of which have had both breakpoint sequences characterized to permit the synthesis of amplification primers,107 as listed in Table 21.4. The remaining δβ-thalassemia and HPFH deletion mutations can be detected and a presumptive diagnosis made by MLPA analysis. A total of three probe sets containing 50 probes have been developed to cover a region of 500 kb of the β-globin gene cluster at an average distance of 10 kb, enabling all large deletions to be detected, including the εγδβ-thalassaemias that leave the βglobin genes intact and are not easily detected by conventional techniques. Hb S Hb S (β Glu → Val) is caused by an A → T substitution in the second nucleotide of the sixth codon of the β-globin gene. The mutation destroys the recognition site for three restriction enzymes, Mnl I, Dde I, and Mst II, the latter being the

672

Genetic Disorders and the Fetus

Figure 21.5 The diagnosis of the sickle cell anemia gene by Dde I digestion of amplified DNA. The diagram shows the location of the two PCR primers used (as listed in Table 21.7) and the sites of the Dde I sites with respect to the βS gene mutation at codon 6. The Dde I site 5′ to codon 6, marked by the dotted arrow, is a rare polymorphic site caused by the sequence change G → A

at position −83 to the β-globin gene. When present, the 175 bp fragment is cleaved to give 153 bp and 27 bp fragments as shown in lane 2. The gel shows DNA fragments from: lane 1, φX174 digested with Hae III; lane 2, AS individual; lane 3, fetal DNA with AS genotype; lane 4, SS individual; lane 5, AS individual; lane 6, AA individual.

enzyme of choice for the detection of the βS allele by Southern blot analysis122 because it cuts infrequently around the β-globin gene, producing large DNA fragments. However, for PCR diagnosis Dde I is used.123 Dde I is a frequent cutter, and several constant sites can be included in the amplified β gene fragment to act as a control for the complete

digestion of the amplified product. The primer sequences currently used in our laboratory are presented in Table 21.9. A Dde 1 analysis of amplified DNA from a normal individual (AA), an individual with sickle cell trait (AS), and a sickle cell homozygote (SS) is shown in Figure 21.5. The βS mutation creates a 321 bp fragment that is absent in the

CHAP T E R 21

normal DNA sample. The βS mutation can also be detected by a variety of other PCR-based techniques, such as ASO/dot blotting or the ARMS method. The primer sequences for the latter method are included in Table 21.6. Hb C Hb C (β6 Glu → Lys) is caused by G → A substitution in the first nucleotide of codon 6 of the βglobin gene. It is found predominantly in West African Negroes and the frequency of heterozygous state has reached 28 percent in some parts of Ghana. The heterozygous state is symptomless and the homozygous state is characterized by a variable hemolytic anemia due to the red cells being abnormally rigid and having a shortened lifespan but is associated with no serious clinical disability.10 Its importance lies in its interaction with the sickle cell gene. The Hb C mutation also occurs inside the recognition sites for Mnl I, Dde I, and Mst II at codon 6. However, it does not abolish the site for Dde I or Mst II because the mutation affects a nonspecific nucleotide in the recognition sequence. Thus, Dde I or Mst II cannot be used to detect the βC mutation, and another method such as allele-specific oligonucleotide hybridization to amplified DNA or the allele-specific priming technique must be used. The primer sequences used for the latter method are included in Tables 21.7 and 21.9. Hb D-Punjab and Hb O-Arab Hb D-Punjab (β121 Glu → Gln) and Hb O-Arab (β121 Glu → Lys) in combination with Hb S give rise to doubly heterozygous conditions that are similar in severity to homozygous sickle cell disease. Hb D-Punjab in combination with βthalassemia trait has very little effect, and the phenotype observed is similar to that of a β-thalassemia heterozygote. In contrast, the Hb O-Arab mutation in combination with a β0-thalassemia gene leads to a moderately severe disorder with a phenotype not dissimilar to that of Hb E thalassemia.10 The Hb D-Punjab and Hb O-Arab mutations abolish an EcoR I site at codon 121123 and their detection is carried out simply by amplification of a fragment containing the site and digesting with EcoR I. Because there are no other EcoR I sites

Prenatal Diagnosis of the Hemoglobinopathies

673

within several kilobases of the β-globin gene, care should be taken always to run appropriate control DNA samples. The primer sequences used for this approach are listed in Table 21.8. Hb E Hb E results from a G → A mutation at codon 26 in the β-globin gene. This point mutation activates the cryptic splice site between codons 24 and 27, resulting in a β-thalassemia phenotype because of the production of two forms of β-globin mRNA. The normally spliced mRNA containing the βE mutation is produced at a low level and leads to a deficiency of βE-globin because the abnormally spliced mRNA does not produce a recognizable β-globin. The heterozygous and homozygous states for Hb E are associated with no clinical disability. The importance of Hb E lies in its interaction with β-thalassemia. The Hb E mutation abolishes a Mnl I site and may be diagnosed by PCR and restriction enzyme analysis.124 The primer sequences for this approach are listed in Table 21.9. However, the Hb E mutation is more commonly diagnosed by the use of ASO probes or ARMS primers, and the sequences for the latter are listed in Tables 21.6 and 21.7.

Diagnostic pitfalls PCR-based techniques now provide a quick and relatively simple method for the carrier detection and prenatal diagnosis of α0-thalassemia, βthalassemia, and sickle cell disease. The techniques have proven to be reliable and accurate as long as careful attention is paid to potential diagnostic pitfalls and best practice guidelines are followed. Best practice guidelines have been prepared following a meeting of the European Molecular Genetics Quality Network (EMQN) in 2002 and can be downloaded from the website www.emqn.org/ emqn.php. Maternal DNA contamination The most important problem is the possible coamplification of maternal DNA sequences. With chorionic villus samples, this is avoided by the careful dissection of maternal decidua from the fetal trophoblast by microscopic dissection,43 as shown by the experience of the Italian groups who

674

Genetic Disorders and the Fetus

reported no misdiagnosis in 457 first-trimester diagnoses for β-thalassemia in the Italian population using the method of dot blot analysis.125 Amniotic fluid cell DNA also may be contaminated with maternal DNA sequences through the contamination of an amniotic fluid sample with maternal blood. In a prenatal diagnosis program for sickle cell disease in the United States, one such misdiagnosis was reported to have occurred in a total of 500 prenatal diagnoses.126 It is essential that, in all diagnoses using cultured amniotic fluid cells in which the fetal genotype is determined to be identical to the maternal genotype, the diagnosis be confirmed using cultured amniocytes or by the inheritance of both maternal and paternal polymorphic markers. It is recommended as good practice to check for any maternal DNA contamination by the analysis of informative DNA polymorphic markers in all prenatal diagnoses. Variable tandem repeat (VNTR) markers were used in the early days of PCR diagnosis42 but most laboratories now use short tandem repeat (STR) markers, using up to 12 STR markers at a time for each case. The polymorphic regions are amplified by gap-PCR using fluorescent oligonucleotide primers flanking the repeat sequence and the products are separated and analyzed according to size on an automated DNA sequencer.127 PCR failure Another potential source of error is the failure to amplify one of the target DNA alleles. In my own experience, this occurred once in a prenatal diagnosis of α0-thalassemia, for which the PCR-based result was different from that obtained by Southern blot analysis because of a failure of amplification of the fetal normal α-globin gene allele. Amplification failure also may result when the hybridization of a primer or probe is compromised by an unexpected change in the target DNA sequence.128 Diagnostic error rate It is important for clinicians to understand that direct detection methods will detect only the particular mutation screened for. A diagnostic error may occur if the fetus inherits an unsuspected mutation as a result of nonpaternity or, as happened in two prenatal diagnoses for sickle cell

disease in our laboratory, when incorrect information was supplied about parental phenotypes. In both cases, the partner in question was not available for testing at the time of the prenatal diagnosis. However, such instances are very uncommon, as revealed by an audit of the accuracy of 3,254 prenatal diagnoses for the hemoglobinopathies in the UK.129 The study revealed a total of 10 nonlaboratory errors as well as 15 errors due to technical problems: eight diagnostic errors associated fetal blood sampling and globin chain synthesis, five errors by Southern blot analysis and two with PCR techniques. The diagnostic error rate for prenatal diagnosis by PCR methods, including nonlaboratory and technical errors, was calculated to be 0.41 percent, confirming it to be a more reliable method than the previous technologies of Southern blotting (0.73 percent error rate) and globin chain synthesis (1.55 percent error rate). In conclusion, the adoption of PCR techniques and the use of best practice guidelines have helped to minimise the misdiagnosis rate but not reduced it to zero and all clinicians should be aware of the slight risk of misdiagnosis and counsel couples undergoing prenatal diagnosis accordingly. Fetal DNA diagnosis: guidelines for best practice • Ensure that fresh parental blood samples are obtained with the fetal sample in order to check the parental phenotypes and to provide fresh control DNA samples. In cases where the father is not available, as often seems to happen with sickle cell prenatal diagnoses, copies of all laboratory results should be seen. • In such cases at risk for a sickle cell disorder, extra tests are carried out when a blood sample from the father is not available. The fetal DNA is always analyzed for βS, βC and common βthalassemia mutations in the parents’ ethnic group when an AS genotype is diagnosed, to avoid an incorrect diagnosis as far as possible. • Ensure that the chorionic villus sample has undergone careful microscopic dissection to remove any contaminating maternal decidua. • Always analyze parental and the appropriate control DNAs with the fetal DNA and always repeat the fetal DNA analysis to double check the result.

CHAP T E R 21

• Whenever possible, use an alternative diagnostic method to confirm the diagnosis. • Check for maternal DNA contamination in every case. Polymorphism analysis by PCR is used routinely to exclude error due to maternal DNA contamination or nonpaternity. • The fetal DNA diagnosis report should detail the types of DNA analysis used and clearly state the risk of misdiagnosis due to technical errors based on current data.

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13. Stamatoyannopoulos G, Grosveld F. Hemoglobin switching. In: Stamatoyannopoulos G, Majerus PW, Perlmutter RM, Varmus H, eds. The molecular basis of blood diseases, 3rd ed. Philadelphia: WB Saunders, 2001:135. 14. Rodgers GP, Steinberg MH, Pharmacologic treatment of sickle cell disease and thalassemia: the augmentation of fetal hemoglobin. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of hemoglobin: genetics, pathophysiology and clinical management. Cambridge University Press. 2001;1028. 15. Charache S, Terrin ML, Moore RD, et al. Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. N Engl J Med 1995;332:1317. 16. Sher GD, Ginder GD, Little J, et al. Extended therapy with intravenous arginine butyrate in patients with beta-hemoglobinopathies. N Engl J Med 1995;332:1606. 17. Wajcman H, Traeger-Synodinos J, Papassotiriou I, et al. Unstable and thalassemic alpha chain hemoglobin variants: a cause of Hb H disease and thalassemia intermedia. Hemoglobin 2008;32:327. 18. Henderson S, Chapple M, Rugless M, et al. Haemoglobin H hydrops fetalis syndrome associated with homozygosity for the alpha2-globin gene polyadenylation signal mutation AATAAA–>AATA–. Br J Haematol 2006;135:743 19. Ko TM, Tseng LH, Hsieh FJ, et al. Prenatal diagnosis of Hb H disease due to compound heterozygosity for south-east Asian deletion and Hb Constant Spring by polymerase chain reaction. Prenat Diagn 1993;13: 143. 20. Henderson S, Pitman M, McCarthy J, et al. Molecular prenatal diagnosis of Hb H hydrops fetalis caused by haemoglobin Adana and the implications to antenatal screening for alpha-thalassaemia. Prenat Diagn 2008; 28:859. 21. Baysal E. The β- and α-thalassemia repository. Hemoglobin 1995;19:213. 22. Thein SL, Hesketh C, Wallace RB, et al. The molecular basis of thalassaemia major and thalassaemia intermedia in Asian Indians: application to prenatal diagnosis. Br J Haematol 1988;70:225. 23. Huang SZ, Wong C, Antonarakis SE, et al. The same TATA box β-thalassaemia mutation in Chinese and US blacks: another example of independent origins of mutation. Hum Genet 1986;74:152. 24. Munro S, Loudianos G, Deiana M, et al. Molecular characterisation of β-thalassaemia intermedia in patients of Italian descent and identification of three novel β-thalassaemia mutations. Blood 1991;77: 1342. 25. Craig JE, Kelly SJ, Basrneston R, et al. Molecular characterisation of a novel 10.3 kb deletion causing β-

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26.

27.

28.

29.

30. 31. 32.

33. 34. 35.

36.

37.

38.

39.

40.

41.

Genetic Disorders and the Fetus thalassaemia with unusually high Hb A2. Br J Haematol 1992;82:735. Thein SL, Hesketh C, Taylor P, et al. Molecular basis for dominantly inherited inclusion body β-thalassemia. Proc Natl Acad Sci USA 1990;87:3924. Fucharoen S. Hb E disorders. In: Steiberg MH, Forget BG, Higgs DR, Nagel RL (eds.) Disorders of Hemoglobin: Genetics, Pathophysiology and Clinical Management., Cambridge: Cambridge University Press. 2001;1139. Steinburg MH. Compound heterozygous and other sickle hemoglobinopathies. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of hemoglobin: genetics, pathophysiology and clinical management. Cambridge University Press. 2001;786. Pagnier J, Mears JG, Dunda-Belkodja O, et al. Evidence of the multicentric origin of the hemoglobin S gene in Africa. Proc Natl Acad Sci USA 1984;81:1771. Powars DR. βS-gene cluster haplotypes in sickle cell anemia. Hematol Oncol Clin North Am 1991;5:475. Old J. Screening and genetic diagnosis of hemoglobin disorders. Blood Rev 2003;17(1):43. Thalassaemia Working Party of the BSCH General Haematology Task Force. Guidelines for the laboratory diagnosis of hemoglobinopathies. Br J Haematol 1998;101:783. Old JM. Screening and genetic diagnosis of haemoglobinopathies. Scand J Clin Lab Invest 2007;67:71. Alter BP. Advances in the prenatal diagnosis of hematologic diseases. Blood 1984;64:329. Old JM, Fitches A, Heath C, et al. First-trimester fetal diagnosis for haemoglobinopathy: report on 200 cases. Lancet 1986;ii:763. Old JM. Fetal DNA analysis. In: Davies KE, ed. Genetic analysis of the human disease: a practical approach. Oxford: IRL Press, 1986:1. Rodeck CH, Nicolaides KH, Morsman JM, et al. A single-operator technique for first-trimester chorion biopsy. Lancet 1983;ii:1340. Ward RHT, Modell B, Petrou M, et al. A method of chorionic villi sampling in the first trimester of pregnancy under real-time ultrasonic guidance. BMJ 1983;286:1542. Smidt-Jensen S, Hahnemann N, Hariri J, et al. Transabdominal chorionic villi sampling for first trimester fetal diagnosis: first 26 pregnancies followed to term. Prenat Diagn 1986;6:125. Brambati B, Oldrini A, Lanzani A. First trimester diagnosis for haemoglobinopathies: report on 200 cases. Lancet 1986;ii:763. Elles RG, Williamson R, Niazi M, et al. Absence of maternal contamination of chorionic villi used for fetal-gene analysis. N Engl J Med 1983;308:1433.

42. Decorte R, Cuppens H, Marynen P, et al. Rapid detection of hypervariable regions by the polymerase chain reaction technique. DNA Cell Biol 1990;9:461. 43. Petrou M, Modell B, Darr A, et al. Antenatal diagnosis: how to deliver a comprehensive service in the United Kingdom. Ann NY Acad Sci 1990;612:251. 44. Camaschella C, Alfarano A, Gottardi E, et al. Prenatal diagnosis of fetal hemoglobin Lepore-Boston disease on maternal peripheral blood. Blood 1990;75:2102. 45. Sekizawa A, Watanabe A, Kimwa T, et al. Prenatal diagnosis of the fetal RhD blood type using a single fetal nucleated erythrocyte from maternal blood. Obstet Gynecol 1996;87:501. 46. Cheung MC, Goldberg JD, Kan YW. Prenatal diagnosis of sickle cell anemia and thalassemia by analysis of fetal cells in maternal blood. Nat Genet 1996;14:264. 47. Lau ET, Kwok YK, Chui DHK, et al. Embryonic and foetal globins are expressed in adult erythroid progenitor cells and in erythroid cell cultures. Prenat Diagn 2001;21:529. 48. Lau ET, Kwok YK, Luo HY et al. Simple non-invasive prenatal detection of Hb Bart’s disease by analysis of fetal erythrocytes in maternal blood. Prenat Diagn 2005;25:123. 49. D’Souza E, Sawant PM, Nadkarni AH, et al. Evaluation of the use of monoclonal antibodies and nested PCR for noninvasive prenatal diagnosis of hemoglobinopathies in India. Am J Clin Pathol 2008;130:202. 50. Kolialexi A, Vrettou C, Traeger-Synodinos J, et. al. Noninvasive prenatal diagnosis of beta-thalassaemia using individual fetal erythroblasts isolated from maternal blood after enrichment. Prenat Diagn 2007; 27:1228. 51. Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350(9076):485. 52. Li Y, Holzgreve W, Hahn S. Size fractionation of cellfree DNA in maternal plasma and its application in noninvasive detection of fetal single gene point mutations. Methods Mol Biol 2008;444:239. 53. Tungwiwat W, Fucharoen S, Fucharoen G, et al. Development and application of a real-time quantitative PCR for prenatal detection of fetal alpha(0)thalassemia from maternal plasma. Ann NY Acad Sci 2006;1075:103. 54. Papasavva T, Kalikas I, Kyrri A et al. Arrayed primer extension for the noninvasive prenatal diagnosis of beta-thalassemia based on detection of single nucleotide polymorphisms. Ann NY Acad Sci 2008;1137:302. 55. Li Y, Finning K, Daniels G, et al. Noninvasive genotyping fetal Kell blood group (KEL1) using cell-free fetal DNA in maternal plasma by MALDI-TOF mass spectrometry. Prenat Diagn 2008;28:203.

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56. Galbiati S, Foglieni B, Travi M, et al. Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of betathalassemia. Haematologica 2008;93:610. 57. Hahn S, Chitty LS Noninvasive prenatal diagnosis: current practice and future perspectives. Curr Opin Obstet Gynecol 2008;20:146. 58. Chui RW, Lau TK, Leung TN, et al. Prenatal exclusion of beta thalassaemia major by examination of maternal plasma. Lancet 2002;360:998. 59. Tungwiwat W, Fucharoen S, Fucharoen G, et al. Development and application of a real-time quantitative PCR for prenatal detection of fetal alpha(0)thalassemia from maternal plasma. Ann NY Acad Sci 2006;1075:103. 60. Li Y, Di Naro E, Vitucci A, et al. Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma. JAMA 2005;293:843. 61. Kanavakis E, Traeger-Synodinos J. Preimplantation genetic diagnosis in clinical practice. J Med Genet 2002;39:6. 62. Chan V, Ng EH, Yam I, et al. Experience in preimplantation genetic diagnosis for exclusion of homozygous alpha thalassemia. Prenat Diagn 2006;26:1029. 63. Deng J, Peng WL, Li J, et al. Successful preimplantation genetic diagnosis for alpha- and beta-thalassemia in China. Prenat Diagn 2006;26:1021. 64. Monni G, Cau G, Usai V, et al. Preimplantation genetic diagnosis for beta-thalassaemia: the Sardinian experience. Prenat Diagn 2004;24:949. 65. Kuliev A, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis and HLA typing for haemoglobin disorders. Reprod Biomed Online 2005;11:362. 66. Alsulaiman A, Hewison J. Attitudes to prenatal and preimplantation diagnosis in Saudi parents at genetic risk. Prenat Diagn 2006;26:1010. 67. Vrettou C, Traegaer Synodinos J, Tzetis M, et al. Realtime PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis. Hum Mutat 2004;23:513. 68. Flint J, Harding RM, Boyce AJ, et al. The population genetics of the haemoglobinopathies. In: Higgs DR, Weatherall, DJ, eds. Baillière’s clinical haematology. International practice and research: the haemoglobinopathies. London: Baillière Tindall, 1993:215. 69. Old JM. Prenatal diagnosis of the haemoglobinopathies. In: Milunsky A, ed. Genetic disorders and the fetus, 5th ed. Baltimore: Johns Hopkins University Press, 2004:663. 70. Dode C, Krishnamoorthy R, Lamb J, et al. Rapid analysis of -α3.7 thalassaemia and αααanti 3.7 triplication by

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enzymatic amplification analysis. Br J Haematol 1992; 82:105. Liu YT, Old JM, Fisher CA, et al. Rapid detection of α-thalassaemia deletions and α-globin gene triplication by multiplex PCRs. Br J Haematol 1999;108:295. Chong SS, Boehm CD, Higgs DR, et al. Single-tube multiplex-PCR screen for common deletional determinants of α-thalassemia. Hemoglobin 1999:95:360. Molchanova TP, Pobedimskaya DD, Postnikov YV. A simplified procedure for sequencing amplified DNA containing the α-2 or α-1 globin gene. Hemoglobin 1994;18:251. Sun CF, Lee CH, Cheng SW et al. Real-time quantitative PCR analysis for alpha-thalassemia-1 of Southeast Asian type deletion in Taiwan. Clin Genet 2001;60:305. Ou-Yang H, Hua L, Mo HQ, et al. Rapid, accurate genotyping of the common alpha (4.2) deletion based on the use of denaturing HPLC. J Clin Pathol 2004; 57:159. Zesong L, Ruijun G, Wen Z. Rapid detection of deletional alpha-thalassemia by an oligonucleotide microarray. Am J Hematol 2005;80:306. Bang-Ce Y, Hongqiong L, Zhuanfong Z, et al. Simultaneous detection of alpha-thalassemia and betathalassemia by oligonucleotide microarray. Haematologica 2004;89:1010. Harteveld Cl, Voskamp A, Phylipsen M, et al. Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. J Med Genet 2005;42:922. Hartveld KL, Heister AJGAM, Giordano PC, et al. Rapid detection of point mutations and polymorphisms of the α-globin genes by DGGE and SSCA. Hum Mutat 1996;7:114. Huisman THJ. Frequencies of common β-thalassaemia alleles among different populations: variability in clinical severity. Br J Haematol 1990;75:454. Baysal E, Indrak K, Bozhurt G, et al. The β thalassaemia mutations in the population of Cyprus. Br J Haematol 1992;81:607. Ristaldi MS, Pirastu M, Rosatelli C, et al. Prenatal diagnosis of β-thalassaemia in Mediterranean populations by dot blot analysis with DNA amplification and allele specific oligonucleotide probes. Prenat Diagn 1989; 9:629. Saiki RK, Walsh PS, Levenson CH, et al. Genetic analysis of amplified DNA with immobilized sequencespecific oligonucleotide probes. Proc Natl Acad Sci USA 1989;86:6230. Maggio A, Giambona A, Cai SP, et al. Rapid and simultaneous typing of hemoglobin S, hemoglobin C and seven Mediterranean β-thalassaemia mutations by

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Genetic Disorders and the Fetus covalent reverse dot-blot analysis: application to prenatal diagnosis in Sicily. Blood 1993;81:239. Sutcharitchan P, Saiki R, Huisman THJ, et al. Reverse dot-blot detection of the African-American bthalassaemia mutations. Blood 1995;86:1580. Sutcharitchan P, Saiki R, Fucharoen S, et al. Reverse dot-blot detection of Thai β-thalassaemia mutations. Br J Haematol 1995;90:809. Gemignani F, Perra C, Landi S, et al. Reliable detection of beta-thalassemia and G6PD mutations by a DNA microarray. Clin Chem 2002;48:2051. Van Moorsel CH, van Wijngaraarden EE, Fokkema IF, et al. beta-Globin mutation detection by tagged singlebase extension and hybridization to universal glass and flow-through microarrays. Eur J Hum Genet 2004; 12:567. Lu Y, Kham SK, Tan PL, et al. Arrayed primer extension: a robust and reliable genotyping platform for the diagnosis of single gene disorders: beta-thalassemia and thiopurine methyltransferase deficiency. Genet Test 2005;9:212. Old JM, Khan SN, Verma IC, et al. A multi-centre study to further define the molecular basis of betathalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system polymerase chain reaction. Hemoglobin 2001;25:397. Tan JAMA, Tay JSH, Lin LI, et al. The amplification refractory mutation system (ARMS): a rapid and direct prenatal diagnostic techniques for b-thalassaemia in Singapore. Prenat Diagn 1994;14:1077. Zschocke J, Graham CA. A fluorescent multiplex ARMS method for rapid mutation analysis. Mol Cell Probes 1995;9:447. Hu H, Li C, Xiong Q, et al. Prenatal diagnosis of betathalassemia by chip-based capillary electrophoresis. Prenat Diagn 2008;28:222. Chehab FF, Kan YW. Detection of specific DNA sequence by fluorescence amplification: a colour complementation assay. Proc Natl Acad Sci USA 1989;86: 9178. Chang JG, Lu JM, Huang JM, et al. Rapid diagnosis of β-thalassaemia by mutagenically separated polymerase chain reaction (MS-PCR) and its application to prenatal diagnosis. Br J Haematol 1995;91:602. Old JM. DNA-based diagnosis of the hemoglobin disorders. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of hemoglobin: genetics, pathophysiology and clinical management. Cambridge: Cambridge University Press, 2001:941. Linderman R, Hu SP, Volpato F, et al. Polymerase chain reaction (PCR) mutagenesis enabling rapid non-

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radioactive detection of common β-thalassaemia mutations in Mediterraneans. Br J Haematol 1991; 78:100. Webster MT, Wells RS, Clegg JB. Analysis of variation in the human beta-globin gene cluster using a novel DHPLC technique. Mutat Res 2002;501:99. Yip SP, Pun SF, Leung KH, et al. Rapid, simultaneous genotyping of five common Southeast Asian betathalassemia mutations by multiplex minisequencing and denaturing HPLC. Clin Chem 2003;49:1656. Su YN, Lee CN, Hung CC, et al. Rapid detection of beta-globin gene (HBB) mutations coupling heteroduplex and primer-extension analysis by DHPLC. Hum Mutat 2003;22:326. Wu G, Hua L, Zhu J, et al. Rapid, accurate genotyping of beta-thalassaemia mutations using a novel multiplex primer extension/denaturing high-performance liquid chromatography assay. Br J Haematol 2003;122: 311. Bournazos SN, Tserga A, Patrinos GP, et al. A versatile denaturing HPLC approach for human beta-globin gene mutation screening. Am J Hematol 2007;82:168. Naja RP, Kaspar H, Shabakio H, et al. Accurate and rapid prenatal diagnosis of the most frequent East Mediterranean beta-thalassemia mutations. Am J Hematol 2004;75:220. Pang L, Li J, Jiang J, et al. DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach. Anal Biochem 2006; 358:99. Faa V, Rosatelli MC, Sardu R, et al. A simple electrophoretic procedure for fetal diagnosis of β-thalassaemia due to short deletions. Prenat Diagn 1992;12:903. Waye JS, Eng B, Hunt JA, et al. Filipino β-thalassaemia due to a large deletion: identification of the deletion endpoints and polymerase chain reaction (PCR)-based diagnosis. Hum Genet 1994;94:530. Craig JE, Barnetson RA, Prior J, et al. Rapid detection of deletions causing δβ thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification. Blood 1994;83:1673. Losekoot M, Fodde R, Harteveld CL, et al. Denaturing gradient gel electrophoresis and direct sequencing of PCR amplified genomic DNA: a rapid and reliable diagnostic approach to beta thalassaemia. Br J Haematol 1991;76:269. Savage DA, Wood NAP, Bidwell JL, et al. Detection of β-thalassaemia mutations using DNA heteroduplex generator molecules. Br J Haematol 1995;90:564. Old JM, Varawalla NY, Weatherall DJ. The rapid detection and prenatal diagnosis of β thalassaemia in the Asian Indian and Cypriot populations in the UK. Lancet 1990;336:834.

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111. Old JM. Haemoglobinopathies: community clues to mutation detection. In: Elles R, ed. Methods in molecular medicine: molecular diagnosis of genetic diseases. Totawa, NJ: Humana Press, 1996:169. 112. Kwok S, Kellogg DE, McKinney N, et al. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type I model studies. Nucleic Acids Res 1990;18:999. 113. Varawalla NY, Old JM, Weatherall DJ. Rare βthalassaemia mutations in Asian Indians. Br J Haematol 1991;79:640. 114. Kazazian HH Jr, Boehm CD. Molecular basis and prenatal diagnosis of β-thalassaemia. Blood 1988;72: 1107. 115. Antonarakis SE, Boehm CD, Diardina PJV, et al. Nonrandom association of polymorphic restriction sites in the β-globin gene cluster. Proc Natl Acad Sci USA 1982;79:137. 116. Chakravarti A, Buetow KH, Antonarakis SE, et al. Non-uniform recombination within the human βglobin gene cluster. Am J Hum Genet 1984;71:79. 117. Orkin SH, Little PFR, Kazazian HH Jr, et al. Improved detection of the sickle mutation by DNA analysis. N Engl J Med 1982;307:32. 118. Old JM, Petrou M, Modell B, et al. Feasibility of antenatal diagnosis of β-thalassaemia by DNA polymorphisms in Asian Indians and Cypriot populations. Br J Haematol 1984;57:255. 119. Kulozik AE, Lyons J, Kohne E, et al. Rapid and nonradioactive prenatal diagnosis of β-thalassaemia and sickle cell disease: application of the polymerase chain reaction (PCR). Br J Haematol 1988;70:455.

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120. Semenza GL, Dowling CE, Kazazian HH Jr. Hinf I polymorphisms 39 to the human β globin gene detected by the polymerase chain reaction (PCR). Nucleic Acids Res 1989;17:2376. 121. Wainscoat JS, Old JM, Thein SL, et al. A new DNA polymorphism for prenatal diagnosis of bthalassaemia in Mediterranean populations. Lancet 1984;2:1299. 122. Old JM, Thein SL, Weatherall DJ, et al. Prenatal diagnosis of the major haemoglobin disorders. Mol Biol Med 1989;6:55. 123. Trent RJ, Davis B, Wilkinson T, et al. Identification of β variant hemoglobins by DNA restriction endonuclease mapping. Hemoglobin 1984;8:443. 124. Thein SL, Lynch JR, Old JM, et al. Direct detection of haemoglobin E with Mnl I. J Med Genet 1987;24:110. 125. Rosatelli MC, Tuveri T, Scalas MT, et al. Molecular screening and fetal diagnosis of β-thalassaemia in the Italian population. Hum Genet 1992;89:585. 126. Wang X, Seaman C, Paik M, et al. Experience with 500 prenatal diagnoses of sickle cell diseases: the effect of gestational age on affected pregnancy outcome. Prenat Diagn 1994;14:851. 127. Stojikovic-Mikic T, Mann K, Docherty Z, et al. Maternal cell contamination of prenatal samples assessed by QF-PCR genotyping. Prenat Diagn 2005;25:79. 128. Chan V, Chan TPT, Lau K, et al. False non-paternity in a family for prenatal diagnosis of β-thalassaemia. Prenat Diagn 1993;13:977. 129. Old J, Petrou M, Varnavides L, et al. Accuracy of prenatal diagnosis in the UK: 25 years experience. Prenat Diagn 2000;20:986.

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Prenatal Diagnosis of Disorders of Bone and Connective Tissue Andrea Superti-Furga and Sheila Unger University of Freiburg, Freiburg, Germany

Molecular constituents of connective tissue not only perform basic structural functions and confer mechanical resistance and stability to soft and hard connective tissues, but also fulfill a variety of other functions such as morphogenesis, signal transduction, maintenance of homeostasis, protection and tissue integrity. It is therefore not surprising that the complex biology of bone and connective tissue is reflected in the myriad types of disorders resulting from defects in its constitutive molecules and metabolic pathways. The tradition of dealing with bone and connective tissue disorders together goes back to V. A. McKusick and his monograph, “Heritable disorders of connective tissues”, in which he discussed heterogeneous conditions such as Marfan syndrome, the Ehlers–Danlos syndrome, the mucopolysaccharidoses, osteogenesis imperfecta and the skeletal dysplasias.1 Key molecules like collagen type 1 and type 2 can result in a variety of conditions that affect the soft connective tissues (skin, tendons and ligaments) as well as the skeletal elements. The nosology of these disorders, however, has expanded dramatically; thus, we distinguish seven types of osteogenesis imperfecta, over 10 types of Ehlers–Danlos syndrome and over 400 distinct genetic skeletal disorders. The clinical diagnostic approach is very different from case to case, and must be tailored to the clinical signs present in each affected individual or family.

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

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The approach to prenatal diagnosis is complex and can be divided into cases in which a fetus is subjected to analysis because of a pre-existing condition in a parent or previous offspring, and those that are found unexpectedly on ultrasound. As a rule, most disorders of soft connective tissue will not be recognizable by ultrasonography and prenatal diagnosis requires pre-existing data of a biochemical or molecular nature on the condition for which the pregnancy is at risk; in this respect, the approach to prenatal diagnosis is similar to that for any other metabolic or genetic disorder. Conversely, many skeletal disorders may present as unexpected morphologic findings during routine ultrasonographic screening of a pregnancy not known to be at risk. The two situations pose quite different challenges; while in the first instance, prenatal diagnosis depends on findings that must have been obtained in an index case (or other family members) prior to the pregnancy, in the second case there is no a priori diagnostic indication and the differential diagnostic process must begin from the ultrasonographic finding. Understandably, the latter situation can be much more difficult.

Prenatal sonographic diagnosis of skeletal dysplasias The prenatal sonographic detection of skeletal dysplasias is a complex subject that needs to be put in perspective. The growing knowledge of the molecular basis of genetic skeletal disorder might theoretically widen the possibility of prenatal molecular

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detection. On the other hand, the extensive genetic heterogeneity behind similar phenotypes and the additional allelic heterogeneity within individual genes make rapid molecular diagnosis a daunting task. Therefore, expert review of the morphologic sonographic data plays a pivotal role in the diagnosis of skeletal dysplasias, even more so in the prenatal period than in the infant and child. Other imaging modalities, such as magnetic resonance imaging (MRI) or fetal computed tomography (CT) with three-dimensional (3D) reconstructions, are usually of little help in the diagnosis of skeletal dysplasias: MRI is not well suited for skeletal elements and fetal CT gives useful images relatively late in pregnancy and is associated with a considerable radiation burden. The morphologic diagnosis of skeletal dysplasias relies on the recognition of a distinct pattern of changes in multiple skeletal elements. This may be easy for conditions such as achondroplasia that are frequent and well known to most pediatric radiologists and clinical geneticists, but remains difficult for the less frequent conditions that make up the majority of cases. Because of these difficulties, diagnostic materials are often referred to regional or national centers of expertise that offer diagnostic review. Prenatal sonographic diagnosis of these disorders requires not only an extensive knowledge of those patterns of skeletal changes that are diagnostic in the child, but also the translational knowledge of how to see and recognize those changes in a different imaging modality – sonography versus radiography. For this reason, successful sonographic diagnosis of a skeletal dysplasia requires close collaboration between the obstetrician as the sonographic expert and the clinical geneticist or radiologist who is familiar with the skeletal changes of specific dysplasias. Unfortunately, the medical literature is inflated with uncritical contributions reporting the “prenatal diagnosis” of specific skeletal dysplasias; in most of these instances the specific diagnosis was made only after termination of pregnancy or after birth. Thus, older reports must be interpreted with caution. Newer studies focus on the development of reliable markers or indices for the diagnosis.2–14 Nevertheless, the field of prenatal sonographic diagnosis of skeletal abnormality remains quite complex, and although individual centers may

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offer outstanding results, most cases will not be evaluated in such centers. Couples may seek genetic counseling and prenatal diagnosis because of a previous pregnancy or child affected by a skeletal dysplasia. If the diagnostic process in the index case has been successful, the results of molecular studies confirming the diagnosis may be available. In such a situation, molecular diagnosis on DNA extracted from a chorionic villus sample (CVS) or early amniocentesis may be the earliest and safest means of prenatal diagnosis. However, in pregnancies with only moderately increased risk for a severe condition (such as that of couples who have had a previous pregnancy with a de novo dominant disorder such as thanatophoric dysplasia or lethal osteogenesis imperfecta), sonography performed at gestational week 14 or 15 may be less invasive and offer good sensitivity albeit with a delay of 2–3 weeks compared to molecular studies. If sonographic diagnosis is to be offered, it is important to have an expert estimation on the degree of severity of the skeletal changes and thus of the earliest possible time point for sonographic recognition during pregnancy. Reviewing the sonographic imaging material from the index pregnancy, if available, is helpful in this respect. In cases where the index case was affected by a skeletal dysplasia but a specific diagnosis has not been secured, or when a skeletal dysplasia is only one of the differential diagnoses, sonographic studies looking for nonspecific signs of skeletal dysplasias may be offered. However, in such cases an effort should be made to obtain the written and imaging records of the index pregnancies and to review them in order to try and specify the possible diagnosis or at least to obtain a better estimation of what might be the signs to be recognized in a possible recurrence. Abnormal fetal morphology as an unexpected finding The implementation of routine sonographic screening during pregnancy has resulted in a steadily increasing number of cases in which abnormal growth parameters and/or abnormal morphology lead to the suspicion of a skeletal disorder in the fetus. One of the most common decisions taken upon the recognition of abnormal growth parameters is to schedule a further sonography session 2

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or more weeks later; however, this common practice may be unfortunate, as a significant growth delay in the early second trimester (assuming the dating of pregnancy is correct) rarely catches up, but usually becomes more marked. The diagnostic evaluation of a pregnancy in which the fetus has significant growth delay should be done in a tertiary center, as the differential diagnosis is so large and complex that it can rarely be mastered by the practicing obstetrician. Some of the items to consider when evaluating growth delay in an early second-trimester fetus include the following. • The most frequent cause of generalized growth delay is maternal and placental factors, such as placental insufficiency. • Aberrant fetal growth in skeletal dysplasias affects primarily the limbs, secondarily the thoracic cage, and rarely the head; thus, significant microcephaly speaks against a purely skeletal dysplasia. • Other malformations (such as neural tube defects, gastroschisis, omphalocele, bladder exstrophy, congenital heart defects, polycystic kidneys, hydronephrosis or other urogenital abnormalities, orofacial clefts, poly- or oligodactyly) must be searched for and excluded (or confirmed), as their presence may point to other diagnoses or, rarely, to specific skeletal dysplasias. • The earlier the diagnosis, the more likely it is to be severe or lethal; thus, the various forms of achondrogenesis, thanatophoric dysplasia or lethal osteogenesis imperfecta are all recognizable around week 14–16, while the common achondroplasia is rarely detectable before week 24. • The diagnostic semiology of skeletal dysplasias is based on radiographic imaging, on the clinical appearance and on additional clinical information. Fetal sonography offers less morphologic detail of skeletal elements and 3D reconstruction (e.g. of the facial features) is at best only a partial substitute for clinical observation after delivery. Under these circumstances, it is difficult to make an unequivocal diagnosis on sonography alone, even for the dysplasia expert. Nonetheless, 3D reconstruction may help the expert eye in identifying diagnostic morphologic features.6 • Given the difficulty in reaching a specific diagnosis, the most relevant issue in the early second trimester is to estimate the viability of the fetus, and in the absence of major organ malformations this

is correlated strongly with the dimensions of the thorax. Thus, the observation of a narrow thorax with protruding abdomen at week 16 may be indicative of a lethal dysplasia even if the differential diagnosis between, for example, thanatophoric dysplasia or a form of short-rib polydactyly syndrome (without polydactyly) cannot be resolved.

Molecular testing during pregnancy Laboratories that offer molecular tests for skeletal dysplasias are often confronted with requests for high-urgency testing of CVS or amniotic cell DNA for a fetus in whom a diagnostic dysplasia is suspected based on sonographic findings. Unfortunately, this rarely results in a more accurate diagnosis for the following reasons. • The only genetically homogeneous skeletal dysplasia is achondroplasia, that is caused by mutation of nucleotide 1138 of FGFR3 in approximately 98 percent of cases. Although achondroplasia is one of the most common diagnoses, it is rarely detectable by ultrasound before pregnancy week 24, and usually only around week 26 and later.11 • Among the most frequent forms of severe or lethal skeletal dysplasias are thanatophoric dysplasia, osteogenesis imperfecta, campomelic dysplasia and achondrogenesis type 2/hypochondrogenesis. Thanatophoric dysplasia is caused by mutations in FGFR3; mutation R248C is relatively frequent (about 50 percent of cases) but the rest are caused by various less frequent mutations.15,16 Osteogenesis imperfecta is genetically quite heterogeneous with mostly “private” mutations in at least four genes – COL1A1 and COL1A2 (dominant forms, about 90 percent)17 and CRTAP and leprecan (recessive forms; about 10 percent).18–20 Campomelic dysplasia is associated with a large allelic heterogeneity within and around the SOX9 locus, and the collagen 2-associated severe dysplasias (achondrogenesis type2/ hypochondrogenesis, Torrance dysplasia, Kniest dysplasia, and severe spondyloepiphyseal dysplasia congenita) are similarly associated with mostly private mutations.21–24 Advances in high-throughput sequencing technologies will make it possible to sequence multiexon genes almost overnight, but wide access will take many years.

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Thus, if the sonographic findings are compatible with thanatophoric dysplasia (normal head, narrow thorax, substernal abdominal protrusion, short limbs, normal bone density, “trident” hand), a directed search for FGFR3 mutation R248C may be a first step; if negative, however, more extensive and thus more time-consuming studies are needed. Moreover, experience shows that the accuracy of the average fetal sonographer in distinguishing between the many dysplasias and thus being able to direct the correct molecular testing is not high. Altogether, when sonography suggests the presence of a severe dysplasia in the fetus it must be carefully considered whether waiting several days or weeks trying to obtain molecular confirmation with a relatively low likelihood of a positive result is a useful option. Estimating the probability of recurrence When providing genetic counseling prior to pregnancy or in the presence of abnormal fetal morphology, it is important not only to look for signs of a specific diagnosis but also to consider the underlying genetic mechanisms; the probability of recurrence ranges from 50 percent for autosomal dominant conditions (such as osteogenesis imperfecta or achondroplasia) when one parent is affected to 25 percent for conditions known to be recessive (such as achondrogenesis type 1A and 1B or severe hypophosphatasia). It is more difficult to obtain precise data on the recurrence probability for conditions that present as de novo dominants, such as thanatophoric dysplasia, achondrogenesis type 2, lethal osteogenesis imperfecta or sporadic achondroplasia; gonadal and/or somatic mosaicism is known for all these conditions but its incidence appears to be very different, highest in OI, intermediate in COL2A1 disorders and lowest in FGFR3 disorders.25–30

Genetic disorders of bone and cartilage: the skeletal dysplasias and dysostoses Skeletal dysplasias are a heterogeneous group of disorders characterized by skeletal deformities and short stature. More than 400 distinct clinical and genetic entities have been described and range

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from perinatal lethal conditions to those that are mild and do not manifest for several years.31 Tissues other than bone and cartilage are often affected. There are several ways to approach and classifiy this large group of disorders. Categories used to classify are lethal/non-lethal, short limb versus short trunk type and, until the 1970s, whether the disorder was clinically recognizable at birth or only later in life.32–35 Today, even those of moderate severity can be recognized before birth with fetal sonography. The molecular defects underlying the manifold genetic disorders of bone are progressively unfolding; not even half of the disorders included in the most recent nosology have been elucidated. The genetic defects uncovered so far have shown an unexpected diversity of molecular mechanisms leading to impaired morphogenesis, development and growth of the skeleton. Mutations may affect structural molecules, enzymes and transporters, signaling pathways and hormones, as well as transcription factors.31,36 As with any other genetic disorder, identification of a pathogenic mutation in an index patient or family may eventually open the possibility of prenatal detection of the mutation in fetal material obtained through CVS or amniocentesis. In practice, this approach is feasible in a subset of pregnancies only, as skeletal dysplasias are often the result of de novo mutations, and because mutation analysis results to be used for prenatal detection are often unavailable. The genetic disorders of the skeleton are so extensive that a thorough discussion of them would not be possible within this chapter. A list of nosologic entities and of the responsible genes, as far as they are known, can be found in the latest Nosology prepared by the International Skeletal Dysplasia Society.31 Given the constant flow of new information on the molecular bases, consultation of online databases such as PubMed and OMIM is advisable. This chapter will focus on a subset of more frequent disorders. Osteogenesis imperfecta Osteogenesis imperfecta (OI) is a heterogeneous disorder involving both bone and connective tissue. The clinical manifestations are of variable severity and include fractures, limb bowing, blue or dark sclerae, hearing loss, dentinogenesis imper-

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fecta, joint laxity, anomalies of the heart valves, and myopia.17,37 The phenotype may range from the stillborn with innumerable fractures and soft bones to the individual with normal stature and only occasional fractures. Inheritance is autosomal dominant in the majority of cases, with a significant proportion of de novo mutations; the common, dominant OI mutations occur in either COL1A1 or COL1A2, the two genes encoding the proa1 and proa2 chains of type I collagen.38 However, somatic and gonadal mosaicism for dominant mutations have been reported, accounting for unexpected recurrences of affected children in families with clinically unaffected or mildly affected parents.25,26,29,39,40 In addition, there is a subset of severe OI cases caused by recessive mutations in any one of three genes involved in posttranslational processing of procollagen: P3H1/ leprecan, CRTAP and PLOD2.18–20,41 Thus, counseling for OI remains quite tricky in cases where the molecular basis has not been established. Although it is clear today that the OI phenotype is a continuous spectrum and that no clinical or radiographic signs reliably distinguish one genetic type from another, the four types of OI originally proposed by Sillence are still generally used as a shorthand clinical classification based on the degree of severity.42 Type I is the mild variant. Patients with type I OI commonly fracture the long bones, ribs and bones of the hands. Fractures may be few or numerous. Since the fractures heal without deformity, the patients obtain a normal or near-normal stature. In most instances, OI type I is due to COL1A1 haploinsufficiency secondary to nonsense-mediated mRNA decay.43 Type II OI is the perinatal lethal variant.17 Infants are often delivered prematurely and have low birthweights. As many as 60 percent die during the first day of life. They have bowed legs, beaded ribs, soft calvarium, dark sclerae, short extremities and flexed hips. The thorax is small. The vast majority of cases represent new dominant mutations. In some families with recurrence, mosaicism has been documented. Type II OI should be detectable by ultrasound during pregnancy, but may be difficult to distinguish from other skeletal dysplasias. The majority of type II OI cases result from point mutations in either COL1A1 or COL1A2, leading to glycine substitutions in the triple helical regions of the proa1(I) or proa2(I) chains. Other muta-

tions include those resulting in nonglycine substitutions, in the carboxyl-terminal portion of the molecule stop codons, splice-site mutations resulting in exon skipping, and small in-frame insertions or deletions. The effects of the mutations may be manifested either through interference with procollagen chain assembly or alternatively by interference with stable triple helical formation following incorporation into procollagen. Type III OI is known as the progressively deforming variant.17 Fractures, short stature and deformity may be recognized in utero. These patients have generalized osteopenia and the highest fracture rate of all OI types. This results in angular bone deformities. Severe kyphoscoliosis can develop, and the lifespan is foreshortened. Most cases are autosomal dominant, although rare recessively inherited cases have been seen. The dominantly inherited COL1A1 and COL1A2 mutations include those resulting in splice-site alterations, glycine substitutions, and single glycine deletions. Type IV is the moderate severity variant with short stature but only mild deformity.17 Fractures of the femurs may also occur in utero. These patients are usually short and may also develop scoliosis or kyphoscoliosis, possibly compromising respiratory functions. Inheritance is exclusively autosomal dominant. Most defined COL1A1 and COL1A2 mutations result in glycine substitutions. Some exon-skipping mutations and in-frame insertions and deletions occur. Types V, VI and VII are rarer and have been suggested to correspond to specific genetic entities unlinked to the collagen 1 genes.44,45 Thus, OI type VII was initially described as a recessive variant of OI46 and subsequently mapped to chromosome 347 and finally shown to be caused by CRTAP deficiency.18 The molecular basis of OI type V and VI is unclear, and their nosologic status still uncertain.

Prenatal diagnosis DNA-based prenatal diagnostic testing is available for OI. The test may be applied in families with defined pre-existing mutations. Because of the heterogeneity of the molecular bases of OI, molecular testing for prenatally suspected de novo cases is theoretically possible but usually not a feasible option (see above). Moderate to severe forms of OI

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in the fetus can be detected by sonography. Sonographic signs of OI include poor mineralization of the skull leading to easy deformability and to unusually good visualization of cranial content; bowing of the limb bones; poor visualization of the cortex of the long bones; and morphologic irregularity changes in the long bones indicating fractures and callus formation. The femurs are the most sensitive indicators of OI, as they are the first bones to fracture and show morphologic changes. Similar changes in the ribs (leading to the appearance of “beaded ribs” in severe cases) are usually an indication of poor prognosis. OI is probably the most frequent cause of bowed limbs in the fetus; it is significantly more frequent than hypophosphatasia, with which it shares some sonographic signs, and also more frequent than campomelic dysplasia which is often the first diagnosis that comes to mind when bowed limbs are observed in utero. Osteopetrosis Osteopetrosis (OP) is characterized by an increase in bone mineral content, and thus in bone density as seen in radiographs or measured by densitometry.48 The increased bone mass does not result in increased mechanical stability, and frequent fractures are a feature of the severe forms of disorder. Osteopetrosis is clinically, pathogenetically and genetically heterogeneous. A common pathogenetic step includes failure of bone resorption by osteoclasts; this can result from a variety of molecular defects, ranging from defect in acidification of osteoclastic lacunae (proton pump deficiency, chloride channel defects or carbonic anhydrase 2 deficiency), defective vacuolar membrane stabilization (OSTM1 defects) and defective vacuolar trafficking (PLEKHM1 defects) to defective differentiation of osteoclasts (RANK and RANKL defects). The severity spectrum of osteopetrosis is extremely wide; thus, several different forms are recognized clinically.48 However, the older clinical classification is not easily adapted to the new molecular data. There is an extremely rare in utero lethal form of osteopetrosis. Affected infants suffer fractures in utero.49 Infantile (previously “malignant”) osteopetrosis is not uncommon and presents during infancy. Patients may present with hypocalcemia in the newborn period because of impaired calcium mobilization from bone. Hepatosplenomegaly reflects extramedullary hematopoiesis and anemia

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may develop due to reduced bone marrow volume. Impaired bone remodeling leads to impaired growth of the cranial basal foramina with progressive optic atrophy; in fact, many children have already severe visual loss at the time of diagnosis in the first months of life. Basal foramina restriction can result in cerebral ischemia. Impaired drainage via the eustachian tubes or sclerosis of the middle ear bones may result in deafness. Fractures are common secondary to impaired bone remodeling. Osteomyelitis is also common. Patients are usually short with a large head and may have genu valgum. The radiographic appearance of osteopetrosis is quite typical, although not specific for one or the other molecular form. Unless this infantile form of osteopetrosis is treated, the lifespan is markedly foreshortened. Definitive treatment consists of bone marrow transplantation which should be performed before irreversible visual loss has occurred. Interestingly, precise molecular characterization of the osteopetrosis variant may influence the indication for bone marrow transplantation.50 At least two separate defective genes cause the malignant infantile recessive form of osteopetrosis. In a larger group with the autosomal recessive malignant variant, mutations in the ATP6i (TCIRG1), encoding the a3 subunit of the vacuolar proton pump, occur.51–53 In a small number of patients, the chloride channel C1C-7 is lost due to mutations in CLCN7.54 CLCN7 mutations have also been defined in intermediate autosomal recessive osteopetrosis.55 Interestingly, CLCN7 gene mutations have also been documented in the benign autosomal dominant variant known as Albers–Schönberg disease (ADO II).56 This is considered to be a common form. Clinical manifestations in ADO II include nontraumatic fractures, palsies of the cranial nerves, and osteoarthritis.48 The CLCN7 mutations in the ADO II form are believed to be manifested in a dominant-negative manner, whereas those in the autosomal recessive malignant or autosomal recessive intermediate forms are believed to represent loss of function.56 Recent results enable delineation into further subtypes of the “osteopetrosis syndromes”. Recessive mutations in OSTM1 are responsible for a severe form of osteopetrosis with primary involvement of the nervous system consisting of cortical dysplasia, demyelinization and cerebral atrophy.57

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A further rare form has been linked to mutations in the PLEKHM1 gene.58,59 Mutations in RANK and in its ligand RANKL affect osteoclast differentiation and result in so-called “osteoclast-poor” osteopetrosis60,61; the RANK-associated form is accompanied by hypogammaglobulinemia because of a defect in immunoglobulin-secreting cells. A further, autosomal recessive variant of osteopetrosis exists that is caused by carbonic anhydrase II deficiency.62,63 Affected individuals do not usually have fractures, but they may be short in stature. They may have failure to thrive, developmental delay with intracranial calcifications, and mild renal tubular acidosis.48,63 Prenatal diagnosis in the malignant recessive forms has been accomplished by ultrasound but this method is not suitable for an early prospective diagnosis. Molecular-based ATP6i prenatal diagnosis has been performed.52,60 Prenatal diagnosis by molecular means is available in principle for all families with defined mutations but because of the genetic heterogeneity, it is imperative to have the studies initiated well in advance of a planned pregnancy.

Disorders due to defects in the cartilage collagens: type II collagen, type X collagen, and type XI collagen Collagen 2 mutations can produce a wide spectrum of chondrodysplasias that range from perinatally lethal to mild or asymptomatic.21–23 Achondrogenesis type II and hypochondrogenesis are the most serious of the COL2A1 collagenopathies. Fetuses have shortened limbs, short neck, prominent abdomen, large heads and flat face. These two disorders are associated with prematurity and hydrops. Stillbirth or perinatal death is the rule in achondrogenesis type 2, while hypochondrogenesis may be compatible with survival. Torrance dysplasia is a variant lethal phenotype caused by a subset of mutations in the C-propeptide of COL2A1.24 Spondyloepiphyseal dysplasia congenita (SEDc) and its variant SEND Strudwick characteristically present with short trunks and prominent proximal foreshortening of the extremities. The neck is also short. Associated findings may include cleft palate

and clubfoot, but the head is normal. Affected individuals may have severe myopia, and retinal detachment can occur. Vertebral bodies are ovoid in newborns, iliac bones are short and square, and the pubic symphysis is poorly ossified. Delayed epiphyseal ossification occurs. SEND Strudwick appears similar at birth but displays more metaphyseal involvement during childhood. In older individuals, metaphyseal involvement is quite pronounced. All SEDC variants are caused by defects in COL2A1.22,64–6 Kniest dysplasia is a severe disorder also presenting with disproportionate short stature.23 The trunk and limbs are short. Cleft palate, clubfoot and inguinal hernias may also occur. Joints are enlarged, the face is flat, and coxa vara is evident. Myopia and hearing loss develop. Numerous COL2A1 mutations have been defined, with a predominance of exon-skipping mutations or other small deletions or insertions that, unlike glycine substitutions, do not affect the regular triplet structure of the collagen triple helix but rather affect the length of one αchain; however, this is a trend and not a strict rule.22,68 Stickler syndrome type I is also due to specific mutations in COL2A1, usually null alleles that affect the quantity of COL2A1 produced but do not affect its structure.69–71 Patients may have cleft palate, micrognathia and severe myopia with retinal detachment, and can develop sensorineural hearing loss and arthritis, but they are not short; in some mutation carriers, there may be no overt clinical phenotype. The phenotype is not detectable in utero, but prenatal molecular diagnosis is available for families with confirmed COL2A1 mutations. In contrast to Stickler syndrome type I, Stickler syndrome type II and the related Marshall syndrome are due to defects in COL11A1.70,72 Finally, the nonocular form of Sticker syndrome, Stickler syndrome type III, is due to dominantly inherited defects in COL11A2.73,74 Most COL11A2 mutations identified in these patients cause splicing defects. The patients may exhibit mild spondyloepiphyseal dysplasia.74 Recessively inherited COL11A2 mutations leading to complete loss of function of COL11A2 result in a severe phenotype. This is termed otospondylomegaepiphyseal dysplasia (OSMED).74,75 Finally, metaphyseal chondrodysplasia, Schmid type (MCS), is caused by dominant mutations

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in the collagen 10 gene, COL10A1. Mutations cluster in the carboxyl terminal nonhelical NC1 domain.76,77 Since this collagen type is expressed only by hypertrophic chondrocytes in the growth plate of long bones, the radiographic changes are detectable only in the metaphyses, mainly of the proximal and distal femur and proximal tibia, with minor changes in the distal tibia and distal radius and ulna. The clinical phenotype is that of progressive genua vara developing after the first year of life (when the child starts to stand and walk). The spine, the upper limbs, the hands, and the head and face are unaffected, and the phenotype is that of short-legged short stature (in adults, height may vary between approximately 130 and 145 cm).

Prenatal diagnosis Fetuses affected by the more severe variants of collagen 2 dysplasias (achondrogenesis type 2, hypochondrogenesis, Torrance dysplasia, Kniest dysplasia, and SEDC) will show abnormalities on prenatal sonography. ACG2, hypochondrogenesis and Torrance dysplasia may present early with short limbs and a prominent nuchal fold. Kniest dysplasia and SEDC will present in the middle of the second trimester with reduced limb length, delayed vertebral maturation, moderately reduced thoracic diameter and clubfeet. The length of hands and feet is usually normal. The findings are, however, nonspecific and in absence of a positive family history, the differential diagnosis is large. Molecular-based prenatal diagnosis for defects in the COL2A1, COL11A1 and COL11A2 is available in cases where the defect has been characterized previously; because of extensive mutational heterogeneity and the large size of the collagen genes, detection of a novel mutation is usually not an option in an ongoing pregnancy, although this may change with the onset of more powerful and rapid sequencing technologies in the future. Prenatal analysis of COL10A1 for Schmid metaphyseal chondrodysplasia has been reported.78 Pseudoachondroplasia and multiple epiphyseal dysplasia Pseudoachondroplasia (PSACH) is an autosomal dominant progressive skeletal dysplasia due to

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mutations in COMP.21,79 A significant proportion of cases of dominantly inherited multiple epiphyseal dysplasia (MED) are caused by COMP mutations, and others by matrilin-3 mutations (MATN3).80,81 Other MED cases are caused by mutations in COL9A1, COL9A2 or COL9A3.82–84 A relatively frequent recessive form of MED is caused by mutations in the diastrophic sulfate transporter gene and is discussed below.85,86 COMP is a pentameric protein present in the extracellular matrix of chondrocytes and is also found in tendons and ligaments. It is a member of the thrombospondin family and it interacts with types I, II, III, and IX collagens.87 Type IX collagen is a member of the fibril-associated collagens with interrupted triple helices. It is believed to act as a bridge between type II collagen and other structural cartilage matrix molecules, including COMP. Matrilin-3 is a member of the oligomeric extracellular matrix protein group. It is highly expressed in bone and cartilage and binds to COMP, type II collagen, and type IX collagen.80 At birth, PSACH infants appear relatively unaffected.21,88 They have both a normal length and head circumference. Skeletal growth decelerates between the first and second year of life. Ultimately, they develop disproportionate short stature with short extremities. Patients also have generalized ligamentous laxity except at the elbows and hips, where mobility is restricted. Additional lower extremity abnormalities lead to early osteoarthritis. The epiphyses, metaphyses, and spine are all involved. Multiple epiphyseal dysplasia also has a delayed onset.79 It is a very heterogeneous disorder caused by mutations in COMP, MATN3, COL9A2, COL9A1, COL9A3 or DTDST/SLC26A2 genes. Patients with COMP mutations can have short stature and major disability caused by joint pain and stiffness. They typically have significant abnormalities of the capital femoral epiphyses and acetabuli.79 Recessively inherited MED is the second most frequent MED variant and is characterized by clubfoot, normal or moderately reduced stature, mild brachydactyly, and epiphyseal flattening on radiographs. Individuals with COL9A1 or COL9A2 mutations have normal or near-normal stature and significant epiphyseal abnormalities in the knees, but the hips are relatively spared.

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MATN3 mutations also result in milder phenotypes than COMP mutations. Both the hips and the knees can be affected. Prenatal analysis can be offered to families with defined mutations in the COMP, DTDST, COL9A1, COL9A2, COL9A3 and MATN3 genes, but is only rarely requested.

Prenatal diagnosis Even in pseudoachondroplasia, ultrasound is not useful for prenatal diagnosis since the skeletal abnormalities are not yet present in utero.21,88 However, prenatal molecular diagnosis is possible in pregnancies at risk. Most defined COMP gene mutations are clustered within exons coding for calcium-binding repeats. The mutations result in amino acid substitutions or are short in-frame deletions or insertions.87,89 Parental germinal mosaicism for COMP mutations has been described repeatedly27 (and personal observation). Similarly, the sonographic prenatal diagnosis of MED variants is not possible, while mutation analysis for COMP, MATN3 or collagen 9 gene mutations is possible in pregnancies where one parents is affected and the defect has been characterized previously. Indeed, a significant proportion of dominant MED cases is familial.79 However, prenatal diagnosis is only rarely requested. For DTDST mutations responsible for rMED, see below.

Disorders due to defects in the diastrophic dysplasia sulfate transporter gene Mutations in the diastrophic dysplasia sulfate transporter (DTDST; SLC26A2) gene are responsible for a family of skeletal dysplasias that include achondrogenesis type 1B (ACG1B), atelosteogenesis type 2 (AO2), diastrophic dysplasia (DTD) and recessive multiple epiphyseal dysplasia (rMED).85,86,90–95 The encoded transmembrane protein is an anion exchanger and functions to transport extracellular sulfate across cell membranes. Mutations in the DTDST gene result in decreased intracellular chondrocyte sulfate and are manifested by decreased or absent cartilage matrix sulfated proteoglycans. The severity ranges from the lethal variants, ACG1B and AO2, to the milder phenotypes, DTD and rMED. This represents an

arbitrary division of disorders since they actually reflect a continuum of phenotypes. Similar to achondrogenesis type 2 (ACG2), ACG1B is a perinatal lethal condition.96 Infants afflicted with both disorders appear phenotypically similar, although the chest is described as barrelshaped in ACG2 and narrow in ACG1B. Another important difference is that the toes and fingers are short and stubby in ACG1B. The conditions can be readily distinguished on radiographs and by histologic examination of cartilage tissue.96 AO2 is also a severe skeletal dysplasia, which is usually lethal. Infants have short limbs, adducted feet and a hitchhiker thumb. Tapering of the distal humerus is also observed.93 Diastrophic dysplasia is a severe dysplasia that is recognizable at birth because of short limbs, contractures and clubfoot, and distinct radiographic changes.93 It is usually not lethal. The trunk and limbs are short, with the limbs being more foreshortened. Bilateral clubfoot, cleft palate, contractures, and “hitchhiker” thumbs and toes are characteristic. Patients with rMED are the mildest affected of those with DTDST mutations.

Prenatal diagnosis Achondrogenesis type 1B is among the most severe skeletal dysplasia phenotypes and can usually be detected at week 14 or 15 with shortened limbs and marked nuchal edema. However, sonographic differentiaton between achondrogenesis types 1A, 1B and 2 and distinction from other severe dysplasias is impossible. Accurate radiographic studies and histologic studies of cartilage tissue may allow for a reliable distinction between these severe dysplasia phenotypes and guide further molecular studies. Diastrophic dysplasia may be first recognized on routine ultrasound because of limb shortening and contractures and the presence of the typical “hitchhiker” thumb. However, even the latter finding is not entirely specific. Except for Finland, where there is a high frequency of the so-called Finnish mutation,90,91 there is extensive mutational heterogeneity, and molecular confirmation of diastrophic dysplasia during pregnancy may or may not be possible. When the mutation has been characterized previously in another affected family member, mutation analysis is possible and allows for early prenatal diagnosis.94

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Larsen syndrome and related disorders Larsen syndrome is a disorder that presents at birth with dislocations of the knees and hips.97,98 The face is flat, there is frontal bossing, and the fingers may show a characteristic deformity consisting of broad and short distal phalanges (“spatulated”). Most cases are sporadic, but dominant inheritance can occur. Diagnostically useful radiographic changes include supernumerary carpal bones with premature carpal ossification as well as broad phalanges and sometimes dislocations at phalangeal joints.97,98 A significant proportion of cases of this disorder are caused by dominant mutations in the gene coding for filamin B (FLNB).99 Mutations in the same gene may give rise to more severe skeletal dysplasias known as atelosteogenesis type 1, atelosteogenesis type 3 and boomerang dysplasia.100 Radiographic signs of these disorders include hypoplastic or radiographically “absent” humeri or femurs, hypoplastic vertebral bodies, and short and broad phalanges. These forms are usually lethal but survival can occur; the phenotype then evolves into a severe Larsen phenotype. Very rarely, recessive mutations in the FLNB gene are responsible for a short trunk dysplasia phenotype called spondylo-carpal-tarsal synostosis. In this form, there is secondary fusion (synostosis) of adjacent vertebral bodies leading to shortening of the trunk and scoliosis.99 A subset of cases of Larsen syndrome does not have mutations in the FLNB gene and is inherited as a recessive trait. Some of these cases have recessive mutations in the gene coding for chondroitin6-sulfotransferase (carbohydrate sulfotransferase 3, CHST3).101 In addition to dislocations of the knees and elbows at birth, these individuals have radiographic changes in the spine that distinguish them from the classic, dominant cases of Larsen syndrome.101 The phenotype evolves into a more severe chondrodysplasia with short trunk and kyphoscoliosis (a phenotype previously called spondyloepiphyseal dysplasia, Omani type). Prior to the identification of CHST3 mutations, individuals had been partly diagnosed as humerospinal dysostosis or “chondrodysplasia with multiple dislocations.”101

Prenatal diagnosis The knee dislocations in dominant or recessive Larsen syndrome can be recognized by prenatal

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sonography, but experience shows that most cases go undetected. The more severe skeletal changes in atelosteogenesis 1/3 are usualy detectable in the second trimester; when pronounced hypoplasia of the humerus or femur is present, a tentative diagnosis can be made but differentiation from other forms of dysostosis is difficult. Molecular prenatal diagnosis in pregnancies at risk for FLNB mutations is available.100 Prenatal diagnosis for recessive Larsen syndrome associated with CHST3 mutations has been performed (Superti-Furga, unpublished).

Skeletal dysplasias and craniosynostosis syndromes associated with fibroblast growth factor receptor mutations These are caused by mutations in fibroblast growth factor receptor genes (FGFR).15,102,103 FGFRs function as tyrosine kinases. All mutations represent gain of function and are inherited in a dominant manner.104–106 Skeletal dysplasias caused by mutations in the fibroblast growth factor receptor gene 3 (FGFR3) These include achondroplasia (ACH), hypochondroplasia, thanatophoric dysplasia (TD), and severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN).15 Achondroplasia represents the most common skeletal dysplasia in humans. Patients have short limbs, midface hypoplasia, and macrocephaly with frontal bossing. Limited elbow extension and a space between the distal phalanges of the third and fourth fingers (“trident hand”) may be present. The long bones are short. Newborns are hypotonic, although this resolves, and the foramen magnum is small. The increased mortality of affected children is attributed to foramen magnum stenosis. The natural history of achondroplasia is well known and may include delayed motor development, recurrent ear infections, development of dorsal gibbus or of severe varus deformity at the knee, pain and neurologic symptoms because of stenosis of the lumbar spinal canal, and obesity.107,108 Mental development is unaffected. Homozygous or double dominant achondroplasia is a usually lethal condition result-

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ing from inheritance of two copies of mutated FGFR3 as may occur in infants born to couples where both parents are affected. Most cases of achondroplasia are due to a Gly380Arg substitution in FGFR3.109,110 Rare cases are associated with the Gly375Cys substitution.111,112 Hypochondroplasia shares phenotypic features with achondroplasia but is milder. Affected individuals are not as short, and facial manifestations are not as severe. The most common defect in FGFR3 is Lys540Asp113 but several other mutations have been reported.114–117 The neurologic complications of achondroplasia usually do not occur in hypochondroplasia. Thanatophoric dysplasia is usually a perinatal lethal condition. Infants have severe limb shortening, very short ribs, and midface hypoplasia. Death is due to either respiratory compromise secondary to the small thorax or neurologic impairment secondary to the small foramen magnum. At least two subtypes correlated with certain mutations are recognized. Type I is associated with curved tubular bones and has numerous different mutations in FGFR3.16,118 Type II has a cloverleaf skull and straight femurs and is often associated with a specific mutation, Lys650Glu.16 Severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN) is a very rare variant caused by a specific FGFR3 mutation (Lys650Met).119 Affected individuals have short stature, midface hypoplasia, developmental delay and mental retardation, and develop acanthosis nigricans. Although all FGFR3-related dysplasias share common features, the distinct phenotypes of TD, ACH and hypochondroplasia are discrete and have little overlap. Occasionally, individuals with hypochondroplasia mutations may present with short limbs already at birth.120 Very rare instances of individuals with TD mutations surviving into the childhood period have been observed; these individuals have a phenotype similar to SADDAN121,122 (and unpublished observation).

de novo, with a relatively strong influence of increasing paternal age. Achondroplasia is not detectable by ultrasound earlier than around week 22–24; at that time femur length will progressively depart from the normal growth curves.123,124 In the third trimester, sonographic signs of achondroplasia may include macrocephaly, frontal bossing with a saddle nose, a “trident” hand, and shortening of the long bones.125,126 In this situation, urgent molecular analysis of the FGFR3 gene may help in confirming the diagnosis but usually has little clinical consequence. Conversely, in a high-risk pregnancy (one affected parent) molecular analysis can detect the presence of the mutation in CVS or amniocyte DNA, and even on fetal DNA extracted from maternal blood.127 Thanatophoric dysplasia can be detected as early as week 15 or 16; severe growth delay will be apparent after week 18. Sonographic signs include marked shortening of the long bones (particularly femur and humerus) with mild bowing, a small thorax with substernal protrusion of the abdomen, relative macrocephaly, and frontal bossing. The bone density is normal. Thanatophoric dysplasia, which occurs exclusively as a de novo dominant, is in fact one of the more frequent severe dysplasias detected on routine ultrasound examination of a low-risk pregnancy. While most cases of hypochondroplasia are still clinically normal at birth, a minority of cases may exhibit growth deceleration in the third trimester128,129 Hypochondroplasia is actually significantly rarer than achondroplasia; the label is often attached to individuals with short stature that is not related to FGFR3 and the diagnosis should always be verified prior to further counseling. The pregnancy of a couple where both parents have achondroplasia or one parent has achondroplasia and the other has a different skeletal dysplasia or short stature condition is not exceptional and requires ad hoc counseling.130 Several examples of children affected by two conditions have been reported, often with a severe or lethal course.131–134

Prenatal diagnosis In pregnancies where one of the parents is affected by achondroplasia or hypochondroplasia and the underlying FGFR3 mutaton is known, prenatal diagnosis can be offered. However, virtually all TD cases and a majority of achondroplasia cases occur

Craniosynostosis syndromes The craniosynostosis syndromes are a group of disorders sharing the premature fusion of one or more sutures of the skull. Often additional anomalies are associated. Many of these disorders are

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caused by defects in the FGFR1, FGFR2 or FGFR3 genes.15,102,103,135,136 The craniosynostoses are autosomal dominant disorders. With the exception of Muenke syndrome and some cases of Crouzon syndrome, in which familial cases are not rare, most cases represent new mutations. It has been shown that de novo mutations in FGFR genes occur mostly on paternal alleles and are related to increased paternal age; a selective advantage for mutation-carrying spermatogonia has been postulated.137,138 Patients with Pfeiffer syndrome have craniosynostosis, with broad thumbs and broad great toes that are deviated medially. Partial syndactyly can occur. Additional findings may be present. Both inherited and sporadic cases occur, with the latter having a more severe phenotype. Mutations have been defined in FGFR1 and FGFR2.139–141 Three subtypes have been distinguished. Patients with Pfeiffer syndrome type 1 have bicoronal craniosynostosis, a normal lifespan, and normal intelligence. These patients exhibit mutations in FGFR1 and FGFR2. Type 2 patients have cloverleaf skulls, severe ocular proptosis, neurodevelopmental delay, elbow ankylosis, and foreshortened lifespans. Type 3 is similar to type 2 but patients do not have cloverleaf skulls. Type 2 and type 3 have mutations restricted to FGFR2. Apert syndrome is a severe form of craniosynostosis with numerous organ systems being affected. Most cases are sporadic and correlate with increased paternal age. The findings include brachycephaly, midfacial hypoplasia, broad thumbs and great toes, and partial or total syndactyly of the hands (“spoon hands”) and feet. Some additional findings may include cleft palate, various structural anomalies of the central nervous system, additional limb abnormalities, vertebral fusion, and some cutaneous manifestations, including acne, hyperhidrosis, hyperkeratosis, and hypopigmentation. Mental retardation may occur. Almost all cases of Apert syndrome are due to specific mutations in FGFR2, Ser252Trp, and Pro253Arg, but there are exceptions.102,142–145 Familial cases are autosomal dominant. Crouzon syndrome is the most common autosomal dominant craniosynostosis. Patients have coronal or multiple suture synostosis, maxillary hypoplasia, a prominent beaked nose, shallow orbits, and ocular proptosis.15 One half of the cases

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are familial, with autosomal dominant inheritance; one half are de novo and correlate with increased paternal age. Intelligence is usually normal and brain abnormalities are rare, although progressive hydrocephalus does occur and may be associated with cerebellar herniation. Vision and hearing deficits can occur. Fusion of C2-C3 can be present, while hands and feet appear normal. Numerous mutations have been identified in FGFR2.102,137,139 Crouzon syndrome with acanthosis nigricans is rare and is associated with a specific mutation in FGFR3 (Ala391Glu).143 Early-onset skin findings include hyperpigmentation and hyperkeratosis, along with verrucous hyperplasia in flexural areas. Interestingly, stature is unaffected with this particular FGFR3 mutation. Patients with sporadic and familial cases of the Muenke syndrome (originally termed “nonsyndromic craniosynostosis”) are often misdiagnosed with another form of craniosynostosis.102 The phenotypic presentation is very variable.15,146 This syndrome is also associated with a specific FGFR3 mutation (Pro250Arg).142,146 Patients can display coronal synostosis, midfacial hypoplasia, downslanting palpebral fissures, and ptosis. Bone abnormalities of the hands and feet occur. Some do not have craniosynostosis, but may have only macrocephaly or even normal-sized heads. Sensorineural hearing loss is seen in about one-third. Intrafamilial variation is great. Inheritance is autosomal dominant. The Jackson–Weiss syndrome is a rarer variant characterized by midface hypoplasia, frontal prominence, and cutaneous syndactyly with varying degrees of medially deviated and enlarged great toes. Some do not have craniofacial anomalies. Intelligence is normal. The clinical presentation is variable. This syndrome is due to a specific FGFR2 mutation, Ala344Gly.139,147

Prenatal diagnosis Sonographic detection of premature craniosynostosis is possible in the third trimester, but difficult, also in view of possible intrafamilial variability. Severe cloverleaf deformation of the skull (“Kleeblattschädel”) can be recognized on sonography. In less severe cases, the detection of an abnormal shape of the skull, accompanied by facial deformation, is possible. The finding of severe syndactyly of the hands can alert the clinician to the

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presence of Apert syndrome. However, most sporadic cases of craniosynostosis of moderate severity, such as classic cases of Crouzon syndrome, usually go undetected before birth. Mutation analysis of the FGFR genes is available in many laboratories, facilitating diagnosis, especially in familial cases of craniosynostosis.

The Ehlers–Danlos syndrome This is a heterogeneous group of connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. Patients may have additional musculoskeletal defects. Several clinical types and molecular subtypes are currently recognized.148 Ehlers–Danlos syndrome (EDS) types I and II: the classic types These are by far the most commonly occurring EDS types. They differ only in the degree of organ system involvement. Patients with EDS I have marked skin involvement, generalized joint hypermobility, and musculoskeletal deformities including dislocations and hypotonia. Prematurity is also characteristic. Patients with EDS I may display some of the more serious findings associated with EDS IV, including a propensity for rupture of the bowel and aorta. EDS II, also known as the mitis type, shares manifestations with EDS I, but the skin is less involved and joint laxity may be confined to hands and feet. Premature births are not increased. Patients with EDS II may remain undiagnosed. Patients with EDS types I and II most commonly have defined mutations in either COL5A1 or COL5A2, but other molecular mechanisms have been described.148–151 Inheritance is usually autosomal dominant in EDS I or II. A few classic EDS cases have been attributed to defects in COL1A1 or COL1A2, sometimes with recessive inheritance.149,150 Rare cases have been associated with tenascin-X deficiency.152,153 Although DNA-based COL5A1 and COL5A2 mutation analysis is available, it has not yet been applied to prenatal diagnosis. COL1A1 and COL1A2 may be analyzed by the same methods applied to OI.

EDS type III: the hypermobility type These patients with generalized joint hypermobility also suffer from frequent dislocations, effusions, and precocious arthritis.148 They often have mitral valve prolapse and velvety, hyperextensible skin. Tissue fragility is not characteristic. The molecular basis remains undetermined. EDS type IV: the vascular type Known also as the “arterial” or “ecchymotic” type, this is the most serious of all EDS types.148,154 Patients can have spontaneous hemorrhage, aneurysms with dissection, and arteriovenous fistulas. Rupture of organs, including the colon and gravid uterus, occurs. Other complications of pregnancy include an incompetent cervix, a prolapsed uterus, and fragile membranes. The skin is thin, fragile, and translucent but not hyperelastic. Wound healing is delayed. Skeletal manifestations may include slight hypermobility of the joints of the hands and feet and hip dislocation. A variety of additional complications may be seen, and the medical literature contains literally hundreds of reports of severe complications in this EDS type. Although complications are rare before puberty, survival is foreshortened.154 Inheritance is autosomal dominant. The disorder is due to defects in type III collagen.154–157 About 50 percent of COL3A1 mutations are de novo. The majority of reported mutations is private. Genetic testing is offered for EDS type IV. DNAbased testing is usually preceded by collagen protein studies. Prenatal diagnosis in at-risk pregnancies has been achieved both by molecular analysis and by direct biochemical analysis of a CVS biopsy.40 EDS type VI: the kyphoscoliotic type There are two subtypes, EDS VIA and EDS VIB.148,158 Those cases characterized as EDS VIA represent the majority and display decreased activity of lysyl hydroxylase, an enzyme required for the hydroxylation of certain lysyl residues in procollagen.148,159 Lysyl hydroxylase is encoded by the PLOD1 gene. A number of mutations have been defined in the PLOD1 gene in EDS VIA.148,158,160,161 The hydroxylated lysines are subsequently substrates in a series of reactions

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resulting ultimately in the intermolecular and intramolecular cross-linking of collagens. Urinary analysis of collagen degradation products may be useful in confirming the diagnosis prior to molecular analysis.162 Inheritance is autosomal recessive. Patients with EDS VI are born with hypotonia and kyphoscoliosis.148,163 The kyphoscoliosis is progressive and decreased pulmonary function often results. Patients have generalized joint laxity. Ocular findings may include scleral fragility, rupture of the globe, microcornea, and retinal detachment. Additional signs of tissue fragility may be manifested by premature rupture of the membranes, arterial rupture, easy bruising, and atrophic scars. Prenatal DNA-based molecular diagnosis has been accomplished in some families with defined mutations.158,160 EDS types VIIA and VIIB: the arthrochalasis types Deletion or skipping of all or a portion of exon 6 in the COL1A1 or COL1A2 genes causes EDS VIIA and EDS VIIB, respectively.148,164,165 Exon 6 in both genes encodes the procollagen N-propeptide cleavage site and a critical lysine residue involved in collagen cross-linking.38,148 These patients display severe, generalized joint hypermobility with dislocations of the hips and sometimes of the knees at birth, and recurrent subluxations. Surgical repair of dislocated hips is difficult and luxation may recur.165 (165). Thoracolumbar scoliosis may result in short stature. The skin is thin and velvety but only moderately extensible. Muscular hypotonia can be a prominent early finding. Interestingly, a few patients have been described with features of mild OI. Inheritance is autosomal dominant.

Prenatal diagnosis This might be based on the sonographic demonstration of hip dislocation and abnormal limb positioning, but experience is lacking. Conversely, diect biochemical analysis of collagen type 1 extracted from a chorion villus biopsy has been used to exclude EDS VIIB in a pregnancy at risk.40 In familial cases, mutation analysis can be used to diagnose EDS VII in the fetus.

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EDS type VIIC: the dermatosparactic type Ehlers–Danlos syndrome type VIIC is caused by a deficiency of the enzyme responsible for cleaving the procollagen N-propeptide, the N-proteinase.148,166–168 In contrast to EDS types VIIA and VIIB, this is a recessively inherited disorder displaying severe skin involvement. Patients are born prematurely, with soft, lax and redundant skin that is friable and tears easily. Although the skin bruises and is avulsed easily, it heals readily without scarring. Additional findings include umbilical hernias and dysmorphic facial features, including micrognathia. Progressive joint laxity is also a feature. The natural history of this disorder is only partially documented.148,168 EDS with defective zinc transport Two independent groups have documented the existence of an autosomal recessive Ehlers-Danlos variant characterized clinically by short stature, thin skin, varicose veins, blueish sclerae, hypodontia, and mild osteodysplastic changes.169,170 This EDS variant is caused by mutations in a zinc transporter gene, ZIP13 (SLC39A13), that is responsible for modulating the zinc levels in the endoplasmic reticulum. The pathogenesis may consist in inhibition of lysyl hydroxylase by competition of zinc with iron (a co-factor of the enzyme) or in a disruption of TGF-β and BMP signaling that is dependent on zinc-sensitive SMADs.169,170 The latter hypothesis is supported by findings in a mouse model of the disorder that precisely recapitulates the findings in affected patients.169

Menkes disease and the occipital horn syndrome These X-linked entities represent different ends of a spectrum of disorders due to defects in a common gene, ATP7A.171–174 This gene encodes an enzyme involved with copper metabolism. The enzyme is a P-type ATPase active in the transport and sorting of copper. Depending on the metabolic state, it is localized to either the plasma membrane or the trans-Golgi network. One function is the delivery of copper into the appropriate cellular compartment for incorporation into copper-requiring enzymes. Copper-requiring enzymes are involved

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in a variety of metabolic reactions and are affected by defects in ATP7A. The occipital horn syndrome (OHS) This is the milder form of ATP7A deficiencyinduced disorders. Patients display manifestations primarily restricted to the connective tissues and skeleton.172,175–180 At birth, umbilical and inguinal hernias may be evident, and the skin can be wrinkled and lax. Jaundice, hypotonia and hypothermia soon develop. Motor development can be delayed. Recurrent urinary tract infections due to bladder diverticuli are common. Patients also have diarrhea. Skeletal manifestations include hypermobile joints, narrow chest, long trunk with kyphosis or scoliosis, pectus deformities, and hyperostosis of the proximal ulna and radius. Occipital exostoses (“occipital horns”) and osteoporosis also develop. Vascular abnormalities including arterial aneurysms may also occur. These connective tissue manifestations are due to secondary deficiencies in lysyl oxidase activity, a copper-containing enzyme. Lysyl oxidase is required for cross-linking of certain collagens and elastin. Lack of lysyl oxidase activity perturbs stable fibrillogenesis. OHS was formerly classified as EDS IX. Menkes disease (MD) Menkes disease represents the more severe form.172,178,180–182 However, overlap forms with the occipital horn syndrome are not rare, depending on the underlying mutation. In Menkes disease, findings in addition to those seen in OHS include spontaneous fractures, progressive neurologic degeneration with retardation of psychomotor development, seizures, failure to thrive, hair changes, marked hypotonia, and dilation and rupture of elastic arteries, including the aorta. These additional manifestations seen in Menkes disease are also considered to reflect secondary deficiencies in other copper-requiring enzymes.172 In addition to lysyl oxidase, they include deficiencies in proteins concerned with cellular respiration such as cytochrome C oxidase, molecules involved in cellular protection against free radicals such as superoxide dismutase, and ceruloplasmin and molecules concerned with the metabolism of neurohormones or transmitters such as dopamine-βhydroxylase and possibly peptidyl α-amidating

mono-oxygenase. In addition, copper accumulation in certain tissues reflects its ineffective efflux. While the severe form of MD is lethal, intermediate forms exist between it and OHS. The severity of disease is somewhat correlated with the genotype as those ATP7A gene mutations resulting in residual activity enzyme are in general associated with a milder phenotype.171,174,180 In patients with milder mutations, copper treatment may be helpful, particularly when initiated early.183–185 Biochemical tests involving the measurement of copper in chorionic villi samples are available, as are copper uptake studies in cultured amniocytes. Both tests have been used successfully for prenatal diagnosis but are reported to have limitations.172 Prenatal DNA-based mutation analysis of the ATP7A gene has been developed.171,173,174 Germline mosaicism has been documented.186

Marfan syndrome and Marfan overlap disorders Marfan syndrome is one of the conditions that gave rise to the concept of “connective tissue disorders.”1 Individuals with classic Marfan syndrome are tall and have long limbs and long fingers. These features usually become apparent during the first years of life, but in severe cases may be already apparent at birth. Skeletal abnormalities can include pectus excavatum or carinatum, pes planus, single or multiple abnormal spinal curvatures, protrusio acetabuli, and joint laxity.187,188 Ocular manifestations include elongation of the globe, corneal flattening and, most importantly, dislocation of the lens leading to ectopia lentis. Findings in the cardiovascular system include mitral valve prolapse with severe regurgitation, dilation of the valvular annulus, and redundancy of the atrioventricular valve leaflets. Arrhythmias may lead to sudden death. Dilation of the aortic root begins in utero and can result in valvular regurgitation. Aortic aneurysm and the potential for dissection have possible life-threatening consequences. The rupture of apical lung blebs may lead to pneumothorax. The single central nervous system manifestation is dural ectasia. Patients may suffer from easy bruising but the most common skin manifestation is striae atrophicae. The so-called neonatal form of Marfan syndrome is a particularly severe form that

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manifests with arachnodactyly, joint contractures, and severe insufficiency of the heart valves in the first weeks of life; there is also a distinct facies, and the prognosis is poor.189–192 Milder forms also occur and in these cases the diagnosis may be difficult; there is overlap with several additional disorders with defined defects in FBN1 that share, but do not meet, the diagnostic criteria for Marfan syndrome.187,193,194 These disorders include familial ectopia lentis, familial aortic aneurysm, and familial aortic dissection. Some with the MASS phenotype (mitral valve prolapse, myopia, mild aortic root dilation, striae, and mild skeletal changes) also have FBN1 mutations. The diagnostic criteria for Marfan syndrome have been well defined and may include molecular findings.195 Special criteria may be needed for the clinical diagnosis in childhood.196 As we know now, most cases are due to mutations in fibrillin 1 encoded by FBN1.187,194,197,198 Fibrillin 1 forms microfibrils that are in turn components of elastic fibers. The effects of defective microfibrils are manifested in numerous organ systems, including the eyes, cardiovascular system, skin, central nervous system, skeleton, lungs, and adipose tissue. The observation of a family with a dominantly inherited Marfan-like syndrome that segregated with the subunit 2 of the TGF-β receptor opened the way to a new nosologic family and contributed significantly to understanding of the pathogenesis of fibrillin-associated Marfan syndrome.199 In summary, mutations in either one of the subunits of the TGF-β receptor my result in a series of phenotypes that recapitulate essential aspects of classic Marfan syndrome. The most severe of these phenotypes is called the Loeys–Dietz syndrome200; milder phenotypes include those previously known as Marfan syndrome type 2 or thoracic ascending aortic aneurysms and dissections.200–202 Approximately 1 in 5 individuals referred with a suspcicion of Marfan syndrome may have a mutation in one of the TGF-β receptor genes.203 Thus, while Marfan syndrome is indeed a disorder of connective tissue, the pathogenesis does not result simply from mechanical failure of elastic fibers but involves a deep disregulation of TGF-β signaling; fibrillin 1 can bind TGF-β and act as a reservoir and thus regulate TGF-β activity, and changes in fibrillin 1 structure and metabolism may affect this signaling.

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Discovery and elucidation of the TGF-β -related Marfan-like syndromes such as the Loeys–Dietz syndrome have significantly contributed to the understanding of classic Marfan syndrome. Most importantly, the most dreaded vascular complications of Marfan syndrome, dilation of the aortic root followed by dissection, can be treated by the administration of pharmaceutical agents that modulate TGF-β signaling.204,205 Pregancy-related aspects and prenatal diagnosis Women affected by the Marfan syndrome have an increased risk of aortic dilation and rupture during pregnancy and should be monitored.188,206,207 Severe Marfan syndrome in the fetus may manifest in the late second or third trimester with femur length above the 90th percentile and/or with aortic root or cardiac dilation.208,209 However, these prenatal observations are rather rare considering the frequency of Marfan syndrome. The TGF-β receptorrelated disorders may also be detected by prenatal sonography.210 Prenatal molecular analysis of FBN1 can be performed in those families in whom Marfan syndrome is suspected or those having a confirmed diagnosis of Marfan syndrome or the Loeys–Dietz syndrome.

Homocystinuria Homocystinuria, due to defects in the enzyme cystathionine-β-synthase, may display findings that may be confused with Marfan syndrome or other connective tissue disorders.211 Ocular findings may include ectopia lentis, myopia, retinal detachment or degeneration, and corneal abnormalities; these findings are important diagnostic hints.212 Reported skeletal manifestations include increased length of long bones, osteoporosis, scoliosis or kyphosis, arachnodactyly, and pectus carinatum or excavatum. In contrast to Marfan syndrome, joint mobility may be restricted. Patients may also exhibit mental retardation or have psychiatric disorders. Thrombotic vascular occlusions are also common.211 Inheritance is autosomal recessive. Numerous mutations have been defined in the cystathionineβ-synthetase gene (CBS).213–215 Most mutations are private and consist of missense mutations. Certain

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mutations are associated with specific ethnicities. Prenatal diagnosis has been accomplished by enzyme assay of cultured amniotic cells or chorionic villi.216 Prenatal DNA-based analysis is possible in families in whom the pre-existing mutations have been defined (see Chapter 15).

Pseudoxanthoma elasticum This is a heritable connective tissue disorder with skin, cardiovascular, and ocular manifestations.217,218 The clinical findings are the result of the progressive fragmentation and calcification of elastic fibers; the increased dermal deposition of extracellular matrix, including proteoglycan, glycosaminoglycans, fibronectin, and vitronectin; and increased production of elastic fibers in affected skin. The skin findings are the most apparent clinical feature and include yellow papules or nodules that coalesce into plaques in flexural sites.217–219 The areas most often affected include the axillae, neck, antecubital and popliteal fossae, and groin. The skin eventually sags, and individuals appear prematurely aged. Ocular involvement can be serious. It is characterized by peau d’orange hyperpigmentation, angioid streaks in the fundi resulting from breaks in calcified elastic lamina of Bruch’s membrane, subretinal membranes with neovascularization, retinal hemorrhages, and central vision loss.217–219 Legal blindness may result. Calcification and degeneration of elastin-rich vessels also occur. Vascular changes may lead to intermittent claudication of the lower extremities, abdominal angina due to celiac artery stenosis, gastric bleeding, and myocardial infarcts at an early age. Endocardial fibroelastosis with mitral valve prolapse is also relatively common.217 Pseudoxanthoma is not usually evident at birth, but may be clinically symptomatic in childhood; considerable clinical variability exists.217,220 The disorder has been linked recently to a series of mutations in ABCC6.219,2211–224 Molecular data have led to the better definition of diagnostic criteria.225 Interestingly, the older notion of a dominantly inherited form of PXE has been refuted.222,224,225 The responsible gene, ABCC6, is a member of the multiple-drug resistance family and is also known as MRP6. ABCC6 encodes a transmembrane transport protein with ATPase activity. Although the exact function remains unknown,

evidence suggests that it may function as an active transporter of anions. The pathophysiology remains speculative, but elegant experiments have shown that the pathologic process in connective tissue is dependent on a humoral, diffusing factor, not on the properties of local cells.226,227 To date, nonsense, missense, deletions, or small and large insertions and exon-skipping mutations have been defined.222,228–230 Affected individuals are homozygotes or compound heterozygotes, although heterozygous carriers may be at risk for partial manifestation. Identification of the defective gene permits prenatal diagnosis in families at risk and allows for genetic counseling and prenatal diagnosis where requested.

Acknowledgments We are very grateful to our colleagues and friends, Drs Leena Ala-Kokko and James Hyland, for allowing us to make use of their previous version of this chapter as a template.

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204. Brooke BS, Habashi JP, Judge DP, et al. Angiotensin II blockade and aortic-root dilation in Marfan’s syndrome. N Engl J Med 2008;358:2787. 205. Habashi JP, Judge DP, Holm TM, et al. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science 2006;312:117. 206. Lipscomb KJ, Smith JC, Clarke B, et al. Outcome of pregnancy in women with Marfan’s syndrome. Br J Obstet Gynaecol 1997;104:201. 207. Pacini L, Digne F, Boumendil A, et al. Maternal complication of pregnancy in Marfan syndrome. Int J Cardiol 2008 (ePub ahead of print). 208. Ramaswamy P, Lytrivi ID, Nguyen K, et al. Neonatal Marfan syndrome: in utero presentation with aortic and pulmonary artery dilatation and successful repair of an acute flail mitral valve leaflet in infancy. Pediatr Cardiol 2006;27:763. 209. Stadie R, Geipel A, Heep A, et al. Prenatal diagnosis of Marfan syndrome. Ultrasound Obstet Gynecol 2007; 30:119. 210. Viassolo V, Lituania M, Marasini M, et al. Fetal aortic root dilation: a prenatal feature of the Loeys–Dietz syndrome. Prenat Diagn 2006;26:1081. 211. Skovby F, Kraus J. The homocystinurias. In: Royce P, Steinmann B, eds. Connective tissue and its heritable disorders. New York: Wiley-Liss, 2002:627. 212. Cruysberg JR, Boers GH, Trijbels JM, et al. Delay in diagnosis of homocystinuria: retrospective study of consecutive patients. BMJ 1996;313:1037. 213. Gaustadnes M, Wilcken B, Oliveriusova J, et al. The molecular basis of cystathionine beta-synthase deficiency in Australian patients: genotype-phenotype correlations and response to treatment. Hum Mutat 2002; 20:117. 214. Miles EW, Kraus JP. Cystathionine beta-synthase: structure, function, regulation, and location of homocystinuria-causing mutations. J Biol Chem 2004;279: 29871. 215. Moat SJ, Bao L, Fowler B, et al. The molecular basis of cystathionine beta-synthase (CBS) deficiency in UK and US patients with homocystinuria. Hum Mutat 2004;23:206. 216. Fowler B, Jakobs C. Post- and prenatal diagnostic methods for the homocystinurias. Eur J Pediatr 1998; 157:S88. 217. Nelder K, Struck B. Pseudoxanthoma elasticum. In: Royce P, Steinmann B, eds. Connective tissue and

218. 219.

220.

221.

222.

223.

224.

225.

226.

227.

228.

229.

230.

its heritable disorders. New York: Wiley-Liss, 2002: 561. Ohtani T, Furukawa F. Pseudoxanthoma elasticum. J Dermatol 2002;29:615. Chassaing N, Martin L, Calvas P, et al. Pseudoxanthoma elasticum: a clinical, pathophysiological and genetic update including 11 novel ABCC6 mutations. J Med Genet 2005;42:881. Sakata S, Su JC, Robertson S, et al. Varied presentations of pseudoxanthoma elasticum in a family. J Paediatr Child Health 2006;42:817. Struk B, Cai L, Zach S, et al. Mutations of the gene encoding the transmembrane transporter protein ABC-C6 cause pseudoxanthoma elasticum. J Mol Med 2000;78:282. Li Q, Jiang Q, Pfendner E, et al. Pseudoxanthoma elasticum: clinical phenotypes, molecular genetics and putative pathomechanisms. Exp Dermatol 2009;18:1. Fulop K, Barna L, Symmons O, et al. Clustering of disease-causing mutations on the domain-domain interfaces of ABCC6. Biochem Biophys Res Commun 2009;379:706. Uitto J. The gene family of ABC transporters – novel mutations, new phenotypes. Trends Mol Med 2005; 11:341. Christen-Zach S, Huber M, Struk B, et al. Pseudoxanthoma elasticum: evaluation of diagnostic criteria based on molecular data. Br J Dermatol 2006;155:89. Uitto J. Pseudoxanthoma elasticum – a connective tissue disease or a metabolic disorder at the genome/ environment interface? J Invest Dermatol 2004; 122:ix. Jiang Q, Endo M, Dibra F, et al. Pseudoxanthoma elasticum is a metabolic disease. J Invest Dermatol 2009;129:348. Ringpfeil F, Lebwohl MG, Christiano AM, et al. Pseudoxanthoma elasticum: mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter. Proc Natl Acad Sci USA 2000;97: 6001. Meloni I, Rubegni P, de Aloe G, et al. Pseudoxanthoma elasticum: point mutations in the ABCC6 gene and a large deletion including also ABCC1 and MYH11. Hum Mutat 2001;18:85. Le Saux O, Beck K, Sachsinger C, et al. A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum. Am J Hum Genet 2001;69:749.

23

Maternal Serum Screening for Neural Tube and Other Defects Aubrey Milunsky1 and Jacob A. Canick2 1 2

Center for Human Genetics, Boston University School of Medicine, Boston, MA, USA Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Neural tube defects (NTDs) are among the most common serious birth defects with significant mortality, long-term morbidity and diminished life expectancy for survivors. Developing countries still have by far the highest prevalence rates, while in developed nations a striking decrease in rates has been reported. Folic acid supplementation, dietary improvements, maternal serum screening for NTDs, and prenatal diagnosis by αfetoprotein and acetylcholinesterase assays and by sonography account for almost all the reduction in prevalence.

Types of neural tube defects Anencephaly and the various forms of spina bifida (SB) are the most common NTDs and they occur with similar prevalence. Other, less common NTDs include exencephaly, iniencephaly, and encephalocele. Anencephaly is a lethal defect in which the cranial vault and varying amounts of brain tissue are absent. SB is associated with varying degrees of mortality and morbidity (see below), depending on the size and location of the lesion and whether it is open or closed. At least 85 percent of SB lesions are open, having only a membrane covering or no covering at all, while closed SB is covered with skin. The SB lesions

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

are made up mainly of meningoceles and myeloceles or combinations thereof (myelomeningoceles). Meningoceles are herniations of the meninges, with the cord remaining in its usual position, and they constitute between 5 and 10 percent of all NTDs.1 In contrast, myeloceles do not involve herniations but neural tissue is exposed. Even when small, myeloceles almost invariably connote serious defects because of involvement of the cord. Myelomeningoceles, which involve both the spinal cord and the nerve roots, are associated with Arnold–Chiari malformations, in which contents of the cranial vault are displaced downward. Women who have had a child with anencephaly may subsequently deliver a child with SB, and vice versa. However, in the vast majority of cases, the lesion is the same as the earlier one.1,2 In a study of 334,262 consecutive births in France, including 360 infants with NTDs, 20.5 percent had associated malformations.3 Those born with encephalocele had more frequent associated malformations (37.5 percent) than infants with anencephaly (11.8 percent) or SB (23.7 percent). Facial clefts, musculoskeletal abnormalities, renal and cardiovascular anomalies were the most frequent.

Prevalence Striking varitions in birth prevalence have been reported over the past 50 years4 in studies done in the 1960s, 1970s, and 1980s. Extremely high rates (exceeding 8 per 1,000 births) were reported for Northern Ireland, Egypt, India, and China5–7 and

705

706

Genetic Disorders and the Fetus

in 2003 a remarkable rate of 13.9 per 1,000 in China.8 Although large geographic and temporal variations in the frequency of NTDs are well known, a marked decrease in the prevalence of NTDs has been observed. In the United States; estimates made in the 1970s varied between 1.4 and 3.1 per 1,000 births.9 US studies in the early 1980s showed a somewhat lower prevalence, between 1.0 and 1.6 per 1,000 births for NTDs and between 0.2 and 0.8 per 1,000 births for SB, with the highest rates being found in southern Appalachia.10,11 A 1995 report from the Centers for Disease Control and Prevention (CDC), studying the birth prevalence in 1985– 1994, put the rate even lower, between 0.4 and 1.0 per 1,000 births.12 In England and Wales, the total prevalence declined from about 3.4 per 1,000 livebirths and stillbirths in 1974 to just under 0.8 per 1,000 by 1994.13,14 The estimated birth prevalence by 1997 was as low as 0.14 per 1,000 (Figure 23.1).15,16 The marked long-term decrease in the prevalence of NTDs antedating folic acid supplementation in the UK and elsewhere17 is most likely due to improved diets and more recently to folic acid supplementation18–21 (discussed below). Secondary prevention with prenatal biochemical screening/ diagnosis and sonography has also significantly led to a decreased prevalence of NTDs.

Table 23.1 Evidence for a putative causal association of genetic factors with neural tube defects Observations implicating genetic factors

Reference

Polygenic nature of most disorders

9

Chromosome defects (see Table 21.4) Bias in sex ratio toward higher number of

5,25

females High percentage of males in low-prevalence

26

areas Increased susceptibility if parent has HLA-DR

27

locus Increased frequency in consanguineous

28

matings Monozygotic twin concordance

29, 25, 26

Racial/ethnic bias in incidence

5, 29a

Familial recurrence pattern: affected parent:

5

affected sibling/aunt/uncle/cousin Consanguinity

30

Increased incidence when there is a previous

31

child with hydrocephaly Increased incidence when there is a previous

32

child with germ cell tumor SNPS and polymorphisms

33

Susceptibility of midline “developmental

34

field” Mitochondrial uncoupling gene

35

Mutations in FOXN1 gene

36

Mutations in PAX-3 gene

37, 38

Abnormalities in folate and/or cobalamin

39–42

metabolism

Etiology and primary prevention Between 70 and 85 percent of NTDs can be avoided with sufficient and timely intake of folic acid.22–24 Other than dietary factors, additional genetic and environmental causes are recognized, some of which may share mechanisms requiring folic acid. The heterogeneous origin of NTDs makes it important to reach a precise diagnosis for genetic counseling. The vast majority of NTDs arise as a consequence of multifactorial inheritance. Putative genetic factors (Table 23.1) as well as many environmental causal associations (Table 23.2) have been suggested. In addition, an NTD may occur in many different syndromes (see examples in Table 23.3) and in association with chromosomal disorders (Table 23.4). The concurrence of a NTD and a chromosomal disorder may be fortuitous or may indicate the locus of a susceptibility gene.

Amorosi et al.178 identified a remarkable case of a fetus with anencephaly and craniorachischisis, absent thymus and abnormal skin, homozyous for a mutation in the FOXN1 gene. The forkhead family of genes is important in patterning the midline of the neural tube. Sever179 hypothesized that environmental factors were of less etiologic importance than genetic influences in a population with a low incidence rate for NTD, compared with areas or time periods with high incidence rates. In support of this hypothesis, Sever found no significant difference between rates by month of birth or conception and no significant association with maternal age or parity for anencephaly. However, he did note a significantly increased frequency with advanced maternal age for SB. Factors that support a genetic origin included a predominance of

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Table 23.2 Reported “environmental” causal associations with neural tube defects Environmental factors

Selected references

Aminopterin

43

Carbamazepine

44

Clomiphene citrate or infertility

45, 46

Copper in drinking water

47

Efavirenz (reverse transcriptase

48

inhibitor) Fetus interaction with residual

49

trophoblast First-trimester surgery

50

Folic acid deficiency

51, 22, 52

Hyperthermia

53, 54

Industrial/agricultural exposure

55, 56

Magnesium or calcium content of

57

drinking water Maternal alcohol ingestion

58

Maternal age 20 or 35 years

59

Maternal diabetes mellitus

60

Maternal health

59

Maternal obesity

61, 62

Maternal weight reduction (early

63

pregnancy) Maternal zinc deficiency

64, 65

Nitrates, nitrites and magnesium

66

salts in foods Oral contraceptives

67

Organic solvents

68

Parity

26

Paternal age

69

Pesticides

70

Potato blight

71

Previous spontaneous abortions/

72, 73

stillbirth Season, epidemics

74

Social class/poverty/illegitimacy

75

Subfertility

76

Tea drinking

77

Thalidomide

78

Twinning

79, 80

Valproic acid

81, 82

Vitamin A

83

Vitamin B12 deficiency

84

Warfarin (coumadin)

85

Zinc deficiency (newborn)

86

Note: Additional references in previous edition 2004.333

females, a higher twin concordance rate, an increased risk in siblings, a declining risk in other family members with increasing genetic distance, and the altered sex ratio in transmitting relatives.180 A cause is identifiable in 6–20 percent of NTDs9,181; most of those recognized are listed in Tables 23.2–23.4. NTDS occur in association with a wide range of numerical and structural chromosome abnormalities.182,183 In a series of 144 fetuses with open NTDS studied in Chile,184 the prevalence of chromsomal abnormality was 16 percent at or before 24 weeks, and 7 percent after that gestational age. Notwithstanding the likelihood that some of the NTD– chromosome defect associations are fortuitous, the concurrence of a NTD with any other malformation would make the offer of prenatal chromosome study mandatory. Earlier authors reported that even with an isolated NTD seen on ultrasound study, 2–6 percent had a chromosomal abnormality.185 The incidence of NTDs in liveborn infants with trisomy 18 is about 6.2 percent.186 The reason for the increased frequency of NTDs in trisomy 18 and triploidy is unknown. Given the occurrence of NTDs in other chromosomal disorders (see Table 23.4), karyotyping is recommended if any additional major fetal defect is evident or when dysmorphic features are noted. Various drugs have been implicated in the etiology of NTDs. Valproic acid187 (dose dependent82) and carbamazepine44 taken during the first 6 weeks of pregnancy have a 2–5 percent risk of causing SB.10,188 Clomiphene citrate to induce ovulation has been associated with NTDs by some authors but not by others.189 It is unclear whether the basic reasons for infertility are not more important. Questions remain about background causes in common, underlying the many apparent associations listed in Table 23.2. The major environmental maternal factors recognized include folic acid deficiency, diabetes mellitus, obesity, hyperthermia, maternal alcohol ingestion, anticonvulsant treatment, and zinc deficiency. Earlier evidence pointed to about a twofold increased risk of NTD in the offspring of obese women.190,191 A meta-analysis of evidence on the relationship between maternal obesity and NTDs concluded that the unadjusted odds ratios (OR) for an affected fetus were 1.22, 1.7 and 3.1 respectively among overweight (not significant), obese

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Genetic Disorders and the Fetus

Table 23.3 Syndromes in which neural tube defects (NTDs) may be a feature Syndrome

Type of

Additional selected clinical features

NTD Acrocallosal88

A

Agenesis corpus callosum; mental retardation; polydactyly

Acromelic frontonasal “dysplasia”89

E

Agenesis corpus callosum; Dandy–Walker malformation;

Amniotic bands/early amnion rupture90

E

Clefting; limb defects Clefting; eye and ear abnormalities

polydactyly; mental retardation Anophthalmia-clefting-neural tube defects91

SB

Anterior encephalocele92

E

Hydrocephalus; eye anomalies

Apert – acrocephalosyndactyly type I93

E

Mental retardation; craniosynostosis; agenesis corpus callosum

Boomerang dysplasia94

E

Short limb dwarfism; omphalocele; ossification defect

Brachydactyly type C95

A

Short stature; brachydactyly; phalangeal anomalies

Carpenter–Hunter96

E

Micromelia; polysyndactyly; fragile bones

Caudal duplication97

SB

Genitourinary and gastrointestinal anomalies

Caudal regression98

SB

Sacral, genitourinary, and anorectal anomalies Mental retardation; craniosynostosis; eye and nasal anomalies

Cerebro-oculonasal99

E

CHILD100

SB

Limb defects; hemidysplasia; ichthyosis

Cleft lip or palate101

SB

Clefting; fusion of eyelids; anal atresia/stenosis

Craniomicromelic syndrome102

E

Craniosynostosis; short limbs; IUGR

Craniotelencephalic dysplasia103

E

Craniosynostosis; agenesis corpus callosum; mental retardation;

Cranium bifidum with neural tube defects104

E, SB

Skull ossification defect; mental retardation; Arnold–Chiari

Currarino triad105

SB

Anorectal and sacral anomalies; urinary reflux

Czeizel106

SB

Split hands and feet; obstructive urinary anomalies;

DiGeorge10717

SB

Mental retardation; immune deficiency; hypoparathyroidism;

Disorganization-like108

A, SB

Tail-like protrusion; accessory limbs; hemangiomas

DK – phocomelia109

E

Radial defect; esophageal atresia; heart defect; anal anomaly;

Donnai/Meckel-like110

E

Cerebellar abnormalities; renal cysts; polydactyly

Durkin–Stamm111

SB

Sacral teratomas; asymmetric lower limbs; lymphomas/

Encephalocele–arthrogryposis – hypoplastic

E

Arthrogryposis; hypoplastic thumbs; normal intelligence; renal

Femoral duplication113

SB

Duplicated femur; imperforate anus; ambiguous genitalia;

Fried114

E

Microcephaly; cleft lip; hypoplastic/absent radii

Frontofacionasal dysplasia115

E

Clefting; mental retardation; coloboma; eye and nasal

Frontonasal dysplasia116

E

Microcephaly; clefting; eye and nasal anomalies

microcephaly malformation

diaphragmatic defect conotruncal cardiac defect

thrombocytopenia

leukemias thumbs112

dysplasia omphalocele

anomalies Fullana117

SB

Caudal deficiency; agenesis corpus callosum; polyasplenia

Gershoni–Baruch118

E

Diaphragmatic agenesis; omphalocele; multiple midline and

Gillessen–Kaesbach119

SB

Microencephaly; polycystic kidneys; brachymelia; heart defects

radial ray defects Goldberg120

SB

Sacral hemangiomas; genitourinary and anorectal anomalies

Goldenhar121

E

Facial, ear, and vertebral anomalies; epibulbar dermoid;

Gollop122

SB

Ectrodactyly; split femur; hydronephrosis

Gonadal agenesis and multiple dysraphic

E, SB

XX-agonadism, omphalocele

mental retardation

lesions123

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Table 23.3 Continued Syndrome

Type of

Additional selected clinical features

NTD Hartsfield124

E

Hegde125

E

Aplasia pectoralis major; limb and renal anomalies

3 H126

SB

Hemihypertrophy; hemihypesthesia; hemiareflexia; scoliosis

Hydrolethalus127

E

Hydrocephalus; osteochondrodysplasia; clefting; limb defects

Ivemark128

E, SB

Asplenia/polysplenia; heart defect; situs inversus

Joubert129

E

Dandy–Walker malformation; microphthalmia; cerebellar

Keutel130

E

Humeroradial synostosis; mental retardation; microcephaly Cervical vertebrae fusion; heart and renal anomalies; deafness

Clefting; holoprosencephaly; ectrodactyly; craniosynostosis

hypoplasia, clefting, retinal dystrophy, renal anomalies Klippel–Feil131

SB

Knobloch–Layer132

E

Detached retina; dextrocardia; scalp defects

Kousseff133

SB

Conotruncal heart defects; sacral and renal anomalies

Lateral meningocele syndrome134

SB

Multiple lateral meningoceles, joint laxity, dysmorphic,

Lehman135

SB

Osteosclerosis; vertebral defects

Lethal branchio-oculofacial136

E

Branchial cleft sinuses; eye and ear anomalies;

Limb/pelvis-hypoplasia/aplasia137

E, SB

Limb deficiency; thoracic dystrophy; pathologic fractures;

Lipomyelomeningocele – familial138

SB

Sacral and vertebral anomalies

Machin139

E

Hydrops; tracheal/laryngeal anomalies/ ear and renal

Marfan140

SB

Aortic, skeletal and ocular abnormalities

Mathias141

SB

Situs inversus; cardiac and splenic anomalies

Meckel–Gruber142

E

Mental retardation; polycystic kidneys; polydactyly

osteosclerosis

holoprosencephaly clefting; normal intelligence

anomalies

Medeira143

A, SB

Clefting; limb reduction; heart defect

Melanocytosis144

SB

Skin hyperpigmentation

Meroanencephaly145

A, E

Skull ossification defects; microcephaly

Morning glory146

E

Clefting; coloboma; optic nerve anomalies

Ochoa147

SB

Hydronephrosis; genitourinary and facial anomalies

Oculocerebrocutaneous148

E

Orbital/cerebral cysts; skin tags; focal dermal effects; mental

Oculoencephalohepatorenal149

E

Mental retardation; ataxia; eye, cerebellar, liver, and kidney

OEIS150

SB

Omphalocele; bladder exstrophy; imperforate anus; spinal

Oral-facial-digital type II151

E

Clefting; deafness; polydactyly; mental retardation

Pallister–Hall152

E

Hypothalamic hamartoblastoma; polydactyly; imperforate

Patel153

SB

Renal agenesis; absent müllerian structures; heart defect

Pentalogy of Cantrell154–156

SB, CR,

Omphalocele, ectopia cordis

retardation anomalies defects

anus

EX Phaver157

SB

Limb pterygia; heart, vertebral, ear, and radial defects

Porphyria, homozygous acute

E

Neurovisceral dysfunction; mental retardation; skin

Renal-hepatic-pancreatic dysplasia159

E

Dandy–Walker malformation; dysplastic kidneys; hepatic

Radial ray anomalies160

A

intermittent158

photosensitivity fibrosis

Roberts161

E

Limb reduction; mental retardation; clefting; eye defects

Rogers162

SB

Anophthalmia/microphthalmia

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Genetic Disorders and the Fetus

Table 23.3 Continued Syndrome

Type of

Additional selected clinical features

NTD Rolland–Desbuquois163

E

Short-limbed dwarfism; vertebral segmentation defects;

Sacral agenesis164

SB

Sacral and vertebral defects

clefting Sacral defects (anterior)165

SB

Absent sacrum; sacral teratoma/tumor

Schisis association166

A, E,

Clefting; omphalocele; diaphragmatic hernia; hypospadias

Short rib-polydactyly type II167

A

Structural brain anomalies; polydactyly; clefting; short ribs

Silverman163

E

Short-limbed dwarfism; clefting; vertebral segmentation

Sirenomelia168

A, SB

Clefting; vertebral segmentation defects; midline anomalies

Spear–Mickle169

SB

Scalp defect; craniostenosis

Tandon170

E

Clefting; colobomas; anogenital and skeletal anomalies

Tactocerebellar dysraphia171

E

Structural cerebellar anomalies; clefting; heart defect

SB

defects

Thoracoabdominal enteric duplication172

SB

Enteric duplication; skeletal anomalies; dextrocardia

Thrombocytopenia – absent radius173

SB

Thrombocytopenia; radial ray defects; heart defects; mental

Velocardiofacial174

SB

Conotruncal heart defects; mental retardation; clefting

Waardenburg37,38

SB

White forelock; deafness; heterochromia iridis; dystopia

Warburg129

E

Hydrocephalus; agyria; eye anomalies; clefting; Dandy–Walker

Weissenbacher-Zweymuller175

E

Skeletal dysplasia; clefting

X-linked neural tube defects176

A, SB

Isolated NTDs

Zimmer177

A

Tetra-amelia; midline anomalies

retardation

canthorum malformation

A, anencephaly; E, encephalocele; SB, spina bifida; CR, craniorachischisis; EX, exencephaly.

and severely obese women.192 Our studies193 and others62 implicate the prediabetic or hyperinsulinemic state, not excluded by most authors. Even increasing prepregnancy maternal weight, independent of folate intake, appears associated with a rising risk of NTD.61 Folic acid and etiology For more than five decades, data4,194 have pointed to the likely key role of folic acid deficiency in the pathogenesis of NTDs.51 In pregnancies with fetal NTDs, levels of serum and red blood cell folic acid and serum vitamin B12 have, on average, been reported to be lower than in pregnancies with normal fetuses.195 Red cell folate is regarded as the better proxy for folate status, given that it reflects the folate turnover during the previous 120 days.84 Daly et al.39 first showed that the risk of NTD is associated with red

cell folate levels in a continuous dose–response relationship (Figure 23.2). Table 23.5 displays the distribution of red cell folate in cases and controls for the risk of NTD in each category. More than an eightfold difference in risk was observed between those with the lowest and those with the highest red cell folate levels. Graphically, Daly et al. demonstrated that the risk of NTD is reduced as red cell folate levels increase well past the point at which levels would have been considered normal. Folic acid is required for methylation reactions in cells, directly involving the biosynthesis of methionine and nucleotides and indirectly influencing the methylation of proteins, DNA, and lipids. When folic acid levels are low, the levels of the precursor of methionine, homocysteine, are high. The discovery that high levels of homocysteine were associated with pregnancies affected by fetal NTDs led researchers to investigate the

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Table 23.4 Examples of neural tube defects associated with chromosomal disorders Chromosomal disorder

Reference

Numerical defects

Chromosomal disorder

Reference

22q11.2 deletion

254a

Trisomy 2

235a, 182

Xp22.1 deletion

255a

Trisomy 7

182

Ring chromosome 13

256a

Trisomy 8

182

Inversion Xq

240

Trisomy 9

236a

Trisomy 13

237a, 238

Trisomy 14

182

Trisomy 15

182

Trisomy 18

237a, 238

Trisomy 16

182

Trisomy 21

230

Triploidy

239a, 240

Trisomy 9 mosaic

241a

Trisomy 11 mosaic

182

Trisomy 20 mosaic

182

Monosomy X

182

Tetroploidy

182

Structural defects

Balanced translocation t(13q14q)

240

2q deletion

183

1p deletion

183

1q duplication

183

3p deletion

183

4p duplication

183

4q deletion

183

5p deletion

183

6p deletion

183

6q duplication

183

6q deletion

183

7p duplication

183

8q duplication

183

1q42–qter deletion

242a

9p deletion

183

2p23–pter duplication

243a, 244a, 245a

10q deletion

183

2q paracentric inversions and insertion

246a

11q deletion

183

3p duplication

247a

12p duplication

183

3q27-3qter deletion

248a

14q duplication

183

4p deletion

237a

14q deletion

183

7q32 deletion

237a

15q deletion

183

11q duplication

249a

18q deletion

183

13q13 deletion

237a

Ring 18

183

13q22 or 31–qter deletion

250a

20 duplication

183

13q33–34 deletion

251a

Isochromosome 20p

183

15q duplication

252a

Xq duplication

183

16q12.1-q22.1 duplication

253a

enzymes central to the interactions between the folic acid and methionine pathways. Allelic variants in a number of these enzymes, in particular 5,10-methylenetetrahydrofolate reductase (MTHFR), have been found to be associated with an increased incidence of NTDs. A common mutation (677CRT) in the MTHFR gene, which results in reduced enzyme activity and impaired homocysteine/folate metabolism, has been found in 5–16 percent of Caucasian cohorts studied and in 2 percent of Japanese.40 This mutation causes mild hyperhomocysteinemia196 and an increased risk of NTDs,39,41 which these authors further demonstrated in a meta-analysis.40 This

mutation is thought to explain a substantial part of the elevated plasma homocysteine levels in mothers of children with NTDs. Individuals who are homozygous for the 677CRT mutation in the MTHFR gene may have higher nutritional folate requirements.197 However, a French study noted no significant difference in the distribution of this mutation in prenatally diagnosed NTDs and controls.198 Although folic acid supplementation clearly reduces the risk of NTDs, the biologic mechanisms remain unclear. Low concentrations of cobalamin and folate in early-pregnancy plasma are regarded as independent risk factors for NTDs.84 Adams

712

Genetic Disorders and the Fetus

Figure 23.1 Decline in the prevalence of anencephaly and open spina bifida births in England and Wales, 1965–

2000. (Courtesy of NJ Wald, Wolfson Institute of Preventive Medicine, University of London, London, UK.)

Figure 23.2 The relationship of red cell folate levels to the risk of neural tube defect (NTD). The solid line shows the predicted risk using logistic regression. The dotted line shows the constant risk assumed beyond a red cell folate level of 1,292 nmol/L (570 ng/mL). The data points and error bars represent the observed risks and their 95 percent confidence intervals at the mean levels of red cell folate. The logistic regression equation is NTD odds5exp (1.646321.21933In[RCF]), where In[RCF] is the natural log of red cell folate measured in nanomoles per liter. If red cell folate is measured in nanograms per milliliter, the constant in the equation becomes 0.6489, but the coefficient of 1.2193 remains the same. (Reproduced with permission from Daly et al.39)

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Maternal Serum Screening for Neural Tube and Other Defects 713

Table 23.5 The distribution of cases and controls and the risk of NTD by red cell folate level Red cell folate

No. of

No. of

Risk of NTD per

95% confidence

(nmol/L (ng/mL))

cases (%)

controls (%)

1,000 births

interval

0–339 (0–149)

11 (13.1)

10 (3.8)

6.6

3.3 to 11.7

340–452 (150–199)

13 (15.5)

24 (9.0)

3.2

1.7 to 5.5

453–679 (200–299)

29 (34.5)

75 (28.2)

2.3

1.6 to 3.3

6880–905 (300–399)

20 (23.8)

77 (29.0)

1.6

1.0 to 2.4

>906 (400)

11 (13.1)

80 (30.0)

0.8

0.4 to 1.5

Source: Daly et al.39

et al.199 reviewed conflicting reports claiming abnormalities in folate metabolism by some authors without confirmation by others in such pregnancies and the fact that women with folate deficiency have not had an increased risk of having offspring with NTDs. They also reviewed evidence of both normal and abnormal cobalamin metabolism in pregnancies with NTDs. They observed increased concentrations of methylmalonic acid, a finding that would be consistent with reports of low plasma cobalamin levels in pregnancies with NTDs. The fact that differences in serum methylmalonic acid, but not cobalamin, were found apparently relates to the fact that methylmalonic acid is a more sensitive measure of cobalamin status than is serum cobalamin.200 Although some studies have noted no difference in mean methylmalonic acid levels in pregnancies with NTDs, the increased plasma homocysteine concentrations observed201 could possibly reflect folate and/or cobalamin deficiencies as well as abnormalities of enzymes required in folate metabolism. Indeed, nonpregnant women with a previous child with an NTD were noted to have elevated serum homocysteine levels after methionine loading. Low activity in either methionine synthase or MTHFR was commonly noted in this group.202 Certainly a defect in methionine synthase could account for the metabolic abnormalities that lead to NTDs, and such a defect might be compensated for by increased intake of folic acid and cobalamin. Steegers-Theunissen et al.202 noted that their abnormal results on methionine loading tests among mothers with methionine synthase abnormalities were corrected by folic acid supplementation. Other evidence implicating abnormalities in

cobalamin metabolism include animal studies showing that increased cobalamin administration may be associated with increased methionine synthase activity in rats203 and that an increased incidence of SB and congenital hydrocephalus was noted in a cobalamin-deficient rat model.204 Adams et al.199 speculated that there is a convergence of evidence of abnormalities in folate and cobalamin metabolism leading to NTDs, suggesting that the cause may be related to methylation. They argued that both folate and cobalamin are involved in critical metabolic pathways providing methyl groups for the methylation of DNA – an essential process in embryogenesis. Oakley205 estimated that worldwide 220,000 children are born with “folic acid preventable SB”. Ray et al.206 presented evidence that serum holotranscobalamin, a sensitive indicator of vitamin B12 status, was low in midtrimester pregnancies with NTDs. Close to a tripling in the risk of a NTD was noted among mothers with low B12 status. Important insights into human NTDs have been gained from animal studies, particularly from reports on more than 10 mouse mutants exhibiting various types of NTDs. The curly-tail mouse has been studied most extensively, the NTDs most closely resembling the human defects in location and form as well as the mode of multifactorial inheritance. Interestingly, the formation of NTDs in curly-tail is not reduced after folate supplementation, nor is the MTHFR gene defective, although plasma homocysteine levels are higher in the mutant than in control C57BL/6 mice.207 Another interesting insight into the mechanisms involved in NTD formation is provided by the curly-tail: the administration of inositol can prevent NTDs in

714

Genetic Disorders and the Fetus

this mutant.208 The mechanism is thought to involve the retinoic acid β receptor found in hindgut endoderm. Emerging from these studies has been a closer understanding of the different mechanisms leading to failures of neural tube closure. For example, in the splotch mouse, abnormal migration of neural crest cells is the fault,209 while in the curly-tail mice, a low proliferation rate of the cells of the gut and notochord in the region of the posterior neuropore produces a ventral curvature apparently preventing neuropore closure, with resulting SB.210 Homozygous splotch mutations have decreased folate available for pyrimidine biosynthesis; supplementation with folic acid will reduce NTDs in the mutant embryos by 40 percent.211 Mutant mice homozygous for the Cart 1 homeobox gene mutation are born with acrania and meroanencephaly – a phenotype with striking resemblance to human NTDs.212 Cart 1 is a transcription factor that regulates downstream target genes and is required for forebrain mesenchyme survival. Its absence disrupts cranial neural tube morphogenesis by blocking the initiation of closure in the midbrain region. Prenatal treatment with folic acid suppressed the acrania/meroanencephaly phenotype.212 Another knockout mouse mutant with a disrupted AP-2 gene exhibits anencephaly, craniofacial defects, and thoracoabdominoschisis.213 The gene-targeted mouse mutant, F52, was fortuitously observed to manifest severe NTDs not associated with other complex malformations.214 This generated mouse mutant identifies a gene whose mutation results in isolated NTDs and is expected to be a valuable experimental model. Evidence from both mouse and human studies clearly suggests that more than one gene is involved in neural tube closure. In addition to genetic evidence already discussed, marked sex differences are recognized according to lesion sites – low sacral lesions are more common among males than females, while the opposite is true for thoracic SB and anencephaly.6,215 The postulate that there is a continuous, bidirectional “zipperlike” process responsible for the pattern of neural tube closure in humans216 has been superseded by data indicating that, as in the mouse, multiple sites of anterior neural tube closure occur.217,218 The co-existence of three NTDs (encephalocele, cervical myelomeningocele, thora-

columbar myelomeningocele) in a child further supports multisite closure of the neural tube.156 Golden and Chernoff218 provided evidence for two mechanisms leading to anterior NTDs: one results from the failure of a closure and the second results from the failure of the two closures to meet. These observations provide additional insight into variations observed in the location, recurrence risk, and causes of anterior NTDs in humans. Mouse models may also contribute important information on different mechanisms for NTDs at different locations.219,219a Folate receptor genes that control maternal– fetal transplacental folate transport have been recognized.220 Their expression and regulation influence cell proliferation. Thus far, however, there has been no association shown between folate receptor gene defects and NTDs.221 Efforts to determine a wide range of other possible associated polymorphisms have not yet identified a molecular signature clearly signaling an increased risk of NTDs. For the vast majority of NTDs, environmental– genetic interaction is causal and most probably depends on dietary folate and susceptibility genes involved in homocysteine, folate, and cobalamin metabolism. The hypothesis that folic acid may have a weak abortifacient effect, accounting for selective loss of affected fetuses,222 has been countered by the suggestion that it maintains pregnancies that would otherwise abort early enough so as not to even be recognized.223 For decades, multiple reports,53,224 in many mammalian species, have associated hyperthermia with NTDs. Inadequate study designs yielded data in humans that were regarded as inconclusive and tenuous. In a very carefully designed, large, broadbased, prospective study, we examined heat exposure (hot tub, sauna, electric blanket) in the first 8 weeks and fever in the first 12 weeks of pregnancy.53 We found that women who used the hot tub in the first 8 weeks of pregnancy had a significant, 2.9 (95 percent confidence interval (CI) 1.4–6.3) relative risk of having a child with an NTD. Use of a sauna was associated with a 2.6 (95 percent CI, 0.7–10.1) relative risk, a finding that was not statistically significant. No association was noted for use of an electric blanket. When the number of heat sources was tallied (excluding electric blanket), risk esca-

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lated with additional sources of exposure. (No data were obtained on the duration, intensity or frequency of these heat exposures.) After exposure to two heat sources, compared with none, the relative risk of having a child with an NTD rose to 6.2 (95 percent CI 2.2–17.2). Others have also observed an increased relative risk of NTD with fever but were unable to disentangle the confounders of infection and medication.225 The mechanism by which heat exposure interferes with neural tube closure is unknown. In mice, however, hyperthermia induces apoptosis by activating the mitochondrial apoptotic pathway.226 Moreover the p53 and p21 genes function to suppress heat-induced malformations.226 Analysis of the same database also focused on analysis of trace elements derived from midtrimester-collected maternal toenail clippings. We observed a clear association between elevated toenail zinc (Zn) and a fetus with a NTD.65 Moreover, a linear trend was evident, relative risk increasing with escalating Zn levels. Our observations reconcile with those showing elevated hair Zn levels in mothers of children with SB.227 It is not known whether Zn sequestration in nails or hair leaves the fetus relatively Zn deficient or that these findings reflect a more fundamental disturbance in Zn metabolism in these mothers. Zn at physiologic concentrations enters both placental syncytiotrophoblast cells and fetal vascular endothelial cells from both protein-bound and nonprotein-bound serum pools.228 In an Indian study Zn deficiency was observed in the serum of newborns with NTDs.229 Given the extensive involvement of Zn in cellular metabolism, a role for this trace element in cells migrating to close the neural tube is highly likely.

Is there a link between neural tube defects and Down syndrome? Some data have linked abnormal folate metabolism with an increased risk of Down syndrome, possibly through an increase in the rate of nondisjunction. This raises the possibility that both Down syndrome and NTD prevalence can benefit from folic acid supplementation. In 1999, a study indicated that increased rate of MTHFR polymor-

phism might be associated with an increased risk of Down syndrome.229a Others have subsequently provided more evidence for that association.230–232 Concurrence of Down syndrome and NTDs in mothers homozygous for the 677TT methylenetetrahydrofolate reductase gene mutation has also been reported.230,233 All the reports234 pointing towards a link between NTDs and Down syndrome are intriguing, but lack the required rigor of epidemologic research.

Biology of α-fetoprotein Human α-fetoprotein (AFP) was recognized as a fetal-specific globulin in 1956,237 and many of its physical and chemical properties have been defined.238,239 Monoclonal antibodies have facilitated purification by immunochromatography. AFP is similar to albumin in molecular weight (about 69,000) and charge238 but has a different primary structure and is antigenically quite distinct.240 The primary structure of AFP is known,240 and the gene on chromosome 4q has been cloned.241 AFP is a glycoprotein and exists in several forms, or isoproteins, with different net charges.238 α-Fetoprotein is synthesized by the yolk sac, the gastrointestinal tract, and the liver of the fetus and is detectable as early as 29 days after conception.242 Both the kidneys and the placenta may produce trace amounts of AFP242 but the fetal liver dominates AFP synthesis. The level of fetal plasma AFP peaks between 10 and 13 weeks of gestation, reaching about 3,000 µg/mL.243 The fetal plasma concentration of AFP declines exponentially from 14 to 32 weeks and then more sharply until term (Figure 23.3). At 32 weeks of gestation, the plasma AFP concentration is about 200 µg/mL. The exponential fall in fetal plasma AFP can be attributed mainly to the dilution effect due to increasing fetal blood volume and the related decline in the amount synthesized by the fetus.244 Synthesis of AFP decreases markedly after 32–34 weeks of gestation. α-Fetoprotein in the fetal plasma enters the fetal urine and from there the amniotic fluid (AF).243 In contrast with other proteins, the primary source of AFAFP appears to be fetal urine,245 where the concentration is higher in AF in early but not in late pregnancy. Peak levels of AFAFP are reached

716

Genetic Disorders and the Fetus

Figure 23.3 Changes in AFP levels in fetal serum, amniotic fluid and maternal serum during gestation. The AFP values (µg/mL) are plotted on the logarithmic y-axis versus completed week of gestation. The levels of AFP in fetal serum, amniotic fluid and maternal serum are

shown by the dotted, dashed and solid curves, respectively. The region enclosed in the rectangle indicates the gestational age range of 15 to 20 weeks. (Figure adapted from Seppala M, Ann N Y Acad Sci, 1974:259:59).

between 12 and 14 weeks of gestation246,247 and then decline by about 13 percent per week during the second trimester,248 being almost nondetectable at term246,247 (see Figure 23.3). The concentration gradient between fetal plasma and AFAFP is about 150–200 : 1,243,244 and the pattern of AFP levels in the two fetal compartments as a function of gestation is similar. The concentration gradient between fetal and maternal serum is about 50,000 : 1.249 Hence, the presence of only a tiny volume of fetal blood contaminating the AF will raise both AF and maternal serum AFP levels, potentially yielding a spurious result. The AFP concentration in maternal serum or plasma during pregnancy rises above nonpregnancy levels as early as the 7th week of gesta-

tion.250 Maternal serum (MS) AFP levels are very much lower than AFAFP levels (see Figure 23.3). The peak level of MSAFP during pregnancy occurs between 28 and 32 weeks of gestation.251 The apparent paradoxic rise in MSAFP when AF and fetal serum levels are decreasing can be accounted for by an increasing placental permeability to fetal plasma protein with advancing gestation252 and to increasing fetal mass and AF volume relative to a constant maternal blood volume. Transport of AFAFP contributes very little to the MSAFP compartment.253 Hay et al.251 noted that higher birthweights were correlated with later attainment of peak MSAFP levels and that peak levels occurred earlier in pregnancy when the fetus was female.

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Maternal Serum Screening for Neural Tube and Other Defects 717

Newborn plasma AFP levels normally decline rapidly, with an average half-life of 5.5 days in the first 2 weeks, reaching adult levels of 1–2 ng/mL by 8 months of age.254 AFP synthesis does not cease entirely after birth, although the concentrations in adult plasma are extremely low,255 about 20,000 times lower than the concentrations found at birth.244 The function of AFP in the fetus remains unknown.235 Because AFP has chemical and physical characteristics similar to those of albumin, it may have an osmotic role in maintaining the intravascular volume of the fetal circulation. Although AFP in the rat and mouse binds estrogens and ureterotropic activity has been described,256 such function in the human seems unlikely.257 Current theory is that AFP is most probably involved in immunoregulation during pregnancy.258 The immune response in the mouse is suppressed by AFP.259 Human lymphocyte transformation induced by mitogens such as phytohemagglutinin is also suppressed by human AFP.260 Immunofluorescence studies have pointed to the presence of AFP receptors on the surface of some T cell lymphocytes in mice. These and other data have therefore formed the basis of a suggestion that human AFP may prevent or be involved in the prevention of the immune rejection of the fetus by the mother. Its possible functions notwithstanding, the presence of AFP during pregnancy does not appear to be necessary for the maintenance of the pregnancy or the well-being of the fetus. Severe AFP deficiency has been reported in two newborns, with no ill effects to the pregnancy or the baby.261 In those cases, the second-trimester AFAFP levels were both less than 0.5 ng/mL. In another case, a maternal serum AFP of 0.00 multiples of the median (MoM) was reported on triple marker screening, and was associated with a finding of trisomy 21.262 The authors cautioned that although congenital absence of AFP is not thought to be associated with adverse obstetric or fetal outcome, very low or undetectable MSAFP levels should still be considered in screening for Down syndrome. In a report of almost 840,000 pregnancies undergoing routine prenatal screening, undetectable levels of maternal serum AFP (defined as 7

MSAFP levels (MoM) oligo

vwd

other

ntd

Figure 23.5 The distribution of anomalies and oligohydramnios as a function of elevated maternal serum AFP (MSAFP). Oligo, oligohydramnios; other, subchorionic bleeding, intra-abdominal echogenicity,

hydronephrosis, echogenic bowel, dilated kidney, heart defect; VWD, ventral wall defect. (Reproduced with permission from Reichler et al.280)

fetal/placental defects. If the pregnancy stage provided by the patient’s menstrual dates is confirmed by the ultrasound study and no defects have been visualized, genetic counseling to discuss the diagnostic options available should be offered as soon as possible. Although an amniocentesis was the primary diagnostic recommendation for many years, more recently patients have been given the option of targeted (level II) ultrasound as an alternative. However, women should be fully informed of the full sequence of possible testing. In our experience, even the very best ultrasonographers may miss SB, especially in the L5–S2 region, making amniocentesis a firm recommendation if the ultrasound is negative and the high MSAFP remains unexplained.281,282 The relative performance of ultrasound and biochemistry is discussed below (see also Chapters 24 and 25). Most laboratories use an upper-limit cut-off of the normal range equivalent to 2.0 or 2.5 MoM. Clearly, the selection of a specific cut-off level

reflects a compromise between missing open NTDs and performing unnecessary amniocenteses or targeted ultrasound scans on pregnancies with normal fetuses. The actual odds that a woman will carry a fetus with open SB have been calculated as a function of the level of MSAFP (see Table 23.6). These odds relate to pregnancies with a single fetus; note that a correction in the gestational age through the use of ultrasound would change the odds that a fetus will be affected. In practice, it is becoming increasingly difficult to calculate the correct patient-specific odds of having a fetus affected with an open NTD. The reason for this is the increase in each person’s folic acid intake in countries that require fortification of flour with folic acid, and with the markedly increased use of preconceptional and prenatal multivitamins in women. This makes the correct assignment of an a priori risk for use in calculating a woman’s odds of an open NTD after screening with MSAFP uncertain. Racial differences in a

722

Genetic Disorders and the Fetus

priori risk may still hold, but those associations also need to be revisited. The use of a fixed MSAFP MoM cut-off in screening for open NTDs obviates the problem of odds calculation, with the understanding that as the MSAFP value increases, the woman’s relative risk also increases. Assay considerations Almost all laboratories use two-site enzyme-linked immunometric assays to measure MSAFP and AFAFP, and the measurement of AFP from dried blood specimens using such assays has been successful.283 In recent years there has been a major shift from assays done manually to assays on automated platforms, having rapid throughput and exhibiting improved precision. This improvement in assay methods has led to a reappraisal of the distribution characteristics of MSAFP and its performance in screening for open NTDs. It is now estimated that when more precise AFP assays are used, the false-positive rate in screening for anencephaly and open SB at a fixed 2.5 MoM cut-off is reduced from 2.0 to 0.8 percent and the detection rate is increased from 81 to 83 percent.284 All laboratories are expected to establish their own gestation-specific reference ranges for MSAFP and AFAFP. Continuous quality control is necessary in the performance of screening and diagnostic assays.285 Guidelines have been issued by the American College of Medical Genetics,286 the American College of Obstetricians and Gynecologists,287 and the Canadian Society of Clinical Chemists,288 and regulations have been promulgated by the College of American Pathologists. This college operates nationwide external proficiency testing in the United States, an essential element in population-based screening programs. In addition, some states restrict screening to only one or a few approved laboratories or require that their own proficiency testing program be followed. Lessons learned from the debacle of uncontrolled newborn screening for phenylketonuria led to this important approach. Factors that influence the interpretation of MSAFP The distribution of MSAFP values in a population of pregnant women is governed by a range of factors. Wald has estimated the impact of many of

these factors on the variance of MSAFP at any given point in gestation.289 He has found that about half the variance is caused by differences between the pregnancies themselves. Another 17 percent is caused by fluctuations in MSAFP within a pregnancy, and about 14 percent is due to assay error. Half of the remaining 18 percent is caused by errors in dating the pregnancy, which leaves less than 10 percent contributed by other factors, such as maternal weight, maternal race, and diabetic status.

Gestational age Accurate assessment of gestational age and the method of dating have a major impact on MSAFP screening. Optimal detection rates are between 16 and 18 weeks, and screening is best confined to 15–20 weeks. Assignment of gestational age is most commonly done by sonographic measurement of the fetus or by self-reporting by the patient of the first day of her last menstrual period (LMP). The most commonly applied ultrasound dating methods are first-trimester crown-to-rump length (CRL), usually measured beginning at about 7 weeks, and second-trimester measurement of biparietal diameter (BPD), femur length, abdominal circumference or a composite of the second-trimester measures. Gestational dating by ultrasound measurement has been shown to improve the performance of screening for NTDs for two reasons. First, ultrasound estimation of gestational age is, on a population basis, more accurate than a woman’s recollection of her LMP.290–292 Certainly, many women have regular menstrual cycles and have very accurate recall of their LMP, but as many as 40 percent of LMP dates are unreliable,290,293 leading to a broadened distribution of AFP MoM values. This difference alone will lead to better screening performance, because the distributions of AFP MoM values for a screened population will be tighter when the population is dated by ultrasound than when it is dated by LMP. Second, ultrasound dating by second-trimester BPD will markedly improve screening for open SB because it will tend to underestimate the gestation of an affected fetus, thus leading to a lower AFP median being used to calculate the AFP MoM. The result is to artificially increase the AFP MoM in

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Maternal Serum Screening for Neural Tube and Other Defects 723

affected pregnancies, enhancing the chances that such pregnancies will have an AFP MoM above the screening cut-off.294 The reason for a smaller BPD in open SB pregnancy is the occurrence in almost all cases of an Arnold–Chiari II malformation, involving movement of the cranial vault contents down the neural tube, leading to the formation of a smaller calvarium and consequently to a small BPD measurement. The average decrease in the apparent gestation in a pregnancy affected by SB and dated by BPD is 16 days, which leads to a marked improvement in performance, estimated to be 11 percentage points (79 percent in LMPdated pregnancies and 90 percent in BPD-dated pregnancies, both for a 5 percent false-positive rate).294

done properly, adjustment of the MoM value according to maternal weight will reduce the variability of the AFP MoM distribution.291 Maternal-weight MSAFP adjustment is also important because of the convincing finding of increased risk of NTDs in obese mothers.61,190,191,300– 302 Obese mothers have an approximately twofold increased risk compared with women of average weight. In addition, obese mothers have an increased risk of another birth defect identified because of elevated MSAFP, fetal omphalocele,190,300 although this is apparently not true for the other major ventral wall defect, gastroschisis.300 Without appropriate maternal weight adjustment of the MSAFP MoM, those with the highest risk would have the lowest average MoM values, and therefore the lowest screening performance.

Maternal weight Maternal serum α-fetoprotein concentrations vary according to maternal weight.295 The heavier the woman, the lower the MSAFP level, as a result of dilution of AFP in a larger blood volume. Adjustment of the MSAFP value for maternal weight increases the detection rate for open SB296 and is especially important when screening for chromosome defects (see also Chapter 24). Dividing the observed MoM by the expected MoM for a given weight enables adjustment for differences in weight. Adams et al.297 described a detailed method for computing risks based on MSAFP values and known variables. Correction for weight up to 250 lb significantly increased the rate of elevated MSAFP results, suggesting overcorrection.238 These authors recommended linear correction of MSAFP up to 200 lb only, with those weighing more being adjusted for this weight only. In contrast, Wald et al. found that an equation comparing maternal weight to the log of the MoM is valid to weights up to about 260 lb.291,298 Published weight equations may not be optimal for some screening programs because of differences in the mean weight of the population being tested. Neveux et al.,299 using a reciprocal-linear equation to describe the association between maternal weight and MoM value, recommended that screening laboratories calculate their own weight correction formulas, based on data from their own population, and monitor the mean maternal weight periodically, updating the formulas when necessary. It is clear that, when

Maternal age The frequency of NTDs has a U-shaped distribution for maternal age. However, no correction has been considered necessary for NTD screening.

Maternal ethnicity Black women have 10–15 percent higher MSAFP levels than nonblacks,29,303,304 requiring automatic adjustments in any formula used to interpret an MSAFP result. This step is especially important, given the lower incidence of NTDs among blacks.305 MSAFP levels are lower in Oriental women than in whites – 6 percent lower in one study.306 Thus far, these lower values have not been regarded as significant enough to require adjustments for MSAFP interpretation. Hispanic women have lower MSAFP values than Caucasians,29 suggesting that women at risk for NTDs might be missed when screening cut-offs for Caucasians are used. In a small study of 3,046 Hispanic and 15,154 Caucasian women, the percentage of Hispanic women with elevated MSAFP was not significantly different from that of Caucasians.29 Adjustment of medians for NTD screening was not recommended (see also Chapter 24). Ultimately, the use of ethnic group-specific medians should yield better risk assessment data.

Maternal insulin-dependent diabetes mellitus Maternal serum α-fetoprotein levels in pregnant women with insulin-dependent diabetes mellitus

724

Genetic Disorders and the Fetus

(IDDM) beginning at or before conception have been reported to be about 20 percent lower than in nondiabetic women during the second trimester,60,307,308 and, for the past 30 years, adjustments have been made in MSAFP interpretation, unless a normal range for IDDM has been established. There is increasing evidence, however, that this effect may no longer be found, most likely because pregnant women with IDDM are now kept in much tighter glycemic control than in years past.309–312 However, some recent studies continue to demonstrate low MSAFP levels in women with IDDM.313,314 Programs that are adjusting MSAFP levels in cases of IDDM should continue doing so until the evidence has been further confirmed and studied. Regardless of its effect on MSAFP levels, IDDM is associated with a much higher frequency of NTDs (10-fold in our study,60 3.5-fold in others315) in the offspring of these patients, which must be noted when the risk of IDDM after screening is calculated. The reason for lower MSAFP values in IDDM remains unclear. Relative intrauterine growth restriction in IDDM probably occurs consistently through the end of the second trimester, resulting in diminished synthesis, secretion, excretion or transport of AFP. First-trimester ultrasound studies in IDDM pregnancies showed that the fetuses were, on average, smaller than normal, with about one-third having even more marked fetal growth restriction.316 Given the twofold to fourfold increase in the frequency of all malformations in the offspring of women with IDDM,317 all such patients should undergo a level II ultrasound study. In one study of 393 women with IDDM who had both MSAFP and ultrasound, 32 had defects at delivery that were detected through both MSAFP screening and ultrasound.318 No malformations missed by ultrasound were detected by MSAFP screening. In this context, a salutary and surprising rate of fetal aneuploidy (13.2 percent) was reported among 53 pregnancies with NTDs,280 supporting AF chromosome study in all cases (see also Table 23.4.)

Multiple pregnancy A level II ultrasound study is recommended at around 16 weeks of gestation in all multiple preg-

nancies, given the increased frequency of fetal defects. The concentration of MSAFP is proportional to the number of fetuses.319 Hence, the upper-limit cut-off in twin pregnancy is twice that for singletons, usually 4.0 or 5.0 MoM. For six triplet and three quadruplet pregnancies, the average MSAFP level was, respectively, three and five times greater than in singleton pregnancies.320 In our prospective study,277 we noted that 53 percent of twin pregnancies had MSAFP values ≥2.0 MoM. Both we and others319 have noted an ominous prognosis in twin pregnancy when MSAFP levels exceeded 5 MoM for singleton pregnancies. Ghosh et al.319 observed that 59 percent of such cases ended in fetal death, stillbirth or a fetus papyraceous in one twin. In 11 of their cases in which twins were discordant for NTDs, the MSAFP value exceeded 5 MoM. One curious and currently unexplained observation is that MSAFP in monozygous twin pregnancies is higher than in dizygous twin pregnancies.321 Brock et al.322 found that in twin pregnancies, low MSAFP levels provided an early signal of growth restriction and high values warned of possible premature delivery. Redford and Whitfield323 found a 40 percent perinatal mortality in twin pregnancy when MSAFP values were ≥4.0 MoM. Cuckle et al.324 studied MSAFP in 46 discordant twin pregnancies with open NTD and 169 unaffected twins between 13 and 24 weeks of gestation. Using a 5.0 MoM cut-off level, their estimated detection rate was 83 percent for anencephaly but only 39 percent for open SB (Table 23.9). They calculated the odds of being affected by an open NTD for individual twin pregnancies (Table 23.10) according to the MSAFP level. For example, at an incidence rate of three SB infants per 1,000 births, an MSAFP level of 5.0 MoM yielded odds of 1 in 73. When MSAFP values are ≥5.0 MoM after sonography, we advocate amniocentesis for AFAFP and acetylcholinesterase (AChE) studies. Fetal sex and MSAFP Maternal serum α-fetoprotein values are, on average, higher when the fetus is male,326 and the effect is most pronounced at levels of 1.5 MoM and higher.289 However, adjustments of MSAFP values for fetal sex are unnecessary.

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Maternal Serum Screening for Neural Tube and Other Defects 725

Table 23.9 The detection rate, false-positive rate, and odds of being affected by anencephaly or open spina bifida, given a positive result (OAPR) for twin pregnancies, according to the level of AFP (assuming a singleton birth prevalence of 1 per 1,000 births for each defect, in the absence of antenatal diagnosis and selective abortion) AFP level (MoM)

Detection rate

OAPR

Anencephaly

Open spina bifida

False-positive rate

Anencephaly

Open spina bifida

>2.0

100%

96%

>2.5

99

89

30

1 : 200

1 : 210

1 : 130

>3.0

98

80

1 : 150

19

1 : 85

>3.5

96

69

1 : 100

12

1 : 55

>4.0

93

58

1 : 76

7.8

1 : 37

>4.5

89

1 : 59

48

5.0

1 : 25

>5.0

1 : 46

83

39

3.3

1 : 17

1 : 37

>5.5

77

31

2.2

1 : 13

1 : 31

>6.0

70

25

1.4

1 : 8.8

1 : 25

4%

Source: Cuckle et al.324 Note: Derived from Gaussian distribution of log10 AFP, using the means and standard deviations specified in Cuckle et al.343 MoM, multiple of the normal median for singletons at the same gestation and laboratory.

Raised MSAFP in sequential pregnancies Wald and Cuckle327 studied MSAFP in 1,717 women between 16 and 21 weeks of gestation in each of two pregnancies, neither with a fetus affected by an NTD. Women with high MSAFP values (≥2.5 MoM) in the first pregnancy had raised values in 6.8 percent of the second pregnancies. In contrast, among women with normal MSAFP values in the first pregnancy, only 1.9 percent had raised AFP in the second. Although this difference was statistically highly significant, the effect was too small to be of practical value. Maternal smoking No significant effect of smoking on MSAFP has been detected.328 MSAFP and adverse pregnancy outcome Maternal serum screening introduces a “noisy” system into physicians’ offices, requiring constant attention, response, documentation, and, most important, time expenditure to address the indisputable new anxieties. Data on MSAFP screening antedated multiple analyte use (see also Chapter 24) and provided additional insight in non-NTD pregnancies. Unexplained elevated MSAFP values

with normal AFAFP denote a probable breach in the integrity of the fetoplacental interface due to either or both placental or membrane pathology.329,330 In our prospective study of such cases, significant observations included undetectable major congenital defects (RR 4.7) excluding NTDs, fetal deaths (RR 8.1), neonatal death (RR 4.7), low birthweight (RR 4.0), oligohydramnios (RR 3.4), and abruptio placentae (RR 3.0). Either high or low MSAFP values (see Table 23.8) were found in 34.2 percent of all major congenital defects, 19.1 percent of all stillbirths or fetal/neonatal deaths, 15.9 percent of serious newborn complications, and 11.0 percent of major pregnancy complications. The probabilities of specific pregnancy outcomes related to MSAFP values are listed in Table 23.8. A summary of reports on MSAFP and adverse pregnancy outcome covering 225,000 pregnancies is shown in Table 23.11.331a Ample evidence of placental pathology (Table 23.12) is consistent with this hypothesis. There is a 20–58 percent likelihood of adverse pregnancy outcome in women with an unexplained raised MSAFP level331a,363,397 (see also Chapter 24 for multiple analyte screening). Hyperechogenic fetal bowel (see also Chapter 25) in association with an elevated MSAFP level is also associated with subsequent pregnancy complica-

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Genetic Disorders and the Fetus

Table 23.10 The odds of being affected by anencephaly or spina bifida for individual twin pregnancies, according to the level of AFP (assuming a singleton birth prevalence of 1 per 1,000 births for each defect in the absence of antenatal diagnosis and selective abortion) AFP level (MoM)

Anencephaly

Open spina bifida

2.0

1 : 48,000

1 : 1,700

2.2

1 : 23,000

1 : 1,200

2.4

1 : 12,000

1 : 830

2.6

1 : 6,900

1 : 620

2.8

1 : 4,100

1 : 470

3.0

1 : 2,600

1 : 370

3.2

1 : 1,700

1 : 300

3.4

1 : 1,100

1 : 240

3.6

1 : 760

1 : 200

3.8

1 : 540

1 : 170

4.0

1 : 390

1 : 140

4.2

1 : 290

1 : 120

4.4

1 : 220

1 : 110

4.6

1 : 170

1 : 93

4.8

1 : 130

1 : 82

5.0

1 : 100

1 : 73

5.2

1 : 82

1 : 65

5.4

1 : 66

1 : 58

5.6

1 : 54

1 : 53

5.8

1 : 44

1 : 48

6.0

1 : 37

1 : 44

Source: Cuckle et al.324

raised MSAFP. A number of major obstetric complications in association with elevated MSAFP are recognized (see Table 23.12) and some need further consideration. Fetal death In 1965, Tatarinov366 first noted high MSAFP after fetal death. We found a relative risk of 8.1 (95 percent CI 4.8–13.4)277 and others367 found a range of relative risk rising from 2.4 to 10.4 with escalating levels of MSAFP for fetal death. Threatened spontaneous abortion has also long been associated with elevated MSAFP levels.338 These authors showed that women with high MSAFP had a tenfold greater likelihood of miscarrying than those with normal values. In women with low MSAFP values, we found a relative risk of 3.3 for fetal death.277 Low birthweight There is a highly significant association between elevated MSAFP and subsequent low birthweight.368 We found a RR of 4.0 (95 percent CI 3.0–5.3) for this association,277 while others369 have pointed out that the risk of having a lowbirthweight infant increases as the MSAFP rises. Placental pathology is the likely reason for this association.

Note: Derived from Gaussian distributions of log10 AFP using the means and standard deviations specified in Cuckle et al.324 MoM, multiple of the normal median for singletons at the same gestation and laboratory.

tions, including intrauterine growth restriction and fetal/neonatal death.364 One study focused on 556 women with high MSAFP levels who had normal sonograms and amniocentesis results. The risk of adverse pregnancy outcome rose from 19 to 29 percent and up to 70 percent when their MSAFP levels were 2.5–2.9, 3.0–5.0 and >5.0 MoM, respectively.365 Especially worrisome are our observations333 and those of others329 showing that after an elevated MSAFP, normal AFAFP, no detectable AChE, and normal ultrasound, a risk approximating 4 percent may remain for the fetus having a serious congenital defect (e.g. hydrocephalus, cardiac defect, limb reduction defect). Associated placental pathology is the likely mechanism for the

Miscellaneous pregnancy complications Pathologic processes that interfere with placental integrity are likely to be associated with elevated MSAFP. It is no surprise, then, that we found statistically significant relative risks for specific pregnancy complications (see Tables 23.7 and 23.8). Fetal loss is very likely if oligohydramnios is associated with a raised MSAFP.87,332,370 Prudence would dictate continuing careful surveillance of all women with high MSAFP (after normal ultrasonographic and amniocentesis results), with special attention in the third trimester and to labor, delivery, and the neonatal period. Fetomaternal hemorrhage may occur without vaginal bleeding and may be signaled by a raised MSAFP level.346 Both transabdominal and transcervical CVS may cause fetomaternal bleeding (see also Chapter 5) with elevations in MSAFP, reported in one study in 18 percent and 5 percent, respectively.371 MSAFP levels were higher after

Table 23.11 Studies evaluating the relation of unexplained elevations of MSAFP and poor pregnancy outcome Authors331a

Brock et al.

Location (year)

Scotland (1977,

Pregnancies

MoM

LBW

IUGR

Premature

Abruption

IUFD

Perinatal

Pre-

screened

cutoff

risk

risk

delivery risk

risk

risk

death

eclampsia

15481

2.3

2.5x

–

–

–

+

+

–

3

4.7x

–

5.8x

–

3.5x

–

–

1979) Wald et al.

England (1977,

3194

1980) 4198

–

–

–

–

–

–

–

–

New York (1978)

6031

2

2x

–

–

–

–

–

–

Gordon et al.

England (1978)

1055

2

–

–

3.5x

–

4.5x

–

9.5x

Smith

England (1980)

1500

2

+

+

+

–

–

–

+

Evans and Stokes

Wales (1984)

2913

2

3x

–

–

+

–

8x

–

Burton et al.

North Carolina

42037

2.5

2x

–

–

–

8x

10x

–

10147

–

(1983, 1988) Persson et al.

Sweden (1983)

2.3

2.8x

–

2x

10x

–

3x

Haddow et al.

Maine (1983)

3636

2

3.6x

–

2x

–

–

–

–

Purdie et al.

Scotland (1983)

7223

2.5

2.5x

–

–

20x

–

–

–

Fuhrmann and

West Germany

50000

2.5

3.5x

–

–

–

8.6x

–

–

Weitzel

(1985)

Williamson et al.

Iowa (1986)

Robinson et al.

California (1989)

1161

–

PO

–

–

–

–

–

–

35787

2

3.5x

–

–

–

–

–

Ghosh et al.

Hong Kong

–

9838

2

+

–

–

–

–

–

Schnittger and

–

Sweden (1984)

18037

2

+

–

–

+

–

+

–

Kjessler Hamilton et al.

Scotland (1985)

10885

2.5

103

2x

>10x

3x

–

8x

–

Doran et al.

Ontario (1987)

8140

2

63

–

–

–

+

–

–

Milunsky et al.

Massachusetts

13486

2

43

–

–

3x

8x

+

2.3x

(1989) Benn et al.331b

Connecticut (2000)

50315

2

1.2x

3.1x

1.2

–

8.9x

+

–

Krause et al.331c

Denmark (2001)

77149

2.5

5.8

2.8x

4.8

–

+

1.9

–

Anfuso et al.331d

Italy (2007)

16747

2

+

5.9x

5.8

7.6

+

–

2.6

Source: Modified from Katz et al., 1990.331a Note: IUFD = intrauterine fetal demise, IUGR = intrauterine growth retardation, LBW = low birth weight, MoM = multiple of the median, + = increased risk but unquantified. * PO, poor outcomes.

Maternal Serum Screening for Neural Tube and Other Defects 727

–

Macri

C H A PTER 23

–

728

Genetic Disorders and the Fetus

Table 23.12 The complications of pregnancy that may be associated with elevated MSAFP in the second trimester of pregnancy Complication

Ref.

Fetal Abdominal pregnancy

334

Ectopic pregnancy

335

Fetal death

329

Multiple pregnancy

323

Oligohydramniosa

277

Polyhydramniosa

277

Pregnancy reduction

336

Rh disease

367

Stillbirth

331a–d

Threatened abortion

338

Placental Abruptio placentaea

277

Chorio-amnionitis (includes chronic villitis)a

339

Choriocarcinomaa

340, 341

Chorionic villus sampling (CVS)

342, 343

Cordocentesis

344

Cytomegalovirus infection (villitis)

345

Fetomaternal hemorrhage

346

HELLP syndrome

347

Hydatidiform mole

348, 349

Intrauterine growth retardationa

277

Low birthweighta

277

Maternal herpes infection with fetal liver

239

necrosis Parvovirus (B19) infection

350

Placental growth impairment

335

Placenta previaa

277

Placenta accreta, increta and percreta

351

Placental chorangioma (hemangioma)

352, 353

Placental vascular lesionsa,b

354–356

Postamniocentesis

357

Pre-eclamptic toxemiaa

277

Premature rupture of membranesa

277

Thrombophilias

358

Triploidyc

359

Umbilical cord hemangioma

360

Uterus – separate/bicornuate/unicornuate

361

Vaginal bleeding

362

Note: Additional references in previous editions.87,265,266,332,333 a

Occurring in the second or third trimester but possibly

preceded by second-trimester elevation in MSAFP. b

Including sonolucency, hemorrhage, thrombosis,

infarction. c

Triploidy with hydropic placental degeneration, may also

be spina bifida.

transabdominal procedures and were also associated with a subsequent increased fetal loss rate. Because fetomaternal bleeding may follow amniocentesis (see also Chapter 2) in 7–15 percent of cases, evaluation of MSAFP should be done before any tap. Cordocentesis frequently results in fetomaternal hemorrhage and in one study, MSAFP was elevated in 30 percent after the procedure.372 The majority of pregnancies with extremely high MSAFP values (≥8 MoM) are associated with major structural fetal defects or fetal death before 20 weeks of gestation. Killam et al.373 analyzed 44 such patients from among 40,676 screened pregnancies. A putative cause was determined in 82 percent at the initial ultrasound, primarily fetal defects, fetal death or placental abnormality. Among pregnancies with a liveborn infant, 88 percent had at least one obstetric complication. Congenital malformations such as septate, unicornuate and bicornuate uteri may be associated with “unexplained” MSAFP elevations.361 These patients are at increased risk for subsequent complications of pregnancy such as placental abruption, uterine rupture, and retained placenta. Other causes of elevated MSAFP levels

Ventral wall, congenital nephrosis and other disorders Certain fetal defects, as well as disorders that may affect either mother or fetus, may be associated with an elevated MSAFP level (Tables 23.13 and 23.14). Some of these maternal disorders may confound routine MSAFP screening. Etiologic overlap may also occur, exemplified by fetal disorders that have associated placental defects. A major Scottish study showed that MSAFP was elevated in 89 percent of fetuses with omphalocele and in 100 percent of those with gastroschisis.391 Congenital nephrosis is an autosomal recessive disorder caused by mutations known thus far in four genes (NPHS1, NPHS2, WT1 and LAMB2) but mainly NPHS1.406 Mutations in NPHS1 have been found in 85 percent of cases.406 Prenatal diagnosis by mutation analysis407,407a would be preceded by parental gene analyses. Pierson syndrome408 (microcoria-congenital nephrosis syndrome)409,410 is caused by mutations in the LAMB2 gene, which facilitates prenatal diagnosis.411 Rarely congenital nephrosis may be associated with a cerebral palsy-

CHAP T E R 2 3

Maternal Serum Screening for Neural Tube and Other Defects 729

Table 23.13 Fetal defects that may be associated with

Table 23.14 Disorders associated with elevated serum AFP

elevated MSAFP in the second trimester of pregnancy

in infants and nonpregnant women

Defect

Reference

Disorder

Acardiac twin fetus

87, 349

Infants

Aplasia cutis congenita

374

Ataxia telangiectasia

392

Arteriovenous fistula (intracranial)

375

Cystic fibrosis (mostly negative reports)

393

Congenital nephrosis

239, 376

Congenital hypothyroidism

381

Cystic adenomatoid malformation

353, 377

Congenital nephrosis

347

Indian childhood cirrhosis

394

(lung)

Reference

Epidermolysis bullosa

378, 379

Neonatal hepatitis

395

Esophageal atresia

380

Polycystic kidney disease

349

Fetal disorders with elevated AFAFP

(See Table 23.21)

Severe neonatal illness

396

Fetal hypothyroidism

381

Tyrosinosis

397

Hydrocephalus

349

Teratoma

398

Limb reduction defect

329, 349

Beckwith–Wiedemann syndrome

399

Multiple acyl-CoA dehydrogenase

382

deficiency NTD (anencephaly; spina bifida;

333, 383

encephalocele; iniencephaly;

Women (nonpregnant) Gastrointestinal cancer

400

Germ cell tumors (includes ovarian:

401

Sertoli–Leydig)

exencephaly) Polycystic kidney disease

349, 384

Renal agenesis

349, 385

Renal cysts

386

Roberts–SC phocomelia syndrome

387

Sex chromosome aneuploidy (includes

333, 388

Hepatic cancer

402

Hereditary persistence of AFP

403, 404

Viral hepatitis

405

Note: Additional references in previous editions.332,333

XXY;XXYY;XO) Simpson–Golabi–Behmel syndrome

389

Triploidy (with spina bifida or

359

placental degeneration) Trisomy 8

390

Trisomy 16 mosaicism Ventral wall defects (omphalocele/

333, 391

exomphalos; gastroschisis; body stalk defect; thoracoabdominal wall defect) Note: Additional references in 2004 edition.333

like syndrome with dystonic features, athetosis and a hearing defect.412 Congenital nephrosis is most common in Finland (about 1 in 8,000 livebirths) and among those of Scandinavian extraction, but cases have been diagnosed throughout the world, with enriched prevalence noted in Malta and among Pennsylvania Mennonites.413,414 Mean survival approximates 8 months; renal transplantation is the only effective treatment.415 Congenital nephrosis is almost invariably associated with markedly elevated AFAFP and MSAFP values.416 In a study

of pregnancies with affected and carrier fetuses417 (Figure 23.6), the median AFP levels in affected pregnancies were very high (8.3 MoM in maternal serum and 33 MoM in AF). A potential confounder to prenatal diagnosis of congenital nephrosis using AFAFP was the finding in the same study that pregnancies with carrier fetuses also had elevated median MSAFP and AFAFP levels in the second trimester (3.2 MoM in maternal serum and 8.9 MoM in AF). Although the AFP levels were, on average, lower in carrier pregnancies, the overlap between AFP levels in carrier and case pregnancies was high. Another potential pitfall is overestimating gestational age and finding a normal AFAFP in an affected fetus. Even with accurate gestational age assessment, neither the AFAFP nor the MSAFP levels may exceed 2.5 MoM. Hyperechoic fetal kidneys, which may or may not also be enlarged with calyceal dilation,418 serve as another nonspecific indicator of possible congenital nephrosis. These sonar observations together with placental enlargement may be seen in the third trimester419 (see also Chapters 25 and 26).

730

Genetic Disorders and the Fetus

Figure 23.6 MSAFP and AFAFP levels in pregnancies in which the fetus was affected with (closed circles) or a carrier for (open circles) congenital nephrosis (Finnish type). Source: Patrakka J, Martin P, Salonen R, et al. Lancet 2002;359:1575. Reprinted with permission from Elsevier.

Fetal teratomas are not usually malignant and may be amenable to surgery soon after birth. Fortuitous diagnosis is the rule through routine ultrasound or elevated AFAFP (see below). In 10 cases of fetal sacrococcygeal teratoma, MSAFP values were not significantly elevated.420 The livebirth prevalence was found to be 1 : 27,000.421 Mild benign fetal obstructive uropathy may be associated with raised MSAFP levels.422 These authors examined 61 consecutive patients with elevated MSAFP and 80 others with normal values. Ultrasonographers were blinded to the MSAFP results. Among male fetuses, 33 percent had pyelectasis, compared with only 5 percent of controls. Among female fetuses, pyelectasis was seen in 16 percent of high-MSAFP cases and in no controls. Fetal proteinuria seems unlikely, but associated placental pathology may explain these observations.

Acardiac twin Pregnancy termination of an apparently normal fetus after the discovery of elevated MSAFP and AFAFP with normal results on ultrasound is rare. One potential pitfall, which may occur despite a diligent ultrasonographic search, is the presence of a subjacent dead acardiac twin. Hereditary persistence of AFP is a rare autosomal dominant disorder due to mutations in the AFP gene promoter423,424 without apparent clinical effects403 that may first be discovered in a healthy woman through routine MSAFP screening. This disorder requires consideration when: • MSAFP values are raised, AFAFP is normal, and high-resolution ultrasound reveals no placental or fetal defects • MSAFP values remain elevated through and after pregnancy

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Maternal Serum Screening for Neural Tube and Other Defects 731

• a previous pregnancy with unexplained MSAFP elevation also resulted in a normal child; inferential diagnosis is clinched if a healthy maternal sibling, parent or previous child has raised MSAFP values (when not pregnant). Management protocol for unexplained elevated MSAFP values After the detection of an elevated MSAFP level, demonstration of no abnormality on a level II ultrasound, and the finding of a normal AFAFP, AChE and fetal karyotype, further and continuing attention is necessary. Ample data, presented above, document the increased risk of adverse pregnancy outcome and probable residual risk of undetected fetal malformation (see also Chapter 23 for risks using multiple analytes). While various professional guidelines remark on unexplained elevated MSAFP, they do not make a specific recommendation.425 A prudent management protocol for these patients would include: • genetic counseling, not only to explain the laboratory and ultrasonographic findings and limitations, but also to evaluate the family pedigree for potential causes or associations • serial level II ultrasound at 16, 19, and 23 weeks of gestation (selective abortion is difficult to obtain after 24 weeks), especially for fetal growth, intracranial anatomy, complete limb and digit evaluation, and AF volume • fetal echocardiogram at 20–23 weeks, given the approximate 1 percent frequency of congenital cardiac defects and their occurrence in patients with elevated MSAFP297,365 • a Kleihauer–Betke test for Rh-negative women, to determine fetomaternal bleeding, to establish a cause for the high MSAFP, and to prevent Rh sensitization • viral titers in maternal sera, when infections (e.g. cytomegalovirus, parvovirus) are suspected, and specific viral DNA analysis of AF cells (see Chapter 32) • lupus anticoagulant, antiphospholipid,426 and factor V Leiden DNA358 studies in women with previous vascular thromboses or unexplained fetal death • third-trimester ultrasound (perhaps best between 28 and 32 weeks) for fetal growth, anatomy, and AF volume

• late third-trimester fetal movement counts, nonstress tests, and biophysical profiles. Although cost–benefit studies of the preceding or similar protocols have yet to be done, the outline presented would reflect prudent care and attention to a pregnancy that is clearly at appreciably higher risk than normal. Amniocentesis or ultrasound for elevated MSAFP levels Amniotic fluid studies for both AFP and AChE result in a detection rate of 95–98 percent for open NTDs and a false-positive rate of 0.4 percent in women with elevated MSAFP levels.427,428 Advances in high-resolution ultrasound have raised detection rates closer to those achieved by AF studies preceded by MSAFP screening. Notwithstanding this progress, small anterior encephaloceles and SB can be missed, and oligohydramnios, maternal habitus, and fetal position may prevent accurate ultrasonographic assessment87,429 (see also Chapter 25). Studies that include detection and false-positive rates for NTDs were assessed430 after the claim431 that high-resolution ultrasound might suffice for NTD diagnosis, rather than dependence on amniocentesis. For anencephaly and SB, the data show overall detection rates of 100 percent and 88 percent respectively, and a false-positive rate of 1.2 percent. A majority of these studies were completed before the recognition of intracranial signs of SB (see also Chapter 25). The data of Nadel et al.431 yielded a 100 percent sensitivity for detection of NTD in their small series. However, ascertainment and selection bias may have confounded these conclusions.432 In a series of 257 pregnancies with elevated MSAFP, 16 fetal defects were detected by AFAFP and AChE study, four (25 percent) of which had been missed by ultrasound.433 In a subsequent paper,385385 these authors, adding to this experience, reported on 36 defective fetuses among 331 women with elevated MSAFP. Six (16.7 percent) fetuses with defects were missed by ultrasound. Despite the authors’ 83 percent sensitivity by ultrasound, they calculated a >1 percent risk of fetal defects when the MSAFP value was >3 MoM and concluded that amniocentesis should be offered routinely when MSAFP elevation is found. In

732

Genetic Disorders and the Fetus

another similar study434 of 681 women with elevated MSAFP, 13 (1.9 percent) fetal defects were detected by amniocentesis study and were missed by ultrasound. In a series of 313 women undergoing amniocentesis for elevated MSAFP levels, Barth et al.435 missed one of four chromosome defects on ultrasound. Early gestational age, small defects, and technical failure were implicated as causes of misdiagnosis. In another study, a risk of chromosomal abnormality in nonselected patients mostly younger than 35 years of age with elevated MSAFP and a normal ultrasound report was close to background expectations (0.6 percent).436 Complementary use of high-resolution ultrasound and amniocentesis after the discovery of an elevated MSAFP remains the safest policy, with SB detection rates exceeding 95 percent with rare false-positive results, plus detection of a few mostly unexpected chromosomal abnormalities. Given the strong trend toward ultrasound follow-up alone, because it poses no risks to the mother or fetus, a reasonable option following a confirmed elevated MSAFP result would first involve a detailed ultrasound examination. If a clear diagnosis of SB or other open fetal defects is apparent, amniocentesis is not required. However, if ultrasound does not reveal an open fetal defect or if the findings are equivocal, the patient should be offered amniocentesis to increase the likelihood that, if a defect is indeed present, it will be found. MSAFP versus ultrasound screening The efficacy and impact of MSAFP screening for NTDs are well documented.333,437 A policy of screening all pregnancies for NTDs using highresolution ultrasound assumes safety of ultrasound; high predictive values, sensitivity, and specificity; availability of the necessary number of skilled and trained individuals; availability of good equipment; and demonstration of cost effectiveness. Harmful effects have not been observed among those born after exposure to ultrasound in utero,438 and consequently routine “anomaly” scans at 18–22 weeks are increasingly offered. In an analysis of the published data, Wald and co-authors found that the performance of two key ultrasound signs in screening rivaled screening with MSAFP.289,439 In 13 studies on the “lemon” sign and eight on the “banana” sign, the compiled SB detec-

tion rates were 86 percent and 93 percent and the false-positive rates were 0.23 percent and 0.05 percent, respectively. However, the authors caution that the results are not likely to be nearly as good in clinical practice, given that the published studies were almost all done on women known to be at high risk of SB. In addition, we have as yet no indication that screening a low-risk general pregnant population by sonographers in the offices of primary care providers will be able to approach the performance of researchers in dedicated, academic ultrasound centers. With the increasing use of first-trimester combined screening for Down syndrome and the 18–22 week fetal anomaly scan for structural fetal abnormalities in the United Kingdom, the National Institute for Health and Clinical Excellence (NICE), part of the NHS in the UK, has recently proposed that the 18–22 week fetal anomoly scan, rather than MSAFP, be used as the primary screening method for detection of NTDs.440 They argue that the anomaly scan shows better screening performance than MSAFP and because it has become part of routine prenatal care, a second-trimester serum draw and MSAFP assay is no longer needed and will result in cost savings. A recent study by Dashe et al.441 supports the view that routine second-trimester ultrasound screening for NTDs performs better than MSAFP. However, it is important to consider that the use of the 18–22 week scan, rather than MSAFP measurement at 16 weeks, will delay screening for a group of serious birth defects by 3–7 weeks. MSAFP screening: policy guidelines Laboratory guidelines on MSAFP screening for NTDs were published by the National Committee for Clinical Laboratory Standards (NCCLS) in 1997442 and by the American College of Medical Genetics in 2003.443 The policy for screening for chromosome defects is discussed in Chapter 24. MSAFP screening for NTDs before 14 weeks There is no recognized correlation between MSAFP and AFAFP values in the first trimester. Experience thus far indicates no likely benefit of early screening for NTDs. One brief report noted normal MSAFP levels at 11–12 weeks in two fetuses with

CHAP T E R 2 3

Maternal Serum Screening for Neural Tube and Other Defects 733

Table 23.15 AFAFP: the detection rate for open neural tube defects and the false-positive rate Gestation

AFP cut-off

Anencephaly

Open spina

Crude false-positive rate

(completed

level (MoM)

(%)

bifida (%)

(all non-NTD singletons) (%)

weeks) 13–15

2.5

100 (21/21)

96 (22/23)

0.7 (24/3,279)

16–18

3.0

99 (96/97)

99 (73/74)

0.7 (58/7,858)

19–21

3.5

99 (69/70)

95 (20/21)

1.0 (15/1,561)

22–24

4.0

94 (32/34)

13–24

as above

98.2 (218/222)

100 (5/5) 97.6 (120/123)

1.5 (6/407) 0.79a (103/13,105)

Source: Adapted from Second Report of the UK Collaborative Study.248 Note: Numbers of pregnancies are shown in parentheses. a

The “practical” false-positive rate (excluding miscarriages and serious malformations) was 0.48 percent (61/12,804).

acrania and one with SB.444 In a prospective set of first-trimester studies of 12 cases with SB and three with anencephaly, only one SB case yielded an elevated MSAFP value at 14 weeks.445 Neither unconjugated estriol nor intact β-human chorionic gonadotropin (β-hCG) was an effective indicator of NTDs in this study. Another prospective study of 19 NTD cases also showed that first-trimester MSAFP screening was of no value, with the median value in the NTD pregnancies only 1.2 MoM.446 Although first-trimester screening with MSAFP is not appropriate, some laboratories have lowered the NTD screening window from 15 to 14 weeks of gestation. The impetus for this is that screening for Down syndrome using triple markers is acceptable at 14 weeks; thus, it would be efficient to screen for NTDs at the same time.447 However, data from the original multicenter study on MSAFP as a screening test for NTDs, the UK Collaborative Study of 1977, clearly showed that screening performance at 14 weeks was inferior to screening from 15 weeks on.271 The estimate from the data in that study is that the detection rate at 14 weeks is no more than 50 percent, using an MoM cut-off of 2.5 MoM, clearly inferior to the 80 percent or higher attainable beginning at 15 or 16 weeks of pregnancy. Amniotic fluid AFP Though AFAFP analysis for the diagnosis of open NTDs has by now become secondary to targeted fetal ultrasound, it should still be offered when the fetal scan is inconclusive. The detailed description of AFAFP analysis, interpretation, and clinical

utility therefore remains important and a useful resource. Brock and Sutcliffe448 first observed elevated AFAFP concentrations when the fetus had an open NTD. The slight overlap in values between affected and unaffected pregnancies was recognized early on.248 These data indicated that the optimal time for amniocentesis for AFP assay, resulting in the least overlap, was 16–18 weeks. To achieve similar false-positive rates for each gestational week, that study concluded that it was necessary to increase the upper cut-off level as pregnancy advanced (Table 23.15). It was also noted that the risk of open SB varies, depending on the concentration of AFAFP, the reason for the amniocentesis, and the background prevalence of open SB (Table 23.16). Table 23.16 also reflects the odds for open SB if MSAFP as well as AFAFP elevations are known. Units used in reporting AFAFP results Amniotic fluid AFP levels decrease logarithmically, at a rate of about 15 percent per week, during the period of 15–22 gestational weeks in which they are used in prenatal diagnosis. For this reason and because of the variability of AFP values from assay to assay, as in screening with MSAFP, the reported AFAFP value is normalized in some way. As with MSAFP, the MoM is the normalizing unit most commonly used in reporting AFAFP results. The MoM cut-offs often used in AFAFP testing are 2.0 and 2.5. However, until about 20 years ago, it was not uncommon to report AFAFP results as the number of standard deviations (SDs) above (or below) the mean level for each week of gestation,

734

Genetic Disorders and the Fetus

Table 23.16 The odds of having a fetus with open SB (compared with a viable fetus without a serious malformation) before and after a positive AFAFP test at 16–18 weeks of gestation, according to the prevalence of open SB and the reason for the amniocentesis Prevalence of open spina bifidaa 1 per 1,000

2 per 1,000

3 per 1,000

Odds of open SBb

Before Reason for amniocentesis

3.0 MoM

AFAFP

3.5 MoM

4.0 MoM

4.5 MoM

26 : 1

test Serum AFP ≥2.53medianc

1:2

9:1

16 : 1

17 : 1

Previous infant with NTD

1 : 100

2:1

4:1

4:1

7:1

Other

1 : 1000

1:4

1:2

1:2

2:3

Serum AFP ≥2.53medianc

1 : 13

18 : 1

32 : 1

35 : 1

52 : 1

Previous infant with NTD

1 : 50

5:1

8:1

9:1

14 : 1

Other

1 : 500

1:2

1:1

1:1

3:2

Serum AFP ≥2.53medianc

1:9

26 : 1

46 : 1

50 : 1

75 : 1

Previous infant with NTD

1 : 33

7:1

13 : 1

14 : 1

20 : 1

Other

1 : 333

2:3

1:1

3:2

2:1

Source: Adapted from Second Report of the UK Collaborative Study.248 a

In the absence of antenatal diagnosis and selective abortion.

b

Odds of having a fetus with open SB after AFAFP found to be at cut-off level

c

At 16–18 weeks of gestation, based on a single serum AFP test, followed by the use of ultrasound to correct

gestational age; if patients with raised levels are tested twice and the average value is used, the odds ratios are increased by about one-third.

with a cut-off of 15.0 SD units or more considered clearly elevated, and 13.0–4.9 SD units considered borderline elevated. The SD method of reporting, similar to that commonly used in clinical chemistry, was phased out in favor of the more stable and better understood MoM unit, already being used in reporting MSAFP results. In both cases, the reference data for AFAFP are gestational age specific. In the case of the MoM, the reference data are completed week-specific median values and in the case of SDs above the mean, the reference data are completed week-specific mean values. Experience with AFAFP assays Amniotic fluid AFP assays have long been routine, and extensive series have been reported248,448–451 and reviewed.333 Tabulated summary data of our first 100,000 cases noted in an earlier edition87 indicated a recurrence rate for a NTD after the birth of a previously affected child as 1.4 percent. Because some samples sent from other states included parents with SB occulta, no reliable recurrence risks could be given for an affected parent. The risk of recurrence after the birth of two previous children with NTDs was 5.9 percent. Routine assays of

AF obtained largely because of advanced maternal age yielded an NTD rate of one per 492 cases studied. This figure from the prefolic acid era is considerably higher than expected, given that these represent only cases with no family history. Among the 499 NTDs that occurred in the first 100,000 cases studied, 29 (5.8 percent) were closed lesions associated with a normal AFAFP. The 317 other congenital defects diagnosed reflect only those with an elevated AFAFP. Among the 100,000 cases, only 779 (0.78 percent) had an elevated (≥5 SD) AFAFP at 99 percent when AChE is also used.87 Fetal blood contamination will lower sensitivity and cannot be corrected for reliability. A specificity of >99 percent for open NTD is achievable. Predictive values will vary with the prevalence of NTD, family history, and cut-off level used. AFAFP ≥2.0 and ≥2.5 MoM yielded risks for open NTD of 24–41 percent and 41–63 percent, respectively.248,449 Multiple pregnancy Given the increased risk of malformations, including NTDs among twins29,466 and speculation about the role of the twinning process in the etiology of NTDs,79 special care is needed in the evaluation for structural defects (see also Chapters 25 and 26). For nonidentical twins or triplets discordant for open NTDs or fetal death, we and others467,468 have repeatedly observed elevated AFAFP and AChE presence in the sac of the affected fetus and normal results in the unaffected twin. There have been occasional exceptions in which dizygous twins discordant for NTDs or fetal death have had elevated AFAFP and AChE presence in both sacs, as also noted by others.469,470

738

Genetic Disorders and the Fetus

Table 23.21 Fetal conditions that may be associated with elevated AFAFP and/or AChE Likely mechanism/condition

Reference

Leakage through skin

Sacrococcygeal teratoma

487, 488

NTDs –anencephaly, spina bifida,

Hamartoma

333

Likely mechanism/condition

Reference

333

encephalocele, exencephaly,

Urinary tract leakage

iniencephaly Anterior abdominal wall defect

455, 333

– omphalocele, gastroschisis, abdominothoracic defect, body stalk abnormality

Congenital nephrosis

333

Denys–Drash syndrome

489

Hydronephrosis

449, 490

Polycystic kidney diseasea

384

Exstrophy of bladder (cloaca)

473, 474

Leakage of placental origin

Epidermolysis bullosa

378, 379, 379a

Fetal blood in amniotic fluid

333

Aplasia cutis congenita

472

Hydatidiform mole

348, 349

Chromosomal defects – trisomies

333 (see also Table 23.4)

Umbilical cord hemangioma

491

21, 18, 13, 8; 45X, 45X/46XX,

Leakage of pulmonary origin

47XXY Cystic hygroma (see text)

333, 449

Cystic adenomatoid

377

malformation of lung

Nuchal cyst

475

Prune belly syndrome

476

? Reduced intestinal AFP clearance or leakage

Median palatoschisis

477

Pharyngeal teratoma

478

Scalp defect

See chromosome defects

Esophageal atresia

492

Amniotic band syndrome

478

Duodenal atresia

472, 493

Fetal death (autolysis)

450, 333

Annular pancreas

494

Twin with cotwin death

479

Intestinal atresia

449, 455

Acardiac twin fetus

455

Meckel syndrome

333

Fetus, papyraceous

480, 481

Hydrops/fetal ascites

455, 481, 482

Lymphangiectasia

483

Bladder neck obstruction

484

(massive distension and death) Urethra absent

485

Rhesus hemolytic disease

337 (see also hydrops)

? Pilonidal sinus

486

Unknown site of “leakage” Multiple congenital defects

333, 449

Hydrocephalus

495

Dandy–Walker malformation

478

Tracheo-esophageal fistula

478

Herpes virus infection (maternal)

477

with fetal liver necrosis Noonan syndrome

333

Tetralogy of Fallot

333

Note: Additional references in previous editions.87,265,266,332,333 a

No AChE detected.

Causes of elevated AFAFP in the absence of NTDs A raised AFAFP without the concomitant presence of AChE poses increased risks for that pregnancy.471 Elevated AFAFP is found in many leaking fetal defects (Table 23.21); the value of the observation is confounded only by fetal blood admixture. Because fetal blood may be present in association with some defects, a level II ultrasound would be recommended in all cases with unexplained elevated AFAFP.

In normal pregnancy, AFP reaches the AF mostly by fetal urination.496 Any fetal skin defects, including NTDs and omphalocele, allow the egress of serum containing AFP into the AF, leading to a quantitative increase in its concentration.497 Other defects causing fetal proteinuria (e.g. congenital nephrosis) could also result in a raised AFP level. Heinonen et al.376 reported an elevated AFAFP after a high MSAFP level in 43 of 44 pregnancies with fetal congenital nephrosis. The range of AFAFP levels was 5.1–43.5 MoM; the sensitivity

CHAP T E R 2 3

Maternal Serum Screening for Neural Tube and Other Defects 739

was 100 percent and the specificity was 99 percent. The one missed case resulted from a normal MSAFP screen. Unexplained high AFAFP levels that are AChE negative may decrease appreciably at a second amniocentesis 2 weeks later if the fetus is likely to be normal or rise if congenital nephrosis or another serious defect is present. Disorders interfering with swallowing or digestion (e.g. esophageal or duodenal atresia) might diminish the turnover of AFP or, through regurgitation of biliary secretions, raise the AFAFP concentration. Although fetal renal agenesis could be expected to involve low or absent AFAFP values, we and others498 have found normal concentrations. Cystic hygromas, which may be found particularly in association with Turner or DS (among others), may also occur in the absence of any chromosome abnormality (see also Chapter 25). A review of 142 cases detected by ultrasound showed 58 percent with Turner syndrome, 28 percent with various chromosomal defects, and 22 percent with normal karyotypes.499 Moreover, elevated AFAFP, with or without positive AChE in such cases, may well be due to direct aspiration of cystic fluid rather than transudation of fetal serum.500 Cystic hygromas may resolve and, with normal karyotypes, may result in normal neonatal outcome.501 Nevertheless, initial detection should be followed by detailed sonographic evaluation of the entire fetus, karyotype, and family history.502,503 Elevated AFAFP levels in association with omphalocele or open ventral wall defects occur in at least 67 percent of cases. When fetal blood contamination has been clearly excluded as the reason for a raised AFP level, our data and those of others450 are similar, in that there is about a two-thirds risk that the fetus has either an NTD or another serious congenital defect. Not all cases of fetal sacrococcygeal teratoma have raised AFAFP or are AChE positive.503a Elevated AFAFP and sonography Highly skilled and superbly equipped experts in obstetric ultrasound occasionally miss an NTD, despite being apprised about an elevated AFAFP level and the presence of AChE. Small sacral NTDs are the lesions mostly commonly missed. However, other cryptic defects may be present (see Table 23.21). Given the precision of the assay for AFP, patient reassurance that all is well in the face of an

unremarkable targeted ultrasound study when AFAFP is elevated (with or without the presence of AChE in a noncentrifuged sample free of fetal blood) is unwarranted. In one series of 263 fetuses with high AFAFP and a normal sonogram, 11 (4.2 percent) had closed central nervous system defects (hydrocephalus in five, Dandy–Walker malformation in two), two had congenital nephrosis, one had tracheo-esophageal fistula, and one had a small omphalocele.478 Meckel syndrome The prenatal diagnosis of Meckel syndrome using AFAFP and AChE has been difficult. Only five of our seven cases had an AFAFP ≥5 SD above the mean. An additional case had a normal AFP but was positive for AFAChE. AChE was positive in five of six cases assayed. Chemke et al.504 described a fetus with this syndrome, with high AFAFP without an NTD, postulating excessive synthesis of AFP by the polycystic kidneys. Johnson and Holzwarth505 reviewed published experience of 32 cases and added three of their own. They again emphasized the variability of the clinical expression of Meckel syndrome.508 In an overall analysis of 79 cases unrelated to prenatal diagnosis, they noted that 57 percent had combined encephalocele, polycystic kidneys, and polydactyly; 16 percent had encephalocele and polycystic kidneys; and 15 percent had polycystic kidneys and polydactyly. Only 3 percent had encephalocele and polydactyly. Most disturbing was the report by Seller506 that 9 percent had only one of these abnormalities. Elsewhere, one of two affected siblings had only urethral atresia and preaxial polydactyly.507 This rare autosomal recessive disorder is genetically heterogeneous and three genes have been mapped, two of which have beeen identified (MKS1 and MKS3).508a Together they are estimated to account for about 14 percent of cases. The third locus at 11q13 (MKS3) can be approached by linkage analysis, pending cloning of the gene. Precise prenatal diagnosis can be anticipated for a majority of cases. Problems and pitfalls

Aspiration of urine In our experience, maternal urine is inadvertently aspirated at amniocentesis in about one in 2,000

740

Genetic Disorders and the Fetus

cases. Most often, this occurs because patients have been requested to have a full bladder for the preceding ultrasound study. For ultrasonically guided amniocentesis, some prefer to keep the bladder distended during the procedure. A sample submitted as AF is usually first suspected as being urine when no AFP is detected on assay. It would seem judicious for obstetricians performing amniocenteses to drop some AF immediately on aspiration onto one of the many types of test strips available that allow for the determination of pH, protein, and sugar, to obviate the problem. Duncan509 suggested the useful routine of testing the urine voided immediately before amniocentesis and keeping the test strip alongside another on which a few drops of AF are placed for comparison. The aspiration of maternal urine instead of AF had led, at least in two cases, to a failure to detect anencephaly and open SB.510,511

Brown or green AF A 1.6 percent frequency of discolored AF obtained during the second trimester has been noted previously.265 When brown AF was associated with an elevated AFP, an untoward outcome of pregnancy was extremely likely (93.6 percent). The experience of Seller512 was very similar. In contrast, brown AF in the second trimester not associated with an elevated AFP does not seem to augur ill for the pregnancy.513 An earlier intrauterine bleed with resulting blood breakdown products is the likely cause for the discolored AF. Green AF shown by spectrophotometric scanning is usually due to meconium. An incidence rate of 1.7 percent in midtrimester AF was reported by Allen,514 with an associated mortality rate of 5.1 percent. AF meconium reflects a characteristic layering effect on ultrasound.515 Amniotic fluid AChE The assay for neuronal-derived AChE516 is a critical adjunct in the prenatal diagnosis of open NTDs.333,427 The most common assay is polyacrylamide-gel electrophoresis (PAGE),517 in which AChE can be distinguished from nonspecific cholinesterase on the basis of mobility in such gels. AChE appears as a faster second migrating band, which can be suppressed by the addition of a specific inhibitor (BW284C51). Normal AF has a single, slowmoving band of nonspecific cholinesterase.

Loft427 compared PAGE, an immunoassay using a monoclonal antibody, to AChE and to a spectrophotometric assay. Although the third was found to be completely unsuitable, almost identical performance in clear AF was obtained for the first two. When fetal blood was present in the AF, the immunoassay was clearly less satisfactory than PAGE, yielding a higher false-positive rate. Better visualization of AChE bands in PAGE has been claimed by using dark-field illumination.518

Experience with AFAChE Extensive clinical use of AChE for prenatal diagnosis of NTDs followed the initial major study. In a review of 20 combined studies, Loft noted detection rates of 98.6, 95.5 and 95.2 percent for anencephaly, for all SB and for encephalocele, respectively.427 These rates are compared with those in the second collaborative study report248 and with our own experience (see Table 23.19). However, case selection differed slightly among some studies, in that AFAFP was considered elevated variably between 2.5 and 3 SD above the mean or at ≥2.5 MoM. Despite this variation, the overall detection efficiency for anencephaly and for SB was 99.7 and 99.4 percent, respectively. Only 68.6 percent of encephaloceles leaked. Although many reports make distinctions between detection rates for open versus closed SB, the clinician counseling a patient before amniocentesis needs to know the overall detection and practical falsepositive rates (see Tables 23.18 and 23.19). Precise interpretation of a PAGE for AFAChE assay depends on gestational age. False-positive rates from AF samples at 12 weeks of gestation have ranged from 4.3 to 33.3 percent.456,484,519,520 False-positive AFAChE rates between 13 and 24 weeks of gestation in the period 1979–1984 were up to 1.8 percent (see Table 23.18). More recently, rates in clear AF samples have been 0.22 percent (see Table 23.18). Given the clear risk of false-positive AChE results before 15 weeks,520,521 high-resolution ultrasound (serially if needed) and occasionally a second amniocentesis about 2 weeks later is recommended. Increased false-positive and false-negative rates have also been noted after 24 weeks.427 Falsenegative AFAChE may also occur rarely with open NTDs.522,524After fetal death, an AChE band

CHAP T E R 2 3

Maternal Serum Screening for Neural Tube and Other Defects 741

is frequently seen in the gel and can sometimes be distinguished from the pattern seen with open NTDs.525,526 The vast majority of disorders associated with elevated AFAFP (see Table 23.21) may also have detectable AChE activity. Some open lesions close later in pregnancy and a few have normal AFAFP and no detectable AChE. The everpossible confounding with fetal blood admixture is now usually resolved by high-resolution ultrasound (see also Chapter 25). Sepulveda et al.457 retrospectively audited 1,737 consecutive AF samples that they obtained for chromosome studies and that included assays for AFP and AChE. In 25 cases elevated AFAFP and/or positive AChE was observed. High-resolution ultrasonography correctly identified all 18 fetuses with defects and associated elevated AFAFP and/or positive ACHE. In the remaining seven fetuses, no anomalies were detected and all appeared normal after birth (a false-positive rate of 0.4 percent). These authors suggested that these biochemical assays would not be cost effective in centers where high-resolution ultrasonography is done before amniocentesis. In combined series, ventral abdominal wall defects without distinguishing omphalocele from gastroschisis were detected in 56.2 percent of cases using AFAFP and AChE (see Table 23.19). The presence of AChE in these cases, as in other leaking lesions, probably reflects transudation of fetal plasma.527 For ventral wall defects, AChE secretion from intestinal nerve plexuses and butyrylcholinesterase secretion from intestinal muscle cells may explain the positive PAGE results.528 Usually, high AChE and low butyrylcholinesterase activities characterize AF from open NTDs; the opposite usually occurs in open ventral wall defects. Ultrasound study immediately after an elevated AFAFP with or without positive AChE is found is recommended, given the need for a precise diagnosis and distinction between gastroschisis and omphalocele (see also Chapter 25). Between 25 and 33 percent of fetuses with omphalocele have other associated defects, including subsequent mental retardation, compared with about 10–36 percent (mainly gut atresias) in those with gastroschisis.333 An abnormal karyotype is frequent with omphalocele, 20–22 percent in two studies529 but as high as 54 percent in another,530 probably reflecting ascertainment bias, with more complex cases being

referred. Prognosis for survival depends primarily on the presence of other anomalies, ranging from 7 percent to almost 100 percent.529 Mode of delivery seems not to have affected survival rates.529 In contrast, gastroschisis is not typically associated with an abnormal karyotype, and survival, while highly likely, is predicated on other factors (e.g. prematurity, sepsis, and associated intestinal atresias).529 Cases in which borderline AFAFP values are found to be positive for AChE and fetal blood admixture require high-resolution ultrasound, serially if necessary. Decisions should be made on the ultrasonographic findings; referral to nomograms531 is useful only when ultrasound facilities are not available. Among 6,183 normal children born after AFAFP was found to be normal, 24 (0.4 percent) in combined series (see Table 23.19) were AFAChE positive.455,511,532–535 In another study of 1,300 AFs,536 faint AChE bands were identified in nine (0.7 percent), none of which was associated with congenital defects. Fetal blood in the AF may well explain even some of these, the AChE source being fetal serum.518 The Collaborative Acetylcholinesterase Study522 showed that a woman with an elevated AFAFP who was AChE positive had a much greater likelihood of carrying a fetus with a serious leaking fetal defect (Table 23.22). In a report of two

Table 23.22 The odds of having a fetus with open SB after positive AFP and AChE tests Birth incidence of SB

1 per 1,000

2 per 1,000

3 per 1,000

Odds of fetus with SB Before

After

Positive

Reason for amniocentesis

AChE

AChE

Raised maternal serum AFP

9:1

144 : 1

Previous infant with NTD

2:1

32 : 1

Other

1:4

4:1

18 : 1

288 : 1

Previous infant with NTD

Raised maternal serum AFP

5:1

80 : 1

Other

1:2

8:1

26 : 1

416 : 1

Previous infant with NTD

Raised maternal serum AFP

7:1

112 : 1

Other

2:3

16 : 1

Source: After Collaborative Acetylcholinesterase Study.522

742

Genetic Disorders and the Fetus

fetuses with esophageal atresia, inexplicable AChEpositive AF in the face of normal AFAFP was noted.518 One other fetus affected by tracheoesophageal fistula was AFAChE negative.

Advantages and disadvantages of the AChE assay Although the AChE assay is also nonspecific, it has a major advantage over AFP in not being dependent on gestational age, at least in the second trimester. Its greater sensitivity than AFP is offset by the need for a second assay with an inhibitor, the use of which is not free of risk to laboratory personnel. It is also less efficient and more expensive than the AFP assay. In addition to the problem of fetal blood contamination, at least two other pitfalls have been encountered. An error resulting in a positive AChE may occur when fetal calf serum is mistakenly introduced into the AF in the process of isolating AF cells. The bovine AChE will then be indistinguishable from the human enzyme. A second error involves old or mishandled AF, in which AChE derives from red blood cells, thereby leading to a false-positive result. The use of the predictive value positive concept has been recommended, especially when borderline results are obtained.537 The advent of AChE and of widely available level II ultrasound has markedly decreased the need for such a risk calculation. Recommendations for prenatal diagnosis of NTDs using AFAFP and AChE assays 1. Couples with an increased risk of having a child with an NTD should be offered (or referred for) genetic counseling and amniocentesis. 2. AF from patients at increased risk should be assayed for both AFP and AChE. 3. AChE assays should be done on all AF samples with AFP values ≥2 MoM (some centers use ≥1.85 MoM). 4. MSAFP screening should not be relied on to exclude a fetal NTD. 5. Accurate ultrasonic fetal age assessment is mandatory immediately preceding amniocentesis because the AFAFP level steadily decreases through the second trimester.

6. Level II ultrasound is recommended for every woman with an increased risk of having a child with a NTD, and increasingly centers are recommending an “anomaly scan” between 18 and 22 weeks in non-high risk pregnancies. 7. Both ultrasound and amniocentesis should be offered at 15–16 weeks of gestation. 8. A 1 mL aliquot of AF is best placed directly into a small tube at the time of amniocentesis, specifically for AFP and AChE assay of a noncentrifuged sample. 9. Obstetricians should discard the first 1–2 mL of AF if the sample contains fresh blood, to minimize problems in the interpretation of the AFP and AChE assays. 10. To exclude the possibility that urine has been obtained inadvertently, 1–2 drops of AF should be placed on a urine testing strip at the time of amniocentesis. 11. If fetal blood is present in the AF and is associated with an elevated AFP level and a normal sonogram, then a second amniocentesis is recommended in about 10 days. 12. Every AF sample obtained for any reason should be assayed for AFP. 13. Every laboratory receiving AF must first have established a normal range of AFP per gestational week and be able to provide accurate and reliable results, including MoMs or the number of SDs above the mean. 14. Fetal hemoglobin should be assayed or fetal red cells should be counted in all samples in which the AFP concentration exceeds the upper-limit cut-off. 15. A clear written policy should exist, showing the AFP level at which an AChE assay is automatically done. 16. If the AFAFP value is raised above the upperlimit cut-off (usually ≥2 MoMs), corroborative evidence should be sought before any decision is made to terminate a pregnancy. If ultrasound studies reveal no fetal abnormalities and the karyotype is normal, a second amniocentesis should be performed in about 10 days, even if the first sample is also AChE positive. This process should also assist in avoiding any sample mix-up. 17. Direct and rapid communication from the laboratory to the physician and from the physician

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Maternal Serum Screening for Neural Tube and Other Defects 743

to the patient should take place if any abnormal assay result is found. 18. Genetic counseling should be urged for all couples when a fetal defect is found or suspected or if laboratory results require further explanation. 19. No pregnancy should be terminated after the observation of an unexplained elevated AFAFP with or without the presence of AChE, without the couple’s full understanding of the possibility that a fetus without apparent anatomic or other abnormality might be aborted. 20. In cases in which only AFAFP values have been high and AChE has been negative, and after parents have elected to abort the pregnancy, careful preparations should be made to obtain fetal kidney tissue for electron microscopy, aimed at determining a diagnosis of congenital nephrosis. It is unacceptable, and indeed negligent, when faced with a potential 25 percent risk of recurrence, to omit proper preparations for light and electron microscopy of the kidneys to diagnose this fatal disorder. Characteristic findings are marked dilation of proximal tubular lumina and Bowman’s spaces and fusion of foot processes of the podocytes of the renal glomeruli.586 Couples who do not receive this thoughtful care and attention will be denied the opportunity of knowing whether they have a 25 percent risk in a subsequent pregnancy and whether they need to consider other options (such as artificial insemination by donor). 21. Renal tissue studies would be unnecessary if DNA analysis of amniotic fluid cells yields definitive mutations in the four known genes (thus far) for congenital nephrosis. 22. When necessary, fetal tissue should be stored frozen (270°F), in anticipation of DNA mutation analysis of the congenital nephrosis genes, for diagnosis and subsequent prenatal diagnosis.

Other techniques to detect neural tube defects The complementary use of AFAFP and AChE, combined with ultrasonography, yields an extremely high degree of accuracy. Any new test challenging the first two biochemical assays would have to exceed their demonstrated complementary value. In particular, any new technique would have

distinct advantages if it could achieve specificity, avoid variation with gestational age, render the effects of maternal or fetal blood contamination of AF irrelevant, and avoid significant overlap between the normal and abnormal ranges. Thus far, these strictures seem to impose too great a challenge for any approach attempting to replace the established tests. In fact, as noted in earlier reviews,98,284 evaluations for a2-macroglobulin, βlipoprotein, β-trace protein, fibrinogen degradation products, glucose, albumin, group-specific component, total protein, brain-specific protein S-100, 5-hydroxyindoleacetic acid, rapidly adherent AF cells, concanavalin A binding, glial fibrillary acidic protein, synaptosomal D2 protein, and various amino acids have all been unsuccessful.

Primary prevention of neural tube defects Genetic counseling Given the heterogeneous etiology of NTDs (see Tables 23.1–23.4), great care should be exercised in providing genetic counseling (see also Chapter 1). Primary prevention of NTDs through risk counseling, however, is extremely limited because about 95 percent of such offspring are delivered by women without a previously affected child. The recurrence risk for first-degree relatives of probands with NTDs of polygenic origin usually parallels the population incidence.538 Nevertheless, many confounding factors exist in assessing recurrence risks. These include a worldwide decline in the incidence of NTDs539 (see discussion below), ethnic differences, and time and space variations. Hence, the range of risk figures in Table 23.23 provides some guidance but will require revision to account for the declining incidence and the wide use of folic acid supplements. Recurrence risks for NTDs will relate directly to etiology. A metaanalysis of randomized trials of folic acid for the prevention of NTDS concluded that an 87 percent reduction in recurrence occurred in women who took supplements prior to conception.540 It is not possible to derive specific risk figures that are uniformly applicable worldwide. Etiologic heterogeneity, use of folic acid and known racial, ethnic, geographic, maternal age, and other factors

744

Genetic Disorders and the Fetus

Table 23.23 The risks of neural tube defect (NTD) according to family history Family history

One previous child with NTD

Risk of NTD

References

USA

UK

Canada

USA

UK

Canada

1.4–3.2

4.6 –5.2

2.4–6.0

449, 450, 538,

5, 587, 588

1, 592, 594

589, 595 Two previous children with NTD

6.4

10–20

4.8

449

589, 590, 591

593

Three previous children with NTD

–

21–25

–

–

590

–

Affected parent and one sibling with

See text

3

4.5

–

590

592

–

13

–

–

591

–

All first cousins

0.26

0–0.6

–

595

–

5

All maternal first cousins

–

–

0.9

–

–

592

All paternal first cousins

–

–

0.5

–

–

592

Affected maternal nephew/niece

0.99

–

0.6–1.3

596

–

1

One child with multiple vertebral

–

3–7

–

–

–

545

One child with spinal dysraphism

–

4

–

–

547

One sibling and a second-degree

–

9

–

–

590

–

7

–

–

590

NTD Affected parent and one sibling with NTD

anomalies

relative affected One sibling and a third-degree relative affected

confound any such effort. All counselors would agree that prenatal studies be recommended to women who have had one or more affected progeny. A similar consensus would lead to highresolution ultrasound for the pregnant siblings of women who have had affected offspring. Because the risks of the siblings of a male who sired a child with an NTD also having an affected child are higher than background, sonography for their partner is recommended. A similar situation applies to the first cousins of both parents of an affected child. All these family members should be offered genetic counseling and should be apprised of their risks and options. In experienced centers, MSAFP screening and targeted ultrasound studies in these family members are likely to be efficacious. Mothers with SB have a 0.5–1 percent risk of having a child with a NTD.541,542 In studies of Irish families with NTDS,543,544 maternal aunts and uncles had more congenital defects (especially NTDS and heart defects) than those on the paternal side. Adverse pregnancy outcomes (including preterm deliveries, miscarriages and stillbirths) were similarly in excess on the

maternal side. The authors caution that confirmatory studes are needed. Other variations require consideration. Congenital vertebral anomalies may involve single or multiple vertebrae and may affect any portion of each bone. Wynne-Davies concluded from both genetic and epidemiologic evidence that multiple vertebral anomalies in the absence of SB were causally related to the NTDs.545 She estimated that after the birth of a child with multiple vertebral anomalies, the recurrence risk is 2–3 percent, while that for an NTD is 3–7 percent. Prenatal studies are therefore clearly recommended in these women at risk. Spinal dysraphism, a disorder in which the conus medullaris is tethered and possibly associated with various anomalies of the cord, vertebrae or overlying skin,546 was also observed to have etiologic associations with NTDs.547 A further illustration of this association is the report of monozygotic twins, one with a lipomyelomeningocele and the other with a tethered cord.547a Spinal dysraphism in one child also provides a clear indication for prenatal studies in subsequent pregnancies.

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Maternal Serum Screening for Neural Tube and Other Defects 745

Whether adults with SB occulta have an increased risk of bearing progeny with NTD remains unsettled. Critical questions of an epidemiologic nature (e.g. ascertainment bias, variations in diagnostic interpretation, age ranges, number of cases) can be leveled at most available studies.333,548 A safe policy in these cases is the offer of MSAFP screening with targeted ultrasound study. The recognition that open and occult SB may occur as autosomal dominant disorders in Mormon83 and perhaps some other families should give rise to caution in counseling. Nutritional supplementation Dietary deficiency, probably of folate, was suspected for many years in the pathogenesis of NTDs.4 In 1989, we published results of the first prospective, broadly based, large (22,776 women) midtrimester study, which examined the relation of multivitamin (with and without folic acid) intake and the risk of NTDs.22 The prevalence of NTDs was 3.5 per 1,000 among women who never used multivitamins before or after conception or who used multivitamins before conception only. The prevalence of NTDs for women who used folic acid-containing multivitamins during the first 6 weeks of pregnancy was significantly lower: 0.9 per 1,000.22 We concluded that multivitamins with folic acid taken when planning pregnancy and for the first 6 weeks after conception provided about 70 percent protection against NTDs. An additional important observation was the strikingly higher prevalence of NTDs in women with a positive family history who did not take supplements (13.0 of 1,000), compared with those with a family history who did (3.5 of 1,000). In 1991, the UK Medical Research Council (MRC) multicountry, randomized, double-blind intervention trial was published.52 The study aimed to determine whether supplementation with folic acid or a mixture of seven other vitamins taken at about the time of conception could prevent a recurrence of NTDs. Analysis of 1,195 women who had at least one previous affected offspring, and for whom pregnancy outcomes were known, revealed a 72 percent protective effect (RR 0.28; 95 percent CI 0.12–0.71). Although a large daily dose (4 mg) of folic acid was used, no harmful effect was noted. Much lower effective doses (0.36 mg daily) were

used by Smithells et al.52a and were recorded by us.22 Subsequently, a major Hungarian doubleblind, randomized intervention trial based on preventing the occurrence of NTDs clearly demonstrated the efficacy of periconceptional folic acid supplementation.549 The authors used multivitamins containing 0.4–0.8 mg folic acid taken at least 1 month before and 3 months after conception. In their nonsupplemented group, the NTD rate was six in 2,104, while no case occurred among the 2,052 women who took supplements. The reason that some 30 percent of cases of NTD occur despite folic acid supplementation is unclear, as is the reported reduced efficacy of folic acid supplementation in Hispanic women.550 Lessons from experiments with curly-tail mice suggest that this folate resistance might be overcome by dietary supplementation with the Bcomplex vitamin myoinositol.551 Apparently, myoinositol treatment stimulates protein kinase C activity and upregulates retinoic acid receptor-β expression, thereby reducing delay in closure of the posterior neuropore. The weight of the evidence supporting periconceptional folic acid supplementation for all women for the purpose of avoiding NTDs led to a call for public health policy. Consequently, the Centers for Disease Control and Prevention (CDC) in the United States issued clear recommendations in 1992 that all women of child-bearing age who are capable of becoming pregnant take 0.4 mg/day of folic acid daily.552 An expert advisory group in the United Kingdom made a similar recommendation,553 including 4–5 mg/day of folic acid for women who had previously had an affected child. The difficulties – economic, educational, and personal – of successful implementation of this recommendation were recognized, and an alternative policy of fortification of grain products with folic acid was approved by the US Food and Drug Administration in 1996.554 However, the level that was set (140 mg/100 g of grain product) is considerably less than the target amount because it will, on average, raise the folic acid intake only by about 100 mg per day.555 Hesitation in implementing an optimal food fortification policy has revolved around the safety issue.556 The lower than optimal amount of folic acid in fortified flour was chosen because that level would avoid ingestion by

746

Genetic Disorders and the Fetus

nontargeted consumers of more than 1,000 mg of folic acid per day. The prime concern is the “masking” of cobalamin deficiency by folic acid, precipitating the neurologic complications of pernicious anemia. Certain medications (e.g. methotrexate, some anticonvulsants, some sulfa drugs) may be less effective for patients taking folic acid. Claims that seizure frequency increased in epileptics taking 5 mg of folic acid three times per day for 1–3 years557 were not supported by the results of other studies, including those that were double blind and randomized.558,559 Other concerns for which data are weak relate to potential folate neurotoxicity, reduced zinc absorption, hypersensitivity to folic acid, and increased susceptibility to malaria.556 In contrast, possible advantages of food fortification with folic acid include a decrease in cardiovascular disease (associated with reduced homocysteine) and in the frequency of cervical and colorectal cancer.560 Mills has questioned how much folic acid is enough.561 Although many are reluctant to advocate higher folic acid intake, Wald et al.24 argued that even the widely recommended 4 mg per day dose is far from optimal for prevention. By analyzing 13 studies in which folic acid intake was correlated with serum folate levels, they demonstrate that daily intake of 5 mg is optimal and would reduce the risk of NTD by about 85 percent.24 As of early 2007, about 54 countries were fortifying grain with folic acid.562 Claims have also been made about the efficacy of periconceptional multivitamin use and the prevention of other congenital defects (e.g. conotruncal and other cardiac defects, cleft lip/palate, and urinary tract).563–567 Confirmation of such claims is needed in additional prospective studies. Because the DiGeorge/velocardiofacial syndrome occurs with typical conotruncal cardiac defects in up to 90 percent of cases, exclusion of these diagnoses by fluorescence in situ hybridization studies is necessary before claims can be credible. Reductions in the frequency of other congenital defects were not observed in our prospective study22 or in the UK MRC trial.52 Notwithstanding the indisputable benefits of folic acid supplementation, public health departments worldwide have failed miserably in educating women in their reproductive years about the

most significant advance ever in the avoidance of one of the most common congenital defects.570a An analysis by the US CDC of a 2007 national survey571 revealed that only 61 percent of women aged 18–24 years were aware of the need for folic acid consumption; only 6 percent knew when it should be taken; only 30 percent actually took daily folic acid. Further evidence of a decline in the NTD birth prevalence During the past few years, two important largescale studies have documented the changing livebirth prevalence of babies with NTDs. The CDC reported that the prevalence of NTDs in the United States between 1996 and 2001 declined by 23 percent (24 percent for SB and 21 percent for anencephaly) among approximately 3.5 million livebirths per year during this period.569 The prevalence decreased from 2.5 to 2.0 per 10,000 during this period. The report included all states except Maryland, New Mexico and New York. The authors commented that these declines have occurred coincidentally with the introduction of folic acid fortification of flour in the United States. More recently, a CDC birth certificate-based (and therefore not optimal) study reported on the birth prevalence of SB in racial/ethnic populations.570 This study examined prefortification, early and mid/ post fortification and recent postfortification of cereal grain products with folic acid over a 10-year period (1995–2005). From the early postfortification period (1999–2000) to the recent period (2003–2005), the birth prevalence of SB for all groups decreased 6.9 percent. While the decrease was 19.8 percent for black mothers, no significant decrease was noted for white and Hispanic mothers. Folic acid fortification in Canada,571 South Africa,572 and Australia592a has led to a reduction in NTDs. Another study done in northern China, in an area with a high rate of NTDs, and in southern China, in an area with a low rate of NTDs, examined changes in the birth prevalence of NTDs between 1993 and 1995 among 130,000 women who took folic acid (400 mg per day) at any time before or during pregnancy and 118,000 who had not taken folic acid.573 The prevalence in the high-risk area declined by almost 79 percent (from 4.8 to 1.0 per 1000) and in the low-risk region, the prevalence declined by 40 percent (from 1.0 to 0.6 per 1000).

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Maternal Serum Screening for Neural Tube and Other Defects 747

These studies provide strong evidence that dietary folic acid supplementation can reduce the prevalence of NTDs. Notwithstanding some success, Oakley has emphasized that over 17 years since the first randomized controlled trial, only 10 percent of the folic acid-preventable NTDs are actually being prevented.574

Patient and family considerations In developed countries, the use of obstetric ultrasound and maternal serum screening has made the unexpected birth of a child with anencephaly a relatively rare event. When prenatal diagnosis has not been made, almost all anencephalics are stillborn or die within hours or days of birth.575 Occasionally, such an infant may survive many months, more especially when parents insist on extreme life-prolonging measures. Serious morbidity and mortality complicate the lives of those children surviving with SB, their outlook depending on the severity of the lesion, its location, and the nature and expertise of the treatment provided. The degrees of handicap among survivors with SB were assessed in studies published 25–30 years ago.333,576 There is a paucity of more recent long-term outcome studies for SB. Results of two major cohorts followed for 20–25 years and up to 38 years are respectively summarized in Table 23.24.577–579 The first study of 117 children born with open SB, first assessed at 16–20 years of age, noted that only eight (7 percent) had little or no disability, while 25 (21 percent) had died within their first year, a total of 48 (41 percent) having died by 16 years of age. Among the 69 (59 percent) who survived to age 16 years, 60 had been shunted for hydrocephalus, two of whom became blind as a consequence. Mental retardation was noted in 22 (19 percent), seizures in 12 (17 percent), 52 (44 percent) with incontinence, and 35 (30 percent) being wheelchair dependent. Lifelong continuous care was required by 33 (28 percent). At 25-year follow-up, some 48 percent had died. In a further follow-up of these patients between 32 and 38 years, 54 percent had died.580 Among 54 survivors, 46 (85 percent) had had a shunt, 39 (72 percent) had an IQ = 80, and only 11 (20 percent) were fully continent. Others have reported fecal or urinary incontinence in 34–90 percent and 61 percent

respectively,544,581 bearing in mind patients’ ages, lesions, presence of hydrocephalus and other factors, let alone issues of case ascertainment. In the second study spanning 20–25 years of 118 children born with SB, about 71 (60 percent) had survived with 19 patients lost to follow-up. The range of complications seen in the first study is, as expected, reflected in the later study, with some notable improvement in the degree of morbidity. However, the high frequency (86 percent) of shunting required for the treatment of hydrocephalus is the same in both studies. In the second study, 41 percent of the shunted study population had 2–3 shunt revisions. Residual fecal incontinence at 25 years (between 8 and 16 percent) and the urinary incontinence (albeit well managed by self-catheterization in most) remained troublesome problems. Being wheelchair bound, mentally handicapped, and requiring lifelong continuous care have remained serious issues. On the positive side, 49 percent achieved university entrance and 45 percent were employed. Thirty-two percent required surgery for a tethered cord and 32 percent developed latex allergy, six of the 23 affected patients experiencing severe, life-threatening anaphylactic reactions. In an Austrian study,582 16 of 35 (46 percent) patients with SB had elevated latexspecific IgE antibodies compared to 5 percent and 8.9 percent in matched children with gastroschisis/ omphalocele and posthemorrhagic/congenital hydrocephalus respectively. The authors’ conclusion reflects the realization that there appears to be a propensity to latex sensitization among those with SB. Latex sensitization is recognized as a significant problem in children with SB.583 Among 32 latex tested and sensitized, 40 percent experienced urticaria, conjunctivitis, angio-edema, asthma or rhinitis.584 In a Canadian study of 104 patients with closed SB lesions, despite a high incidence of ankle/foot abnormalities, most walked and did not require a wheelchair.585 Fractures, especially of femurs and tibias, are not uncommon. Among 221 children, adolescents and adults with SB, fractures occurred in 23/1,000, 29/1,000 and 18/1,000 respectively.586 Functional independence is most influenced by the level of the lesion and the presence/absence of hydrocephalus. A Netherlands study found that 165 patients with SB (mean age 20 years 9 months)

748

Genetic Disorders and the Fetus

Table 23.24 Selected complications in two original cohorts of 117 and 118 patients with SB with an extended followup (%)577–580 Original

Survivors 16

Survivors

Mean age

Original

cohort (%)

years (%)

mean age

35 years (%)

cohort

25 years (%)

(32–38 years)

Deceased b

Survivors 20–25 years

Number with SB

117

69 (59)

61 (52)

54

118a

–

Died by age 1 year

25 (21)

–

–

–

–

28 (24)

–

Died by age 16 years

48 (41)

–

–

–

–

–

–

Died by age 25 years

5 (48)

71 (60)

–

–

–

–

–

–

Hydrocephalus and shunt

–

60 (87)

52 (85)

46 (85)

–

24 (86)

61 (86)

Visual defect (blind)

–

2b (6)

27 (44)

2 (3.7)

–

–

–

Mental retardation

–

22 (32)

–

18 (30)

–

12 (17)

Epilepsy

–

32 (17)

14 (23)

–

–

–

16 (23)

Urinary incontinence

–

52 (75)

45 (74)

–

–

–

60 (85)c

Wheelchair dependent

–

35 (51)

41 (67)

7 (13)

–

–

29 (41)

Lifelong continuous care

–

33 (48)

33 (54)

20 (37)

–

–

13 (18)

Chronic pressure sores

–

32 (46)

19 (31)

30 (55.6)

–

–

–

Unsatisfactory urinary

–

19 (28)

14 (23)

–

–

–

153

–

–

9 (15)

–

–

–

– –

15 (27.8)

(IQ = 80)

incontinence control Hypertension on treatment Depression on treatment

–

–

Obesity

–

23 (33)

16 (26)

4 (7)

–

–

–

30 (55.6)

–

–

Fecal incontinence

–

24 (30)

5 (3)

–

–

–

–

11 (16)

High school

–

–

College

–

–

–

–

–

–

26 (36)

–

–

–

–

Special education

–

35 (49)

–

–

–

–

–

Employed

26 (37)

–

–

–

13 (241)

–

–

33 (45)

Scoliosis

–

–

–

–

–

–

25 (49)

Tethered cord

–

–

–

–

–

–

23 (32)

Latex allergy

–

–

–

–

–

–

23 (32)

Cervical decompression/

–

–

–

–

–

19 (68)

11 (16)

–

–

–

–

–

–

tracheostomy/and/or gastrostomy Respiratory support a

2 (3.7)

Blind = 2.

b

19 patients lost to follow-up.

c

All maintained on clean intermittent catheterization of their bladders, 90% doing their own.

and hydrocephalus with a lesion at L2 or above were dependent as regards to sphincter control (98 percent), locomotion (79 percent), self-care (54 percent) and communication (15 percent).597 Erectile dysfunction and infertility are common and also dependent on the level of the lesion.598 Both males and females with SB may achieve reproduction.185 Those confined to a wheelchair are at risk

of becoming obese and developing the metabolic syndrome. A third of 34 with SB (ages 11–20 years) in one study developed this syndrome.599 Sutton et al.600 reported on survival and disability in 623 infants with NTDs born in the Dublin area between 1976 and 1987. Only 41 percent of the livebirths were still alive at 5 years. Among those who survived beyond 5 years, 75 percent had

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Maternal Serum Screening for Neural Tube and Other Defects 749

a disability, 56 percent being severely disabled; 51 percent had mobility limitations, 59 percent were incontinent, 42 percent had hydrocephalus and 17 percent had intellecutal disability. Many factors have an impact on survival for those born with open SB,594,600 and reported survival rates vary significantly. In Glasgow, Scotland, 71 percent survived to 5 years, whereas in the Atlanta region, cumulative survival was calculated to reach 84 percent.601,602 Although there is clear evidence of improving survival rates,602 death rates continue to climb through early adulthood; the two most common causes of death are unrecognized shunt malfunction and renal failure. Endstage renal failure in 25 patients with SB after hemodialysis and renal transplantation yielded 5-year survival rates around 80 percent.602 Patients with SB appear to be at an increased risk of bladder cancer. Austin et al.603 reported on 19 patients with a median survival of 6 months. Young age and advanced cancer at presentation were typical. While less frequent than SB (0.8–4 per 10,000 livebirths),604 encephaloceles are also associated with significant adverse consequences. A Canadian report of 85 patients showed 41 (48 percent) with normal development, nine (11 percent) with mild delay, 14 (16 percent) with moderate delay, and 21 (25 percent) with severe delay. Hydrocephalus, epilepsy, microcephaly, other brain abnormalities and the presence of brain tissue were associated with poor outcome. The multidisciplinary team in Seattle reported life expectancy data on their enormous experience with 904 of 1054 patients seen with SB between 1957 and 2000. Survival rates to 16 years of age prior to and after 1975 were 54 percent and 85 percent respectively. At age 16, survival with or without a shunt for hydrocephalus was not significantly different. However, at 34 years, those without shunts605 had a survival probability of 94 percent compared to 75 percent with shunts. The moral, ethical, and medicolegal aspects of care of the defective newborn have been thoroughly debated.606–610 The effects on a marriage of having a child with SB have also been repeatedly studied.611 The 10-year longitudinal study by Tew et al.612 demonstrated clear deterioration in the marital relationships of families who had at least one child with a major NTD, including a divorce rate twice

that of the general population. Although other studies have drawn similar conclusions concerning marital disharmony, not unexpectedly, some have noted little or no negative effect of having severely affected children in these families.613 Perhaps the least attention has been devoted to the degree of suffering and quality of life of the affected children, especially those most severely affected, who died before reaching their 10th birthday.614,615 Minimal attention has been paid to the long-range effects of a severely myelodysplastic child on his or her siblings. Shurtleff and Lamers’ observation of the remarkable rate of abandonment by the parents of children with SB is both poignant and telling, and one that has implications for the affected child and the unaffected siblings.616

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507. Wright C, Healicon R, English C, et al. Meckel syndrome: what are the minimum diagnostic criteria? J Med Genet 1994;31:482. 508. Sepulveda W, Sebire NJ, Souka A, et al. Diagnosis of the Meckel–Gruber syndrome at eleven to fourteen weeks’ gestation. Am J Obstet Gynecol 1997;176:316. 508a. Khaddour R, Smith U, Baala L, et al. Spectrum of MKS1 and MKS3 mutations in Meckel syndrome: a genotype-phenotype correlation. Mutation in brief #960. Online. Hum Mutat 2007;28:523. 509. Duncan SLB. Antenatal misdiagnosis of neural-tube defects. Lancet 1975;2:709. 510. Field B, Kerr C. Antenatal diagnosis of neural-tube defects. Lancet 1975;2:324. 511. Brock DJH. Biochemical and cytological methods in the diagnosis of neural tube defects. In: Steinberg AG, Bearn AG, Motulsky AG, et al., eds. Progress in medical genetics. Philadelphia: WB Saunders, 1977:1. 512. Seller MJ. Amniotic fluid alpha-fetoprotein and Turner’s syndrome. Lancet 1977;1:995. 513. Hankins GD, Rowe J, Quirk JG, et al. Significance of brown and/or green amniotic fluid at the time of second trimester genetic amniocentesis. Obstet Gynecol 1984;64:353. 514. Allen R. The significance of meconium in mid-trimester genetic amniocentesis. Am J Obstet Gynecol 1985; 152:413. 515. Benacerraf BR, Gatter MA, Ginsburgh F. Ultrasound diagnosis of meconium-stained amniotic fluid. Am J Obstet Gynecol 1984;149:570. 516. Chubb IW, Pilowsky PM, Springell HJ, et al. Acetylcholinesterase in human amniotic fluid: an index of fetal neural development? Lancet 1979;1:688. 517. Smith AD, Wald NJ, Cuckle HS, et al. Amniotic-fluid acetylcholinesterase as a possible diagnostic test for neural-tube defects in early pregnancy. Lancet 1979; 1:685. 518. Holzgreve W, Golbus MS. Amniotic fluid acetylcholinesterase as a prenatal diagnostic marker for upper gastrointestinal atresias. Am J Obstet Gynecol 1983; 147:837. 519. Drugan A, Syner FN, Belsky R, et al. Amniotic fluid acetylcholinesterase: implications of an inconclusive result. Am J Obstet Gynecol 1988:159:469. 520. Campbell J, Cass P, Wathen N, et al. First-trimester amniotic fluid and extraembryonic coelomic fluid acetylcholinesterase electrophoresis. Prenat Diagn 1992; 12:609. 521. Drugan A, Syner FN, Greb A, et al. Amniotic fluid alpha-fetoprotein and acetylcholinesterase in early genetic amniocentesis. Obstet Gynecol 1988;72:35. 522. Collaborative Acetylcholinesterase Study. Amniotic fluid acetylcholinesterase electrophoresis as a second-

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ary test in the diagnosis of anencephaly and open spina bifida in early pregnancy. Lancet 1981;1:321. Brock DJH, Barron L, van Heyningen V. Prenatal diagnosis of neural tube defects with a monoclonal antibody specific for acetylcholinesterase. Lancet 1985;1:5. Boogert A, Aarnoudse JG, de Bruijn HWA, et al. Falsenegative amniotic fluid acetylcholinesterase in a case of meningo-encephalocele. Prenat Diagn 1989;9:133. Voigtländer T, Friedl W, Cremer M, et al. Quantitative and qualitative assay of amniotic-fluid acetylcholinesterase in the prenatal diagnosis of neural tube defects. Hum Genet 1981;59:227. Coombes EJ, Wood PJ, Spencer K, et al. Improved discrimination in the detection of neural tube defects: five biochemical tests compared. Clin Chim Acta 1982;122:249. Burton BK. Positive amniotic fluid acetylcholinesterase: distinguishing between open spina bifida and ventral wall defects. Am J Obstet Gynaecol 1986;155:984. Appleyard ME, Smith AD. Secretion of acetylcholinesterase and butyrylcholinesterase from the guineapig isolated ileum. Br J Pharmacol 1989;97:490. Heydanus R, Raats MAM, Los FJ, et al. Prenatal diagnosis of fetal abdominal wall defects: a retrospective analysis of 44 cases. Prenat Diagn 1996;16:411. Gilbert WM, Nicolaides KH. Fetal omphalocele: associated malformations and chromosomal defects. Obstet Gynecol 1987;70:633. Wald NJ, Cuckle HS. Nomogram for estimating an individual’s risk of having a fetus with open spina bifida. Br J Obstet Gynaecol 1982;89:598. Milunsky A, Sapirstein VS. Prenatal diagnosis of open neural tube defects using the amniotic fluid acetylcholinesterase assay. Obstet Gynecol 1982;59:1. Crandall BF, Kasha W, Matsumoto M. Prenatal diagnosis of neural tube defects: experiences with acetylcholinesterase gel electrophoresis. Am J Med Genet 1982;12:361. Read AP, Fennell S, Donnai D, et al. Amniotic fluid acetylcholinesterase: a retrospective and prospective study of the qualitative method. Br J Obstet Gynaecol 1982;89:111. Barlow RD, Cuckle HS, Wald NJ. False positive gelacetylcholinesterase results in blood-stained amniotic fluids. Br J Obstet Gynaecol 1982;89:821. Goldfine C, Haddow J, Hudson G, et al. Densitometry as an aid in amniotic fluid gel acetylcholinesterase analysis. Am J Obstet Gynecol 1983;145:317. Sheffield LJ, Sackett DL, Goldsmith CM, et al. A clinical approach to the use of predictive values in the prenatal diagnosis of NTDs. Am J Obstet Gynecol 1983;145:319. Janerich DT, Piper J. Shifting genetic patterns in anencephaly and spina bifida. J Med Genet 1978;15:101.

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539. Elwood JM, Mousseau G. Geographical, secular and ethnic influences in anencephalus. J Chronic Dis 1978; 31:483. 540. Grosse SD, Collins JS. Folic acid supplementation and neural tube defect recurrence prevention. Birth Defects Research (Part A) 2007;79:737. 541. Erata M, Grover S, Dunne K, et al. Pregnancy outcome and complications in women with spina bifida. J Reprod Med 2000;45:743. 542. Shurtleff DB. Epidemiology of neural tube defects and folic acid. Cerebrospinal Fluid Res 2004;1:5. 543. Byrne J. Birth defects in uncles and aunts from Irish families with neural tube defects. Birth Defects Res A Clin Mol Teratol 2008;82(1):8. 544. Vande Velde S, van Bjervliet S, van Rentergham K, et al. Achieving fecal continence in patients with spina bifida: a descriptive cohort study. J Urol 2007;178(6): 2640. 545. Wynne-Davies R. Congenital vertebral anomalies: aetiology and relationship to spina bifida cystica. J Med Genet 1975;12:280. 546. Anderson FM. Occult spinal dysraphism: a series of 73 cases. Pediatrics 1975;55:826. 547. Carter CO, Evans KA, Till K. Spinal dysraphism: genetic relation to neural tube malformations. J Med Genet 1976;13:343. 547a. Spacca B, Buxton N. Spina bifida occulta and monozygotic twins. J Neurosurg Pediatr 2008;2:258. 548. Field B, Kerr C. Offspring of parents with spina bifida occulta. Lancet 1975;2:1257. 549. Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med 1992;327:1832. 550. Shaw GM, Schaffer D, Velie EM, et al. Periconceptional vitamin use, dietary folate, and the occurrence of neural tube defects. Epidemiology 1995;6:219. 551. Green ND. Inositol prevents folate-resistant neural tube defects in the mouse. Nat Med 1997;3:60. 552. Centers for Disease Control. Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. Morb Mortal Wkly Rep 1992;41:1. 553. Tucker KL, Mahnken B, Wilson PWF, et al. Folic acid fortification of the food supply. JAMA 1996;276:1879. 554. Food and Drug Administration. Food standards: amendment of standards of identity for enriching grain products to require addition of folic acid. Fed Regist 1996;61:8781. 555. Centers for Disease Control and Prevention and Prevention Working Group on Folic Acid. Position paper on folic acid food fortification and the prevention of spina bifida and anencephaly (SBA). Atlanta: Centers for Disease Control and Prevention, 1993.

556. Campbell NRC. How safe are folic acid supplements? Arch Intern Med 1996;156:1638. 557. Reynolds EH, Wales MB. Effects of folic acid on the mental state and fit-frequency of drug-treated epileptic patients. Lancet 1967;1:1086. 558. Norris JW, Pratt RF. A controlled study of folic acid in epilepsy. Neurology 1971;21:659. 559. Brown RS, DiStanislao PT, Beaver WT, et al. The administration of folic acid to institutionalized epileptic adults with phenytoin-induced gingival hyperplasia: a double-blind, randomized, placebo-controlled, parallel study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1991;71:565. 560. Beresford SAA. How do we get enough folic acid to prevent some neural tube defects? Am J Public Health 1994;84:348. 561. Mills JL. Fortification of foods with folic acid: how much is enough? N Engl J Med 2000;342:1442. 562. Centers for Disease Control and Prevention (CDC). Trends in wheat-flour fortification with folic acid and iron – worldwide, 2004–2007. Morb Mortal Wkly Rep 2008;57:8. 563. Czeizel AE. Prevention of congenital abnormalities by periconceptional multivitamin supplementation. BMJ 1993;306:1645. 564. Shaw GM, Lammer EJ, Wasserman CR, et al. Risks of orofacial clefts in children born to women using multivitamins containing folic acid periconceptionally. Lancet 1995;346:393. 565. Li DK, Daling JR, Mueller BA, et al. Periconceptional multivitamin use in relation to the risk of congenital urinary tract anomalies. Epidemiology 1995;6:212. 566. Botto LD, Khoury MJ, Mulinare J, et al. Periconceptional multivitamin use and the occurrence of conotruncal heart defects: results from a populationbased, case-control study. Pediatrics 1996;98:911. 567. Czeizel AE, Toth M, Rockenbauer M. Populationbased case control study of folic acid supplementation during pregnancy. Teratology 1996;53:345. 568. Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med 1992;327:1832. 569. Mathews TJ, Honein MA, Erickson JD. Spina bifida and anencephaly prevalence – United States, 1991– 2001. MMWR Recomm Rep 2002;51:9. 570. Centers for Disease Control and Prevention (CDC). Racial/ethnic differences in the birth prevalence of spina bifida – United States, 1995–2005. Morb Mortal Wkly Rep 2009;57:1409. 570a. Brent RL, Oakley GP. The folate debate. Pediatrics 2006;117(4):1419. 571. Centers for Disease Control and Prevention (CDC). Use of supplements containing folic acid among

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women of childbearing age – United States, 2007. Morb Mortal Wkly Rep 2008;57:5. Sayed AR, Bourne D, Pattinson R, et al. Decline in the prevalence of neural tube defects following folic acid fortification and its cost-benefit in South Africa. Birth Defects Res A Clin Mol Teratol 2008;82:211. Berry RJ, Li Z, Erickson JD, et al. Prevention of neuraltube defects with folic acid in China. N Engl J Med 1999;341:1485. Oakley GP Jr. Elimination of folic acid-preventable nerual tube defects. Am J Prev Med 2008;35:606. Kalucy M, Bower C, Stanley F, et al. Survival of infants with neural tube defects in Western Australia 1966– 1990. Paediatr Perinatol Epidemiol 1994;8:334. McLaughlin J, Shurtleff D, Laners J, et al. Influence of prognosis on decisions regarding the care of newborns with myelodysplasia. N Engl J Med 1985;312:1589. Hunt GM. Open spina bifida: outcome for a complete cohort treated unselectively and followed into adulthood. Dev Med Child Neurol 1990;32:108. Hunt GM, Poulton A. Open spina bifida: a complete cohort reviewed 25 years after closure. Dev Med Child Neurol 1995;37:19. Bowman RM, McLone DG, Grant JA, et al. Spina bifida outcome: a 25-year prospective. Pediatr Neurosurg 2001;34:114. Hunt GM, Oakeshoff P. Outcome in people with open spina bifida at age 35: prospective community based cohort study. BMJ 2003;326:1365. Verhoef M, Lurvink M, Barf HA, et al. High prevalence of incontinence among young adults with spina bifida: description, prediction and problem perception. Spinal Cord 2005;43(6):331. Eiwegger T, Dehlink E, Schwindt J, et al. Early exposure to latex products mediates latex sensitization in spina bifida but not in other diseases with comparable latex exposure rates. Clin Exp Allergy 2006;36(10):1242. Pires G, Morais-Almeida M, Gaspar A, et al. Risk factors for latex sensitization in children with spina bifida. Allergol Immunopathol (Madr) 2002;30:5. Ausili E, Tabacco F, Focarelli B, et al. Prevalence of latex allergy in spina bifida: genetic and environmental risk factors. Tur Rev Med Pharmacol Sci 2007;11(3):149. Ross M, Brewer K, Wright FV, et al. Closed neural tube defects: neurologic, orthopedic, and gait outcomes. Pediatr Phys Ther 2007;19(4):288. Dosa NP, Eckrich M, Katz DA, et al. Incidence, prevalence, and characteristics of fractures in children, adolescents, and adults with spina bifida. J Spinal Cord Med 2007;30(suppl 1):S5. Carter CO, David PA, Laurence KM. A family study of major central nervous system malformations in South Wales. J Med Genet 1968;5:81.

588. Owens JR, Simpkin JM, Garris F. Recurrence rates for neural tube defects. Lancet 1985;1:12. 589. Carter CO, Roberts JA. The risk of recurrence after two children with central nervous-system malformations. Lancet 1967;1:306. 590. Smith C. Implications of antenatal diagnosis. In: Emery AEH, ed. Antenatal diagnosis of genetic disease. London: Churchill Livingstone, 1973:137. 591. Nevin NC, Johnston WP. Risk of recurrence after two children with central nervous system malformations in an area of high incidence. J Med Genet 1980; 17:87. 592. Lippman-Hand A, Fraser FC, Cushman Biddle CJ. Indications for prenatal diagnosis in relatives of patients with neural tube defects. Obstet Gynecol 1978;51:72. 592a. Chan AC, van Essen P, Scott H, et al. Folate awareness and the prevalence of neural tube defects in South Australia, 1966–2007. Med J Aust 2008;189:566. 593. McBride ML. Sibling risks of anencephaly and spina bifida in British Columbia. Am J Med Genet 1979;3:377. 594. Bamforth SJ, Baird PA. Spina bifida and hydrocephalus: a population study over a 35-year period. Am J Hum Genet 1989;44:225. 595. Toriello H, Higgins JV. Occurrence of neural tube defects among first-, second-, and third-degree relatives of probands: results of a United States study. Am J Med Genet 1983;15:601. 596. Zackai EG, Spielman RS, Mellman WJ, et al. The risk of neural tube defects to first cousins of affected individuals. In: Crandall BF, Brazier MAB, eds. Prevention of neural tube defects: the role of alpha-fetoprotein. New York: Academic Press, 1978:99. 597. Verhoef M, Barf HA, Post MW, et al. Functional independence among young adults with spina bifida, in relation to hydrocephalus and level of lesion. Dev Med Child Neurol 2006;48(2):114. 598. Bong GW, Royer ES. Sexual health in adult men with spina bifida. Scientific World J 2007;7:1466. 599. Nelson MD, Widman LM, Abresch RT, et al. Metabolic syndrome in adolescents with spinal cord dysfunction. J Spinal Cord Med 2007;30(suppl 1):S127. 600. Sutton M, Daly LE, Kirke PN. Survival and disability in a cohort of neural tube defect births in Dublin, Ireland. Birth Defects Res A Clin Mol Teratol 2008; 82:701. 601. Dastgiri S, Gilmour WH, Stone DH. Survival of children born with congenital anomalies. Arch Dis Child 2003;88:391. 602. Patrick GM, Mahony JF, Disney AP. The prognosis for end-stage renal failure in spinal cord injury and spina bifida: Australia and New Zealand. Aust NZ J Med 1994;24:36.

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603. Austin JC, Elliott S, Cooper CS. Patients with spina bifida and bladder cancer: atypical presentation, advanced stage and poor survival. J Urol 2007;178(3 Pt 1):798. 604. Lo BW, Kulkarni AV, Rutka JT, et al. Clinical predictors of developmental outcome in patients with cephaloceles. J Neurosurg Pediatr 2008;4:254. 605. Davis BE, Daley CM, Shurtleff DB, et al. Long-term survival of individuals with myelomeningocele. Pediatr Neurosurg 2005;41:186. 606. Duffs RS, Campbell AGM. Moral and ethical dilemmas in the special-care nursery. N Engl J Med 1979; 289:890. 607. Hayden PW, Shurtleff DB, Broy AB. Custody of the myelodysplastic child: implications for selection for early treatment. Pediatrics 1974;53:254. 608. Venes JL, Juttenlocher PR, Paxson CL Jr, et al. Management of the infant with unmanageable disease. N Engl J Med 1974;290:518. 609. Robertson JA. Discretionary non-treatment of defective newborns. In: Milunsky A, Annas GJ, eds. Genetics and the law. New York: Plenum Press, 1976:451.

610. Burt RA. Authorizing death for anomalous newborns: ten years later. In: Milunsky A, Annas GJ, eds. Genetics and the law, vol. III. New York: Plenum Press, 1985:259. 611. Hare EH, Laurence KM, Payne H, et al. Spina bifida and family stress. BMJ 1966;2:757. 612. Tew BJ, Payne H, Laurence KM. Must a family with a handicapped child be a handicapped family? Dev Med Child Neurol 1974;16:95. 613. Li HR, Borjeson M-C, Lagerkvist B, et al. Children with myelomeningocele: the impact of disability on family dynamics and social conditions: a Nordic study. Dev Med Child Neurol 1994;36:1000. 614. Tew BJ, Laurence KM. The effects of admission to hospital and surgery on children with spina bifida. Hydrocephalus Spina Bifida 1976;37(suppl):119. 615. Herskowitz J, Marks AN. The spina bifida patient as a person. Dev Med Child Neurol 1977;19:413. 616. Shurtleff DB, Lamers J. Clinical considerations in the treatment of myelodysplasia. In: Crandall BF, Brazier MAB, eds. Prevention of neural tube defects: the role of alpha-fetoprotein. New York: Academic Press, 1978:103.

24

Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities Howard S. Cuckle1 and Peter A. Benn2 1

Department of Obstetrics and Gynecology, Columbia University, New York, NY, USA, and School of Medicine, University of Leeds, UK, 2 Diagnostic Human Gentics Laboratories, University of Connecticut Health Center, Farmington, CT, USA

In the past, antenatal screening for chromosomal abnormalities was a simple matter of noting the maternal age and referring for invasive prenatal diagnosis those regarded by local policy or national convention as having increased risk. A family history of aneuploidy also was generally regarded as sufficient grounds for prenatal diagnosis. But the discovery in the early 1980s that secondtrimester maternal serum α-fetoprotein (AFP) levels were reduced on average in pregnancies affected by fetal aneuploidy led to a sea change in clinical practice. Routine screening, based on testing maternal serum for multiple markers together with the determination of one or more ultrasound markers, can now obtain a 4–5-fold increase in the proportion of affected pregnancies detected antenatally and a considerable decrease in the extent of invasive testing. However, the screening methods needed to achieve this benefit are complex, involve statistical manipulation and are expressed in unfamiliar terms. The focus here is to clarify such screening by revealing step-by-step the underlying principles and explaining the terminology as well as demonstrating the relative efficiency of different screening policies.

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

Chromosomal abnormalities Aneuploidy is a common event in pregnancy with a wide spectrum of medical consequences from the lethal to the completely benign. Most affected embryos abort spontaneously early in the first trimester, many of them even before there are clinical signs of pregnancy. Those that survive into the second trimester also experience high late intrauterine mortality and increased risk of infant death. Viability and clinical outcome vary according to the genotype and this chapter will concentrate on the more common forms of aneuploidy which are sufficiently viable to survive to term in relatively large numbers and are amenable to screening. By far the most frequent of these is Down syndrome (DS), with a birth prevalence in the absence of prenatal diagnosis and therapeutic abortion of 1–2 per 1,000 in developed countries. Consequently it is considered first and more extensively than Edwards and Patau syndromes, which have respectively about one-tenth and one-twentieth the birth prevalence, and the sex chromosome aneuploidies which are common but relatively benign. Screening and prenatal diagnosis There is a fundamental difference between screening and diagnostic tests, despite the fact that the same terms are used to describe their results: “true positive,” “false positive,” “true negative” and “false

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negative.” The aim of screening is limited to the identification from among apparently healthy individuals of those at high enough risk of a specific disease to warrant further investigation. In the context of chromosomal abnormalities these investigations involve hazardous invasive procedures – chorionic villus sampling (CVS), amniocentesis and occasionally percutaneous umbilical cord sampling – to obtain material for prenatal diagnosis. Thus screening does not aim to make a diagnosis, but rather to ration the use of diagnostic procedures that would be too hazardous, and tests that would be too expensive, to offer without prior selection. Screening tests based on multiple markers are provided unselectively to all women. The principal markers are continuous variables whose distribution of values is higher or lower on average in affected pregnancies. Typically, screening markers have considerable overlap in the distribution of results between affected and unaffected individuals. In contrast, the distribution of values for a variable used in diagnosis would have virtually no overlap. The potential utility in screening of a given marker depends on the extent of separation between the two distributions. This can be expressed as the absolute difference between the distribution means divided by the average standard deviation for the two distributions, a form of Mahalnobis distance.* For continuous variables the choice of a cut-off level that determines whether a value is positive or negative is arbitrary as there is no intrinsic division between the distributions. The choice will be influenced by the perceived relative importance of three factors. They are the detection rate (DR), the proportion of affected pregnancies referred for prenatal diagnosis, the false-positive rate (FPR), the proportion of unaffected pregnancies referred, and the positive predictive value (PPV), the chance of being affected given that the result is positive. The prior risk in those screened will influence all three factors so published values from case–control studies or estimated when screening a high-risk population are not generally applicable. *The Mahalnobis distance of a value v is (v-m)/s, where m and s are the distribution mean and standard deviation. In this chapter we use the term to refer to |ma − mu|/((sa + su)/2), were ma, mu, sa and su are the affected and unaffected means and standard deviations.

Widely used markers Of the more than 50 maternal blood, maternal urine or ultrasound markers of DS, seven are widely used in routine multi-marker screening today. These are maternal serum human chorionic gonadotropin (hCG), the free β-subunit of hCG, α-fetoprotein (AFP), unconjugated estriol (uE3), inhibin A and pregnancy associated plasma protein (PAPP)-A, and ultrasound nuchal translucency (NT). Maternal serum AFP was the first analyte to be used in DS screening after the observation that levels were reduced on average in pregnancies with chromosome abnormalities in general1 and DS in particular,2 in both the first and second trimester. Umbilical cord serum and amniotic fluid (AF) AFP levels are also lower than normal. The biology of AFP was discussed in Chapter 23. The reason for decreased AFP synthesis is unknown, but in the second trimester it may reflect hepatic immaturity. Histologic study has revealed undervascularization of the placentae of fetuses with various chromosomal defects, possibly representing placental immaturity with arrested or delayed angiogenesis.3 In the first trimester AFP is predominantly of fetal yolk sac origin. Maternal serum hCG4 and free α-hCG5 levels are increased on average in DS pregnancies, the latter in both the first and second trimester although the extent of increase is greater as pregnancy progresses. Gonadotropins are glycoproteins with epitopes on the protein surface and hCG is a 39.5 kDa glycoprotein made up of two nonidentical α- and β-subunits that exist either free or bound to each other. Free α-hCG levels are also increased in DS but the marker is not widely used in screening. Abnormality in cytotrophoblast differentiation may be the basis of the elevated hCG levels in DS pregnancies.6 Six different genes code for the β-subunit of hCG, while only one α-subunit gene is known thus far. Not all the factors involved in hCG secretion are known, but cyclic adenosine monophosphate (cAMP), prolactin, corticosteroids, and gonadoliberin influence release, while polyamines, estradiol, and progesterone inhibit release. Total maternal urinary estriol excretion in the third trimester of DS pregnancies is lower than in unaffected pregnancies7 and subsequently the level of maternal serum uE3 was also found to be lower

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than average8 in both the first and second trimester. In the DS fetus there is adrenal hypoplasia and the adrenal cortex produces dehydroepiandrosterone sulfate (DHEAS), which the fetal liver hydroxylates. The newly formed product, 16-αhydroxy-DHEAS, is formed in the fetal liver by hydroxylation of DHEAS and transported to the placenta, where it undergoes desulfation and aromatization into estriol. Inhibin levels have been shown to be increased in DS pregnancies, using assays that detect all species9 and those specific for inhibin-A.10 These increases are not as marked before 13 weeks gestation as they are later. Inhibin is a dimer of 32 kDa with an α-subunit and one of two similar but distinguishable β-subunits. Of two mature forms, dimeric inhibin-A and inhibin-B, only the former is present in pregnancy sera. Inhibin is considered to have a role in the regulation of gonadotropin biosynthesis and secretion, ovarian and placental steroidogenesis, and oocyte maturation. Inhibin is regarded as a member of the transforming growth factor β super family and is characterized by its ability to suppress follicle-stimulating hormone secretion. PAPP-A levels are reduced in first-trimester DS pregnancies11 but this reduction diminishes as pregnancy progresses and there is little or no difference by the second trimester. PAPP-A is a 750 kDa α2 mobile glycoprotein containing 16 atoms of zinc and has a high affinity for heparin. In maternal serum PAPP-A is complexed with the proform of eosinophil major basic protein. PAPP-A is a protease for insulin-like growth factor binding protein 4 and may therefore play a role in regulating fetal growth and trophoblast proliferation. The reason for the low levels in DS is not known but it is likely to be connected with placental insufficiency and maybe the same mechanism that leads to low levels in nonviable pregnancies.12 NT is increased in DS pregnancies13 but there is a narrow window at 11–13 weeks (crown–rump length (CRL) 45–85 mm) when subcutaneous edema can readily be measured in the fetal neck. NT is visualized in the sagittal section used for crown–rump length and it is recommended that a standardized technique is adopted for measurement.14 The reasons for the increased edema in DS are not known but the most plausible explanations are: altered composition of the cellular matrix,15

abnormal or delayed development of the lymphatic system and cardiac insufficiency. Additional ultrasound markers Four additional ultrasound markers can be determined at the same time as NT measurement. These are absence of the fetal nasal bone (NB), abnormal blood flow in the ductus venosus (DV), tricuspid regurgitation (TR), both requiring pulse-wave Doppler, and the frontal-maxillary facial angle (FMF), requiring three-dimensional scanning (see Chapter 25). Currently, relatively few centers are sufficiently proficient to determine these markers routinely but this is likely to change in the near future. In particular, many centers performing NT now have experience with NB. The “anomaly scan” or “genetic sonogram” routinely carried out in the second trimester can also be used in screening. The presence of a major anomaly is a risk factor and there are a number of so-called “soft” markers that can be determined at this time (see Chapters 6 and 25). These include increased nuchal skinfold (NF), short femur and humerus lengths, hydronephrosis, echogenic intracardiac focus and echogenic bowel. Currently, the genetic sonogram is only used in the post hoc modification of risk among women considering amniocentesis, but second-trimester ultrasound markers could be formally incorporated into routine multimarker screening policies outside specialist centers. NF is the marker most suited to this and it can be readily coupled with two other facial profile markers, nasal bone length (NBL) and prenasal thickness (PT), as well as the long bone measurements. Allowing for gestation All seven widely used markers are continuous variables whose levels in unaffected pregnancies change with gestation. For the serum markers this is allowed for by the use of multiples of the gestationspecific median (MoMs) for unaffected pregnancies. Early ultrasound studies of NT did not allow for gestation at all, but levels are now being reported either in MoMs or deviations from the gestation-specific median. The best results are obtained when the unaffected medians are calculated to the day of gestation using regression curves. For NT some practitioners use center- or operator-specific curves.16

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Meta-analysis of all the published literature is probably the most reliable way to estimate, for each marker, the relative increase or decrease on average in DS pregnancies and the extent of separation between affected and unaffected distributions. The advantage of meta-analysis is that it produces the most robust estimate of the mean and by combining the results from a wide range of centers, it reflects the average experience likely to be achieved in practice. Parameters from a single study are subject to considerable sampling error as even the largest study to date includes no more than about 100 affected pregnancies. Nonintervention studies produce estimates of the means for cases present at term. Intervention studies introduce “viability” bias that will skew the results towards the extreme. This bias arises because a proportion of those with extreme marker levels who have a termination of pregnancy would have been destined to miscarry anyway whereas nonviable affected pregnancies with normal screening results will not be known to the investigators. In our analysis the average MoM for secondtrimester serum markers was derived from two published meta-analyses of nonintervention studies: hCG, free β-hCG, uE3 and AFP,17 and inhibin.18 Gestational age was largely based on ultrasound biometry. First-trimester gestationspecific average MoMs for serum mrkers were obtained from a published meta-analysis of nonintervention studies of PAPP-A only,19 combined with the results of a second meta-analysis that included some cases obtained by intervention20 and two more recent large studies that included an intervention component but not until the second trimester: SURUSS21 and FASTER.22,23 The means at each completed week were derived from the weighted average value observed in the four sources and then subjected to log-quadratic regression. The data in the second meta-analysis were from intervention studies that used NT as well as serum markers and it can be estimated that this will have led to a 1.5 percent reduction in the mean PAPP-A level, 1 percent increase in the mean free β-hCG and 0.5 percent in hCG. The observed means were therefore adjusted by these proportions. The average DS MoM for NT was derived from a meta-analysis of intervention and nonintervention studies,24 updated to include the latest

Table 24.1 Mean level in DS for each widely used marker, according to gestation and the Mahalnobis distance Marker

Gestation

DS cases

MoM

(weeks) NT

distance

11

2.30

2.02

2.10

1.87

1.92

1.65

0.40

1.31

0.45

1.14

12

0.53

0.90

13

0.65

0.61

1.66

0.76

1.86

0.94

12

2.01

1.05

13

2.09

1.11

2.30

1.33

1.03

0.05

1.18

0.32

12

1.41

0.68

13

1.77

1.14

12

962

13 PAPP-A

10 11

Free β-hCG

892

10 11

14–18 hCG

Mahalnobis

626

477

10 11

467

14–18

850

2.02

1.15

AFP

14–18

1,140

0.73

0.79

uE3

14–18

613

0.73

0.83

Inhibin A

14–18

603

1.85

1.12

FASTER data which included measurements on cases with septated cystic hygromas, previously excluded.23 To overcome viability bias, all studies were used to estimate the rate of change in NT with gestation in a weighted within-study regression, but the intercept was estimated using the weighted average among the intervention studies adjusted for the extent of viability bias previously reported.25 The sources of data used to estimate the standard deviations needed for the Mahalnobis distance are specified in the Likelihood Ratios section below. Table 24.1 shows the average MoM for each marker together with the Mahalnobis distance, based on these meta-analyses. As a guide, maternal age, which is a poor DS screening variable, has a Mahalnobis distance of about 1 and AFP when used to screen for spina bifida has a value of about 2.4. NT is by far the single best individual marker. Among the serum markers, PAPP-A is the most discriminatory but the

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 775

Mahalnobis distance declines rapidly with increasing gestation. Free β-hCG is more discriminatory at 14–18 weeks than at 10–13 weeks, although there is a gradual change in Mahalnobis distance between 10 and 18 weeks. At 14–18 weeks’ gestation, hCG is less discriminatory than free β-hCG and before 13 weeks it is a poor marker. At 14–18 weeks inhibin A is of comparable discriminatory power to hCG. AFP and uE3 are not very discriminatory markers. Risk screening It can be shown statistically that the optimal way of interpreting the multi-marker profile is to estimate the risk of DS from the marker levels.26 This is done by modifying the prior risk, that pertaining to the situation before testing, by a factor known as the “likelihood ratio” derived from the marker profile. This posterior risk is then compared to a fixed cut-off risk. If the risk is greater than the cutoff the result is regarded as positive, otherwise it is negative. This approach will yield a higher detection rate for a given false-positive rate than any other method of test interpretation developed to date. It also provides a way of encapsulating the result for the purposes of counseling. The method is flexible enough to provide a risk even if a single marker is used and can incorporate both physical or biochemical markers. The prior risk of DS, based on maternal age and family history can be expressed as a probability, say p, or a rate of 1 in 1/p, and needs to be converted into an odds of p:(1-p) or 1:(1-p)/p. The posterior risk is calculated by multiplying the left-hand side of the odds by the likelihood ratio from the marker profile (x). The result re-expressed as the rate of 1 in 1 + (1-p)/px, probability px/(1 + p(x-1)), or odds 1:(1-p)/px. The prior risk can be expressed as the chance of having a term pregnancy with the disorder or the chance of the fetus being affected at the time of testing. Insofar as the aim of screening is to reduce birth prevalence, the former is the most appropriate. But screening is also about providing women with information on which to base an informed choice about prenatal diagnosis, in which case it can be argued that the latter is more relevant. This calculation assumes that the marker levels and age are independent determinants of risk, and

that the marker levels are unrelated to the probability of intrauterine survival. There is, however, evidence that extreme values of some of the biochemical and ultrasound markers are associated with an increased rate of fetal demise (see below). Age-specific risk at term The best available estimate is obtained by metaanalysis of published birth prevalence rates for individual years of age that were carried out before prenatal diagnosis became common. Four metaanalyses have been published based on 11 different maternal age-specific birth prevalence series. The studies differed in the number of series included, the method of pooling series, the type of regression equation and the extent to which the maternal age range was restricted. In the first, all eight series published at that time were included with a total of 4–5,000 DS and more than 5 million unaffected births.27 For each year of age, data were pooled by taking the average birth prevalence rate across the series weighted by the number of births. A three-parameter additiveexponential regression equation was used of the form y = a + exp(b + cx) where y is prevalence and x is age. A single regression was performed over the entire age range. In the second study the same eight series were included but a separate analysis was carried out for the two series that the authors regarded to be most complete.28 Pooling was by summation of the birth prevalence numerators and denominators. Two different additiveexponential regression equations were used, the linear equation above and a five-parameter version with a cubic exponential component. The maternal age range was restricted in four ways (15–49, 20– 49, 15–45, 20–45). The third study included four series comprising the two “most complete” series above, extended by more recent data, and two newer series.29 A separate analysis was carried out after excluding one of the new series. Pooling was by summation. Three-, five- and six-parameter additive-exponential regression equations were used, the latter having a quartic exponential component. There was no age restriction. The last study included nine series, six of the original eight, with the updated recent data, the two additional series used in the third study and a further new series.30 A separate analysis was carried out after

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Genetic Disorders and the Fetus

excluding one of the original series. Pooling is by the use of a weighting factor which estimates the proportional underascertainment in each series. The regression analysis simultaneously estimates the curve parameters and this proportion. A threeparameter logistic regression equation is used of the form y = a + (1-a)/(1 + exp(-b-cx)) where a is between 0 and 1. There was no age restriction. From the perspective of overall detection rates and false-positive rates, there is little practical difference between the 19 regression curves published in the different meta-analyses over the 15–45 year age range. The real differences emerge at older ages: for example, at age 50 the risks range from 1 in 5 to 1 in 18. There is no simple way of deciding which of the curves is the most accurate since the age-specific rates differ between the component series of the meta-analyses, partly due to underascertainment, use of assisted reproductive technologies by older women, and possibly due to real underlying differences between the populations. Another curve has been published using data on about 11,000 cases from the UK National Down Syndrome Cytogenetic Register (NDSCR), a very complete national database.31 The estimates differ from those obtained by previous meta-analyses: significantly higher at ages 36–41 and considerably lower after age 45. These discrepancies may be due to the fact that 45 percent of NDSCR cases were diagnosed prenatally and 82 percent of these ended in termination of pregnancy, whereas the previous series were collected before antenatal screening and prenatal diagnosis became widespread. To estimate birth prevalence, it was necessary to allow for intrauterine survival following prenatal diagnosis and the authors used the same survival rate at all ages and indications for prenatal diagnosis. However, there is evidence that survival is age dependent in both unaffected and DS pregnancies (see below). Risk at the time of the test Some screening programs report the risk of DS at the time of the test rather than the risk of a term affected pregnancy. This can be calculated from the intrauterine survival rates of DS from the first and second trimesters, say p1 and p2. The relative risk in the first trimester, second trimester and at birth is 1/p1:1/p2:1. Studies of prenatal diagnosis are used

to estimate fetal loss rates, either by comparing the observed number of cases with that expected from birth prevalence, given the maternal age distribution, or by follow-up of individuals declining termination of pregnancy, using direct or actuarial survival analysis. Published prevalence studies include a total of 341 DS cases diagnosed by CVS and 1,159 at amniocentesis.32 There are three published follow-up series including 110 cases diagnosed at amniocentesis33 and a series of 126 cases from the NDSCR which has been analyzed according to the gestational age at prenatal diagnosis.34 However, the Register study is biased as some miscarriages may have occurred in women who did intend to have a termination, thus inflating the rates. An actuarial survival analysis of the Register data has now been carried out35 which overcomes the bias and is more data efficient since all cases contribute to the estimate, not just those where termination was refused. Actual and potential heterogeneity between the various studies precludes a grand meta-analysis to estimate the fetal loss rates. But an informal synthesis has been carried out and reached the conclusion that about one-half of DS pregnancies are lost after first-trimester CVS and one-quarter after midtrimester amniocentesis.36 Formulae have been published from a large series of more than 57,000 women having invasive prenatal diagnosis because of advanced maternal age alone37; a recent reanalysis of the data has yielded the quadratic formula for the DS survival rate: 0.739286-0394765*x-000524864*x2, where x is gestation in completed weeks (R. Snijders and K. Nicolaides, personal communication). These calculations assume that fetal loss rates do not vary with maternal age.38 However, the studies used to calculate the overall rates are largely based on women aged over 35 so this cannot be readily examined. Since the fetal loss rate in general increases markedly with maternal age39 it is likely that this will also happen in DS pregnancies, and this appears to be confirmed in a NDSCR actuarial survival analysis based on 5,116 registered DS pregnancies, of which 271 ended in a livebirth and 149 in a fetal loss; the remainder were terminated.40 The overall estimated fetal loss rates from the time of CVS and amniocentesis were close to other studies, but these rates increased steadily with

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 777

maternal age, from 23 percent and 19 percent at age 25 to 44 percent and 33 percent at age 45. One caveat, though, is that the observed maternal age effect is confounded by differences in marker levels. A large proportion of the prenatally diagnosed cases will have been detected because of a positive result following routine antenatal screening. But the marker distribution in screen positives varies according to maternal age. Thus in young women their marker profile will tend to be extreme whereas in older women, even those with moderate profiles can have a screen-positive result because of the contribution of their advanced age to the risk. Since extreme values of most of the markers are associated with impending or actual fetal loss, there must be some confounding. This would tend to mask some of the underlying effect and the rate of increase in losses with age may be even greater than that observed. On the other hand, NDSCR has underascertainment of DS pregnancies in younger women which will have an effect in the opposite direction. The bias will be present because younger women have lower screening DRs and less uptake of invasive prenatal diagnosis, so that more affected pregnancies that miscarry never come to attention. Likelihood ratios All seven widely used markers follow an approximately log Gaussian distribution over most of their range for both DS and unaffected pregnancies. These Gaussian distributions are defined by the marker means and standard deviations after log transformation. The likelihood ratio (LR) for a single marker is calculated by the ratio of the heights of the two overlapping distributions at the specific level. For extreme results that fall beyond the point where the data fit a Gaussian distribution, it is standard practice to use the LR at the end of the acceptable range. The method is the same for more than one marker except that the heights of multivariate log Gaussian distributions are used. These are defined, in addition to means and standard deviations, by the correlation coefficients between markers within DS and unaffected pregnancies. The standard deviations and correlation coefficients are probably best derived by meta-analysis. The most accurate results are obtained, where pos-

sible, by tailoring the variance/co-variance matrices, derived from the standard deviations and correlation coefficients, to the population being tested. Briefly, this involves using meta-analyses to derive the difference in variance/co-variance matrix between DS and unaffected pregnancies. Then the latter is added to the matrix for unaffected pregnancies in the local population. In the current analysis we use published metaanalyses, which had been derived by tailoring, for the standard deviations and within-trimester correlation coefficients of first- and second-trimester serum markers.17–19 For NT, the standard deviations were from four large prospective studies combined41 giving a single value in DS and gestation-specific values in unaffected pregnancies; no correlation with serum markers was assumed. The between-trimester correlation coefficients were from a meta-analysis24 updated to include more recent data.23 The acceptable limits of each marker are from the SURUSS study.21 The parameters are shown in Table 24.2. In this chapter we use a single set of parameters but in practice two sets are needed for the serum markers. The variance/co-variance matrices are different in women whose gestational age is based on menstrual dates and those where ultrasound biometry has been used. Although an individual scan estimate of gestation has wide confidence limits, on average scanning is the more precise method and leads to a reduction in variance. In contrast, the mean marker profile should not differ according to the dating method. Infants with DS are growth restricted at term42 but early biometric measures other than long bone measurements do not appear to be altered in early pregnancy. An international multicenter collaborative study has investigated possible bias in the two main biometric measures of gestation, crown–rump length and biparietal diameter (BPD).43 In 55 case–control sets using the former and 146 for the latter, the median difference in measurements was zero for both measures. Modeling performance Two widely used methods of estimating DS screening detection and false-positive rates are numerical integration and Monte Carlo stimulation. Numerical integration uses the theoretical log Gaussian

778

Genetic Disorders and the Fetus

Table 24.2 Standard deviations and correlation coefficients of log10 MoM values for the widely used marker in DS and unaffected pregnancies, according to gestation Marker

Gestation (weeks)

DS

Unaffected pregnancies

Standard deviation NT

11 12

0.132 0.229

13

0.116 0.112

PAPP-A

10–13

0.326

0.285

Free β-hCG

10–13

0.290

0.287

14–18

0.302

0.244

10–13

0.225

0.210

14–18

0.282

0.247

AFP

14–18

0.181

0.165

uE3

14–18

0.192

0.138

Inhibin A

14–18

0.265

0.213

PAPP-A & free β-hCG

10–13

0.13

0.11

PAPP-A & hCG

10–13

0.27

0.23

AFP & free β-hCG

14–18

0.16

0.06

AFP & hCG

14–18

−0.01

0.12

AFP & uE3

14–18

0.37

0.21

hCG

Correlation coefficient*

AFP & inhibin

14–18

0.08

0.16

free β-hCG & uE3

14–18

−0.14

−0.14

free β-hCG & inhibin

14–18

0.37

0.32

hCG & uE3

14–18

−0.22

−0.09

hCG & inhibin

14–18

0.44

0.38

uE3& inhibin

14–18

−0.13

0.01

PAPP-A & AFP

10–13/14–18

0.11

0.08

PAPP-A & free β-hCG

10–13/14–18

−0.30

0.16

PAPP-A & hCG

10–13/14–18

−0.11

0.10

PAPP-A & uE3

10–13/14–18

0.24

0.09

PAPP-A & inhibin

10–13/14–18

−0.16

0.11

free β-hCG & AFP

10–13/14–18

−0.04

0.04

free β-hCG & free β-hCG

10–13/14–18

0.78

0.76

free β-hCG & hCG

10–13/14–18

0.42

0.56

free β-hCG & uE3

10–13/14–18

−0.22

−0.02

free β-hCG & inhibin

10–13/14–18

0.31

0.34

hCG & AFP

10–13/14–18

0.11

0.07

hCG & free β-hCG

10–13/14–18

0.57

0.72

hCG & hCG

10–13/14–18

0.69

0.72

hCG & uE3

10–13/14–18

−0.17

0.03

hCG & inhibin

10–13/14–18

0.25

0.32

* All correlations with NT assumed to be zero.

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 779

distributions of each marker in DS and unaffected pregnancies.26 The theoretical range is divided into a number of equal sections, thus forming a “grid” in multidimensional space. The Gaussian distributions are then used to calculate for each section (square for two markers, cube for three, etc.): the proportion of DS and unaffected pregnancies in the section and the LR. It is then a matter of applying these values to a specified maternal population. At each maternal age the number of DS and unaffected pregnancies is estimated from the age-specific risk curve. The distributions of DS risks are then calculated from the grid values. Monte Carlo stimulation also uses the Gaussian distributions but instead of rigid summation over a fixed grid, it uses a random sample of points in multidimensional space to simulate the outcome of a population being screened. Other models have been fitted to the marker distributions but none have been found to be as effective as Gaussian. For example, in recent years an empirical model of NT values has been promoted41 but the group behind this has now moved to a Gaussian approach, albeit using two sets of distributions for DS pregnancies whose proportions differ according to gestational age (the socalled “mixture model”).44 It remains to be seen if this improves on a simple Gaussian approach. The model-predicted detection and false-positive rates are highly dependent on the maternal population specified, usually a national population whose maternal age structure has been published. An alternative is to standardize for age by using a standard female population and a set of agespecific fertility rates45 or simply to use a Gaussian distribution of maternal ages.46 We use the latter in this analysis with mean age 27 and standard deviation 5.5 years. Whichever method is used, the comparison of performance between policies is reasonably robust regardless of the population specified. When assessing the relative benefits of different policies it is best to either fix the false-positive rate (e.g. 1 percent or 5 percent) and compare the detection rates or fix the detection rate (e.g. 75 percent or 85 percent) and compare the false-positive rates. However, when changing policy it would be confusing to alter the cut-off risk in order to maintain the DR or FPR as before. So in practice

it is common to retain the cut-off (e.g. 1 in 250 at term or 1 in 270 at mid trimester) and allow both DR and FPR to vary. In this chapter performance is presented using all three methods.

Current multi-marker policies Most experience with serum screening is in the second trimester, where it became a natural extension to established AFP screening programs for neural tube defects (NTDs), largely at 15–19 weeks’ gestation. At this time most centers use a twomarker combination of either hCG or free β-hCG and AFP, a three-marker set that adds uE3 or four markers with the inclusion of inhibin-A. It has become common to refer to these different combinations of second-trimester markers by the shorthand “double”, “triple” and “quadruple” tests. Although this may be convenient, it can be misleading and restrictive. The ordinal implies that the screening efficiency of the triple is necessarily better than the double and, worse, lower than the Quadruple. And then there is an implication of uniqueness. Thus “triple” has become solely usable for the particular second-trimester combination of three markers for which it was first coined. Other three-marker combinations also have high screening potential but they, or indeed the same second trimester combination performed in the first trimester, cannot be called triple tests, and this may restrict their use. Over the last decade there has been a gradual realization of the benefits of moving prenatal screening from the second to the first trimester. These include earlier diagnosis, less traumatic and safer termination of pregnancy if requested, and earlier reassurance. Using serum alone, this would be limited to PAPP-A plus either hCG or free βhCG, which are weaker combinations than any of the second-trimester protocols. Some centers initially used ultrasound NT alone but the best results are obtained by the combination of serum markers and NT. There is an important practical constraint influencing the design of such policies, namely that the results of a scan can be reported to the patient immediately whereas a serum result will not usually be available for a number of days. The reason for the delay is that biochemical assays are normally

780

Genetic Disorders and the Fetus

done in batches, which, to avoid unnecessary expense, include about 50–100 samples. However, new techniques have been developed which allow single samples to be tested economically and with results available in an hour. This means that if the test equipment is installed close to the ultrasound unit, combined serum and ultrasound results can be reported together (sometimes known as OSCAR – one-stop risk assessment). Concurrent screening can also be performed without such equipment provided a blood sample is obtained a few days before the scheduled scan appointment and arrangements made to ensure that the serum MoMs are available for risk calculation as soon as the NT is measured (sometimes known as IRA – instant risk assessment). The combination of serum markers and NT is commonly referred to as the “combined” test, which is again misleading because other combinations of markers are possible. Indeed, the combination of first- and secondtrimester serum markers with and without NT will yield even better results than combinations within the first trimester. One approach is to measure all markers when they are most discriminatory, namely PAPP-A and NT in the first trimester but delay hCG or free β-hCG measurement until the second trimester, and also testing the second sample for AFP, uE3 and inhibin.21 This six-marker combination, known as the “integrated” test, requires nondisclosure of any intermediate risk based on the PAPP-A and NT levels. A serum-only version, the “serum integrated” test, has also been proposed. Some regard the nondisclosure to be unethical, or at least impractical due to the difficulty for the professional of not acting on intermediate findings which would of themselves be abnormal – particularly the NT – and any increase in detection is paid for by sacrificing early diagnosis and reassurance. Alternative two-stage sevenmarker strategies have been suggested to overcome these limitations. One approach is the “stepwise sequential” test where the first stage is the same as the combined test and women with risks below the cut-off are offered the same second-trimester markers as the quad test with the final risk based on all markers.22 To avoid a very high false-positive rate, this is best done using a much higher than usual combined test cut-off. The “contingent” test is similar except that only women whose risk is

borderline after the first stage are offered the second-stage markers.47 Another approach, the “independent sequential” test, albeit statistically invalid, is being practiced to some degree by default, namely by carrying out a combined test followed by a quad test and calculating separate risks from each.48 Independent sequential screening is invalid since the second-trimester test risk does not incorporate all the available risk-related information, and therefore this approach should be avoided. In this chapter we estimate the performance of each policy and despite the caveats above, for ease of communication, do adopt the commonly used names.

Model predictions Table 24.3 shows the model predicted DRs and FPRs for second-trimester double, triple and quad tests. For a fixed 5 percent false-positive rate, the detection rate ranges from 56 percent to 71 percent. Combinations using free β-hCG yielded a higher DR than those using total hCG. A greater increase in detection was seen between the triple and quad tests compared to that between the double and triple tests. For a fixed 75 percent detection rate, the FPR approximately halved from the best to the worst combination. Table 24.4 shows the predicted rates for NT alone and combined tests according to gestation. The detection rate for a fixed FPR declines with advancing gestation, but even with NT alone at 13 weeks it is comparable with the best DR using secondtrimester serum markers. The combined test performs considerably better than NT alone at all gestations. As with the second trimester, the use of free β-hCG improves detection compared with total hCG when a combined test is carried out before 13 weeks. Despite this, another modeling exercise claims that there is no material difference.49 The model used parameters from the FASTER trial together with hCG levels based on the retrospective assaying of stored serum samples from only 79 DS and 395 unaffected pregnancies. Larger data sets are needed before concluding that there is no difference between free β-hCG and total hCG.50 Table 24.5 shows the predicted rates for the sequential test strategies. The combined test can achieve a higher DR than the serum integrated test

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 781

Table 24.3 Second-trimester screening policies, according to hCG type hCG type

DR for FPR 1%

FPR for DR 5%

DR & FPR for cut-off risk

75%

85%

Term 1 in 250

Mid trimester 1 in 270

Double Free β-hCG

37%

61%

12%

22%

62% & 5.2%

67% & 7.4%

hCG

33%

56%

16%

29%

56% & 5.2%

62% & 7.6%

Free β-hCG

42%

65%

20%

64% & 4.7%

69% & 6.7%

hCG

39%

60%

26%

59% & 4.6%

64% & 6.8%

Free β-hCG

50%

71%

6.9%

15%

68% & 4.2%

73% & 5.9%

hCG

46%

67%

9.3%

20%

64% & 4.3%

69% & 6.0%

Triple 9.9% 14%

Quad

Table 24.4 First-trimester screening using NT alone and combined with serum markers, according to gestation hCG type

Gestation (serum/NT)

DR for FPR 1%

FPR for DR 5%

75%

DR & FPR for cut-off risk 85%

Term 1 in 250

Mid trimester 1 in 270

NT alone −/11

64%

77%

3.8%

12%

73% & 2.9%

76% & 4.1%

−/12

62%

75%

4.8%

15%

70% & 2.7%

73% & 3.8%

−/13

57%

71%

7.7%

22%

66% & 2.8%

69% & 4.1%

10/11

74%

87%

1.1%

3.6%

82% & 2.4%

84% & 3.2%

11/11

74%

87%

1.2%

3.8%

81% & 2.4%

84% & 3.3%

11/12

73%

86%

1.3%

4.6%

80% & 2.4%

82% & 3.4%

12/12

72%

84%

1.5%

5.3%

79% & 2.5%

82% & 3.5%

12/13

68%

82%

2.4%

7.3%

76% & 2.7%

79% & 3.8%

13/13

66%

80%

2.9%

8.8%

75% & 2.8%

78% & 4.0%

10/11

71%

85%

1.5%

4.8%

80% & 2.5%

82% & 3.4%

11/11

71%

84%

1.6%

5.3%

79% & 2.5%

82% & 3.5%

11/12

70%

83%

1.9%

6.5%

77% & 2.5%

80% & 3.5%

12/12

70%

83%

1.9%

6.6%

77% & 2.5%

80% & 3.5%

12/13

65%

80%

3.0%

9.1%

74% & 2.7%

77% & 3.9%

13/13

67%

81%

2.4%

7.4%

76% & 2.7%

79% & 3.8%

Combined Free β-hCG

hCG

but the full integrated test would increase detection for a fixed 5 percent FPR by more than 10 percent. However, both the stepwise sequential and contingent tests have a predicted rate comparable with the integrated test. Given the human and practical benefits, and lower costs, the contingent test should be the sequential strategy of choice. The table also

clearly shows another reason why the independent sequential test is to be avoided.

Prospective intervention studies In general, statistical modeling may be a useful technique for comparing competing policy options

782

Genetic Disorders and the Fetus

Table 24.5 Sequential screening according to strategy, according to first-trimester hCG type and gestation* First-trimester

GA

hCG type

DR for FPR 1%

FPR for DR 5%

75%

DR & FPR for final cut-off risk# 85%

Term 1 in 250

Mid trimester 1 in 270

Integrated Serum only

Serum & NT

11

61%

78%

3.7%

10%

74% & 3.2%

77% & 4.5%

13

55%

73%

5.7%

14%

70% & 3.7%

74% & 5.2%

11

85%

93%

0.3%

1.1%

87% & 1.6%

89% & 2.1%

13

79%

89%

0.6%

2.5%

84% & 2.0%

86% & 2.7%

11

85%

94%

0.4%

1.0%

89% & 1.7%

91% & 2.2%

13

80%

91%

0.6%

1.9%

86% & 2.1%

88% & 2.8%

11

86%

94%

0.4%

0.9%

89% & 1.6%

90% & 2.1%

13

80%

91%

0.6%

1.9%

85% & 2.0%

87% & 2.6%

11

85%

92%

0.4%

1.0%

88% & 1.6%

89% & 2.0%

13

79%

88%

0.7%

2.3%

84% & 1.9%

85% & 2.4%

11

84%

90%

0.4%

1.2%

86% & 1.4%

87% & 1.8%

13

79%

88%

0.6%

2.5%

83% & 1.8%

85% & 2.3%

Stepwise sequential Free β-hCG

hCG

Contingent Free β-hCG

hCG

Independent Free β-hCG

hCG

11

74%

84%

1.5%

6.1%

83% & 4.5%

85% & 6.3%

13

72%

84%

1.5%

5.7%

83% & 4.5%

86% & 6.3%

11

74%

84%

1.5%

5.7%

84% & 4.5%

86% & 6.4%

13

73%

84%

1.5%

5.4%

84% & 4.5%

86% & 6.3%

* All strategies use first-trimester PAPP-A and second-trimester AFP, free β-hCG, uE3 and inhibin and apart from the integrated test, they also use first-trimester free β-hCG; the stepwise sequential, contingent and independent tests use a 1 in 50 at term first stage cut-off (equivalent to 1 in 38 at mid trimester) and the contingent test uses a 1 in 1500 lower cut-off (1 in 1200 at mid trimester). # Cut-off for the integrated test, the second stage of the sequential and contingent tests, based on all first- and second-trimester markers included, and for the independent test based on the second-trimester markers alone.

but models rest on many assumptions and need to be validated. In the current context there are two questions to be addressed: how reliable is the model-predicted DR and FPR values and how accurate are the individual risk estimates? There are sufficient published results to show that both aspects of modeling are robust but two problems need to be considered before this can be demonstrated. Firstly, the observed detection rate in DS screening intervention studies is necessarily an overestimate of the true rate because of the nonviability bias described above in relation to mean NT esti-

mation. One unbiased estimate derived from the observed numbers of DS cases (screen-detected terminated (n1) or not (n2), missed by screening but terminated subsequently (n3) or born (n4)), using the formula (n1*p + n2)/(n1*p + n2 + n3*p + n4), where p is the intrauterine survival rate for DS at the time of prenatal diagnosis. Another approach is to calculate the expected number of DS births, given the maternal age distribution of screened women, e, and use the formula 1 − (n2 + n4)/e. Secondly, the confidence limits on a DR estimate in even the largest intervention study will be

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 783

quite large and meta-analysis of all published studies would seem to be the best option. Whilst screening protocols differ markedly in terms of marker combination, cut-off and maternal age distribution, pooling the results, with suitable adjustment for viability bias, is a guide to actual performance. Nevertheless, detailed comparisons, say between centers using two markers and those with three, are probably precluded. There are 25 large second-trimester serum studies that can be analyzed; 20 of the 21 cited in51, 52, one of which has been updated,53 and seven published more recently.54–62 This includes results for a total of 234,000 women having the double test including 322 observed with DS which yields an observed DR of 66 percent, equivalent to 59 percent after allowance for bias using the overall survival rate, and a FPR of 5.0 percent. For 612,000 women having a triple test, of whom 1,021 were observed with DS, the DR was 72 percent, equivalent to an unbiased value of 67 percent, and FPR 6.5 percent. One large study reported a mixture of double and triple tests on 854,000 women, including 977 DS, with rates of 73 percent, 68 percent and 6.8 percent. There have so far only been three quad test prospective studies, totalling 86,000 women, 160 DS cases, and rates of 83 percent, 79 percent and 7.4 percent. Of the many studies using NT without serum markers, only six expressed the results in terms of risk.63–68 The combined results include a total of 142,000 screened women of whom 643 were observed to have a DS fetus. This yielded an observed DR of 84 percent, equivalent to 72 percent after allowance for bias using the overall survival rate, and a FPR of 8.4 percent. There have so far been 15 studies of the combined test69–82: 145,000 women, 638 DS, 89 percent observed and 81 percent unbiased detection rate, with 5.9 percent FPR. Prospective intervention studies of the serum integrated, integrated and stepwise sequential tests have now been published.83–87 Serum integrated testing was performed on 11,159 women though only 79 percent had both steps, including 16 DS cases; the observed DR was 87 percent and FPR 3.2 percent.84 In one study integrated testing was only completed by 75 percent of women85; among 2,332 screened including 12 DS cases, the observed DR

was 83 percent and false-positive rate was 2.9 percent.86 In a second study 92 percent completed both steps and of the 32,227 screened including 86 DS cases, the observed DR was 88 percent and false-positive rate 3.1 percent.83 Stepwise sequential testing was offered to 1,528 women and 78 percent of those with negative results at the first stage had second-trimester tests, there were only three DS cases and all were identified; the overall FPR was 6.9 percent.87 The shortfall in uptake of the second stage in these sequential strategies is a particular problem for the integrated test as a proportion of women will not have been given any risk assessment. In general, the prospective studies confirmed model predictions, in essence showing that whatever the choice of cut-off risk, the performance is consistent with model predictions. The firsttrimester studies yielded DRs lower than the model predictions for the optimal 10 and 11 weeks’ gestations but in keeping with modeling at 12 and 13 weeks. However, this does not say anything about accuracy of individual risks, which could be quite imprecise, whilst the performance for the whole population is acceptable. In nine studies the results were published in such a way that the validity of individual risk estimation could be assessed as well as overall performance.63,88–95 These studies broke down the results into groups according to the estimated risk used on the test report. For each group, the average risk was given together with the observed DS prevalence, adjusted for viability bias. They all found that the numbers of affected cases within each group were close to the expected number based on the reported risks. The impact of screening can also be judged to some extent by national trends in birth prevalence. In the UK data from the NDSCR show that the number of pregnancies terminated following prenatal diagnoses of DS increased steadily from 290 in 1989 to 767 in 2006 whilst the number of livebirths remained relatively static (www.wolfson. qmul.ac.uk/ndscr/reports) despite the expected increase due to an aging pregnant population. Birth certificate data for the US are also consistent with steady rates of DS births nationally despite demographic changes,96 and regional data with close to complete ascertainment would also appear to indicate the same trends.97

784

Genetic Disorders and the Fetus

The retrospective analysis of data in a nonintervention study can sometimes also be used to evaluate policy. The FASTER trial directly demonstrated that the combined test could achieve a better performance than the quad test22 and a recent reanalysis has been used to compare contingent, stepwise sequential and integrated tests.23 Marker levels from women who completed both the first and second stages of the trial – intervention was in the second stage – were used to calculate DS risks. There were 86 DS and 32,269 unaffected pregancies. The DR for the contingent test was 91 percent and FPR 4.5 percent; the initial DR was 60 percent, 1.2 percent initial FPR, and 23 percent had borderline risks. Stepwise testing had 92 percent DR and 5.1 percent FPR; integrated screening 88 percent and 4.9 percent respectively. This clearly confirms the model prediction of similarity between the three. It does not, however, provide a reliable estimate of DRs for any of them. The rates are underestimated because some early detected cases, particularly those with cystic hygromas, were excluded.

Further multi-marker strategies So far, only the widely used markers have been considered, but it is feasible to use additional ultrasound markers in the first and second trimester, either routinely or contingently. The possibility of using additional biochemical markers can also be considered as well as variants on the more standard policies. Contingent combined test The concept of contingent screening is appealing and has stimulated the development of other related strategies. The simplest version is to carry out the serum screening stage of a combined test on all women but restrict the NT stage to those who have relatively high DS risks after serum testing.98 This would be useful in a center with limited equipment or operators with adequate training in NT measurement. Modeling shows that in these circumstances a contingent combined test would yield a higher DR than other non-NT approaches. Thus modeling predicts that testing for PAPP-A and free β-hCG at 10 weeks and contingently measuring NT at 11 weeks on the one-

third of women with the highest risks would only reduce the detection rate for a 5 percent FPR from 87 percent to 82 percent, still considerably more than the 71 percent provided by a quad test. Raising the cut-off so that NT was offered to the one-fifth with high risks would yield a 77 percent DR. Another type of contingent combined approach is to carry out the NT scan and, depending on the results, then do the serum testing. In the FASTER trial the presence of cystic hygroma was sufficient to offer immediate invasive diagnostic testing because of the extremely high risk of aneuploidy and general poor prognosis. However, this is equivalent to a high NT MoM cut-off or very high NT-based risk since in most, though not all, firsttrimester cases of cystic hygroma there is increased NT. For example, in one series of 42 cystic hygroma cases found in nearly 7000 routine first-trimester scans, 35 had an NT of 3 mm or more.99 Three-stage contingent test The small loss of detection with the contingent combined test can be completely recouped by the contingent determination of second-trimester serum markers just like a standard sequential contingent test.100 The practicality of this approach has been confirmed in a 6-month trial carried out under the auspices of the UK National Screening Committee (www.pi.nhs.uk/screening/downs). Additional first-trimester ultrasound markers Several studies have found that absence of the nasal bone on ultrasound examination at 11–13 weeks was a highly discriminating DS marker. In one meta-analysis of nine studies there was absent NB in 69 percent of 397 cases and 1.4 percent of 12,652 unaffected pregnancies.101 From this, the LR for NB absence would be 49 and for presence 0.31. However, a simple LR might not be appropriate because of correlations with gestation, NT and ethnicity. A logistic regression formula is, however, available to calculate LR, taking account of these factors.101 Broadly, for NT ≤1.6 MoM at CRL of 45–64 mm, the LRs for absent and present NB are 26 and 0.37 respectively; for higher NT they are 12 and 0.29.102 The corresponding LRs at CRL of 65–84 mm are 72 and 0.43 and 35 and 0.33 respectively.

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 785

Table 24.6 Addition of ultrasound nasal bone determination* to three first-trimester policies, according to hCG type and gestation First-trimester

GA

hCG type

DR for FPR

FPR for DR

DR & FPR for cut-off risk#

1%

5%

75%

85%

Term 1 in 250

Mid trimester 1 in 270

11

80%

90%

0.8%

2.0%

85% & 2.1%

87% & 2.8%

13

76%

87%

0.9%

3.2%

83% & 2.5%

84% & 2.9%

11

86%

94%

0.2%

0.8%

89% & 1.5%

90% & 2.2%

13

82%

91%

0.4%

1.6%

86% & 1.8%

87% & 2.4%

11

84%

93%

0.4%

1.1%

88% & 1.7%

89% & 2.2%

13

83%

92%

0.4%

1.4%

86% & 1.8%

88% & 2.3%

11

92%

93%

0.1%

0.3%

91% & 0.8%

92% & 0.9%

13

89%

90%

0.2%

0.5%

89% & 1.0%

89% & 1.1%

11

91%

92%

0.1%

0.4%

90% & 0.8%

90% & 0.9%

13

89%

91%

0.2%

0.5%

89% & 0.9%

89% & 1.1%

NT & NB

Combined & NB Free β-hCG

hCG

Contingent & NB Free β-hCG

hCG

# Final cut-off for Contigent & NB based on all first- and second-trimester markers included. * First-stage cut-offs for Contigent & NB 1 in 50 and 1 in 1500.

Table 24.6 shows the results of modeling with these parameters when NB is added to NT alone, the combined test and the contingent test. There is a substantial increase in detection, which is greatest for NT alone, and it is so large that it reduces the relative benefits of different isoforms of hCG and gestation. The modeling assumes that NB is uncorrelated with the serum markers. A small reduction in PAPP-A and an increase in free β-hCG have been reported in affected pregnancies with absent NB compared with other DS cases where the NB could be seen, but these differences were not statistically significant.101,103 With a subjective marker like absent NB, there is the possibility of bias when carried out prior to invasive prenatal diagnosis. The screening marker profile is likely to influence the interpretation which could easily account for the observed results. A problematic aspect of NB is quality assurance since, unlike continuous variables such as NT, there is no satisfactory way of providing external quality control. It is relatively simple to establish whether an individual operator can identify the NB landmarks. But, since absent NB is a relatively rare event, the frequency with which the operator misclassifies absent NB as present or vice versa cannot

be easily determined. This consideration suggests a cautious approach to interpretation for the inexperienced operator. In women with high DS risk based on other testing, when there is absent NB it is reasonable to use an LR to increase the risk, since there is no great penalty for misclassification. Similarly in women with intermediate or borderline risks (see below). However, in women with high DS risk who apparently have NB presence, it may be prudent not to reduce the risk, if this would make the final result negative. First-trimester DS fetuses tend to have abnormal Doppler ductus venosus blood flow demonstrated by reduced end-diastolic velocities correlating with atrial contraction (A-wave) which may display reversed or absent velocities, or increased pulsatility index for veins (PIV). The seven studies summarized by Borrell104 and a more recent paper105 all found that a large proportion of DS fetuses had abnormal flow but the corresponding proportion in unaffected fetuses was variable. A contributor to this variability will be the qualitative assessment of the A-wave result: PIV is likely to be more reproducible. The recent study used PIV expressed in MoMs and yielded good results: DS median of 1.70 MoM and SDs similar in magnitude to NT105

786

Genetic Disorders and the Fetus

Some studies showed a correlation with NT but they used the subjective A-wave and so may have been biased by knowledge of the NT result. Modeling with the recent paramenters predicts that the addition of DV to NT alone or a combined test is of similar benfit to the addition of NB.105 Another trisomy marker that can be seen at the NT scan is tricuspid regurgitation. In a series of 742 singleton pregnancies, the tricuspid valve was examined by a specialist cardiologist and could be reliably seen in 718 cases.106 There was regurgitation in 65 percent of the 126 with DS. As with nasal bone, there was correlation with gestation and NT. Also among unaffected pregnancies, those with structural cardiac defects had much higher TR (47 percent) than those without (5.6 percent). The FMF angle was first investigated in a series of 3-D ultrasound volumes of the fetal profile.107 The angle was measured between the top of the maxilla and the bony forehead or the unfused metoptic suture with apex at the front of the maxilla. Images from 100 DS pregnancies and 300 controls were obtained before CVS; in 69 percent of cases the FMF angle was above the 95th centile of the controls, 40 percent were greater than the highest control and only two cases had an angle below the normal median. FMF angle appears to be independent of gestation, NT and NB. First-trimester contingent test At present the expertise required to carry out NB, DV, TR and FMF angle measurements is limited to expert fetal medicine units and it may be some time before these markers can be used routinely. This suggests a form of the contingent test whereby women with borderline risks based on firsttrimester serum markers and NT are immediately referred to a specialist center for the more advanced markers rather than waiting until the second trimester for further serum markers. This has been modeled predicting a 92–94 percent DR and 2.1– 2.7 percent FPR, depending whether NB, DV or TR is used in the 16 percent of women with borderline risks.108,109 Additional first-trimester serum markers The combined test might be improved by the addition of further first-trimester serum markers and

one possibility is the existing second-trimester markers AFP, uE3 and inhibin which also have some discriminatory power in the first trimester. We modeled this combining first-trimester gestationspecific DS means from a meta-analysis using reports with regression,20,21,110 inhibin means from a meta-analysis,111 other parameters using all firsttrimester gestations combined for AFP and uE3 in a meta-analysis,19 and for inhibin, and correlations with hCG from SURUSS.21 Table 24.7 shows that the additional markers each increase the DR for a 5 percent FPR by 1–3 percent; using more than one additional marker will improve detection further. Second-trimester combined test The routine combination of serum and ultrasound markers can considerably improve secondtrimester screening, just as it does in the first trimester. One approach is to use markers that can be readily measured when carrying out a BPD measurement. These are nuchal skinfold, nasal bone length, prenasal thickness and either humerus or femur length. A thick nuchal pad is a phenotypic feature of DS and is seen in most affected newborns. Since an increase in NF among DS pregnancies was first shown over 20 years ago,112 studies have generally not reported values in MoMs. In a meta-analysis of five studies in which MoMs were either reported or could be derived from a figure in the publication, the average was 1.45 MoM, a Mahalnobis distance of about 1.0.113 The only consistent correlation between NF and serum markers with hCG or free β-hCG is in DS pregnancies: an average of 0.32 over three studies.113–115 Table 24.8 shows, using the parameters from this meta-analysis, the model predictions of adding ultrasound NF to the quad test markers. There is an estimated 9–11 percent increase in DR for a 5 percent FPR. Nasal bone measurement rather than absence per se is also a marker, but not until the second trimester. In a first trimester series of 79 cases, 54 of which had absent NB, the remaining 25 had normal NB length (NBL).116 In the second trimester only a small proportion have absent NB but the remainder have reduced NBL.117,118 One possibility is to assign a NBL value at the lower truncation limit of the range when NB is absent and to use Gaussian methods to calculate risk. Modeling with

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 787

Table 24.7 Addition of further serum markers to the combined test, according to hCG type and gestation hCG type

GA

DR for FPR 1%

FPR for DR 5%

75%

DR & FPR for cut-off risk 85%

Term 1 in 250

Mid trimester 1 in 270

Combined alone Free β-hCG

hCG

11

74%

87%

1.2%

3.8%

81% & 2.4%

84% & 3.3%

13

66%

80%

2.9%

8.8%

75% & 2.8%

78% & 4.0%

11

71%

84%

1.6%

5.3%

79% & 2.5%

82% & 3.5%

13

67%

81%

2.4%

7.4%

76% & 2.7%

79% & 3.8%

11

74%

88%

1.1%

3.5%

82% & 2.3%

84% & 3.2%

13

66%

81%

2.6%

7.7%

76% & 2.7%

79% & 3.9%

11

71%

85%

1.5%

5.1%

79% & 2.5%

82% & 3.4%

13

68%

82%

2.3%

7.1%

76% & 2.5%

79% & 3.6%

11

75%

88%

1.0%

3.3%

81% & 2.1%

84% & 2.9%

13

68%

82%

2.2%

7.0%

76% & 2.5%

79% & 3.5%

11

72%

86%

1.3%

4.5%

79% & 2.2%

82% & 3.0%

13

70%

84%

1.8%

6.0%

77% & 2.3%

80% & 3.3%

11

75%

88%

1.0%

3.4%

82% & 2.3%

85% & 3.2%

13

70%

84%

1.7%

5.6%

79% & 2.7%

82% & 3.8%

11

73%

86%

1.3%

4.3%

80% & 2.4%

83% & 3.3%

13

71%

84%

1.6%

5.3%

79% & 2.6%

82% & 3.6%

Combined & AFP Free β-hCG

hCG

Combined & uE3 Free β-hCG

hCG

Combined & inhibin Free β-hCG

hCG

the parameters used by Maymon et al.118 predicts that this will achieve a further modest increase in detection when added to NB and the quad test markers (see Table 24.8), making the value of this test comparable to that for a first-trimester combined test. Modeling predicts an even greater increase when another facial feature, prenasal thickness (PT), is added, using parameters from the initial series of 21 DS cases and 500 controls118 which has now been confirmed in 26 DS cases and 135 controls.119 The short stature associated with children with DS is reflected in utero by smaller than average long bones measured by ultrasound. There have been proposals to incorporate into serum screening protocols either humerus length (HL)120 or femur length (FL), HL and NF.115 There are five papers from which FL or FL/BPD in MoMs can be derived,115,121–125 yielding an overall mean of

0.94 MoM and Mahalnobis distance 0.80. Small correlations between FL and uE3 in DS and between FL and AFP in unaffected pregnancies need to be confirmed.115 Based on the assumption of no correlations modeling predicts that adding FL to NF and the quad test markers increases detection by less than 2 percent.115 There is a high degree of correlation between FL and HL, and using both will increase detection by only a small amount. FMF angle could also be used in the second trimester, albeit with a different technique, and it yields results similar to the first trimester. Digitally stored images of fetal profiles of 34 DS and 100 normal fetuses were obtained prior to amniocentesis.126 The FMF angle was above the 95th centile in 79 percent of cases; changing the angle to include the skin over the forehead – taking advantage of the prenasal edema118 – the proportion detected increased to 88 percent. Unlike other markers,

788

Genetic Disorders and the Fetus

Table 24.8 Addition of ultrasound NF, NBL and PT to the quad test, according to hCG type hCG type

DR for FPR 1%

FPR for DR 5%

75%

DR & FPR for cut-off risk 85%

Term 1 in 250

Mid trimester 1 in 270

Quad alone

50%

71%

6.9%

15%

68% & 4.2%

73% & 5.9%

Free β-hCG

46%

67%

9.3%

20%

64% & 4.3%

69% & 6.0%

Free β-hCG

64%

80%

3.0%

8.4%

75% & 2.9%

78% & 4.1%

hCG

62%

78%

3.7%

10%

73% & 3.0%

76% & 4.2%

Free β-hCG

69%

84%

1.8%

5.5%

78% & 2.6%

81% & 3.5%

hCG

68%

83%

2.2%

6.7%

77% & 2.6%

80% & 3.7%

Free β-hCG

83%

93%

0.3%

1.3%

88% & 1.9%

90% & 2.6%

hCG

81%

92%

0.4%

1.5%

87% & 2.0%

89% & 2.7%

hCG Quad & NF

Quad, NF & NBL

Quad, NF, NBL & PT

there was no association between FMF angle and second-trimester gestational age.127 Sequential genetic sonogram In the second trimester, it has become common practice to provide an anatomic survey of the fetus prior to an amniocentesis and the results are used in a post hoc modification of the patient-specific risk.128 Likelihood ratios have been developed for specific anatomic abnormalities, or combinations of abnormalities, while an entirely normal ultrasound is associated with a risk reduction, typically by 50 percent.128,129 Consistent with this, a prospective study of women who had high risks based on a triple test found abnormal ultrasound findings or markers in 53 percent of 245 affected pregnancies and 14 percent of 8,707 unaffected pregnancies (positive LR 3.7, negative LR 0.55).130 Risk modification using this approach assumes that the serum screening test results and the ultrasound findings are uncorrelated. When the genetic sonogram is sequentially offered only to women who have high DS risk based on an initial DS screening protocol, the net effect will be a reduction in the FPR, but there will also be a reduction in DR, i.e. the sonogram gives false reassurance. The extent of these changes will

depend on the maternal age distribution in the population, the type of initial screening test, and cut-off, but typically the expected FPR reduction will be over one-third with a DR reduction of 5–6 percent.131 Among women who have their risk reduced sufficiently to make a positive test result negative, about 1 in 180–260 will actually have an affected pregnancy. This loss in detection can be offset by expanding the numbers of women who are referred for the genetic sonogram, to include those with borderline risks, as in a contingent test.131 Repeat measures markers and highly correlated markers Some markers will show a difference between affected and unaffected at one time point in pregnancy but not at other times. For example, hCG levels in DS pregnancies are essentially indistinguishable from unaffected pregnancies at 10 weeks but are raised on average in the second trimester. Conversely, PAPP-A levels are reduced on average in first-trimester affected pregnancies but gradually become normal as pregnancy progresses. Repeat measures in both trimesters will not only provide the results at the time when there is an informative difference but will also capture the

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 789

changing profile of the markers. It is possible that the characteristic change in marker concentrations over time provides some of the most useful information for screening. For populations of affected and unaffected pregnancies, the means, standard deviations at the two time points together with the between-measures correlation coefficients will record all the information needed to incorporate the changing marker concentrations into a screening algorithm. The benefit of repeat measures in aneuploidy screening was first pointed out by Wright and Bradbury.132 Based on modeling with SURUSS parameters, they demonstrated potentially highly effective screening using repeat measures, particularly with PAPP-A measured in the first and second trimester. A small case–control study on stored specimens demonstrated the value of adding a second-trimester PAPP-A assay to various screening protocols that included a first-trimester PAPP-A assay.133 Repeat measure screening is attractive not only because of the potentially impressive performance but also because it can be easily introduced into laboratories that are already providing sequential screening protocols. Repeat measures of a marker will generally show very high between-measure correlation coefficients for both affected and unaffected pregnancies. High correlations are not necessarily confined to repeat measures and when they are encountered, providing both tests should not necessarily be dismissed on the basis that a second test will yield little new information. For example, hCG and free β-hCG are highly correlated but it has been pointed out that the provision of both tests early in the first trimester could be advantageous.134 One caveat to this approach is that when tests are highly correlated, the correlation coefficients need to be very accurately established because the calculated risks can be strongly influenced by small differences in the values of these parameters.

growth. An early report documented very low levels of this protein in the serum from women with DS pregnancies at 6–11 weeks.135 A series of subsequent reports (reviewed elsewhere136) confirmed that the marker was low in early firsttrimester affected pregnancies although the levels were not as low as initially suggested. Despite a strong correlation with PAPP-A, modeling indicated that the addition of ADAM12s to PAPP-A and free β-hCG at 9 weeks followed by NT measurement at 12 weeks would substantially improve screening performance. However, by 12–13 weeks, the time at which most first-trimester screening is currently performed, ADAM12s levels in DS cases are close to normal and there are weak correlations with existing markers.137 In the second trimester, ADAM12s appears to be elevated on average in affected pregnancies138,139 and a modest improvement can be achieved when this marker is added to second-trimester screening protocols. Based on these observations, ADAM12s would appear to be a potentially valuable marker for very early screening and its temporal pattern in affected pregnancies may make it particularly useful in repeat measures protocols. Additional data are needed. Pregnancy-specific glycoprotein (SP)-1 Maternal serum SP-1 levels are reduced on average in the first trimester and increased in the second. The average level in a total of 111 published cases at 10–14 weeks’ gestation was 0.81 MoM and in 379 at 15–22 weeks was 1.47 MoM.140 The use of SP-1 as a fifth first-trimester serum marker would only increase the detection rate by about 1 percent whereas in the second trimester there would be a 2–4 percent increase.141 The first-trimester results would probably be better if samples were taken earlier than 11 weeks’ gestation as there is a tendency for the average level in DS pregnancies to become closer to 1 MoM as the first trimester advances.

Other markers A disintegrin and metalloprotease (ADAM)12s This is a placentally derived glycoprotein that digests insulin growth factors and may control fetal

Urinary hCG species There are several markers of DS in maternal urine. Although there is the additional complication of standardizing for concentration, as determined by

790

Genetic Disorders and the Fetus

the creatinine level, some of them have screening potential and a combination of urine and serum screening could be considered. The urine marker that has been most studied so far is the β-core fragment of hCG, its major metabolic product. In the second trimester of pregnancy the mean based on a meta-analysis of seven studies,142 extended to include two further studies,21,143 is 3.70 MoM with a Mahalnobis distance of 1.51. In the first trimester levels are also raised but to a much lower extent. Other urinary hCG species, intact hCG, free β-hCG and hyperglycosylated hCG, also known as invasive trophoblast antigen (ITA), are also elevated on average in affected pregnancies whilst maternal urine total estrogen and total estriol levels are reduced. When all the hCG species are measured in the same samples, ITA appears to be the most discriminatory21 and by itself might detect half the DS cases for a 5 percent FPR.21,144,145 A pre-amniocentesis study estimated the screening efficiency of combining second-trimester serum AFP, uE3 and hCG with urinary ITA and β-core hCG plus ultrasound NT, HL and anomalies.146 Among 568 women, 17 of whom had fetuses with DS, the detection rate for 5 percent false positives was 94 percent. Another study estimated that for an 85 percent DR, the FPR of first- and second-trimester serum combinations with and without NT would be reduced by about one-third if urinary ITA was also measured.21 However, caution is needed in interpreting urine results as there is significant heterogeneity between the published studies, probably due to differences in assay method, study design and the integrity of urine samples during transport and storage. Serum ITA For first-trimester screening, it would appear that ITA could substitute for free β-hCG and provide comparable screening efficacy.147,148 But used as an additional marker in the combined test, the incremental gain in detection would be only about 2 percent for a 5 percent FPR. Similarly, in the second trimester, ITA provides an alternative to hCG148,149 but as additional marker in the quad test the incremental gain would be 3–4 percent.

No fully prospective data are available on the use of ITA for DS screening and its utility for detecting other aneuploidy has not been assessed. Consequently, this marker has not gained widespread acceptance as a standard component of routine prenatal screening. DNA, RNA and fetal cells in maternal circulation Maternal plasma contains DNA and during pregnancy 3–6 percent of this is derived from the conceptus.150,151 Conceptus DNA is thought to be from trophoblasts but is commonly referred to as free fetal (ff-)DNA.152 The concentration of ff-DNA varies between pregnant women and according to gestation.153 Most of the ff-DNA exists as short segments of 313 bp or smaller.154 The ff-DNA has already been used for fetal sexing, Rhesus typing, and detection of paternally derived mutations154–157 see Chapter 30). RNA derived from the conceptus is also present in maternal plasma. There is considerable interest in using ff-DNA or RNA either for screening or noninvasive diagnosis of fetal aneuploidy. Several approaches are being pursued: • quantification of DNA at multiple single nucleotide polymorphism (SNP) sites to detect differences in maternally and paternally derived copies of chromosomes158 • quantification of maternally and paternally derived polymorphic DNA for genes that have methylation differences between material of placental and maternal blood origin159 • quantification of maternally and paternally derived polymorphic mRNA from genes that are transcribed in the placenta and not in maternal blood cells160 • digital polymerase chain reaction (PCR), which relies on multiple separate amplifications of DNA that has been diluted to less than one copy of a sequence per aliquot – the number of successful amplifications corresponds to the number of DNA molecules in the original specimen161 • a shotgun sequencing approach, whereby parallel sequencing is carried out at a very large number of sites across multiple chromosomes – the numbers of sequence tags are counted to determine if specific chromosomes are over- or underrepresented.162

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These methods are technically challenging because of the limited amount of ff-DNA or RNA available. However, they are highly amenable to automation. They do have the potential to replace all current approaches to screening and diagnosis of fetal aneuploidy (see Chapter 30). Little progress has been made with whole fetal cells found in maternal blood163 and the advent of new molecular methods may eclipse this approach (Chapter 30).

Clinical factors There are a large number of clinical factors that need to be taken into account when interpreting an individual screening test result and they can alter performance. Here we review the more important factors. Maternal age The DR and FPR figures in Tables 24.3–24.6 predict screening performance for the population as a whole. This is important for public health planners who need to know the best or at least most costeffective policy. But for pretest counseling the individual woman needs to know the DR and FPR specific for her age, and since the prior risk increases with age, it necessarily follows that for any risk cutoff both the DR and FPR will also increase. Table 24.9 shows the values for NT alone, the combined test and the contingent test at three maternal ages. The effect of maternal age is less for the more efficient screening policies as it is an increasingly minor variable in the risk calculation. Twins In spontaneous pregnancies the rate of twinning increases with age and, depending on the maternal age distribution, the extent of assisted reproduction in the population, and race or ethnicity. Overall, some 2–3 percent of pregnancies are twins. The interpretation of a DS screening test in twins differs from singletons and at all ages the screening efficiency is lower. Despite the presence of two fetuses, the a priori risk of an affected pregnancy appears to be the same as in singletons. The birth prevalence of DS can be estimated by meta-analysis of five cohort studies including a total of 106 DS twins.164,165 The

estimate is only 3 percent higher than the prevalence in singletons. None of the studies was stratified for maternal age. Therefore, the observed small increase in the crude DS prevalence rate among twins implies a reduction in the age-specific prevalence. However, until there is a more precise estimate of these rates it is reasonable to assume that the prior term risk for twin pregnancies does not differ from that of singletons. This assumption implies that the a priori risk of one or both of the fetuses being affected is about the maternal agespecific risk in singletons since most twin pregnancies are dizygotic. There are no data on the prior risk during pregnancy and in the absence of data to the contrary, loss rates during pregnancy are assumed to be the same as for singletons. Table 24.10 shows the median MoM value for each marker in unaffected twins, from a published meta-analysis166 updated to include a more recent paper on inhibin.167 The table also shows the expected means for twin pregnancies where one or both of the fetuses have DS. These means cannot be reliably estimated directly as there are insufficient published data and an indirect method is used.168 This is based on the assumption that each fetus contributes the expected amount for an affected or an unaffected singleton and that the same deviation from expectation seen in unaffected twins also applies. Therefore only the mean in DS pregnancies needs to be estimated. For example, the median AFP level in affected discordant twins would be (1 + 0.73)*(2.23/2) or 1.93 MoM and (0.73 + 0.73)*(2.23/2) or 1.63 MoM for concordant twins, since the singleton median for DS is 0.73 MoM. These estimates do not take into account possible differences according to chorionicity; one study found that the median PAPP-A level was significantly lower in monochorionic twins compared to dizygotic twins.169 The marker standard deviations and correlation coefficients for all combinations in twins appear to be similar to singletons.170,171 The estimated DS means are consistent with the few direct data that are available. Thus in 20 second-trimester cases in one report,166 the median AFP was 1.85 MoM; 15 were concordant and five discordant, so from the above the expected weighted mean was 1.87 MoM. The corresponding values for free b-hCG were 3.35 MoM and

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Genetic Disorders and the Fetus

Table 24.9 DR and FPR given final cut-off risk# for three policies at three selected maternal ages, according to hCG type and gestation hCG type

GA

Age 20 Term 1 in 250

Age 30 Mid trimester 1

Age 40

Term 1 in 250

in 270

Mid trimester 1

Term 1 in 250

in 270

Mid trimester 1 in 270

Quad Free β-hCG

53%, 2.0%

58%, 2.8%

62%, 3.9%

66%, 5.0%

90%, 29%

92%, 34%

hCG

49%, 2.0%

52%, 2.7%

56%, 3.6%

62%, 5.3%

88%, 31%

91%, 40%

11

73%, 1.3%

76%, 1.8%

77%, 2.1%

80%, 2.9%

92%, 14%

94%, 18%

13

64%, 1.3%

67%, 1.9%

69%, 2.4%

72%, 3.4%

90%, 20%

92%, 27%

11

70%, 1.3%

73%, 1.8%

75%, 2.2%

78%, 3.1%

91%, 16%

93%, 21%

13

65%, 1.3%

69%, 1.8%

70%, 2.2%

74%, 3.2%

91%, 19%

93%, 25%

11

81%, 0.8%

83%, 1.0%

85%, 1.4%

87%, 1.8%

95%, 9.2%

96%, 11%

13

76%, 0.9%

77%, 1.2%

81%, 1.6%

82%, 2.1%

94%, 12%

95%, 15%

11

79%, 0.7%

80%, 0.9%

83%, 1.3%

84%, 1.6%

95%, 9.1%

95%, 11%

13

75%, 0.9%

76%, 1.1%

80%, 1.6%

81%, 2.1%

93%, 12%

94%, 15%

Combined Free β-hCG

hCG

Contingent Free β-hCG

hCG

# For Contingent based on all first- and second-trimester markers included.

Table 24.10 Mean level in unaffected twins and the expected mean in DS twins for each widely used marker, according to gestation Serum marker PAPP-A

Gestation (weeks)

707

1.83

DS, one fetus

DS, both fetuses

1.28

0.73

1.33

0.82

12

1.40

0.97

13

1.51

1.19

10

2.76

3.45

11

2.98

3.87

12

hCG

Mean (MoM)

10 11

Free (-hCG

Unaffected

3.13

4.18

13

4961

2.08

3.22

4.36

14–18

3.43

4.79

10

1.91

1.93

11

2.04

2.21

12

2.26

2.65

13

3312

1.88

2.60

3.32

14–18

2.84

3.79

AFP

14–18

9959

2.23

1.93

1.63

uE3

14–18

1569

1.61

1.39

1.17

Inhibin

14–18

287

2.03

2.89

3.75

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Multi-Marker Maternal Serum Screening for Chromosomal Abnormalities 793

3.73 MoM. In 16 first-trimester cases in that report,166 the median PAPP-A was 1.25 MoM; 13 were concordant and three discordant, and the median gestation was 12 weeks, so the expected value was 1.30 MoM. Similarly, for 19 first-trimester free b-hCGs the observed and expected values were 2.91 MoM and 3.19 MoM. Two different methods have been used to estimate the DS risk for a twin pregnancy based on serum markers. The initial approach was to calculate a so-called “pseudo-risk” dividing the observed MoMs by the medians for unaffected twins (see Table 24.10) and calculate the risk as if the pregnancy were a singleton. The object of this manipulation was to achieve a false-positive rate not markedly different from that in singleton pregnancies.172 An alternative approach is to calculate risks using the estimated DS and unaffected means as in Table 24.10, with standard deviations and correlation coefficients the same as in singletons.168 Two likelihood ratios are calculated, one for concordant and one for discordant twins, and the geometric mean taken, weighted for the proportion of twin pregnancies that are monozygous, which reduces with age.173,174 In a twin pregnancy achieved by assisted reproduction technology, only the discordant twin LR need be used. Zygosity can also be determined by ultrasound examination of the fetal membranes; a so-called “lambda” sign, caused by invasion of the intertwin membrane by chorionic villus, is evidence of dizygosity175 so only the discordant LR need be used. Otherwise a “T” sign is seen, indicating a monochorionic twin pregnancy which can be assumed to be monozygotic. When NT is determined, the DS risk for a twin pregnancy can be estimated using the same parameters as a singleton pregnancy. The average NT measurement in twins appeared to be similar to singletons176–178 and does not differ between those conceived by assisted reproduction or spontaneously.178,179 For proven monochorionic pregnancies, the average of the NT MoMs is used in the risk calculation. Otherwise, separate risks are estimated for each fetus and possibly combined to estimate the chance that at least one fetus is affected. There is a strong correlation between the NT MoMs in unaffected pregnancies but not in discordant DS twins.180 Therefore, a twin fetus with a given MoM whose co-twin has a similar value is more likely to be unaf-

fected than one whose co-twin has a very different value. The risk can be calculated using bivariate log Gaussian NT distributions for the proband fetus and the co-twin MoMs; there are two DS distributions, for the discordant and concordant cases. When both NT and serum markers are available, the fetus-specific risk is estimated by applying the appropriate NT LR to half the serum-based risk rather than half the maternal age-specific risk. Table 24.11 shows examples of the age-specific DR and FPR for different screening policies in twins, based on the actual risk of a DS pregnancy rather than pseudo-risk. Rates were modeled separately for concordant and discordant affected twins and the weighted average taken using the proportion of twins in US whites that are monozygous: 16 percent, 13 percent and, 10 percent at age under 20, 30 and 40 respectively.173 For all policies, screening efficiency is much lower in twins than singletons and is extremely low for policies that do not involve NT. A commonly encountered problem, particularly in the first trimester, is the detection of fetal demise of a co-twin – spontaneous reduction to a singleton pregnancy or “vanishing twin.” In one study, for cases where the demise was thought to have occurred within 4 weeks of testing, PAPP-A and free b-hCG were both significantly elevated relative to singleton pregnancies.181 Another study failed to identify any significant differences in serum marker levels in cases with a vanishing twin.182 Presumably, increased marker concentrations could arise when there is residual trophoblast activity from the deceased twin or slow clearance of the proteins from the maternal circulation. There is a relatively strong chance that the deceased twin had a chromosome abnormality, given the very high aneuploidy rate in early singleton abortuses. In this situation, it is probably prudent not to use serum markers but additional ultrasound markers may be helpful. Previous affected pregnancy Women who have had a DS pregnancy are at increased risk of recurrence. Some will consider the risk sufficiently high to warrant invasive prenatal diagnosis without screening. Others will want to have their risk assessed by screening before making this decision.

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Genetic Disorders and the Fetus

Table 24.11 Twins: DR and FPR given final cut-off risk# for three policies at three selected maternal ages, according to hCG type and gestation hCG type

GA

Age 20 Term 1 in 250

Age 30 Mid trimester 1

Age 40

Term 1 in 250

in 270

Mid trimester 1

Term 1 in 250

in 270

Mid trimester 1 in 270

Quad Free β-hCG

17%, 0.7%

21%, 1.3%

24%, 1.8%

32%, 3.5%

85%, 48%

92%, 66%

hCG

19%, 0.9%

24%, 1.6%

26%, 2.1%

33%, 3.9%

85%, 55%

93%, 74%

11

63%, 1.4%

66%, 2.0%

67%, 2.4%

71%, 3.5%

86%, 20%

89%, 27%

13

55%, 1.2%

58%, 1.8%

60%, 2.2%

63%, 3.2%

81%, 23%

86%, 36%

11

66%, 1.3%

69%, 1.9%

70%, 2.2%

74%, 3.2%

88%, 18%

91%, 24%

13

57%, 1.1%

61%, 1.7%

63%, 2.1%

65%, 3.0%

85%, 22%

89%, 33%

11

65%, 1.3%

68%, 2.0%

70%, 2.4%

72%, 3.2%

88%, 18%

90%, 25%

13

57%, 1.0%

59%, 1.4%

61%, 1.7%

65%, 2.8%

84%, 21%

88%, 29%

11

71%, 0.9%

74%, 1.3%

76%, 1.7%

78%, 2.4%

91%, 13%

93%, 17%

13

63%, 0.8%

66%, 1.1%

68%, 1.5%

71%, 2.3%

85%, 20%

88%, 28%

11

69%, 0.8%

72%, 1.1%

74%, 1.5%

76%, 2.1%

90%, 12%

92%, 15%

13

61%, 0.6%

63%, 0.9%

65%, 1.2%

68%, 1.8%

87%, 15%

90%, 19%

NT alone

Combined Free β-hCG

hCG

Contingent Free β-hCG

hCG

# For Contingent based on all first- and second-trimester markers included.

In a small proportion of cases there will be a parental structural chromosome rearrangement and the recurrence risk can be quite high, depending on the specific parental karyotype. The most frequent is a heterozygous Robertsonian balanced translocation involving chromosome 21 and for female carriers the risk is great enough to dwarf the age-specific risk at most ages (see Chapter 6). If a woman has had a previous pregnancy with DS and the additional chromosome 21 was apparently noninherited, there is still an increased risk of recurrence. There are three available estimates of excess risk. In an unpublished study of more than 2,500 women who had first-trimester invasive prenatal diagnosis, the excess risk compared with the maternal age-specific expected risk was 0.75 percent (K. Nicolaides, personal communication). In a meta-analysis of second-trimester amniocentesis results in 4,953 pregnancies, the excess was 0.54 percent.183 A meta-analysis of 433 livebirths had five recurrences, an excess risk of

0.52 percent.184 The weighted average of these rates, allowing for fetal losses, is 0.77 percent in the first trimester, 0.54 percent in the second and 0.42 percent at term and can be added to the agespecific risk expressed as a probability. The recurrence risk is relatively large for young women but by the age of about 40 it is not materially different from the risk in women without a family history. There is also evidence that the risk for a potentially viable aneuploidy is increased for women who have had a previous different aneuploidy. Therefore, an alternative approach to incorporating a prior history of aneuploidy is to calculate their screening risks based on a maternal age of 38–39 (see Chapter 6). Table 24.12 shows the model-predicted detection and false-positive rates for women with a previous DS pregnancy. Modeling was carried in the same way as for sporadic DS pregnancies, except that the prior risk of being affected was calculated by adding the excess risk, as above. As expected, for

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Table 24.12 Previous DS pregnancy: DR and FPR given final cut-off risk# for three policies at three selected maternal ages, according to hCG type and gestation hCG type

GA

Age 20 Term 1 in 250

Age 30 Mid trimester 1

Age 40

Term 1 in 250

in 270

Mid trimester 1

Term 1 in 250

in 270

Mid trimester 1 in 270

Quad Free β-hCG

82%, 17%

86%, 21%

84%, 18%

87%, 24%

92%, 36%

95%, 46%

hCG

80%, 18%

83%, 23%

81%, 20%

85%, 27%

92%, 43%

94%, 52%

11

89%, 8.6%

91%, 12%

90%, 9.4%

92%, 12%

94%, 19%

96%, 24%

13

85%, 12%

88%, 16%

85%, 12%

88%, 17%

93%, 28%

95%, 36%

11

87%, 9.3%

89%, 13%

88%, 10%

90%, 14%

93%, 22%

95%, 28%

13

85%, 11%

89%, 15%

86%, 12%

89%, 16%

93%, 26%

95%, 33%

11

93%, 5.7%

94%, 6.9%

93%, 6.1%

94%, 7.5%

96%, 12%

97%, 15%

13

91%, 7.6%

92%, 9.4%

91%, 8.1%

93%, 10%

95%, 18%

96%, 21%

11

92%, 5.6%

93%, 6.9%

93%, 5.9%

94%, 7.3%

96%, 12%

96%, 15%

13

90%, 6.7%

92%, 8.8%

91%, 7.2%

92%, 9.3%

95%, 16%

96%, 20%

Combined Free β-hCG

hCG

Contingent Free β-hCG

hCG

# For Contingent based on all first- and second-trimester markers included.

all screening policies both rates will be higher than for singletons and the difference in efficiency according to maternal age will be reduced. There is evidence that some mothers of infants with DS have abnormal folate and methyl metabolism, as well as mutations in folate genes, features in common with NTDs. A relatively high DS risk might be expected in women who are at increased NTD risk. In a study of 493 such families, 445 with a history of NTD and 48 with isolated hydrocephalus, there were a total of 11 DS cases among 1,492 at-risk pregnancies, compared with 1.87 expected on the basis of maternal age.185 On the basis of this series the age-specific risk is increased 5.9-fold in families with NTD. This is consistent with the observation in the same study of seven NTDs among 1,847 pregnancies in 516 families at high risk of DS compared with 1.37 expected. However, two follow-up studies failed to confirm the association between NTD and DS in the same families186,187 and additional data are required before concluding that an adjustment is needed to allow for a prior history of an NTD.

Assisted reproduction When a nonspontaneous pregnancy has been achieved in a subfertile couple, often after a long waiting period and with some difficulty, there is additional reason to avoid the hazards of invasive prenatal diagnosis. Such couples need to have the maximum number of markers tested in order to produce the best available DS risk. There is no reason to believe that the age-specific risk of DS is higher in pregnancies conceived by in vitro fertilization (IVF) than for spontaneous pregnancies. The DS prevalence in the combined data from four age-matched or age-standardized studies was 0.23 percent.188–191 The average for the controls, weighted according to the number of cases, was 0.21 percent in spontaneously conceived pregnancies. Similarly, the results from three large series of pregnancies achieved by intracytoplasmic sperm injection (ICSI) are consistent with no increased risk. Among 1,244 women having prenatal diagnosis after ICSI, the risk was 0.32 percent (four cases) compared with the expected rate of 0.23 percent for women aged 33,

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Genetic Disorders and the Fetus

the average in this series.192 In one total series of 1,003 infants born after ICSI, the rate was 0.10 percent (one case), compared with 0.13 percent (7/5,446) in a conventional IVF series collected in the same country.193 And in a series of 643 women, of whom 158 had prenatal diagnosis, the rate was 0.47 percent (three cases) compared with an expected rate of 0.17 percent for their age and gestation.194 When calculating the age-specific risk of DS in pregnancies achieved by IVF, whether conventional or using ICSI, care is needed over the maternal age. If a donor egg was used the maternal age at term is calculated from the age of the donor at the time of sampling plus 266 days, the time from conception to term. A similar calculation is done if the woman’s own egg was used and it was frozen after sampling. These calculations assume that risk relates to the age of the donor rather than the recipient and that storage has no effect on risk. On average, first- and second-trimester hCG and free β-hCG levels are raised and PAPP-A levels reduced in pregnancies conceived by IVF, ICSI or other forms of assisted reproductive such as intrauterine insemination or following ovulation induction alone. In the combined results of all published series195–211g, the overall mean value for all hCG isoforms was 1.08 MoM and for PAPP-A 0.91 MoM. However, there is considerable heterogeneity between the series, possibly due to the method of gestational assessment, the cause of infertility or the type of therapy, for example whether the oocytes are donated or obtained from the patient, frozen or fresh. The specific hormone treatments or infertility conditions that presumably are the underlying cause of the alteration in the marker levels remains unclear. Given the uncertainties about when to correct the marker levels, the relatively small numbers of women involved, and the practicality of collecting detailed information about the ART procedures used, most programs currently do not adjust for ART. This is likely to result in overestimation of risks. Obstetricians should be aware that individual patient-specific risk figures may be less accurate for ART patients.

Maternal diabetes In the past, women with insulin-dependent diabetes mellitus (IDDM) were found in several series to have reduced second-trimester maternal serum AFP levels, by about 20 percent on average.140 These early studies excluded gestational diabetes. In more recent series the effect was much smaller, possibly due to better diabetic control,212 and some authors have cautioned against adjustment.213 In a recent meta-analysis the second-trimester serum markers other than AFP were altered to a small extent.214 NT measurements appear to be the same for maternal IDDM compared with controls but there are conflicting data for PAPP-A and firsttrimester free β-hCG.215 One study has shown that in gestational diabetes both AFP and free β-hCG are reduced216 and although it is generally assumed that the prior DS risk is not altered in this condition, there is some evidence of increased aneuploidy risk.217–219 There is a lack of data on maternal serum marker levels in diabetic patients receiving newer oral hypoglycemic drugs for management such as metformin (Glucophage). In the absence of better data, we recommend applying analyte adjustments when the patient: (1) was insulin dependent prior to, or around, the time of conception; (2) was receiving oral hypoglycemic agents at, or around, the time of conception, and would have received insulin if the newer drugs were not available; (3) should have been receiving treatment but was noncompliant with physicians’ recommendations. Correction is not performed if the patient: (4) had diabetes prior to, or around, the time of conception but was being successfully managed by dietary control alone; (5) developed diabetes in the second or third trimester of pregnancy.

Renal transplant High hCG and free β-hCG levels have been reported in women who had had a renal transplant220,221 and/or were in end-stage renal failure and on dialysis222 when they were screened. The dialysis study found a strong positive correlation between free β-hCG and serum creatinine. From a table in the publication, it can be estimated that the expected hCG MoM is 0.0125 times creatinine

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raised to the power 1.070, which might be used to adjust the level. Previous false-positive result The chance of having a false-positive result is increased among those who have had a false-positive result in a previous pregnancy. Maternal age alone will necessarily produce a correlation in risks between pregnancies but the phenomenon is also due to a degree of consistency in marker levels between pregnancies to the same woman. Positive correlations have been found for AFP,223,227–229 hCG.224,225,227–229 free β-hCG,226,228,230 uE3,224,227–229 and PAPP-A.228,230 There have been two studies of NT: one found no effect230,231 and whilst the second reported a significant correlation, the results were not expressed in MoMs.232 Tables have been published for use in counseling women about the relative increase in the positive rate given a previous screen-positive result.224,226,230 The relative increase declines with age since in older women age per se becomes a dominant reason for a positive result. Also the relative increase is lower for combinations using NT since this marker is not correlated between pregnancies. A method has been proposed to use the observed MoMs from an initial pregnancy to adjust the results of a subsequent pregnancy228 and modeling suggests that this could reduce recurrent falsepositives.233 A retrospective analysis of women with two or more singleton pregnancies screened by the second-trimester triple test confirmed an improvement in screening efficacy. However, implementing this prospectively is problematic. In addition to linkage of records, good pregnancy outcome information is required for the initial pregnancy to ensure that the initial result was not attributable to abnormality, pregnancy complications, inaccurate dating, twins, etc. A change in maternal health or smoking habits could also confound the adjustment. Smoking In the second trimester both hCG and free β-hCG levels are reduced on average in smokers with a median of 0.79 MoM in 10 studies combined.234–243 In the first trimester free β-hCG levels may not be reduced but PAPP-A levels are, and to a similar

extent to second-trimester hCG.244,248 Inhibin levels appear to be increased to an even greater extent than both these markers240,243,249 but levels are not materially altered in the other serum markers140 or NT.250 Taken together, there are strong grounds for adjusting both first- and second-trimester marker values to take account of maternal smoking. Several studies have reported that smoking is less common in the mothers of infants with DS. However, smoking habits are subject to strong birth cohort effects so it is important to take full account of maternal age and whilst most of the data come from age-matched case–control studies or where age is adjusted for in the analysis, the method of age adjustment in some studies was based on broad age bands and this may not be adequate. This was demonstrated in one study which found a relative risk of 0.87 with broad age grouping, 0.89 adjusting for additional variables and 1.00 when age adjustment, together with the additional variables, was in single year bands.251 The latest overview on this topic concludes that there is no difference in risk.243 Information collected on smoking during pregnancy is often inaccurate, particularly the level of consumption. With the exception of PAPP-A,248 there does not appear to be an obvious “dose– response” relationship between intake and the alteration in marker levels. Adjustment for smoking is generally based on self-reporting that the woman was a smoker at the time of testing. As a practical matter, it is generally not possible to stratify on the basis of the reported number of cigarettes smoked daily, although taking this into account may result in more accurate risk assessment.248 Ethnicity In women of Afro-Caribbean origin or AfroAmericans, second-trimester AFP and intact hCG are increased on average, with medians of 1.15 and 1.18 MoM respectively in one meta-analysis, as is second-trimester free β-hCG, with median 1.12 MoM.252 Inhibin-A is decreased to 0.92 MoM252 whilst uE3 levels are not materially altered.140 In women of South Asian origin residing in the UK, uE3 and total hCG levels appear to somewhat higher – 1.07 and 1.06 MoM respectively – than for Caucasian women.252 Differences in

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Genetic Disorders and the Fetus

second-trimester serum marker levels may also exist for other populations253–255 and these can be gestational age dependent.256 Population differences have also been noted for first-trimester markers, notably PAPP-A. In AfroCaribbean women in the UK, PAPP-A median MoM was reported to be 55–57 percent higher than that seen in Caucasians257,258 although a smaller difference was noted for Afro-Americans.259 Free β-hCG are also higher for Afro-Caribbean and Afro-American women257–259 while NT measurements may be somewhat lower.257,260 The firsttrimester marker profile may also differ in women of Oriental and Asian origin.257,258,260–262 These differences do not matter in ethnically homogeneous populations, since centers establish their own population-based median values, but those that serve an ethnically mixed population must allow for this in risk calculation. Programs with large enough minorities can convert concentrations to MoMs with ethnic-specific medians, otherwise an ethnic multiplication factor might be used to adjust ordinary MoMs. The adjustment factors for ethnicity are not necessarily the same across all gestational ages.256 The prevalence of DS at birth is generally considered to be similar for all populations. However, there are many individual reports of relatively high or low birth prevalence in different ethnic groups. A meta-analysis using data from countries with reliable systems for collecting information on maternal ages found that two groups had some evidence for rates greater than Europeans.263 These are those of Mexican and Central American origin in California (standardized indices 1.19 and 1.30 in two studies) and Jews of Asian or African origin in Israel (1.27). The standardized indices were markedly reduced in some populations, including three studies in Africans, but the authors conclude that this is likely to be due to incomplete ascertainment. Maternal weight All the serum markers used in DS screening demonstrate a negative correlation between the level, expressed in MoMs, and maternal weight. This is usually explained in terms of dilution. A fixed mass of chemical produced in the fetoplacental unit is diluted by a variable volume in the maternal unit.

But this cannot be the only factor involved since the extent of correlation differs between the markers. The correlation is almost twice as great for PAPP-A as for AFP or hCG; inhibin has a weaker correlation than these two and for uE3 there is hardly any association at all, particularly in the first trimester. It is standard practice to adjust all serum marker levels for maternal weight, dividing the observed MoM by the expected value for the weight derived by regression. The best regression formula is an inverse regression curve.264 Although log-linear curve does not differ markedly from the inverse curve for most women,265 it considerably underadjusts for weight in light women and overadjusts in those at the higher end of the weight range. NT levels are not strongly correlated with maternal weight.266,267 The impact of weight adjustment on individual patient risks will depend on the combinations of markers used and adjustment of some markers will affect risks in opposite directions, for example second-trimester AFP and hCG. Adjustment for weight does not introduce any bias since the average maternal weight is similar in DS and unaffected pregnancies.164 In order to provide the most accurate risks, weight adjustment should routinely be performed. Other factors There are several other factors known to be associated with one or more markers but they are not used to formally adjust levels. This is because the association is weak, the factor is subjective or is impractical to assess. There is a weak association between most markers and gravidity or parity (for a review see 140). Vaginal bleeding can lead to a high AFP level, presumably because of fetomaternal transfusion, and is associated with increased DS risk.268 However, it is an extremely variable and subjective factor, ranging from “spotting,” which is very common in early pregnancy, to threatened abortion. Throughout pregnancy, maternal serum hCG levels are higher in pregnancies where the fetus is female and in the second trimester AFP is lower.269,270 Gender has not hitherto been taken into account when interpreting screening results but this may change now that it can be determined with reasonable accuracy by ultrasound.271

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Edwards syndrome (trisomy 18) Many centers have extended their multiple marker screening program for DS to include trisomy 18. This involves calculating the risk of both disorders from the maternal age and marker profile using a multivariate Gaussian model.272–275 The maternal age-specific risk of trisomy 18 can be taken to be a fixed fraction of the corresponding DS risk: one-ninth, one-quarter and one-thir at term, mid trimester and in late first trimester respectively. These fractions were obtained by the relative frequency of 85 DS and 10 trisomy 18 cases in series of routinely karyotyped neonates,184,276 1,086 DS and 241 trisomy 18 cases in large amniocentesis series277,278 and 211 DS and 67 trisomy 18 cases in large chorionic villus sampling series.279–281 The newborn studies did not include many cases with trisomy 18 so the factor of one-ninth is not very secure. However, it is consistent with studies of the late fetal loss rates for the two disorders showing that about two-thirds of trisomy 18 cases spontaneously abort from mid trimester to term33 and about three-quarters from late first trimester to term.33,282 In trisomy 18 pregnancies, the mean serum AFP, uE3, hCG or free β-hCG, inhibin and PAPP-A levels are 0.68, 0.44, 0.31, 0.81 and 0.14 MoM respectively, based on two published meta-analyses272,283 extended to include more recent data.273,284– 294 There was no material difference in the mean

MoM value between hCG and free β-hCG and there was no material difference in means for either marker between the first and second trimesters. The median NT in two prospective studies was 3.27 MoM283 and 3.21 MoM.295,296 Allowing for viability bias, which is even stronger than for DS, the best estimate of the mean is 2.77 MoM. The standard deviations and most of the correlation coefficients were derived from the weighted mean of those in one of the meta-analyses272 and eight other series273,283,284,286–288,292,294 the remainder were assumed to be the same as in unaffected pregnancies. A large proportion of trisomy 18 cases are detected as a result of a high DS risk in a combined test but the proportion is much lower for scondtrimester screening tests. Table 24.13 shows the estimated detection rates for DS screening policies, together with the rates when DS screening is extended to include explicit trisomy 18 screening. The trisomy 18 detection rate is particularly high in the first trimester even without explicit screening because most cases are associated with raised NT. Second-trimester screening cannot achieve such high detection even with explicit screening.

Other conditions associated with altered markers Some centers interpret tests with low risk of DS, trisomy 18 or NTD results as screen positive when

Table 24.13 Trisomy 18 detection rate using a DS risk cut-off alone and with an explicit trisomy 18 risk cut-off, with two policies hCG type

GA

DS cut-off only Term 1 in 250

DS/ES cut-off Mid trimester 1

Term 1 in

Mid trimester 1

Term 1 in

Mid trimester 1

in 270

250/50

in 270/50

250/100

in 270/100

Quad Free β-hCG

31%

36%

48%

53%

52%

55%

hCG

29%

35%

32%

38%

39%

45%

11

81%

83%

81%

83%

81%

83%

13

80%

82%

81%

83%

82%

84%

Combined Free β-hCG

hCG

11

87%

88%

87%

89%

87%

89%

13

78%

80%

81%

83%

83%

85%

800

Genetic Disorders and the Fetus

one or more of the markers has an extremely high or low value. Some marker values or combinations of results do select a very high-risk group and warrant further investigation. These are discussed below. Other chromosome abnormalities It has been suggested that first-trimester aneuploidy screening be extended to include trisomies 13, 18 and 21.297 In the second trimester uE3 levels are reduced298 and preliminary results indicate that inhibin is increased286 but the marker profile is more extreme in the first trimester, with extremely high NT coupled with very low free β-hCG and AFP.299 The proposed algorithm uses one set of parameters to calculate the combined risk of trisomy 18 or trisomy 13. Model predictions are that 95 percent of cases with one or the other type of aneuploidy can be detected for a 0.3 percent false-positive rate. In the absence of prenatal screening and diagnosis, trisomy 13 has a birth prevalence about one-quarter that of trisomy 18 (see Chapter 6). Among the cases present at 12 weeks’ gestation, approximately one-half would be expected to end in miscarriage or stillbirth.282 Other common aneuploidies have abnormal marker profiles and whilst an explicit risk screening has not been suggested, they are often detected as part of DS, trisomy 18 or NTD screening. In triploidy there are two distinct types of secondtrimester marker profile: (1) grossly elevated AFP, hCG and inhibin with low to normal uE3 and (2) very low hCG, uE3 and inhibin with low to normal AFP.300 The same distinction has now been observed in the first trimester and additionally with type 1, NT is increased whilst with type 2, PAPP-A is extremely reduced.301 There are also two distinct patterns for Turner syndrome with and without hydrops; both types have reduced uE3 but hCG levels are increased with hydropic disease and reduced when there is no hydrops.302–304 On average, PAPP-A levels are low and NT levels are very high when Turner syndrome is present.305 There are also case reports and small series (see for example 305) where screening preferentially identified other sex chromosome abnormalities. However, the results are subject to strong bias since the cases were generally diagnosed after a procedure carried out because of abnormal screening results while screen-negative cases would remain

unrecognized. Viability bias also distorts case reports and marker profiles for lethal chromosomal disorders. There are other chromosome abnormalities that may be associated with an abnormal marker patterns. These are discussed in Chapter 6. X-linked ichthyosis X-linked ichthyosis (XLI) is found in approximately 1 in 2000 males and is characterized by scaly dark skin on the scalp, trunk and limbs. It is caused by deficiency of the steroid sulfatase (STS, also known as placental sulfatase). Most cases are caused by a deletion of the gene at Xp22.32 arising through nonhomologous recombination of sequencies that show some homology and that are located either side of the STS gene. A small number of cases are due to point mutations within the gene. Other cases involve larger deletions and these can result in Kallman syndrome, mental retardation and other abnormalities (“contiguous gene deletion syndromes”). XLI is identified through second-trimester serum screening because STS deficiency causes abnormal estriol biosynthesis, resulting in extremely low maternal serum uE3 levels. For example, in one series of nine pregnancies with low or absent second-trimester maternal serum uE3 levels, six were found to have a complete and one a partial deletion of the steroid sulfatase deficiency gene.306 Other second-trimester serum markers show normal levels. STS deficiency can often be definitively established from a known history of ichthyosis or through fluorescence in situ hybridization (FISH) testing of either maternal lymphocytes or amniotic fluid cells. In apparently de novo cases, additional testing to rule out a contiguous gene deletion syndrome may be necessary. Isolated XLI has generally been considered to be a mild condition. However, one report suggests that the disorder can be associated with attention deficit hyperactivity disorder even when the mutation is limited to the common XLI gene deletion.307 If confirmed, this may significantly complicate the counseling of this relatively common finding. Smith-Lemli-Opitz syndrome Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder in which a defect in cholesterol biosynthesis causes mental retardation,

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skeletal, genital, cardiac, pulmonary and renal malformations. There can be considerable clinical diversity with some patients showing only mental retardation and mild dysmorphism while other cases are associated with severe anatomic abnormalities and in utero death. In this disorder mutation of the 3β hydroxysterol-Δ7-reductase gene causes accumulation of 7-hydrocholesterol (7DHC) and detection of this product is the basis of diagnostic testing of amniotic fluid and various tissues. The incidence of SLOS at birth has been estimated to be approximately 1 per 20,000 to 1 per 40,000 births. Because cholesterol is a precursor of estriol, affected pregnancies are characterized by low second-trimester maternal serum uE3.308 AFP and hCG also appear to be reduced and an algorithm has been proposed for second-trimester screening for SLOS.309 In a prospective trial involving over 1 million pregnancies, using a second-trimester risk cut-off of 1 in 50, 0.29 percent of all women screened were found to be screen positive for SLOS.310 Over two-thirds of the women who were screen positive for SLOS were also screen positive for some other disorder and the screening for SLOS therefore only added 0.07 percent to the overall rate of women with positive prenatal screening tests. Based on the theoretical detection rate, an initial estimate for the incidence of the disorder (1 in 20,000), and the number of women screened, approximately 32 SLOS-affected pregnancies should have been identified as being screen positive. In fact, only five severely affected cases were screen positive and only one additional case was identified in a screen-negative woman. Although SLOS screening did not detect many SLOS-affected pregnancies, it did identify a high number of other abnormalities. Confining the analysis to women who only had a false-positive SLOS result and were screen negative for all other prenatal screening categories, 3.3 percent had a chromosome abnormality, 5.8 percent an anatomic abnormality, and 5.0 percent other conditions.311 Based on the excess of males identified, it can be estimated that approximately 28 percent of the remaining false positives can be attributed to steroid sulfatase deficiency. Given the relatively low number of additional positive screening tests (less than 0.1 percent), the minimal additional costs of including SLOS screen-

ing, and the high frequency of significant abnormalities detected, SLOS screening would appear to be useful. However, counseling women who are SLOS screen positive can be complex because of the diverse reasons for a positive result and the complexity of the options for follow-up testing. Management of women who are screen positive should include ultrasound to rule out simple explanations, such as fetal death and nonpregnancy, to look for anatomic findings consistent with chromosome abnormality or SLOS, and also to determine fetal gender. Steroid sulfatase deficiency can often be established from a known history of ichthyosis (see above). Once the more common reasons for a SLOS screen-positive result have been excluded, few women will have an indication for diagnostic testing for SLOS by 7DHC testing of amniotic fluid. Diagnostic testing may also be possible using a maternal urine or serum specimen.312 Cornelia de Lange syndrome (CdLS) This is a fetal abnormality characterized by mental retardation and severe limb reduction. In a series of 18 second-trimester pregnancies the median maternal serum PAPP-A level was 0.21 MoM; free β-hCG and inhibin levels were also reduced with medians of 0.67 and 0.62 MoM respectively.313 There have also been four case reports of increased NT or cystic hygroma.314–317 Cardiac abnormalities Several studies have reported increased NT in pregnancies with major cardiac abnormality.318–327 From some of the studies it is possible to derive an observed detection rate: 61 percent,324 40–56 percent,322 51 percent,327 27–36 percent,325 12–15 percent,326 11 percent.323 These differences may reflect referral, ascertainment and viability biases and the studies used a variety of NT cut-off levels. A meta-analysis showed that, using a 2.5 mm cutoff for NT, the detection rate for major cardiac defects was 38 percent for a 4.9 percent false-positive rate.328 Among those with screen-positive results, it is expected that cardiac defects would be found at a rate of 18 per 1000. The prevalence of major cardiac defects in chromosomally normal fetuses with NT exceeding 3.5 mm is 78 per 1000. Given these rates, it is standard practice to offer follow-up fetal echocardiography to women with

802

Genetic Disorders and the Fetus

increased NT, regardless of the serum marker results.329 Moles and placental mesenchymal dysplasia Maternal serum AFP and uE3 levels are essentially undetectable for complete hydatidiform molar pregnancies because there is no fetus which is the usual source of AFP and uE3 precursors.330 HCG and inhibin A levels are very high in these pregnancies.331 Partial moles are associated with a triploid karyotype and the markers seen with triploidy are discussed in the Other Chromosome Abnormalities section. Placental mesenchymal dysplasia is a separate entity that is characterized by cystic villi but no trophoblast hyperplasia and the presence of a substantially normal fetus on ultrasound examination. AFP and hCG have been reported to be high in these pregnancies but there are few precise data and levels for other markers have not yet been described.332 In the first trimester hydatidiform mole, ectopic pregnancy and impending or actual fetal loss are frequent findings in women with extremely low PAPP-A levels.333–335 In the second trimester this occurs, albeit to a lesser extent, with low uE3.330,336 Fetal demise In the first trimester, low PAPP-A is associated with a subsequent fetal demise or pregnancy loss.333,335,337–343 Either low free β-hCG338,340,341 or high free β-hCG338 also appear to be more common in those pregnancies that will spontaneously abort. A very large NT measurement or cystic hygroma is also likely to be found in nonviable pregnancies.22,338,341,344,345 Second-trimester markers are also associated with fetal death. These associations exist for elevated or low AFP346,347 (see Chapter 23), low uE3342,347 high hCG,347–349 and elevated inhibin-A.347 Combinations of abnormal markers can be associated with higher risks of fetal demise, for example, elevated AFP and low uE3,336 elevated AFP and hCG349 and elevated AFP and inhibin A.347 Although these assocations exist, specifically screening for risk of fetal loss would not be highly efficient using the current markers.342 The patterns of markers associated with fetal demise do suggest that both

first- and second-trimester screening will preferentially identify DS pregnancies that are at the greatest risk for spontaneous abortion. However, there are few data available to assess this nonviability bias. In the second trimester, DS fetuses with hydrops which are at very high risk for fetal demise appear to show marker patterns that are more extreme.350 There is also some evidence of higher second-trimester hCG levels in affected pregnancies with anatomic abnormalities.351 The use of second-trimester ultrasound will also be a factor in the overall identification of those affected pregnancies with the most serious major malformations. Pregnancy complications and other adverse outcomes Preterm birth is the most important cause of perinatal morbidity and mortality with an incidence that appears to be increasing in many developing countries. For the US, in 2005, 13 percent of all births and 11 percent of singletons were preterm.352 Maternal race, weight, socio-economic status, family health, and genetic factors all appear to affect rates.353 First-trimester markers that are associated with preterm birth include low PAPPA,339,340,343,354–357 increased NT355,356 and increased inhibin-A.358 Second-trimester low AFP346 or high AFP, hCG and inhibin A have all been reported in pregnancies ending in a preterm birth.346,347 A similar set of changes in markers is seen in pregnancies with fetal growth restriction (IUGR) or small for gestational age (SGA) fetuses, particularly low first-trimester PAPP-A334,335,340,343,354–356,358 and low free-β hCG.334,355,356 First-trimester ADAM12 may also be informative in helping to identify IUGR.359 In the second trimester, elevated AFP, hCG and inhibin-A but low uE3 have been reported.346,347 Pre-eclampsia complicates 5–7 percent of pregnancies and although there are no good preventive measures, early diagnosis is extremely important because of the associated mortality and morbidity.360 Again, there are associations with existing DS screening markers, including first-trimester low PAPPA334,335,340,354,361,362 and second-trimester elevated AFP,347,363,364 hCG365–367 and inhibin-A.347,366–368 Additional reports describe similar associations between the markers and “hypertensive disorders”

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which includes patients without all the criteria for the diagnosis of pre-eclampsia. Combinations of the second-trimester markers do not appear to result in particularly effective screening for preeclampsia.369 However, previous pregnancy history, combinations of first- and second-trimester DS screening markers, uterine Doppler ultrasonography370 and a number of additional serum markers show considerable potential371–373 and it is reasonable to think that an effective screening protocol for pre-eclampsia will soon be developed.

Planning a program There is now a wide range of possible DS screening strategies and detailed policies. The tables in this chapter can serve as a guide to health planners as to the relative efficiency of the competing approaches. However, whilst efficiency is important, other determinants of choice include the human and financial costs, as well as organizational matters. Currently, many women continue to receive only second-trimester screening using 2–4 serum markers but, as we have shown, a much greater detection rate can be achieved by other policies, particularly those involving NT. This is now widely accepted by planners and there is a rapid shift in practice towards the combined test. The only practical limitation is the lack of adequate ultrasound facilities and experience with CVS in some localities. In this situation, a gradual shift could be considered initially using either a contingent combined test strategy or restricting the combined test to twin pregnancies, women who have had a previous DS pregnancy, those having assisted conception, and those at the most advanced maternal ages. In some healthcare settings there may not be sufficient resources to carry out both the combined test and a separate AFP test at 16–18 weeks to screen for NTDs. But it should be noted that the NTD detection rate is much higher using a second-trimester ultrasound examination compared to AFP screening. The human advantages of first-trimester screening are obvious: earlier reassurance and, if termination of pregnancy is chosen, it can be completed before fetal movements are felt. The early termination of those DS pregnancies that are destined to

miscarry is an advantage since it prevents a late miscarriage and diagnosis yields information on recurrence risk. Termination of pregnancy is safer in the first trimester than later in pregnancy.374 [A recent Cochrane Review included 9,000 pregnancies from three large randomized trials and the fetal loss rate was one-third higher for CVS.375 However, a subsequent NIH randomized trial of almost 4,000 women found an absolute increase of just 0.26 percent.376,377 Furthermore the Cochrane Review took no account of nonrandomized studies such as the WHO-sponsored Registry, which after the first 139,000 procedures registered concluded that “CVS is a safe procedure with an associated fetal loss rate comparable to that of amniocentesis”378 (see Chapter 2). Sequential screening strategies are even more efficient than the combined test. The biggest practical problem is the extended period of several weeks between initiating the process and its completion, and the consequent anxiety. Some women will find this unacceptable and would rather have a test with a lower DR. The addition of new ultrasound markers such as NB will improve first-trimester screening efficiency to such an extent that sequential screening will become unnecessary. Meanwhile a contingent screening strategy within the first trimester is a sound alternative option. Guidelines from the American College of Obstetrics and Gynecology379 and the American College of Medical Genetics380 emphasize patient autonomy in decision making during the provision of prenatal screening and diagnosis. Early screening with full disclosure of the results and the option of additional (sequential) screening and diagnosis therefore needs to be available. A high level of patient autonomy can be entirely consistent with the most efficacious protocols because the policies that provide the highest detection rates and lowest false-positive rates are also those that provide some of the highest patient-specific risks in affected pregnancies and lowest risks for unaffected pregnancies. Effective counseling can help minimize unnecessary screening and diagnostic tests. A difficulty associated with sequential strategies is the fact that patients will receive different and sometimes conflicting risk figures at each step. However, in the normal course of pregnancy it is usual for obstetricians to gather increasing information

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Genetic Disorders and the Fetus

about maternal and fetal health and they are well able to deal with changing situations as the pregnancy progresses. Development of national strategies and guidelines, for example, recommended cut-offs for referring patients for the second step in contingency screening and elements to be included in a second-trimester anomaly scan, would be helpful.381 A systematic review of economic evaluations of antenatal screening included 10 studies of DS screening, seven using biochemistry and two based solely on the anomaly scan.382 The reviewers concluded that serum screening was cost effective but pointed out that the incremental cost of adding additional markers rather than the average cost was not generally reported and this was critical for health planners. First-trimester screening was not considered by the papers reviewed but this has been assessed in eight subsequent publications381,382,389 which show an overwhelming economic benefit of a change from second-trimester screening to first-trimester protocols. One study compared the different sequential strategies and concluded that contingent screening was the most cost-effective.381 Unit costs vary in different localities390 and healthcare systems and planners wishing to use the published calculations of incremental costs may need to substitute their own unit costs.

Conclusion Since multi-marker serum screening for DS was first introduced there has been a steady increase in the detection rate, in relatively small increments, as new markers have been added. The incorporation of ultrasound markers has continued and accelerated the process as well as raising the level of complexity, as has the concept of sequential screening. Today detection rates in excess of 90 percent are achievable and at a lower false-positive rate than in the past. Thus a minimum standard can now be expected. The requisite components are: first-trimester ultrasound that includes NT; laboratories that can perform both first- and second-trimester tests in a manner appropriate for the generation of accurate risk estimates; and second-trimester ultrasound that can identify major anatomic abnormalities. All these elements need to be integrated into pro-

grams where a priori risks and the associations of markers with other disorders are understood, genetic counseling and patient education resources are available, and there is access to fully diagnostic invasive testing. In the near future DS screening will be expanded to include other fetal disorders and pregnancy complications. The links that have been built between obstetric services, laboratories, geneticists and others provide a solid foundation for this expansion in prenatal screening.

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11. Brambati B, Lanzani A, Tului L. Ultrasound and biochemical assessment of first trimester pregnancy. In: Chapman M, Grudzinskas G, Chard T, eds. The embryo: normal and abnormal development and growth. New York: Springer-Verlag, 1991;181. 12. Westergaard JG, Sinosich MJ, Bugge M, et al. Pregnancy-associated plasma protein A in the prediction of early pregnancy failure. Am J Obstet Gynecol 1983; 145:67. 13. Nicolaides KH, Azar G, Byrne D, et al. Fetal nuchal translucency: ultrasound screening for chromosomal defects in first trimester of pregnancy. BMJ 1992;304: 867. 14. Snijders RJ, Thom EA, Zachary JM, et al. First-trimester trisomy screening: nuchal translucency measurement training and quality assurance to correct and unify technique. Ultrasound Obstet Gynecol 2002; 19:353. 15. von Kaisenberg CS, Krenn V, Ludwig M, et al. Morphological classification of nuchal skin in human fetuses with trisomy 21, 18, and 13 at 12–18 weeks and in a trisomy 16 mouse. Anat Embryol 1998;197:105. 16. Logghe H, Cuckle H, Sehmi I. Centre-specific ultrasound nuchal translucency medians needed for Down’s syndrome screening. Prenat Diagn 1995;23:389. 17. Cuckle HS. Improved parameters for risk estimation in Down’s syndrome screening. Prenat Diagn 1995;15: 1057. 18. Cuckle H. Updated modeling parameters for Down syndrome screening. Balkan J Med Genet 2003;6:101. 19. Cuckle HS, van Lith JMM. Appropriate biochemical parameters in first trimester screening for Down’s syndrome. Prenat Diagn 1999;19:505. 20. Spencer K, Crossley JA, Aitken DA, et al. Temporal changes in maternal serum biochemical markers of trisomy 21 across the first and second trimester of pregnancy. Ann Clin Biochem 2002;39:567. 21. Wald NJ, Rodeck C, Hackshaw AK, et al. First and second trimester antenatal screening for Down’s syndrome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). Health Technol Assess 2003;7:1. 22. Malone FD, Canick JA, Ball RH, et al. First- and Second-Trimester Evaluation of Risk (FASTER) Research Consortium. First trimester or second-trimester screening, or both, for Down’s syndrome. N Engl J Med 2005;353:2001. 23. Cuckle HS, Malone FD, Wright D, et al, for the FASTER Research Consortium. Contingent screening for Down syndrome – results from the FaSTER trial. Prenat Diagn 2008;28:89. 24. Cuckle H, Benn P, Wright D. Down syndrome screening in the first and/or second trimester: model pre-

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chorionic gonadotropin concentrations and nuchal translucency are associated with obstetric complications. A population-based screening study (The FASTER Trial). Am J Obstet Gynecol 2004;191:1446. Spencer K, Cowans NJ, Avidou K, et al. First trimester ultrasound and biochemical markers of aneuploidy and the prediction of impending fetal death. Ultrasound Obstet Gynecol 2006;28:637. Dugoff L, Cuckle HS, Hobbins J, et al, for the FASTER Research Consortium. Prediction of patient-specific risk for fetal loss using maternal characteristics and first and second trimester maternal serum Down syndrome markers. Am J Obstet Gynecol 2008;199:290. Barrett SL, Bower C, Hadlow NC. Use of the combined first-trimester screen result and low PAPP-A to predict risk of adverse outcomes. Prenat Diagn 2008;28:28. Souka AP, Krampl E, Bakalis S, et al. Outcome of pregnancy in chromosomally normal fetuses with increased nuchal translucency in the first trimester. Ultrasound Obstet Gynecol 2001;18:9. Malone FD, Ball RH, Nyberg DA, et al. FASTER Trial Research Consortium. First-trimester septated cystic hygroma: prevalence, natural history, and pediatric outcome. Obstet Gynecol 2005;106:288. Krause TG, Christens P, Wohlfahrt J, et al. Secondtrimester maternal serum alpha-fetoprotein and risk of adverse pregnancy outcome. Obstet Gynecol 2001; 97:277. Dugoff L, Hobbins JC, Malone FD, et al. FASTER Trial Research Consortium. Quad screen as a predictor of advese pregnancy outcome. Obstet Gynecol 2005;106: 260. Wenstrom KD, Owen J, Boots LR, et al. Elevated second-trimester human chorionic gonadotropin levels in association with poor pregnancy outcome. Am J Obstet Gynecol 1994;171:1038. Benn PA, Horne D, Briganti S, et al. Elevated secondtrimester maternal serum hCG alone or in combination with elevated alpha-fetoprotein. Obstet Gynecol 1996;87:217. Benn PA, Egan JFX, Ingardia CJ. Extreme secondtrimester serum analyte values in Down syndrome pregnancies with hydrops fetalis. J Mat Fetal Neonatal Med 2002;11:1. Tanski S, Shulman Rosengren S, Benn PA. Predictive value of the triple screening test for the phenotype of Down syndrome. Am J Med Genet 1995; 85:123. Martin JA, Hamilton BE, Sutton PD, et al. Births: final data for 2005. Natl Vital Stat Rep 2007;56:1. Pennell CE, Jacobsson B, Williams SM, et al. Genetic epidemiologic studies of preterm birth: guidelines for research. Am J Obstet Gynecol 2007;196:107.

354. Smith GC, Stenhouse EJ, Crossley JA, et al. Early pregnancy levels of pregnancy-associated plasma protein A and the risk of intrauterine growth restriction, premature birth, preeclampsia, and stillbirth. J Clin Endocrinol Metab 2002;87:1762. 355. Krantz D, Goetzl L, Simpson JL, et al. First Trimester Maternal Serum Biochemistry and Fetal Nuchal Translucency Screening (BUN) Study Group. Association of extreme first-trimester free human chorionic gonadotropin-β, pregnancy-associated plasma protein A, and nuchal translucency with intrauterine growth restriction and other adverse pregnancy outcomes. Am J Obstet Gynecol 2004;191:1452. 356. Pihl K, Sørensen TL, Nørgaard-Pedersen B, et al. First trimester combined screening for Down syndrome, prediction of low birth weight, small for gestational age and pre-term delivery in a cohort of non-selected women. Prenat Diagn 2008;28:247. 357. Spencer K, Cowans NJ, Molina F, et al. First-trimester ultrasound and biochemical markers of aneuploidy and the prediction of preterm or early preterm delivery. Ultrasound Obstet Gynecol 2008;31:147. 358. Tul N, Pusenjak S, Osredkar J, et al. Predicting complications of pregnancy with first trimester maternal serum free-β hCG, PAPP-A and inhibin-A. Prenat Diagn 2003;23:990. 359. Cowans NJ, Spencer K. First-trimester ADAM12 and PAPP-A as markers for intrauterine fetal growth restriction through their roles in the insulin-like growth factor system. Prenat Diagn 2007;27:264. 360. Wagner LK. Diagnosis and management of preeclampsia. Am Family Physician 2004;70:2317. 361. Spencer K, Cowans NJ, Nicolaides KH. Low levels of maternal serum PAPP-A in the first trimester and the risk of pre-eclampsia. Prenat Diagn 2008;28:7. 362. Kang JH, Farina A, Park JH, et al. Down syndrome biochemical markers and screening for preeclampsia at first and second trimester, correlation with the week of onset and the severity. Prenat Diagn 2008;28: 704. 363. Räty R, Koskinen P, Alanen A, et al. Prediction of preeclampsia with maternal mid-trimester total rennin, inhibin A, AFP and free β-hCG levels. Prenat Diagn 1999;19:122. 364. Yaron Y, Cherry M, Kramer RL. Second trimester maternal serum screening, maternal serum αfetoprotein, β-chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome. Am J Obstet Gynecol 1999;181:968. 365. Pouta AM, Hartikainen AL, Vuolteenaho OJ, et al. Mid trimester N-terminal proatrial natriuretic peptide, free beta hCG and alpha fetoprotein in predicting preeclampsia. Obstet Gynecol 1998;91:940.

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366. Aquilina J, Maplethorpe R, Ellis P, et al. Correlation between second trimester maternal serum inhibin-A and human chorionic gonadotrophin for the prediction of pre-eclampsia. Placenta 2000;21:487. 367. Lambert-Messerlian GM, Silver HM, Petraglia F, et al. Second trimester levels in maternal serum human chorionic gonadotrophin and inhibin A as predictors of pre-eclampsia in the third trimester of pregnancy. J Soc Gynecol Investig 2000;7:170. 368. Cuckle H, Sehmi I, Jones R. Maternal serum inhibin A can predict pre-eclampsia. Br J Obstet Gynaecol 1998;105:1101. 369. Wald NJ, Morris JK, Ibison J, et al. Screening in early pregnancy for pre-eclampsia using Down syndrome quadruple test markers. Prenat Diagn 2006;26:559. 370. Spencer K, Yu CKH, Savvidou M, et al. Prediction of pre-eclampsia by uterine artery Doppler ultrasonography and maternal serum pregnancy associated plasma protein-A, free β-human chorionic gonadotropin, activin A and inhibin A at 22 + 0 to 24 + 6 weeks’ gestation. Ultrasound Obstet Gynecol 2006;27: 658. 371. Conde-Agudelo A, Villar J, Lindheimer M. World Health Organization systematic review of screening tests for preeclampsia. Am J Obstet Gynecol 2004; 104:1367. 372. Levine RJ, Maynard SE, Qian C, et al. Circulating angiogenic factors and the risk of pre-eclampsia. N Engl J Med 2004;350:672. 373. Spencer K, Cowans NJ, Chefetz I, et al. Secondtrimester uterine artery Doppler pulsatility index and maternal serum PP13 as markers of pre-eclampsia. Prenat Diagn 2007;27:258. 374. Lawson HW, Frye A, Atrash HK, et al. Abortion mortality, United States, 1972–1987. Am J Obstet Gynecol 1994;171:1365. 375. Alfirevic Z, Gosden CM, Neilson JP. Chorionic villus sampling versus amniocentesis for prenatal diagnosis. Cochrane Library, Issue 4. Oxford, Update Software, 2002. 376. Rhoads GG, Jackson LG, Schlesselman SE, et al. The safety and efficacy of chorionic villus sampling for early prenatal diagnosis of cytogenetic abnormalities. N Engl J Med 1989:320:609. 377. Jackson LG, Zachary JM, Fowler SE, et al. A randomized comparison of transcervical and transabdominal chorionic-villus sampling. The U.S. National Institute of Child Health and Human Development Chorionic-Villus Sampling and Amniocentesis Study Group. N Engl J Med 1992;327:594.

378. Kuliev A, Jackson L, Froster U, et al. Chorionic villus sampling safety. Report of World Health Organization/ EURO meeting in association with the Seventh International Conference on Early Prenatal Diagnosis of Genetic Diseases, Tel Aviv, Israel, May 21, 1994. Am J Obstet Gynecol 1996;174:807. 379. American College of Obstetrics and Gynecologists and American College of Medical Genetics. Screening for fetal chromosome abnormalities. ACOG Practice Bulletin No. 77. Obstet Gynecol 2007;109:217. 380. Driscoll DA, Gross SJ. American College of Medical Genetics Practice Guidelines. First trimester diagnosis and screening for fetal aneuploidy. Genet Med 2008; 10:73. 381. Ball RH, Caughey AB, Malone FD, et al. First- and second-trimester evaluation of risk for Down syndrome. Obstet Gynecol 2007;110:10. 382. Petrou S, Henderson J, Roberts T, et al. Recent economic evaluations of antenatal screening: a systematic review and critique. J Med Screen 2000;7:59. 383. Vintzileos AM, Ananth CV, Smulian JC, et al. Costbenefit analysis of prenatal diagnosis for Down syndrome using the British or the American approach. Obstet Gynecol 2000;95:577. 384. Gilbert RE, Augood C, Gupta R, et al. Screening for Down’s syndrome: effects, safety, and cost effectiveness of first and second trimester strategies. BMJ 2001; 323:423. 385. Caughey AB, Kuppermann M, Norton ME, et al. Nuchal translucency and first trimester biochemical markers for Down syndrome screening, a cost-effectiveness analysis. Am J Obstet Gynecol 2002;187:1239. 386. Christiansen M, Olesen Larsen S. An increase in costeffectiveness of first trimester maternal screening programmes for fetal chromosome anomalies is obtained by contingent testing. Prenat Diagn 2002;22:482. 387. Cusick W, Buchanan P, Hallahan TW, et al. Combined first-trimester versus second-trimester serum screening for Down syndrome: a cost analysis. Am J Obstet Gynecol 2003;188:745. 388. Macones GA, Odibo A. First trimester screening: economic implications. Semin Perinatol 2005;29:363. 389. Kott B, Dubinsky TJ. Cost-effectiveness model for firsttrimester versus second-trimester ultrasound screening for Down syndrome. J Am Coll Radiol 2004; 1:415. 390. Roberts T, Henderson J, Mugford M, et al. Antenatal ultrasound screening for fetal abnormalities: a systematic review of studies of cost and cost effectiveness. Br J Obstet Gynaecol 2002;109:44.

25

Prenatal Diagnosis of Fetal Malformations by Ultrasound Yves G. Ville and Durata Nowakowska Department of Obstetrics and Gynecology, CHI Poissy Saint Germain, Université de Versailles SaintQuentin-en-Yvelines, Poissy, France

Ultrasound is the key to the prenatal diagnosis of most fetal malformations. This safe prenatal investigation is offered mostly to pregnant women at the optimal gestations of 11–14 weeks and 20–24 weeks. It allows an examination of the external and internal anatomy of the fetus and the detection of not only major malformations but also subtle markers of chromosomal abnormalities and genetic syndromes. The first fetal malformation to be detected antenatally by ultrasonography leading to the termination of pregnancy for medical indication was anencephaly.1 Subsequently, thousands of reports have appeared in the scientific literature describing the diagnosis of an ever-expanding range of fetal structural and functional abnormalities. This chapter provides an overview of the prenatal diagnosis of some of these defects and their associated abnormalities. Special emphasis is placed on the diagnosis of fetal abnormalities during the first trimester of pregnancy. Indeed, improvement of the techniques and wider access to ultrasound examination for pregnant women have moved the challenge of prenatal diagnosis of fetal abnormalities, especially chromosomal disorders, to the first trimester. The new developments of an old technique, fetoscopy, which has recently been rediscovered because of the miniaturization of the

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

instruments and the development of endoscopic fetal surgery, will also be highlighted. Routine ultrasound screening of the whole population has the potential advantage of detecting most major fetal malformations. For this reason, the Royal College of Obstetricians and Gynaecologists in Great Britain recommended that (1) more facilities for high-quality ultrasound machines and for training of personnel be provided to all obstetric departments, and (2) all pregnant women be offered a proper ultrasound scan at approximately 20 weeks of gestation for fetal biometry but also for a systematic search for major and minor defects.2 The usefulness of screening low-risk populations by ultrasound has been challenged.3 Six large series examined the value of a detailed ultrasound examination before 24 weeks of gestation in populations in which the incidence of major abnormalities ranged from 1.4 percent to 2.5 percent.3–8 Authors reported heterogeneous ranges of sensitivity and positive predictive values (17–71 percent and 75–98 percent, respectively), but excellent specificity and negative predictive values (99.9 percent and 98–99.5 percent, respectively). However, more skeptical opinions about the value and cost effectiveness of ultrasound screening for fetal abnormalities have been posited.3 Unfortunately, this large study of 15,151 women focused on a selected low-risk population (representative of 37 percent of the general population) and reported a lower rate of detection than most of the other studies; the power of this analysis was also too low to make conclusions about many secondary outcomes (e.g.

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perinatal mortality and morbidity, survival rate if a major anomaly is present, termination of pregnancy for major fetal abnormalities). Studies in which a detailed second-trimester scan was conducted at 20–22 weeks gestation seem to report a higher detection rate than those in which this was done earlier. Another factor to take into account is the minimal standard required for fetal cardiac examination when this is included in the routine scan: a four-chamber view alone is likely to be less accurate than a combined view of the short axis of the heart and imaging of the crossing of the great arteries. The usefulness and cost effectiveness of prenatal ultrasound are likely to depend on the standards required for prenatal care and the competence of the operators and therefore reflect to a great extent a social choice rather than technical limitations.

Common defects amenable to prenatal diagnosis Craniospinal defects

Neural tube defects (NTDs) Incidence and prevalence,9 etiology,10 morbidity and mortality,11 and prevention12 are fully discussed in Chapter 23. Ultrasonographically, the diagnosis of anencephaly can be made as early as 12 weeks’ gestation. When the cephalic pole of the fetus is situated deeply in the pelvis, transvaginal rather than transabdominal sonography can be used. Absence of the cranial vault and cerebral hemispheres are constant findings. However, the facial bones and brainstem and portions of the occipital bones and midbrain are usually present. Associated spinal lesions are found in up to 50 percent of cases. Encephaloceles are recognized as cranial defects with herniated fluid-filled or brain-filled cysts. They are most commonly found in an occipital location (70–75 percent of cases), but alternative sites include the frontoethmoidal and parietal regions. Associated abnormalities include hydrocephaly, Dandy–Walker malformation, and Meckel syndrome. The prognosis for encephaloceles is inversely related to the amount of herniated cerebral tissue.13 For the diagnosis of spina bifida (SB),14 each neural arch from the cervical to the sacral region

must be examined transversely, longitudinally, and in a frontal plane. In the transverse scan, the normal neural arch appears as a closed circle with an intact skin covering whereas in SB, the arch is U-shaped and there is an associated bulging meningocele or myelomeningocele. The extent of the defect and any associated kyphoscoliosis are best assessed in the longitudinal and frontal scans. The prognosis for the lesion is assessed by applying the same criteria as those used by Lorber15 postnatally. However, limb movements may appear to be normal even with major lumbosacral lesions and are therefore of no prognostic significance. The ultrasonographic diagnosis of fetal open spina bifida (OSB) has been greatly enhanced by the recognition of associated abnormalities in the skull and brain.16,17 These abnormalities include cerebral ventriculomegaly, microcephaly, frontal bone scalloping (lemon sign; Figure 25.1), and obliteration of the cisterna magna with either an “absent” cerebellum or abnormal anterior curvature of the cerebellar hemispheres (banana sign; see Figure 25.1). Nyberg et al.17 suggested that the presence of a lemon sign is related to gestational age. Among their 50 cases with OSB, they noted a lemon sign in 89 percent of the 27 fetuses examined before 24 weeks, in 50 percent of the 16 fetuses examined between 24 and 34 weeks, and in none of the seven fetuses examined after 35 weeks. Van den Hof et al.18 evaluated the incidence and diagnostic accuracy of the lemon and cerebellar ultrasonic markers, as well as head size and ventriculomegaly in their study of 1,561 patients at high risk for fetal NTDs. In the 130 fetuses with OSB, there was a relationship between gestational age and the presence of each of these markers (Table 25.1). The lemon sign was present in 98 percent of fetuses at ≤24 weeks gestation but in only 13 percent of those at >24 weeks gestation. Cerebellar abnormalities were present in 95 percent of fetuses irrespective of gestation; however, the cerebellar abnormality at 24 weeks gestation was predominantly the banana sign (72 percent), whereas at gestations >24 weeks, it was cerebellar “absence” (81 percent). Both growth restriction and cerebral ventriculomegaly significantly worsened with gestation, while the head circumference remained disproportionately small throughout gestation.

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A

B Figure 25.1 Transverse section of the fetal head (A) at the level of the septum cavum pellucidum, demonstrating the “lemon” sign (scalloping of the frontal bones), and suboccipital bregmatic view (B), demonstrating the

“banana” sign (anterior curvature of the cerebellar hemispheres and obliteration of the cisterna magna), in a 21-week fetus with OSB (arrow), visible on a frontal plane (C).

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Genetic Disorders and the Fetus

C Figure 25.1 Continued

Table 25.1 Ultrasound findings in 130 fetuses with OSB in relation to gestational age Feature

Gestation (weeks) 24 weeks

(n5107)

(n523)

Lemon-shaped skull

105 (98%)

3 (13%)

Abnormal cerebellum

103 (96%)

21 (91%)

Banana-shaped

74 (69%)

4 (17%)

Absent

29 (27%)

17 (74%)

Va/H: >97.5th centile

61 (57%)

18 (78%)

Vp/H: >97.5th centile

79 (74%)

19 (83%)

HC: 5 mm at 20–30 weeks, and >7 mm at 30–40 weeks.146,149 Transient hydronephrosis may be due to relaxation of smooth muscle of the urinary tract by the high levels of circulating maternal hormones or maternal–fetal overhydration.150 However, if the renal pelvis measures more than 10 mm with a pelvis-tokidney diameter ratio of more than 50 percent with rounded calyces, the disease is usually progressive.151 Similarly, during the first 24 hours of life, there may be transient disappearance of mild and moderate hydronephrosis due to relative dehydration and decreased glomerular filtration rate. Therefore, postnatal assessment of the baby should be delayed until 48 hours after birth. Ureteropelvic junction obstruction is usually sporadic, and although in some cases there is an anatomic cause, such as ureteral valves, in most instances the underlying cause is thought to be functional. In 70–90 percent of cases, the condition is unilateral.151 Prenatal diagnosis is based on the demonstration of hydronephrosis in the absence of dilated ureters and bladder. Occasionally, perinephric urinomas and urinary ascites may be present. Postnatally, renal function is assessed by serial isotope imaging studies, and if there is deterioration, pyeloplasty is performed. However, the majority of infants have moderate or good function and can be treated conservatively.152 Ureterovesical junction obstruction is characterized by hydronephrosis and hydroureter in the presence of a normal bladder. The causes are diverse, including ureteric stricture or atresia, retrocaval ureter, vascular obstruction, valves, diverticulum, ureterocele, and vesicoureteral reflux.153 Ureteroceles are usually found in association with duplication of the collecting system. In ureteral duplication, the upper pole moiety characteristically obstructs and the lower one refluxes. The dilated upper pole may enlarge to displace the nondilated lower pole inferiorly and laterally. Vesicoureteric reflux is suspected when intermittent dilation of the upper urinary tract over a short period is seen on ultrasound scanning. Occasionally, in massive vesicoureteric reflux without

obstruction, the bladder appears persistently dilated because it empties but rapidly refills with refluxed urine.154 Primary megaureter can be distinguished from ureterovesical junction obstruction by the absence of significant hydronephrosis.

Urethral obstruction Urethral obstruction can be caused by urethral agenesis, persistence of the cloaca, urethral stricture, or posterior urethral valves. Posterior urethral valves occur only in males and are the commonest cause of bladder outlet obstruction.129 The condition is sporadic and is found in 1–2 per 10,000 boys.155 With posterior urethral valves, there is usually incomplete or intermittent obstruction of the urethra, resulting in an enlarged and hypertrophied bladder with varying degrees of hydroureters, hydronephrosis, a spectrum of renal hypoplasia and dysplasia, oligohydramnios, and pulmonary hypoplasia. In some cases, there is associated urinary ascites from rupture of the bladder or transudation of urine into the peritoneal cavity. Megacystis–microcolon–intestinal hypoperistalsis syndrome is a rare condition of uncertain cause that also presents with dilated bladder, ureters, and pelvicalyceal system, but in the absence of urinary tract obstruction.156 The fetuses are usually female, and the amniotic fluid volume is normal or increased. There is associated shortening and dilation of the proximal small bowel, and microcolon with absent or ineffective peristalsis. In approximately 7 percent of cases, there is omphalocele.156 The disease is usually fatal, caused by bowel and renal dysfunction. In fetal lamb, ureteric ligation during the first half of gestation results in dysplastic kidneys, whereas in the second half of pregnancy, ureteric ligation is associated with the development of hydronephrosis but preservation of renal architecture.157 Harrison et al.158,159 showed that ligation of the urethra and urachus in fetal lambs at 95–105 days of gestation causes severe hydronephrosis and pulmonary hypoplasia. Decompression of the obstructed fetuses by suprapubic cystostomy at 120 days’ gestation improves survival. Furthermore, with decompression, there is significant resolution of the urinary tract dilation and the newborns have less respiratory difficulty (see Chapter 31). Glick et al.160 demonstrated that ure-

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teric ligation at 65 days’ gestation produces renal dysplasia and that subsequent decompression before term prevents renal dysplasia and produces reversible postobstructive changes. At term, the degree of pathologic changes seen in the obstructed and then decompressed kidneys was proportional to the length of time the obstruction existed. Encouraged by the results of these animal studies, and on the assumption that unrelieved obstruction causes progressive renal and pulmonary damage, several investigators have performed in utero decompression of the urinary tract in the human, either by open surgical diversion161 or by the ultrasound-guided insertion of suprapubic vesicoamniotic catheters162,163 (see Chapter 31). The most common underlying pathology was posterior urethral valves, which had a survival of 74 percent. This compares favorably with the reported mortality of 45 percent in untreated posterior urethral valves.164 However, Reuss et al.165 reported that 8/9 fetuses with posterior urethral valves who had no antenatal intervention survived. This technique carries a significant fetal/neonatal morbidity; indeed, vesicoamniotic shunts become obstructed or displaced in up to 25 percent of cases,166 requiring additional interventions to replace the shunt, with their attendant complications, such as preterm labor and delivery and chorioamnionitis. Less common complications include abdominal wall defect. These problems could potentially be avoided by intrauterine surgery aiming at curing the malformation (posterior urethral valves in utero). The experience in humans is mainly that of one team.167 The feasibility of fetal cystoscopy in humans was assessed in a population of 13 fetuses referred for sonographic suspicion of lower obstructive uropathy and serial vesicocenteses were performed for urine biochemistry. This was possible in 11 of 13 cases at 20 (16–28) weeks. Small fiberoptics could be introduced through an 18 gauge needle. The bladder mucosa was characterized (trabeculation, edema, hemorrhage), and dilation of the ureteral orifices was performed. The urethra was successfully cannulated in three of 11 cases; four did not meet the criteria and three decided to terminate the pregnancy. These results fail to demonstrate conclusively that in utero intervention improves renal or pul-

841

monary function beyond what can be achieved by postnatal surgery. However, they expose the need for (1) the investigation of these fetuses with the aim of excluding associated nonrenal abnormalities, and (2) the development of reliable criteria for the prenatal diagnosis of irreversible renal and pulmonary damage. The limited experience in human fetal surgery and the potential technical and developmental problems that arise from endoscopic fetal surgery of posterior urethral valves make it necessary to work on an animal model before any further therapeutic attempt in the human fetus. In the antenatal evaluation of obstructive uropathy, the ultrasonographic finding of multicystic kidneys is associated with renal dysplasia. In hydronephrosis, both the degree of pelvicalyceal dilation and the volume of amniotic fluid are poor predictors of outcome; urodochocentesis or pyelocentesis with measurement of sodium, calcium, urea, and creatinine provides useful information for more accurate counseling of the parents. Furthermore, fetal urinary biochemistry provides a rational basis for selecting patients who may benefit from vesicoamniotic shunting or other intrauterine urinary diversion procedures and allows evaluation of the possible effectiveness of such therapeutic interventions. Poor fetal renal function can be inferred from high urinary sodium and calcium levels and from low urea and creatinine (Figure 25.5).168,169 Serial measurements of the fetal urine biochemical parameters are likely to lead to a more objective assessment of the fetal renal function and should ideally be undertaken before any definite decision has been made about the management of the pregnancy.170 Skeletal dysplasias There is a wide range of rare skeletal dyplasias, each with a specific mode of inheritance, genotype, phenotype, recurrence risk, and implications for neonatal survival and quality of life (see Chapter 22).171,172 Gene discovery (see Chapter 22) has made accurate prenatal diagnosis a reality in some cases. The incidental discovery of a skeletal dysplasia on routine ultrasound screening in a pregnancy not known to be at risk for a specific syndrome necessitates a systematic examination to arrive at the correct diagnosis. All limbs must be evaluated

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Genetic Disorders and the Fetus

Figure 25.5 Urinary sodium (left) and calcium (right) in fetuses with obstructive uropathy, plotted on the appropriate reference ranges (mean, 95th and 5th centiles) with gestation. In 20 cases, the fetuses survived

and/or did not have any evidence of renal dysplasia (open circles); the remaining 40 fetuses died and/or there was histologic evidence of renal dysplasia (closed circle).

for length, shape, mineralization, and movement, and associated abnormalities in other systems, particularly the head, thorax, and spine, should be sought (Table 25.5). A putative diagnosis may then allow definitive confirmation via mutation analysis. The majority of bones of the appendicular system can be imaged in the early second trimester, and several nomograms relating the length of long bones to menstrual age or biparietal diameter have been published.173,174 The severe limb reductions associated with osteogenesis imperfecta type II, achondrogenesis, and thanatophoric, diastrophic, and chondroectodermal dysplasias can be detected by a single measurement at 16–18 weeks gestation (Figure 25.6). In the case of achondroplasia, however, the diagnosis may not become obvious until 22–24 weeks; therefore, serial measurements are necessary. Mutation analysis may be indicated if there is uncertainty (see Chapter 11). Homozygous achondroplasia, which is usually lethal, manifests abnormally short limbs earlier than the heterozygous form. Syndromes vary in the degree of severity to which the proximal (rhizomelic dwarfism, e.g. achondroplasia) or distal (mesomelic

Table 25.5 Limb reduction deformities: associated abnormalities Feature

Example

Cranium Megalocephaly

Achondroplasia

Brachycephaly

Achondrogenesis

Prominent forehead

Thanatophoric dysplasia

Microcephaly

Chondrodysplasia punctata

Chest Short, barrel-shaped

Achondrogenesis

Long, narrow

Asphyxiating thoracic dystrophy

Narrow, pear-shaped

Thanatophoric dwarfism

Rib fractures

Osteogenesis imperfecta

Spine Lumbar lordosis

Achondroplasia

Scoliosis

Diastrophic dwarfism

Flattened vertebrae

Thanatophoric dwarfism

Unossified bodies

Achondrogenesis

dwarfism, e.g. chondroectodermal dysplasia) long bones are affected. The femur, however, is abnormally short even in mesomelic dwarfism; therefore, in our routine fetal abnormality screening, we tend

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843

Figure 25.6 Ultrasonographic picture of the arm, forearm, and hand (arrow) of a fetus with a lethal skeletal dysplasia at 15 weeks.

to confine limb measurement to the femur. When dealing with pregnancies at risk for a skeletal dysplasia, both segments of all limbs are measured. A minor degree of lateral curvature of the femur is commonly seen in normal fetuses. Pronounced bowing, however, is observed in association with campomelic dysplasia, thanatophoric dwarfism, autosomal dominant osteogenesis imperfecta, achondrogenesis, and hypophosphatasia. In the latter, fractures and callus formation may also be detected.175 Reduced echogenicity of bones, suggestive of hypomineralization, is seen in disorders such as hypophosphatasia, osteogenesis imperfecta, and achondrogenesis. The virtual absence of ossification of the spine, characteristic of achondrogenesis, may lead to the erroneous diagnosis of complete spinal agenesis. Similarly, the pronounced clarity with which the cerebral ventricles are imaged, as a result of the poorly mineralized globular cranium in cases of hypophosphatasia, may result in the misdiagnosis of hydrocephalus.

Care must be exercised, however, because lesser degrees of hypomineralization may not be detectable. Isolated limb reduction deformities such as amelia (complete absence of extremities), acheiria (absence of the hand), phocomelia (seal limb) or aplasia–hypoplasia of the radius or ulna are often inherited as part of a genetic syndrome (Holt– Oram syndrome, Fanconi pancytopenia, thrombocytopenia with absent radii syndrome) and are readily diagnosable by ultrasonography in an atrisk fetus. Other causes of focal limb loss include the amniotic band syndrome, thalidomide exposure, and caudal regression syndrome. Ultrasonography can aid in the diagnosis of conditions characterized by limitation of flexion or extension of the limbs such as arthrogryposis and multiple pterygium syndrome. Fetal fingers and toes can be seen and, with meticulous examination, abnormalities of numbers, shape, movement, and attitudes can be recognized.

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Abnormalities of the amniotic fluid volume Ultrasonographically, the diagnosis of polyhydramnios or oligohydramnios is made when there is excessive or virtual absence of echo-free spaces around the fetus.

Polyhydramnios The incidence of polyhydramnios is 1–3 percent using the definition of a vertical deepest pool (VDP) of at least 8 cm.176,177 Severe polyhydramnios, defined by a VDP > 15 cm, represents 5 percent of all cases and is most often associated with other fetal abnormalities. Polyhydramnios is often associated with maternal diabetes, monozygotic twin pregnancies, and fetal malformations. The rate of reported abnormalities varies with the criteria used for the diagnosis of polyhydramnios, and they may be detected in up to 50 percent of cases.176–178 Craniospinal defects (such as anencephaly), facial tumors, gastrointestinal obstruction, compressive pulmonary disorders (such as pleural effusions or asphyxiating thoracic dystrophy), and arthrogryposis produce polyhydramnios by interfering with fetal swallowing. Maternal diabetes mellitus or fetal diabetes insipidus causes fetal polyuria. In most of these conditions, the polyhydramnios develops in the late second or the third trimester. Acute polyhydramnios at 18–24 weeks is seen mainly in association with twin-to-twin transfusion syndrome. Testing for maternal diabetes, detailed sonographic examination for anomalies, and fetal karyotyping should constitute the cornerstones of the diagnostic protocol in the investigation of these cases. The aim is to reduce the risk of very premature delivery and the maternal discomfort that often accompanies severe polyhydramnios. Treatment will obviously depend on the diagnosis, and will include better glycemic control of maternal diabetes mellitus, antiarrhythmic medication for fetal hydrops due to dysrhythmias, and thoracoamniotic shunting for fetal pulmonary cysts or pleural effusions. For the other cases, polyhydramnios may be treated by repeated amniocenteses every few days and drainage of large volumes of amniotic fluid. However, the procedure itself may precipitate premature labor. An alternative and effective method of treatment is the administration of

indomethacin to the mother.179 However, this drug may cause fetal ductal constriction, and close monitoring by serial fetal echocardiographic studies is necessary. In twin-to-twin transfusion syndrome, development of acute polyhydramnios before 28 weeks’ gestation is associated with a high perinatal mortality rate, primarily due to spontaneous abortion or very premature delivery of growth restricted or hydropic babies. This subject is analyzed in detail below.

Oligohydramnios Oligohydramnios in the second trimester is usually the result of preterm premature rupture of the membranes, urinary tract malformations, and uteroplacental insufficiency, and it is associated with a high perinatal mortality. Although diligent ultrasonographic search for fetal malformations is essential, it should be emphasized that, in the absence of the “acoustic window” normally provided by the amniotic fluid and the “undesirable” postures often adopted by these fetuses, confident exclusion of a fetal malformation may be impossible. Nevertheless, in cases of preterm prelabor rupture of the membranes, detailed questioning of the mother may reveal a history of chronic leakage of amniotic fluid. Furthermore, in uteroplacental insufficiency, Doppler blood flow studies will often demonstrate the characteristically high impedance to flow in the placental circulation and redistribution of the fetal circulation in favor of the brain at the expense of the viscera.180 In the remaining cases, intra-amniotic instillation of normal saline may help improve ultrasonographic examination and lead to the diagnosis of fetal abnormalities such as renal agenesis. Fetal blood sampling for diagnosis of chromosomal abnormalities, fetal infection, and fetal hypoxia provides additional information in the prenatal evaluation of these cases (see Chapter 2).

Detection of abnormalities in the first trimester of pregnancy Ultrasonography When fetal development is not a limiting factor, most abnormalities detectable in the second trimester can be described at 12–14 weeks’ gestation.

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The most common abnormalities amenable to an early diagnosis as a result of a routine examination performed at 10–14 weeks will be described. Many of the abnormalities have been diagnosed when a detailed scan has been performed after the finding of an unusual aspect of the fetal nuchal area. The nuchal translucency represents the fluidlike collection in the subcutaneous tissue at the back of the fetal neck that is present during the first trimester (Figure 25.7). Nuchal translucency can be measured successfully by transabdominal ultrasound examination in about 95 percent of cases; in the others, it is necessary to perform vaginal sonography.181

Anencephaly In anencephaly, the primary defect is absence of the cranial vault (Figure 25.8), with subsequent disruption of the cerebral cortex.182 Prenatal ultrasonographic diagnosis of anencephaly during the second and third trimesters is based on the dem-

845

onstration of absent cranial vault and cerebral hemispheres. During the first trimester, anencephaly presents with acrania and varying degrees of cerebral degeneration. In normal fetuses, mineralization of the skull, and therefore hyperechogenicity in comparison to the underlying tissues, occurs at around the 10th week of gestation.183 In a multicenter study involving 55,237 pregnancies at 10–14 weeks’ gestation, there were 47 fetuses with anencephaly (prevalence, about 1 in 1,200).184 During the first phase of the study, 34,830 fetuses were examined, and in eight of the 31 (25.8 percent) with anencephaly, the diagnosis was not made at the 10–14 week scan. Following the audit, 20,407 fetuses were examined, and in all 16 with anencephaly, the diagnosis was made at the 10–14 week scan. These findings demonstrate that anencephaly can be reliably diagnosed at the routine 10–14 week ultrasound scan, provided the specific sonographic features are searched for and recognized.

Figure 25.7 Ultrasonographic picture demonstrating subcutaneous nuchal translucency (between the calipers). In some cases, the translucency extends over a wide area of the fetus but is most prominent behind the neck.

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Genetic Disorders and the Fetus

Figure 25.8 Sagittal view of a 12-week fetus with exencephaly (arrow).

Cardiac defects In a study involving pathologic examination of the heart and great arteries after surgical termination of pregnancy in 112 chromosomally abnormal fetuses identified by nuchal translucency (NT) screening, the majority had abnormalities of the heart and great arteries.185 The most common cardiac lesion seen in trisomy 21 fetuses was an atrioventricular or ventricular septal defect. Trisomy 18 was associated with ventricular septal defects and/or polyvalvular abnormalities. In trisomy 13, there were atrioventricular or ventricular septal defects, valvular abnormalities, and narrowing of either the isthmus or truncus arteriosus. Turner syndrome was associated with severe narrowing of the whole aortic arch. In all four groups of chromosomally abnormal fetuses, the aortic isthmus was significantly narrower than in normal fetuses and the degree of narrowing was significantly greater in fetuses with high NT thickness. It is postulated that narrowing of the aortic isthmus may be the basis of increased NT thickness in all four chromosomal abnormalities.

In a study of 1,389 chromosomally normal fetuses with increased nuchal translucency at 10–14 weeks of gestation, the prevalence of major cardiac defects (diagnosed either by postmortem examination following termination of pregnancy, intrauterine death, or neonatal death or by clinical examination and appropriate investigation of livebirths) was 17 per 1,000.185 The prevalence of cardiac defects increased with NT thickness.186 Two fetal echocardiographic studies at 10–16 weeks gestation reported that 16 of the 20 fetuses with cardiac defects had abnormal collection of nuchal fluid.187,188 Chromosomally normal fetuses with increased NT thickness, particularly more than 3.5 mm, should be rescanned at 16 and 20 weeks, and special attention should be given to the examination of the heart and great arteries.

Omphalocele (exomphalos) Ultrasound studies examining the association between fetal abnormalities and chromosomal defects often fail to take into account the maternal

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847

Figure 25.9 Sagittal view of a 12-week fetus with omphalocele (arrow).

age and gestational age distribution of their population and inevitably report a wide range of results; the reported frequency of chromosomal defects in fetuses with omphalocele varies from 10 to 66 percent (see Chapters 6 and 23). Omphalocele, or herniation of abdominal viscera into the base of the umbilical cord, can be diagnosed at any gestation if liver is involved. In cases in which only bowel is involved, it is essential that the minimum crown-to-rump length (CRL) of 45 mm is considered; otherwise, this can be mistaken for the physiologic herniation of bowel (Figure 25.9). In a study involving 15,726 singleton pregnancies at 11–14 weeks of gestation, the data were used to calculate both the prevalence of omphalocele and the risk of associated chromosomal defects, mainly trisomy 18, at different stages of pregnancy.189 The estimated prevalence of omphalocele in a population with the maternal age distribution of all deliveries in England and Wales, which is very similar to that of the United States (median age, 28 years), is 7.4 per 10,000 at 12 weeks gestation, and

this decreases to 3.5 at 20 weeks and 2.9 in livebirths. Similarly, the estimated frequency of chromosomal defects in fetuses with omphalocele decreases from 40 percent at 12 weeks’ gestation to 28 percent at 20 weeks and 15 percent in livebirths. These findings are not surprising, because omphalocele is a common feature of chromosomal defects that are associated with a high rate of intrauterine lethality.

Megacystis The fetal bladder is visible in only 50 percent of fetuses at 10 weeks but in nearly all cases if the CRL is more than 67 mm.126,190,191 At 10–14 weeks’ gestation, the longitudinal diameter of the fetal bladder (BL) increases with gestation to a maximum of 6 mm or BL-to-CRL ratio of 10 percent. In a study of 24,492 singleton pregnancies, there was megacystis in 15 cases (prevalence of about 1 in 1,600).191 In three of the 15 cases with megacystis, there were chromosomal abnormalities. In the chromosomally normal group, there were seven cases with spontaneous resolution, whereas

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Genetic Disorders and the Fetus

Figure 25.10 Frontal view of a 12-week fetus with megacystis (arrow) that proved to be related to prune-belly syndrome.

in four cases, there was progression to severe obstructive uropathy (Figure 25.10). The BL was 8–12 mm in the seven cases with resolution and in one of the four with progressive megacystis; in the other three with progressive obstruction, the BL was >16 mm. Therefore, severe megacystis (BL, >16 mm) evolves into severe second-trimester oligohydramnios and renal dysplasia. With mild-tomoderate megacystis (BL, 8–12 mm), usually but not invariably, there is spontaneous resolution.

Other abnormalities and genetic syndromes In the vast majority of fetuses with increased NT and normal karyotype, the translucency resolves and the babies are normal. However, in some cases, increased NT may be associated with an underlying abnormality, such as cardiac defect, diaphragmatic hernia, skeletal dysplasia, renal defect, obstructive uropathy, or omphalocele. In some cases, especially those with NT > 3.5 mm, the babies may have a rare genetic syndrome such as Jarcho–Levin syndrome or Smith–Lemli–Opitz syndrome.192 The

prevalence of these syndromes is less than 1 per 20,000, and it is impossible at present to know what percentage of affected fetuses actually have increased NT at 10–14 weeks. It is therefore important that centers participating in the multicenter study have good documentation of all pregnancy outcomes so that we can identify as quickly as possible which genetic syndromes are associated with increased NT. If the NT at 10–14 weeks is >3.5 mm and fetal karyotype is normal, a very detailed scan should be carried out at 20 weeks and attention should be given to the detection of not only major defects but also minor dysmorphic features. Genetic syndromes reported in association with increased NT include the following: • arthrogryposis or multiple pterygium syndrome193 • amnion disruption sequence • Noonan syndrome • Jarcho–Levin syndrome • Smith–Lemli–Opitz syndrome

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Prenatal Diagnosis of Fetal Malformations by Ultrasound

• Stickler syndrome • various skeletal dysplasias.

Fetoscopy/embryoscopy Direct visualization of the embryo and the fetus can be achieved by embryoscopy and fetoscopy, respectively. Both techniques rely on the visualization of the conceptus through an optical device made of lenses or fiberoptics. These were considered vital tools for prenatal diagnosis in the 1970s and early 1980s but became obsolete with improving ultrasound technology in the late 1980s. Embryoscopy is historically the oldest technique for visualizing the embryo and is best used for genetic syndromes known to be expressed in the same families by constant external fetal structural abnormalities (e.g. Ellis–van Creveld, Smith–Lemli–Opitz, DOOR syndrome, Apert, Rothmund–Thomson, Baller–Gerold).194 The procedure can be performed only before 10 weeks when the extracelomic space disappears, ideally at 9 weeks’ gestation. A 1.5–2 mm diameter rigid endoscope is introduced via the cervix through the still thick villus chorion and is put in contact with the amnion; the embryo is examined through this translucent membrane. The risk of miscarriage is around 12.5 percent.194 When the parents can bear delaying the diagnosis until 12–14 weeks, first-trimester ultrasound examination can suspect or even diagnose most of these conditions. However, complete examination of the fetal anatomy by ultrasound is very unlikely, and lethal or complex anomalies as well as isolated structural defects can be associated with other abnormalities that may not be recognized by ultrasound examination. One option is to wait for a detailed ultrasound examination in the second trimester, but this is rarely welcomed by the parents, whose anxiety calls for rapid and complete fetal evaluation, especially when termination of the pregnancy is an option. Confirmation of prenatally diagnosed anomalies is critical for effective counseling. However, when termination of the pregnancy is requested in the first trimester, some patients will decline the burden of a mutilator induced by prostaglandins, and dilation-aspiration techniques seriously limit postmortem examination.

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Further development and refinement of this technology have allowed direct visualization of the fetus with a fiberoptic endoscope that could be directed in the amniotic cavity through a 19–20 gauge needle.195 Precise assessment of the fetal anatomy should be carried out before evacuation, and transabdominal fetoscopy is another option. Various anomalies such as facial clefts (Figure 25.11), encephalocele, and Smith–Lemli–Opitz and Meckel–Gruber syndromes have been diagnosed with this technique.196 There are several concerns regarding the use of this method of investigation. 1. Care should be taken in diagnosing fetal anomaly in the first trimester. Fetoscopy offers an incomplete evaluation of the external fetal anatomy and must be carried out under ultrasound control. 2. Human data on the safety of fetoscopic white light for the developing retina are still limited. 3. The risk of abortion following the procedure is at present unknown. Before the development of high-resolution ultrasonography, transabdominal fetoscopy was carried out with more invasive instruments and was associated with a fetal loss rate of 4–8 percent. The procedure-related risk of miscarriage for the present technique is probably not much greater than that of early amniocentesis (see Chapter 2). An endoscope196 is passed through an 18 gauge needle and adds an extra minute to the procedure of amniocentesis, which can be performed at the same time. Fetoscopy should therefore not add much to the background risk of amniocentesis performed at the same gestational age. However, this remains to be demonstrated, and patients should be counseled accordingly, especially if this technique finds widespread application in early fetal diagnosis and therapy.

Ultrasonographically detectable markers of fetal chromosomal defects The methods of screening to identify the high-risk group are maternal age, ultrasound findings at 11–14 weeks and/or in the second trimester and maternal serum biochemical testing at 11–14 weeks and/or in the second trimester. To calculate the individual risk, it is necessary to take into account the background risk (which depends on maternal

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Figure 25.11 Fetoscopic diagnosis of cleft lip (thick arrow) at 12 weeks, as opposed to the normal lower lip (thin arrow), using a 1 mm endoscope through a 1.3 mm needle.

age and gestational age) and multiply this by a series of factors, which depend on the results of ultrasound findings and maternal serum biochemical tests carried out during the course of the pregnancy. Every time a test is carried out, the background risk is multiplied by the test factor to calculate a new risk, which then becomes the background risk for the next test. This process is called “sequential screening.”197 In 1866, Langdon Down reported that the skin of individuals with trisomy 21 appears to be too large for their body.198 In the 1990s, it was realized that this excess skin can be visualized by ultrasonography as increased NT in the third month of intrauterine life.199

Ultrasonographic detection of chromosomal markers in the first trimester of pregnancy Fetal nuchal translucency Measurement of the fluid-like collection in the subcutaneous tissue at the back of the fetal neck, NT, which is present only during the first trimester, can be achieved by transabdominal or transvaginal examination (see Figure 25.7). Fetal NT thickness increases with CRL; therefore, in determining whether a given NT thickness is increased, it is essential to take gestation into account. In a fetus with a given CRL, every NT measurement represents a factor that is multiplied

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by the background risk to calculate a new risk.200 The larger the NT, the higher the multiplying factor becomes and therefore the higher the new risk. In contrast, the smaller the NT measurement, the smaller the multiplying factor becomes and therefore the lower the new risk.201 There are numerous prospective studies examining the implementation of NT measurement in screening for trisomy 21 (Table 25.6).201–214 Although different cut-offs were used for identifying the screen-positive group, with consequent differences in the false-positive and detection rates, they all reported high detection rates. The combined results on a total of 174,473 pregnancies, including 728 with trisomy 21, demonstrated a

851

detection rate of 77 percent for a false positive rate of 4.7 percent. Trisomy 21 is associated with increased maternal age, increased fetal NT thickness, increased maternal serum free β-hCG and decreased serum pregnancy-associated plasma protein-A (PAPP-A) concentration. Studies have previously estimated that the most effective method of screening for trisomy 21 would be by a combination of maternal age, fetal NT and serum biochemistry at 11–14 weeks’ gestation (Table 25.7) (see Chapter 24). It was predicted that for a false-positive rate of 5 percent the detection rate of trisomy 21 by this method would be about 90 percent, which is superior to the 30 percent achieved by maternal age

Table 25.6 Studies examining the implementation of fetal nuchal translucency (NT) screening Study Pandya et al.

Gestation no. 201a

Weeks

Cut-off

FPR

DR trisomy 21 3 of 4 (75%)

1,763

10–14

NT ≥2.5 mm

3.6%

Szabo et al.201b

3,380

9–12

NT ≥3.0 mm

1.6%

28 of 31 (90%)

Taipale et al.202

6,939

10–14

NT ≥3.0 mm

0.8%

4 of 6 (67%)

Hafner et al.203

4,371

10–14

NT ≥2.5 mm

1.7%

4 of 7 (57%)

Pajkrt et al.204

1,547

10–14

NT ≥3.0 mm

2.2%

6 of 9 (67%)

96,127

10–14

NT ≥95th centile

4.4%

234 of 327 (72%)

2,281

11–14

NT ≥99th centile

0.4%

6 of 8 (75%)

Snijders et al.201 Economides et al.205 Schwarzler et al.205

4,523

10–14

NT ≥2.5 mm

2.7%

8 of 12 (67%)

Theodoropoulos et al.207

3,550

10–14

NT ≥95th centile

2.3%

10 of 11 (91%)

Zoppi et al.208,208a

12,311

10–14

NT ≥95th centile

5.0%

52 of 64 (81%)

Gasiorek-Wiens et al.209

23,805

10–14

NT ≥95th centile

8.0%

174 of 210 (83%)

Brizot et al.210

2,996

10–14

NT ≥95th centile

5.3%

7 of 10 (70%)

Audibert et al.211

4,130

10–14

NT ≥95th centile

4.3%

9 of 12 (75%)

Wayda et al.212

6,750

10–12

NT ≥2.5 mm

4.3%

17 of 17 (100%)

Source: Adapted from Nicolaides, 2003.200 FPR, false-positive rate; DR, detection rate.

Table 25.7 Detection rate for trisomy 21 and false-positive rate of screening tests Screening test

Detection rate

False-positive rate

Maternal age (MA)

30% (or 50%)

5% (or 15%)

MA + serum β-hCG and PAPP-A at 11–14 weeks

60%

5%

MA + fetal nuchal translucency (NT) at 11–14 weeks

75% (or 70%)

5% (or 2%)

MA + fetal NT and nasal bone (NB) at 11–14 weeks

90%

5% 5% (or 2%)

MA + fetal NT and serum β-hCG and PAPP-A at 11–14 weeks

90% (or 80%)

MA + fetal NT and NB and serum β-hCG and PAPP-A at 11–14 weeks

97% (or 95%)

5% (or 2%)

MA + serum biochemistry at 15–18 weeks

60–70%

5%

Ultrasound for fetal defects and markers at 16–23 weeks

75%

10–15%

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Genetic Disorders and the Fetus

alone, the 65 percent by maternal age and secondtrimester serum biochemistry and the 75 percent by maternal age and first-trimester fetal NT.215–217 Pregnancy outcome, including karyotype results or the birth of a phenotypically normal baby, was obtained from 14,383 consecutive cases managed with this protocol.218 The median maternal age of these cases was 34 (range 15–49) years and in 6,768 (47.1 percent) the age was 35 years or greater. The median gestation at screening was 12 (range 11– 14) weeks and the median fetal CRL was 64 (range 45–84) mm. The estimated risk for trisomy 21 based on maternal age, fetal NT and maternal serum free β-hCG and PAPP-A was 1 in 300 or greater in 6.8 percent (967/14,240) normal pregnancies, in 91.5 percent (75/82) of those with trisomy 21 and in 88.5 percent (54/61) of those with other chromosomal defects. For a fixed falsepositive rate of 5 percent the respective detection rates of screening for trisomy 21 by maternal age alone, maternal age and serum free β-hCG and PAPP-A, maternal age and fetal NT, and by maternal age, fetal NT and maternal serum biochemistry were 30.5 percent, 59.8 percent, 79.3 percent, and 90.2 percent, respectively. Consequently, with this method of screening and invasive testing for all screen-positive pregnancies, one chromosomally normal fetus will be lost for every 18 abnormal fetuses that are detected. Alternatively, healthcare planners may recommend that the minimum detection rate should be 60 percent, which can be achieved with screening by fetal NT and serum biochemistry at 11–14 weeks at a false-positive rate of less than 1 percent and a risk cut-off for invasive testing of 1 in 9. In this case, one chromosomally normal fetus will be lost for every 213 abnormal fetuses that are detected. In these calculations it is assumed that the doctors performing CVS are appropriately trained, in which case the procedure-related risk of miscarriage would be 1 percent, which is the same as for second-trimester amniocentesis (see Chapter 5). Recently, Wright et al. demonstrated that fetal NT measurements follow two distributions, one dependent on CRL and the second one independent from CRL.219 They postulated that in normal healthy pregnancies the majority of fetuses show an increase in NT with CRL and only a minority

have relatively large NT independent from CRL. However, in chromosomally abnormal fetuses, NT was increased and independent from CRL. In this so called mixture-model of NT distributions, 95 percent of the trisomic fetuses follow the CRLindependent distribution. Detection rate of trisomy 21 based on maternal age combined with the mixture model reached 80 percent sensitivity with 5 percent false-positive rate (FPR) in 56,000 normal pregnancies and 395 cases with Down syndrome.220 These new estimates were based on multiple regression to adjust for free β-hCG and PAPP-A maternal concentration and pregnancy characteristics at 11–14 weeks.221 In an alternative strategy, maternal blood was obtained at 10 weeks and NT measurement was performed at 12 weeks (Box 25.1). The detection rates were different by 2–4 percent for the same FPR.221 In counseling women, an alternative approach is to accept that decisions made by healthcare planners based on arbitrary equations of the burdens of miscarriage to those of the birth of a chromosomally abnormal baby are contrary to the basic principle of informed consent. Our responsibility is to assess the risk of a pregnancy being affected using the most accurate method and to allow the parents to decide for themselves for or against invasive testing. This provides evidence that currently the most effective method of screening for chromosomal defects is that provided by a combination of maternal age, fetal NT, and maternal serum free β-hCG and PAPP-A at 11–14 weeks and supports the view that the time has come for a total shift to first-trimester screening (see Chapter 24). With the advent of rapid immunoassays, it has become possible to provide pretest counseling, biochemical testing of the mother, ultrasound examination of the fetus and post-test counseling of a combined risk estimate, all within a 1-hour visit to a multidisciplinary one-stop clinic for assessment of risk (OSCAR) for fetal anomalies.222

Three- and four-dimensional ultrasound Fetal face The detection rate of fetal facial anomalies in utero is increased when ultrasound examination of the face includes an analysis in the three traditional

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Box 25.1 Fetal Medicine Foundation protocol for the measurement of nuchal translucency (NT)

• The fetal CRL should be between 45 and 84 mm. • A good sagittal section of the fetus must be obtained, with the fetus horizontal on the screen. The correct view is that of a clearly visualized fetal profile. • The fetus should be in a neutral position, with the head in line with the spine, not hyperextended or flexed. • Ideally, only the fetal head and upper thorax should be shown. The magnification should be as large as possible and ALWAYS such that each slight movement of the callipers produces only a 0.1 mm change in the measurement. • The widest part of the translucency should be measured. • Measurements should be taken with the inner border of the horizontal line of the callipers placed ON the line that defines the nuchal

translucency thickness – the crossbar of the calliper should be such that it is hardly visible as it merges with the white line of the border, not in the nuchal fluid. However, when tissue harmonic imaging (THI) is used, the calipers should be placed slightly inside the NT lines rather than on the lines as THI might thicken the lines. • When magnifying the image it is important to turn the gain down. This avoids the mistake of placing the calliper on the fuzzy edge of the line that causes an underestimate of the nuchal measurement. • Care must be taken to distinguish between fetal skin and amnion. • During the scan more than one measurement must be taken and the maximum one that meets all the above criteria should be recorded. It is good practice to retain at least one image for your patient records.

Table 25.8 Fetal face anomalies as best featured at three ultrasound planes (according to Rotten and LeVaillant224) Plane

Focus on facial features

Anomalies

Midsagittal

Dysmorphologies

Hypoplasia or agenesis of the nasal bones, protruding maxillary prolabium, displacement forwards or backwards of the fetal tongue, retrognathia

Nose-mouth-coronal

Clefts

Labial and palatal clefts

Staged-axial

Facial anomalies

Hypo- or hypertelorism, abnormal orbital diameter, cataract, cleft lip, cleft alveolus with disruption and anteroposterior shift in the alveolar ridge, hypo- or hypertrophy of fetal tongue, hypoplasia or agenesis of the maxilla or mandible

Source: Adapted from Nicolaides, 2003.200

sonographic examination planes, i.e. sagittal, coronal and axial. Integrated approach of 2D and 3D ultrasound with both orthogonal and multislice view modes significantly improved the prenatal detection rate for cleft palate compared to 2D ultrasound alone (88.9 percent versus 22.2 percent).223 The 3D/4D ultrasound is currently an integral part of both screening for facial clefts and cleft

analysis and is a time-saving procedure. The multiplanar reconstruction mode allows the simultaneous analysis of the three reference planes.224 Once the midsagittal plane is obtained, the volume dataset is acquired (in 4D, one of the recorded blocks of data is selected). The surface-rendering mode can be used to identify facial dysmorphologies and clefts (analysis of the lips, nasal bridge, and eyelid obliquity) (Table 25.8). The multiplanar

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slicing mode shows the three reference planes: sagittal, axial and coronal.225 Surface-rendering mode acquired in the coronal plane is used to show the features of soft tissue surface of the fetal face and allows rapid evaluation of facial dysmorphology or clefting. Three-dimensional multiplanar imaging allows standardization of the section planes, simultaneous visualization of the three reference orthogonal planes, and easy analysis of each of the planes.225 Three-dimensional multiplanar reconstructions give more precise information, especially on posterior coronal views. Three-dimensional imaging allows precise evaluation of cleft anatomy and cleft extension and 3D ultrasound is the only modality that can evaluate the secondary palate. Analysis of palate integrity can be facilitated by using skeletal mode, mainly in the coronal plane.225 Campbell et al. also evaluated the clinical value of reverse face 3D ultrasound technique and showed it to be useful in the assessment of the integrity of the hard palate.226 Similarly to 2D, 3D ultrasound analysis is difficult when the fetus is leaning against the uterine wall or the placenta, or when the amount of amniotic fluid volume is insufficient. Fetal facial anomalies can be isolated or associated with chromosomal anomalies or various multiple malformation conditions.227,228 The use of 3D and 4D ultrasound imaging facilitates and allows precise evaluation of the fetal face. Recently, Paladini et al. reported on the use of 3D volume contrast imaging mode in the coronal plane (VCI-C) for visualization of the fetal anterior fontanelle and showed that it was significantly enlarged in fetuses with Down syndrome.229 Fetal central nervous system (CNS) Volume scanning (3D sonography) allows the operator to scan transabdominally or transvaginally, not only in all three classic scanning planes, i.e. axial, coronal and sagittal, but also in any other plane.230 Several postprocessing methods are useful in the evaluation of CNS anomalies. The developing fetal brain can be examined from the 7th week of gestation.231 The IUSOG guidelines published in 2007 have defined the three most useful planes including the axial plane which is acquired by transabdominal ultrasonography showing transventricular, transthalamic and transcerebellar views. The coronal and sagittal planes are easier to

obtain by transvaginal sonography when the fetus is in cephalic presentation. The four sections to be described here from anterior to posterior are: the transfrontal, transcaudate, transthalamic and transcerebellar planes.231 The two clinically significant sagittal sections are the median plane and the two left and right oblique or parasagittal planes.231 Finally, 3D ultrasonography allows three scanning planes that can be displayed on one screen (orthogonal planes).231 Both 2D and 3D techniques allow imaging of the corpus callosum from 18–20 weeks’ gestation and at its final form at 28 weeks gestation. This anechoic structure can be visualized together with the pericallosal arteries using power Doppler. The cavum septi pellicidi should be seen in all fetuses at 18 and 37 weeks’ gestation. Absence of cavum septi pellicidi can be a sign of agenesis of the corpus callosum, holoprosencephaly, septo-optic dysplasia, schizencephaly, porencephaly/hydranencephaly, basilar encephaloceles and severe hydrocephaly.231 Dilatated cavum septi pellicidi has been linked with chromosomal anomalies. Dilation of the lateral ventricles can be a clue to several anomalies such as SB and agenesis of the corpus callosum. The size of the lateral ventricles is influenced aslo by fetal gender, being higher in normal male fetuses than in females. The transcerebellar plane allows evaluation of the posterior fossa inluding the size of the cerebellomedullary cistern, the cerebellar hemispheres, as well as the cerebellar vermis. During the third trimester of pregnancy, cortical development leads to the appearance of the gyri and sulci.231 Acquisition, storage and display are the three basic steps in performing a 3D US study.231 Several displays are available to enhance certain features and help visualize anomalies. The tomographic display allows visualization of multiple parallel slices of the volume. The volume contrast imaging (VCI) and the thick slice display edges and the image is given more depth, and transform some slices of the volume into a 2D image. 3D acquisition of a volume while a power or color Doppler is used for selective imaging of blood vessels is useful in the diagnosis of agenesis of the corpus callosum showing the absence of pericallosal arteries. In the inversion mode, fluid-filled areas of interest can be “inverted” and seen as volumes.

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Phenotypic expression of fetal aneuploidies in the second trimester In the first trimester, a common feature of many chromosomal defects is increased NT thickness. In later pregnancy each chromosomal defect has its own syndromic pattern of abnormalities. Fetal trisomy 21 is associated with a tendency to brachycephaly, mild ventriculomegaly, nasal hypoplasia, nuchal edema (or increased nuchal fold thickness), cardiac defects, mainly atrioventricular septal defects, duodenal atresia and echogenic bowel, mild hydronephrosis, shortening of the femur and more so of the humerus, sandal gap, clinodactyly and midphalanx hypoplasia of the fifth finger. Trisomy 18 is associated with a strawberry-shaped head, choroid plexus cysts, absent corpus callosum, enlarged cisterna magna, facial cleft, micrognathia, nuchal edema, heart defects, diaphragmatic hernia, esophageal atresia, exomphalos, usually with bowel only in the sac, single umbilical artery, renal defects, echogenic bowel, myelomeningocele, growth restriction and shortening of the limbs, radial aplasia, overlapping fingers and talipes or rocker-bottom feet. In trisomy 13, common defects include holoprosencephaly and associated facial abnormalities, microcephaly, cardiac and renal abnormalities with often enlarged and echogenic kidneys, exomphalos, and postaxial polydactyly. Triploidy, in which the extra set of chromosomes is paternally derived, is associated with a molar placenta; pregnancy rarely persists beyond 20 weeks. When there is a double maternal chromosome contribution, the pregnancy may persist into the third trimester. The placenta is of normal consistency but thin, and the fetus demonstrates severe asymmetric growth restriction. Commonly there is mild ventriculomegaly, micrognathia, cardiac abnormalities, myelomeningocele, syndactyly, and “hitch-hiker” toe deformity. The lethal type of Turner syndrome presents with large nuchal cystic hygromata, generalized edema, mild pleural effusions and ascites, cardiac abnormalities, and horseshoe kidney, which are suspected by the ultrasonographic appearance of bilateral mild hydronephrosis.

855

The incidence of abnormalities in common chromosomal defects The incidence of various abnormalities detected by ultrasound examination during the second and third trimesters in fetuses with trisomies 21, 18, and 13, triploidy and Turner syndrome is shown in Table 25.9. For example, in trisomy 21, the most commonly found abnormalities are nuchal edema, mild hydronephrosis, relative shortening of the femur, and cardiac abnormality. The combined data from Nyberg et al. and Bromley et al. are summarized in Table 25.9.231,232 The incidence of each marker in trisomy 21 pregnancies can be divided by their incidence in chromosomally normal pregnancies to derive the appropriate likelihood ratio (Table 25.10). For example, an intracardiac echogenic focus is found in 28.2 percent of trisomy 21 fetuses and in 4.4 percent of chromosomally normal fetuses, resulting in a positive likelihood ratio of 6.41 (28.2–4.4) and a negative likelihood ratio of 0.75 (71.8–95.6). Consequently, the finding of an echogenic focus increases the background risk by a factor of 6.41, but at the same time, absence of this marker should reduce the risk by 25 percent. The same logic applies to each one of the six markers in Table 25.9. Thus, in a 25-year-old woman undergoing an ultrasound scan at 20 weeks’ gestation, the background risk is about 1 in 1,000. If the scan demonstrates an intracardiac echogenic focus but the nuchal fold is not increased, the humerus and femur are not short and there is no hydronephrosis, hyperechogenic bowel or major defect; the combined likelihood ratio should be 1.1 (6.41 × 0 .67 × 0.68 × 0.62 × 0.85 × 0.87 × 0.79), and consequently her risk remains at about 1 in 1,000. The same is true if the only abnormal finding is mild hydronephrosis, which has a combined likelihood ratio of 1.0 (6.77 × 0.67 × 0.6 8 × 0.62 × 0.75 × 0.87 × 0.79). In contrast, if the fetus is found to have both an intracardiac echogenic focus and mild hydronephrosis but no other defects, the combined likelihood ratio should be 8.42 (6.41 × 6.77 × 0.67 × 0.68 × 0.62 × 0.87 × 0.79) and consequently the risk is increased from 1 in 1,000 to 1 in 119. Prefumo et al.233 scanned 7,686 normal singleton pregnancies and determined that first-trimester NT is associated with isolated cardiac echogenic

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Genetic Disorders and the Fetus

Table 25.9 Incidence of ultrasound abnormalities in 461 fetuses with chromosomal defects examined at the Harris Birthright Research Center for Fetal Medicine Fetal abnormality

Chromosomal

Defect Trisomy

21

18

13

Triploidy

Turner

(%; n5155)

(%; n5137)

(%; n554)

(%; n550)

(%; n565)

Strawberry-shaped head

–

54

–

–

–

Brachycephaly

15

29

26

Microcephaly

–

1

24

Ventriculomegaly

16

14

9

Holoprosencephaly

–

3

39

–

2

–

–

–

–

Skull/brain

Choroid plexus cysts Absent corpus callosum

8 –

47 7

–

Posterior fossa cyst

1

10

15

Enlarged cisterna magna

7

16

25

10

32

–

5 18

2 –

6 –

– –

Face/neck Facial cleft

1

10

39

2

–

Micrognathia

1

53

9

44

–

Nuchal edema

38

5

22

1

2

–

Cystic hygromata

4 –

6 88

Chest Diaphragmatic hernia

–

10

6

2

Cardiac abnormality

26

52

43

16

31

17

– 48

Abdomen Omphalocele Duodenal atresia Absent stomach Mild hydronephrosis Other renal abnormalities

– 8

–

2

2 –

– –

3

20

2

2

30

16

37

4

– 8

7

12

24

6

6

Other Hydrops

20

4

7

2

80

Small for gestational age

20

74

61

100

55

Relatively short femur

28

25

9

60

59

Abnormal hands/feet

25

72

52

76

3

30

11

8

Talipes

2 –

n, number of cases.

foci. They concluded that risk calculations for trisomy 21 based on NT should not use cardiac foci as an independent marker.233 In estimating the risk in a pregnancy with a marker, it is logical to take into account the results of previous screening tests. For example, in a 39-year-old woman at 20 weeks’ gestation (background risk for trisomy 21 of about 1 in 100), who had a 11–14 week assessment by fetal NT and serum free β-hCG and PAPP-A that

resulted in a tenfold reduction in risk (to about 1 in 1,000) after the diagnosis of a short femur but no other abnormal findings at the 20-week scan (likelihood ratio of 1.6; see Table 25.10), the estimated new risk is 1 in 625. Notwithstanding the foregoing discussion, a meta-analysis by Smith-Bindman et al.233a evaluated the use of second-trimester ultrasound markers to detect fetal Down syndrome. They

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Prenatal Diagnosis of Fetal Malformations by Ultrasound

857

Table 25.10 Incidence of major and minor defects or markers in the second-trimester scan in trisomy 21 and chromosomally normal fetuses in the combined data of two major series231,232 T21

Normal

Positive LR

Negative LR

LR for isolated marker

Nuchal fold

107/319 (33.5%)

59/9331 (0.6%)

53.05 (39.37–71.26)

0.67 (0.61–0.72)

9.8

Short humerus

102/305 (33.4%)

136/9254 (1.5%)

22.76 (18.04–28.56)

0.68 (0.62–0.73)

4.1

Short femur

132/319 (41.4%)

486/9331 (5.2%)

7.94 (6.77–9.25)

0.62 (0.56–0.67)

1.6

Hydronephrosis

56/319 (17.6%)

242/9331 (2.6%)

6.77 (5.16–8.80)

0.85 (5.16–8.80)

1.0

Echogenic focus

75/266 (28.2%)

401/9119 (4.4%)

6.41 (5.15–7.90)

0.75 (0.69–0.80)

1.1

Echogenic bowel

39/293 (13.3%)

58/9227 (0.6%)

21.17 (14.34–31.06)

0.87 (0.83–0.91)

3.0

Major defect

75/350 (2.4%)

61/9384 (0.65%) 32.96 (23.90–43.28)

0.79 (0.74–0.83)

5.2

Source: Adapted from Nicolaides, 2003.200 Note: From these data, the positive and negative likelihood ratios (with 95% confidence interval) for each marker can be calculated. In the last column is the likelihood ratio for each marker found in isolation. LR, likelihood ratio.

analyzed 56 papers encompassing 1,930 fetuses with Down syndrome and 130,365 unaffected fetuses. In their determination of sensitivity, specificity, and positive and negative likelihood ratios, they assessed the following markers: choroid plexus cyst, thickened nuchal fold, echogenic intracardiac focus, echogenic bowel, renal pyelectasis, and humeral and femoral shortening. Their main conclusion was that individual markers alone were inefficient in discriminating between fetuses with and without Down syndrome, and hence should not be used as indicators for amniocentesis except when associated with other structural abnormalities.

Fetal abnormalities with chromosomal defects Brain abnormalities

Ventriculomegaly In 14 published series on fetal ventriculomegaly,25–28,31,234–239 the mean incidence of chromosomal defects was 13 percent; the incidence was 2 percent for fetuses with no other detectable abnormalities and 17 percent for those with additional abnormalities. The most common chromosomal defects were trisomies 21, 18, and 13 and triploidy.

Holoprosencephaly In the published studies40–43,238,240,241 on fetal holoprosencephaly, the mean incidence of chromo-

somal defects among 93 cases was 33 percent; the incidence was 4 percent for fetuses with apparently isolated holoprosencephaly and 39 percent for those with additional abnormalities. The commonest chromosomal defects were trisomies 13 and 18.

Microcephaly In a series of 2,086 fetuses that were karyotyped because of fetal malformations or growth restriction, the diagnosis of microcephaly was made if the head circumference was below the 5th centile and the ratio of head circumference to femur length was below the 2.5th centile.238 There were 52 cases of microcephaly, and eight (15 percent) of these had chromosomal defects. Eydoux et al.242 reported chromosomal defects in five (25 percent) of 20 cases. In the combined data from these two series, 12 of the 13 chromosomally abnormal fetuses had additional abnormalities, and the most common chromosomal defect was trisomy 13. Others have emphasized the heterogeneity, variability, and complexities involved in the prenatal detection of microcephaly.242a These authors found microcephaly with chromosomal abnormality in 23.3 percent of 30 cases.

Choroid plexus cysts (see Figure 25.2) Several reports233,233a,238,241,243–264 have documented an association between choroid plexus cysts and chromosomal defects, particularly trisomy 18 (13 percent). The mean incidence of chromosomal

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Genetic Disorders and the Fetus

defects in the various published series was 8 percent, with an incidence of 1 percent for apparently isolated lesions and 54 percent for those with additional abnormalities. In a meta-analysis of 13 prospective studies encompassing 24,654 second-trimester cases, including 1,346 fetuses with isolated choroid plexus cysts, Yoder et al.264a noted that seven had trisomy 18 and 5 had trisomy 21. The likelihood of trisomy 18 was 13.8-fold greater than the a priori risk in fetuses with isolated choroid plexus cysts. The likelihood of trisomy 21 was not significantly increased. A subsequent meta-analysis of eight prospective trials aimed to determine the incidence of trisomy 18 in women 48 h could be equally well predicted by means of the Bishop score or cervical length measurement alone.276

Intrapartum use of ultrasound Intrapartum ultrasound has been utilized for several indications.277 Transabdominal ultrasonography appears to be more accurate than digital vaginal examination in establishing the exact position of the fetal head during labor.278 Engagement of the fetal head is normally determined by means of physical examination. Transabdominal sonographic evaluation of engagement of the fetal head consists of determining whether the fetal biparietal diameter is depicted below (engaged) or above the pelvic outlet (not engaged).279 In a different approach, midsagittal translabial ultrasonography showed a close correlation with abdominal palpation regarding engagement of the fetal head.280 Also, the position of the fetal head has been studied by intrapartum ultrasonography, demonstrating that most occipitoposterior positions of the fetal head rotate to the anterior position even near full dilation.281 Arrest of the second stage of labor may result in vaginal instrumental delivery. Problems were encountered during vacuum extraction when intrapartum ultrasound had established lack of descent of the fetal head.282 Intrapartum Doppler ultrasound has provided some interesting data on fetal blood flow.277 A marked reduction in downstream impedance at the level of the fetal middle cerebral artery was

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Genetic Disorders and the Fetus

found in fetuses during uterine contractions and relaxations.283 Marked blood flow changes were also observed in the fetal ductus venosus in women during labor, with a significantly raised ductus venosus Pulsatility Index284 and reduced late diastolic flow velocities coincident with fetal cardiac atrial contraction during uterine contractions.285 Finally, estimation of maternal urinary bladder volume may help in determining postvoid residual volume during labor and postpartum.286

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239. Forrester MB, Merz RD. Population-based study of small intestinal atresia and stenosis, Hawaii, 1986– 2000. Public Health 2004;118:434. 240. Shawis R, Antao B. Prenatal bowel dilatation and the subsequent postnatal management. Early Hum Dev 2006;82:297. 241. Grosfeld JL, Rescorla FJ. Duodenal atresia and stenosis: reassessment of treatment and outcome based on antenatal diagnosis, pathology variance, and long-term follow-up. World J Surg 1993;17:301. 242. Hausler MC, Berghold A, Stoll C, et al. Prenatal ultrasonographic detection of gastrointestinal obstruction: results from 18 European congenital anomaly registries. Prenat Diagn 2002;22:616. 243. Cohen-Overbeek TE, Grijseels EWM, Niemeijer N, et al. Isolated or non-isolated duodenal obstruction: perinatal outcome following a prenatal diagnosis or a diagnosis only at birth. Ultrasound Obstet Gynecol 2008;32:784. 244. Fratelli N, Papageorghou AT, Bhide A, et al. Outcome of antenatally diagnosed abdominal defects. Ultrasound Obstet Gynecol 2007;30:266. 245. Pryde PG, Bardicef M, Treadwell MC, et al. Gastroschisis: can antenatal ultrasound predict infant outcomes? Obstet Gynecol 1994;84:505. 246. Cohen-Overbeek TE, Hatzman TR, Steegers EA, et al. The outcome of gastroschisis after a prenatal diagnosis or adiagnosis only at birth. Recommendations for prenatal surveillance. Eur J Obstet Gynecol Reprod Biol 2008;139:21. 247. Puligandla PS, Janvier A, Flageole H, et al. Routine cesarian delivery does not improve the outcome of infants with gastroschisis. J Pediatr Surg 2004;39:742. 248. Ergun O, Barksdale E, Ergun FS, et al. The timing of delivery of infants with gastroschisis influences outcome. J Pediatr Surg 2005;40:424. 249. Barisic I, Clementi M, Hausler M, et al. Evaluation of prenatal ultrasound diagnosis of fetal abdominal wall defects by 19 European registries. Ultrasound Obstet Gynecol 2001;18:309. 250. Lakasing L, Cicero S, Davenport M, et al. Current outcome of antenatally diagnosed exomphalos: an 11 year review. J Pediatr Surg 2006;41:1403. 251. Helder AL, Strauss RA, Kuller JA. Omphalocele: clinical outcome in cases with normal karyotypes. Am J Obstet Gynecol 2004;190:135. 252. Cohen-Overbeek TE, Tong WH, Hatzmann TR, et al. Omphalocele: comparison of the perinatal outcome following a prenatal diagnosis or a diagnosis at birth. Ultrasound Obstet Gynecol 2008;in press. 253. De Veciana M, Major CA, Porto M. Prediction of an abnormal karyotype in fetuses with omphalocele. Prenat Diagn 1994;14:487.

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Prenatal Diagnosis and Management of Abnormal Fetal Development with Emphasis 909

254. Brantberg A, Blaas HG, Haugen SE, et al. Characteristics and outcome of 90 cases of fetal omphalocele. Ultrasound Obstet Gynecol 2005;26:527. 255. Elder JS. Antenatal hydronephrosis: fetal and neonatal management. Pediatr Urol 1997;44:339. 256. Degani S, Leibovitz Z, Shapiro I, et al. Fetal pyelectasis in consecutive pregnancies: a possible genetic predisposition. Ultrasound Obstet Gynecol 1997;10:9. 257. Herndon CD, McKenna PH, Kolon TF, et al. A multicenter outcomes analysis of patients with neonatal reflux presenting with prenatal hydronephrosis. J Urol 1999;162:1203. 258. Sairam S, Al Habib A, Sasson S, et al. Natural history of fetal hydronephrosis diagnosed on mid-trimester ultrasound. Ultrasound Obstet Gynecol 2001;17:191. 259. Chudleigh PM, Chitty LS, Pembrey M, et al. The association of aneuploidy and mild fetal pyelectasis in an unselected population: the results of a multicenter study. Ultrasound Obstet Gynecol 2001;17:197. 260. Cohen-Overbeek TE, Wijngaard-Boom P, Ursem NTC, et al. Mild renal pyelectasis in the second trimester: determination of cut-off levels for postnatal referral. Ultrasound Obstet Gynecol 2005;25:378. 261. Aviram R, Pomeran A, Sharony R, et al. The increase of renal pelvis dilatation in the fetus and its significance. Ultrasound Obstet Gynecol 2000;16:60. 262. Ismaili K, Hall M, Donner C, et al. Results of systematic screening for minor degrees of fetal renal pelvis dilatation in an unselected population. Am J Obstet Gynecol 2003;188:242. 263. Hecher K, Henning K, Spernol R, et al. Spontaneous remission of urinary tract obstruction and ascites in a fetus with posterior urethral valves. Ultrasound Obstet Gynecol 1991;1:426. 264. Morris RK, Quinlan-Jones E, Kilby MD, et al. Systematic review of accuracy of fetal urine analysis to predict poor postnatal renal function in cases of congenital urinary tract obstruction. Prenat Diagn 2007;27:900. 265. Zaccara A, Giorlandino C, Mobili L, et al. Amniotic fluid index and fetal bladder outlet obstruction. Do we really need more? J Urol 2005;174:1657. 266. Salam MA. Posterior urethral valve: outcome of antenatal intervention. Int J Urol 2006;13:1317. 267. Kitagawa H, Pringle KC, Koike J, et al. Is a vesicoamniotic shunt intrinsically bad? Shunting a normal bladder. J Pediatr Surg 2007;42:2002. 268. Sago H, Hayashi S, Chiba T, et al. Fetal urethrotomy for obstructive uropathy can be achieved in utero using an endoscopic laser approach. Fetal Diagn Ther 2008; 24:92. 269. To MS, Skentou CA, Royston P, et al. Prediction of patient-specific risk of early preterm delivery using maternal history and sonographic measurement of cer-

270.

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277. 278.

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vical length: a population-based prospective study. Ultrasound Obstet Gynecol 2006;27:362. Celik E, To M, Gajewska K, et al. Cervical length and obstetric history predict spontaneous preterm birth: development and validation of a model to provide individualized risk assessment. Ultrasound Obstet Gynecol 2008;31:549. Alfirevic Z, Allen-Coward H, Molina F, et al. Targeted therapy for threatened preterm labor based on sonographic measurement of the cervical length: a randomized controlled trial. Ultrasound Obstet Gynecol 2007;29:47. Palacio M, Sanin-Blair J, Sanchez M, et al. The use of variable cut-off value of cervical length in women admitted for preterm labor before and after 32 weeks. Ultrasound Obstet Gynecol 2007;29:421. Vankayalapati P, Sethna F, Roberts N, et al. Ultrasound assessment of cervical length in prolonged pregnancy: prediction of spontaneous onset of labor and successful vaginal delivery. Ultrasound Obstet Gynecol 2008;31:328. Mowa N, Jesmin S, Sakuma I, et al. Characterization of vascular endothelial growth factor (VEGF) in the uterine cervix over pregnancy: effects of denervation and implications for cervical ripening. J Histochem Cytochem 2004;52:1665. Rovas L, Sladkevicius P, Strobel E, et al. Reference data representative of normal findings at three-dimensional power Doppler ultrasound examination of the cervix from 17 to 41 weeks. Ultrasound Obstet Gynecol 2006;28:761. Rovas L, Sladkevicius P, Strobel E, et al. Three-dimensional ultrasound assessment of the cervix for predicting time to spontaneous onset of labor and time to delivery in prolonged pregnancy. Ultrasound Obstet Gynecol 2006;28:306. Sherer DM. Intrapartum ultrasound. Ultrasound Obstet Gynecol 2007;30:123. Dupuis O, Ruimark S, Corrine D, et al. Fetal head position during the second stage of labor: comparison of digital and vaginal examination and transabdominal ultrasonographic examination. Eur J Obstet Gynecol Reprod Biol 2005;123:193. Sherer DM, Abulafia O. Intrapartum assessment of fetal head engagement: comparison between transvaginal digital and transabdominal ultrasound determinations. Ultrasound Obstet Gynecol 2003;21:430. Dietz HP, Lanzarone V. Measuring engagement of fetal head: validity and reproducibility of a new ultrasound technique. Ultrasound Obstet Gynecol 2005;25:824. Akmal S, Tsoi E, Howard R, et al. Investigation of occipitoposterior delivery by intrapartum sonography. Ultrasound Obstet Gynecol 2004;24:425.

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282. Henrich W, Dudenhausen J, Fuchs I, et al. Intrapartum translabial ultrasound (ITU): sonographic landmarks and correlation with successful vacuum extraction. Ultrasound Obstet Gynecol 2006;28:753. 283. Li H, Gudmundsson S, Olofsson P. Acute centralization of blood flow in compromised human fetuses evoked by uterine contractions. Early Hum Dev 2006;82:747. 284. Krapp M, Denzel S, Katalinic A, et al. Normal values of fetal ductus venosus blood flow during the first stage of labor. Ultrasound Obstet Gynecol 2002;19:556.

285. Szunyogh N, Zubor P, Dokus K, et al. Uterine activity and ductus venosus flow velocity patterns during the first stage of labor. Int J Gynaecol Obstet 2006;95: 18. 286. Weiniger CF, Wand S, Nadjari M, et al. Post-void residual volume in labor: a prospective study comparing parturients with and without epidural anaethesia. Acta Anaesthesiol Scand 2006;50:1297.

27

Prenatal Diagnosis by Fetal Magnetic Resonance Imaging Nadine Girard1 and Kathia Chaumoitre2 Department of 1Neuroradiology and 2Medical Imaging, Hôpital Nord, CHU Marseille, France

Introduction While ultrasound (US) is currently the primary screening technique for imaging the fetus1,2 (Chapter 25), magnetic resonance imaging (MRI) is playing an increasingly important role in the evaluation of fetal genetic disorders and malformations. MRI has the potential to improve prenatal diagnosis of genetic disorders when combined with ultrasonography and prenatal genetic testing. Although fetal MRI was introduced in the 1980– 90s, the indications for MRI increased as soon as single-shot T2-weighted images (WI) were available.3 Currently T2, T1 and diffusion WI are obtained in reasonable acquisition time, from 15 s to 1 min 40 s.4 Assessment of the fetal central nervous system (CNS) is the major indication for fetal MRI.

Magnetic resonance imaging of the fetal central nevous system Magnetic resonance imaging provides a highly accurate depiction of the morphologic changes of development in the normal brain5–14 and in fetal brain disorders.15–20 Thus, MR imaging can often provide useful information when ultrasonography (US) is inconclusive. MR can provide improved

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

anatomic resolution when US is limited by patient size, fetal presentation or oligohydramnios. Another advantage of MR is that intracranial brain imaging is not impacted by the calvarium, which allows clear identification of the cortex, subarachnoid space7,18 and posterior fossa.21 MRI can be performed several times during the course of pregnancy, which permits the documentation of the natural history of brain injury over gestational age.22 Postmortem MR imaging, which has been described as an alternative to autopsy,23,24 may be a valuable adjunct to autopsy for fetuses with central nervous system (CNS) and non-CNS anomalies. However, although MRI can be considered as a macroscopic analysis, it cannot replace the microscopic and immunologic information given by autopsy. Moreover, although able to depict brain, chest and abdominal malformations, postmortem MRI is not a useful tool for cardiac analysis or for the detection of superficial anomalies that need visual examination (e.g. cutaneous anomalies, dysmorphology, imperforate anus). Technical issues Magnetic resonance imaging does not use ionizing radiation but fetal safety concerns are related to teratogenesis and acoustic damage. A cautious approach uses MRI only during the second and third trimesters. The risk of acoustic damage to the fetus is negligible according to previous reports.25,26 Optimal MR imaging technique is necessary in order to collect as much information as possible about the fetal brain condition. Aside from conventional sequences, diffusion images can also be

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used routinely27–29 to look for vasogenic and cytotoxic edema. Diffusion tensor imaging (DTI) is also available, with long acquisition time (from 5 min on). Monovoxel proton magnetic resonance spectroscopy (MRS) can also be performed in utero, but this technique is used in research protocols and is not yet employed in a routine clinical protocol.30,31 Technical limitations exist in utero compared to the postnatal period. Indeed, there is no coil devoted to the brain in utero with expected loss of signal. However, employment of phased array coils, techniques of parallel acquisition and of movement synchronization help improve the signal.7 No high-resolution three-dimensional (3D) T1 WI can be performed in utero so that small foci of malformation of the cortex such as polymicrogyria may be missed. The lack of a cerebrospinal fluid (CSF) flow sequence prevents complete evaluation of hydrocephalus or cystic malformations. Contrast media IV administration (gadolinium) crosses the placenta and is not approved for use during pregnancy.32,33 Gadolinium injections are therefore not used in utero, which impedes identification of abnormal blood vessels and the rupture of the blood–brain barrier. The protocol for evaluating the fetal brain includes T2 WI following the three planes of the fetal head, axial and coronal T1 images, and axial diffusion images. Additional sequences are performed when necessary as MR angiography in vascular malformations, and gradient echo T2 WI to detect calcifications in cases of congenital infections. Maternal sedation/relaxation with flunitrazepam may be necessary to decrease maternal anxiety and fetal movements, with consequent improvement of the image quality.

malformation (31 percent of cases) or brain injury.9,14,34 Obviously those conditions can overlap since an increased ventricular size may result from injury, malformation or hydrocephalus with or without injury. MRI is extremely helpful in the evaluation of ventriculomegaly because, compared to US, it has greater sensitivity in the detection of associated brain lesions.35 Magnetic resonance imaging is also commonly performed after a normal brain ultrasound within the following contexts11,34: • familial disorders (X-linked hydrocephalus, tuberous sclerosis, neurofibromatosis type 1, siblings with malformation of cortical development, siblings with an inborn error of metabolism) • maternal (acute gestational/maternal event, infections, coagulation disorders) • fetal (twin pregnancy, fetuses presenting with extracerebral multiple malformations that can be associated with brain lesions such as the association of thoracic lymphangioma and megalencephaly, and cardiac malformation that can lead to leukomalacia). Magnetic resonance imaging is not usually indicated in cases of intrauterine growth restriction (IUGR). However, it can provide useful information when IUGR is associated with progressive microcephaly or other abnormalities, such as fetal hydrops or arthrogryposis. Ideally, MRI should be performed at a neuroradiologic unit at a tertiary care facility after US has been performed by a dedicated neurosonographer.36 Because intracranial anomalies can be missed in the second trimester, MRI is optimally performed in the late second or third trimester.37

Fetal brain MRI: when and why? Magnetic resonance is usually performed from 18 to 20 weeks or later. Below this gestational age, MRI is not necessary because severe malformations are well identified by US. Furthermore, the cerebral mantle is thin with MRI anatomic resolution not being high enough to detect subtle signal changes within the cerebral parenchyma. Magnetic resonance imaging is commonly performed because of abnormal US findings. Ventricular dilation is the most frequent indication (40 percent of cases) followed by suspicion of CNS

Anatomic and maturational effects on the MR signal change with gestational age, which correspondingly changes the pathologic features detected by MRI.18–20,22 Thus, an inconclusive MR examination should be repeated, showing the natural history of a pathologic disorder. Moreover, the image itself may be confusing, especially in young fetuses at 20–25 weeks of gestation, because different disorders can display similar images. Brain development is characterized by changes in brain morphology and in brain composition. Changes in brain morphology include increase in

Developing brain

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brain volume and weight, alterations in surface configuration due to the developing sulcation, changes in ventricular shape, and decrease in volume of the subarachnoid spaces.38 These changes are mostly seen during the fetal period and are well illustrated by fetal brain MRI11,39 (Figure 27.1). From midgestation through infancy, brain

growth reflects neuronal differentiation and synapse formation, glial cell differentiation and formation of myelin, programmed cell death, neurotransmitter development and vascular development. The ventricular size is quite constant throughout pregnancy from 20 to 40 weeks and the normal ventricular size at the atrial level on the

Figure 27.1 Axial T2 WI (A, C) and T1 WI (B, D) at 24 weeks (A, B) and 32 weeks (C, D). At 24 weeks the brain is smooth with a multilayered pattern of the cerebral mantle. The germinal matrix is thick and the basal ganglia display bright signal on T1 WI and low signal on

T2 WI due to the high cell density. At 32 weeks the brain is convoluted. The intermediate layer of migrant cells is not identified at this stage. The germinal matrix is not visible.

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axial plane is known from US studies to be 7.6 ± 0.6 mm. The upper limit generally admitted is 10 mm. The subarachnoid spaces are also prominent in young fetuses. Decrease in volume is seen from 30 weeks on. However, prominence of the subarachnoid spaces still persists in some fetuses at the parieto-occipital level; it can be associated with mild uni- or bilateral ventriculomegaly and these aspects are thought to reflect the vacuolization of the primary meninges which is known to occur from ventral to dorsal and posterior to anterior, leading to posterior accumulation of cerebrospinal fluid (CSF).40 Sulcation changes dramatically throughout the last half of gestation, progressing from an agyric brain to a convoluted pattern. Gyri appear in regular sequence on the cerebral surface. The sylvian fissure is the last to be achieved and is dependent upon the development of the frontal and temporal operculum. An open insula can be considered as a marker of different clinical conditions such as prematurity, aminoacidopathies and abnormal cortical development. The convoluted pattern of the cerebellum is well seen from 30 weeks on and is always identified beyond 33 weeks. The cerebellar surface appears quite smooth until 31–32 weeks. Note that in vivo MR evaluation of gyration shows discrepancy with histology,38 with time delay on MRI. The germinal matrices volume increases between 13 and 26 weeks. One-half of its volume is lost between 26 and 28 weeks, and gradually regresses. Note that this 2-week period is a time of high risk for hemorrhage of the germinal matrix together with its high fibrinolytic activity at that time. The most rapid changes in brain composition and myelination occur between midgestation and the second postnatal year. Two partially overlapping stages can be identified: a period of oligodendrocyte proliferation and differentiation, and a period of rapid myelin synthesis and deposition. The effects of brain composition changes on the MR signal are a shortening of T1 (bright signal on T1 WI) and a shortening of T2 (dark signal on T2 WI). Primary mechanisms responsible for these effects are the decrease of water content mainly in the white matter, the cell density and the MR properties of lipids of myelin. The high cell density and the cell packing observed in the

cortex, the basal ganglia and the germinal matrix are responsible for a multilayered pattern of the cerebral hemisphere seen in utero. The intense proliferation of astrocytes to guide neuronal migration and of oligodendrocytes before the onset of myelination (the so-called myelination gliosis) is depicted as an intermediate layer within the white matter. This layer of migrating cells is transient and is seen up to 30 weeks. From 30 weeks on, some residual nests of cells can persist and appear as periventricular nodules, predominantly in the frontal areas, that should not be confused with nodules of leukomalacia. Absence of the intermediate layer prior to 30 weeks on fetal MRI coincides with white matter injury, whatever the cause of the damage.11 In contrast, a persistent subcortical layer beyond 30 weeks is seen in agyria-pachygyria. The subplate is an important transient structure because it is a temporary goal of afferent fibers originating from the thalamus, brainstem nuclei and the contra- and ipsilateral hemispheres. The subplate also acts as a reservoir for maturing neurons and transient synapses. The subplate is thick at 29 weeks and thus well identified on fetal brain MRI immediately below the cortical ribbon. It regresses after 31 weeks and disappears after birth, and thus seems to coincide with expansion of gyration. The germinal matrix is also highly cellular in young fetuses and appears as a thick layer on fetal MRI up to 29–30 weeks. Disruption or nodular appearance of the ventricular wall coincides with ependymal reactions to injury, especially ventricular dilation, infection or inflammation. Signal changes from brain myelination are detected early in utero and are seen in the posterior brainstem at 20 weeks, in the posterior limb of the internal capsule after 33 weeks, in the optic tracts and in the white matter underlying the central area after 35 weeks.

Developmental abnormalities Brain malformations are characterized by their specific morphologic changes, whereas brain injury may display an abnormal signal, irregular ventricular wall, lack of brain layering, absence of the normal signal of cortex and white matter and/or absence of maturation milestones.10,11,20,22,34

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However, these criteria may overlap because injury of the brain may be associated with a malformation. Vascular malformation can lead to brain injury. Some malformations show abnormal signal such as lipoma, tuber and white matter lesions in tuberous sclerosis (Bourneville disease). CNS malformations Many CNS malformations can be identified in utero. Some malformations, however, may be difficult to identify such as micropolygyria (MPG) below 24 weeks, lobar holoprosencephaly, partial commissural agenesis, and histogenetic disorders of the posterior fossa.18 Malformations are usually classified following the different steps in brain development: disorders of neurulation, of diverticulation, commissural agenesis, disorders of histogenesis, and miscellaneous anomalies that include extracerebral cyst, vascular malformations, and craniosynostosis. However, some points merit emphasis. Corpus callosum agenesis (CCA) is the more frequent malformation (Figure 27.2A). Absence of the corpus callosum and of the other commissures is a nonspecific finding that is part of more than 70 syndromes.41 The important feature is the absence, or the defect, of the corpus callosum and of the associated hippocampal commissure. The resulting deformity of the ventricular complex (lateral ventricles away from each other and from the midline, posteriorly enlarged) is characteristic and easy to identify on fetal brain MRI.42 However, it may be very difficult to achieve the overall evaluation of CCA by MR until after delivery. This limitation is particularly true in cases of partial agenesis involving an interhemispheric cyst that, through its mass effect, can prevent the detection of associated malformations of the cortical development (MCD).11 The associated malformations may be more difficult to identify and should be carefully sought. Many abnormalities may be found in association with the commissural agenesis: ocular malformations, septo-optic dysplasia, hypothalamo-pituitary defects, cystic malformations of the posterior fossa, and craniofacial clefts. Within the brain tissue, other developmental disorders should be looked for such as cortical dysplasia or gray matter heterotopias.

Absence of the septum is also part of commissural agenesis. Septo-optic dysplasia includes absence of the septum, hypoplastic optic nerves, hypoplasia/aplasia of olfactory bulbs, and sometimes pituitary gland abnormalities. This abnormality is usually diagnosed when assessing mild ventriculomegaly. However, hypoplasia of optic nerves and olfactory bulbs may be difficult to identify. Among neural tube defects, myelomeningoceles are easily identified by US and in most cases it is not necessary to proceed with MRI, except in institutions in which prenatal surgery is performed.43 MRI is also performed to look for another malformation besides the Chiari II, complications of hydrocephalus, and to assess cervical and dorsal myelomeningocele before planning neonatal surgery. In contrast, MRI is always performed in encephaloceles and pure meningoceles in order to evaluate the content of the meningocele. Disorders of diverticulation include holoprosencephalies and posterior fossa cysts. Holoprosencephalies are classified as alobar, semilobar (Figure 27.2B) and lobar. Alobar holoprosencephaly is characterized by absence of division of the cortical mantle, with a single vesicle resulting in a single ventricular cavity instead of the third and lateral ventricles, with no septum pellucidum, a common posterior tela choroidea, and fusion of thalami and anterior basal ganglia. In the semilobar form, the interhemispheric fissure is present posteriorly with fusion of the anterior hemispheres. Semilobar holoprosencephaly is characterized by the presence of a cleavage of the posterior hemispheres. In lobar holoprosencephaly, the cleavage is almost complete but fusion of the cortex is seen either at the level of the frontobasal area or at the level of the vertex. This latter form is called syntelencephaly or middle interhemispheric variant of holoprosencephaly.44 Lobar holoprosencephaly is the most difficult form to identify in utero compared to the alobar and semilobar forms. Magnetic resonance imaging is very helpful in cystic malformations of the posterior fossa, in which it is better able than US to detect whether the dural structures, mostly the tentorium, are normally positioned or not.42 Posterior fossa cyst is a frequent indication for MRI in utero because

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Figure 27.2 A, Corpus callosum agenesis at 24 weeks, axial T2 WI: the interhemispheric commissure is absent. The lateral ventricles are widely separated with parallel course. B, Semilobar holoprosencephaly at 32 weeks, axial T2 WI: ventriculomegaly with absence of the septum.

Fusion of the frontal lobes. C, Posterior fossa cyst at 23 weeks, sagittal T2 WI: enlarged posterior fossa with absence of the inferior part of the vermis. D, Tuberous sclerosis at 32 weeks, axial T1 WI: subependymal nodules display a bright signal on T1 WI (arrow).

the CSF spaces in the posterior fossa are normally large. The Dandy–Walker malformation (Figure 27.2C), with either close or open cyst, is characterized by an elevation of the tentorium (well above the inion), the bulging of the parieto-occipital vault, and partial or total absence of the vermis. The retrocerebellar pouch (expansion of the Blake’s pouch) also shows a tentorium that is too high with a normal development of the vermis and is part of the Dandy continuum.21 In contrast, a small posterior fossa is seen in Chiari II malformation. A normally positioned tentorium is seen in malformations within a posterior fossa of normal size, such as histogenetic disorders of the

posterior fossa. Dandy–Walker malformation is associated with other developmental anomalies of the CNS in 50 percent of cases. Numerous chromosomal abnormalities and up to 40 syndromes have been reported in association with this malformation. The presence of distal limb abnormalities (polydactyly) is highly suggestive of a genetic condition.45 The cisterna magna is a dorsal expansion of the fourth ventricle lumen, in which tufts of choroid plexus can be found. The term mega cisterna magna indicates a large cisterna magna within normal anatomic limits and a normally attached tentorium. In some cases the mega cisterna magna decreases and even disappears after birth, con-

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founding the determination of etiology and prognosis. Mega cisterna magna is considered a normal variant but is also seen in cases of mental retardation such as in mutations of the oligophrenin 1 gene,46 trisomy and some inborn errors of metabolism. Magnetic resonance imaging identification of histogenetic disorders, which are rarely suspected on US, facilitates genetic counseling for future pregnancies.47 These abnormalities can be summarized as: • disturbance in cell proliferation with abnormal cell differentiation leading to microlissencephalies, cortical dysplasia with balloon cells, tuberous sclerosis, hemimegalencephaly • disturbance in cell migration resulting in heterotopia, lissencephalies (agyria-pachygyria), and congenital muscular dystrophy • disturbance in late migration and organization of the cortex leading to micropolygyria, schizencephaly and focal dysplasia without balloon cells. Microcephaly describes a small head and brain. Diagnosis is usually made in the last trimester provided the head circumference is three standard deviations below the mean. The frontal lobes are underdeveloped, with obliquity of the lateral ventricles, and a simplified cortical pattern. Abnormal development of the frontal lobes is difficult to depict in early pregnancy since the normal development of the frontal lobes is achieved around term. The sporadic focal cortical dysplasia of Taylor’s type and hemimegalencephaly are extremely rare in utero.18 Tuberous sclerosis is a form of focal cortical dysplasia that associates brain abnormalities as cortical tubers, subependymal nodules and lesions across the white matter, with visceral tumor-like lesions such as cardiac rhabdomyoma that is the primary feature in fetuses. Brain lesions usually appear bright on T1 WI (Figure 27.2D) and of low signal on T2 WI48 and are usually missed on US. Absence of a brain lesion, however, does not rule out the diagnosis of tuberous sclerosis. In contrast, numerous cortical tubers and nodules are considered poor prognostic signs in terms of epilepsy, cognitive and mental development. Heterotopia diagnosed in utero is not common. The indicative sign on US can be ventricular dilation. US is not highly sensitive in cases of agyria or pachygyria that is usually detected on MRI

performed because of familial history. Diagnosis by MRI, however, may be difficult, especially below 32 weeks of gestation. Particular attention is given to the presence of a large subcortical band instead of the subplate in fetuses below 30 weeks of gestation with poor or absent segregation between the cortical plate and the underlying white matter (that appears dark on T2 WI).18 Gyration is not developed before 25 weeks so the diagnosis is extremely difficult at that gestational age. Beyond 30 weeks, special attention is given to the gyration that is not compatible with gestational age. Lissencephaly is also described in association with cerebellar hypoplasia, microcephaly, and corpus callosum agenesis. Overmigration of neurons is seen in the Walker– Warburg syndrome and in congenital muscular dystrophies. Walker–Warburg lissencephaly is rare and manifests as a “cobblestone” cortex and is usually suspected because of hydrocephalus and familial history. Zellweger syndrome is also part of cortical malformations characterized by pachygyria and micropolygyria related to peroxisomal disorder. Of the malformations of the cortex, MPG is the most frequent malformation encountered in utero. From the middle to the end of the third trimester, MRI features are familiar and similar to what is known from the ex utero period. MPG appears on MRI as packed and serrated microgyri, with an irregular cortex–white matter junction. Aberrant sulci, atrophy and white matter abnormalities such as gliosis are also seen. MPG is often perisylvian, but not exclusively, and may be uni- or bilateral. A pseudopachygyric appearance can be seen but careful analysis of the cortex–white matter junction always shows an irregular appearance.18,19 In young fetuses, however, identification of the malformation is difficult and even impossible around 20–21 weeks. MRI appearances include the absence of the normal signal of the cortex (especially on T1 WI), the presence of sulci at the surface of the brain that are not expected according to the gestational age, and the irregular surface of the cerebral hemisphere18,19 (Figure 27.3A,B). It may be necessary in early gestation to repeat the MRI several weeks after the initial referral in order to obtain more familiar images of MPG. MPG is usually detected because of mild ventricular dilation on US or because of the history of infection

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Figure 27.3 A, B, Axial T2 WI at 27 weeks, toxoplasmosis: irregular cerebral surface related to micropolygyria (arrow in A), with parenchymal abscess (arrow in B). C, Old hemorrhage at 30 weeks, axial T2 WI: note the ventriculomegaly with focal thickening of the ventricular

wall (arrow) due to an old hemorrhagic ependymal lesion. D, Acute hemorrhage at 32 weeks, axial T1 WI: severe ventriculomegaly with intraventricular and parenchymal hemorrhage that displays a bright signal on T1 WI.

or hypoxia-ischemia before 22 weeks. MPG is encountered in infectious cases (especially cytomegalovirus infection), in hypoxia-ischemia (such as in twin-to-twin transfusion syndrome) and in genetic disorders, especially when bilateral. MPG, however, is very often idiopathic.

Schizencephaly is a cleft extending from the ependyma to the surface of the brain with either an open or closed lip lined with dysplastic cortex, usually of micropolygyric type. Gray matter heterotopia can also be seen. The defect may be unior bilateral. The septum may be absent, especially

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when the defect is frontal or perisylvian.49 The dysplastic cortex is difficult to identify in early gestation. Abnormal arrangements of sulci are also encountered in dwarfism, especially of thanatophoric type, with horizontally oriented sulci in the temporal lobes associated with a deformation of the cranial vault.18,19 Abnormal sulcation of the medial temporal lobes is described in patients with an FGFR3 mutation.50 Different FGFR3 mutations are responsible for diseases with skeletal dysplasia and short stature, including thanatophoric dysplasia (type I and II), achondroplasia, hypochondroplasia and SADDAN (severe achondroplasia with development delay and acanthosis nigricans).51 FGFR3 mutations are also reported in cases with normal stature,52 as in Crouzon disease, sagittal craniosynostosis and lacrimo-auriculo-dento-digital syndrome. Disturbances of histogenesis in the posterior fossa are not common,18,21 possibly because US is not able to detect these types of malformation. The posterior fossa is usually of normal size, in contrast to the cystic malformations in which the posterior fossa is enlarged, and to the neural tube defects, in which the posterior fossa is small. An enlarged cisterna magna may be a revealing sign on US. Extremely severe pontocerebellar hypoplasia is easy to identify with poor development of the cerebellar hemispheres and persistent brainstem flexure that mimic an arrested brain at the embryonic period. Severe hypoplasia, which suggests a poor prognosis, manifests as small cerebellar hemispheres with shallow brainstem and absence of the anterior bulging of the pons. Cerebellar hypoplasia may be seen with a normal bulge of the pons, especially when unilateral, making the distinction from necrosis challenging. The cortical ribbon of cerebellar hemisphere is usually absent in necrosis, whereas it is most likely seen in hypoplasia, whether irregular or not. Vermian agenesis is a more common malformation of the posterior fossa and easy to identify. Disorders resulting in cerebellar hypoplasia include metabolic diseases, chromosomal abnormality (especially trisomy 18), and fetal alcohol syndrome. Cerebellar cortical dysplasia is uncommon, very often missed on US, and is difficult to identify on MRI. Rhombencephaloschisis and rhombencepha-

losynapsis are very rare. The concern in the posterior fossa is usually about the size of the cisterna magna: diagnostic uncertainties are between a mega cisterna magna, that is considered an anatomic variant, cerebellar dysplasia, which can be associated with mental retardation, cerebellar injury, and a metabolic degenerative disease, all with extremely different prognoses. Vascular malformations usually encountered in utero are the vein of Galen aneurysm (VGAM) and dural sinus malformation (DSM). VGAM develops at the end of the embryonic period so that US usually enables a diagnosis of the malformation during the second trimester. The role of MRI is therefore to evaluate the cerebral parenchyma. The most severe form produces early brain ischemia. Fetal angio-MR also identifies high-flow fistulae. DSM includes pial and nongalenic arteriovenous malformation and is extremely rare in utero. This type of vascular malformation is difficult to identify. Several diseases are known to be hereditary such as Rendu–Osler–Weber disease caused by mutations in either of two genes (ENG or ALK1). Ventriculomegaly and genetic disorders Ventriculomegaly is a major indication for MRI of the fetal CNS. It may be caused by malformation, brain injury and, less commonly, by tumors (see Chapters 25 and 26). Ventricular dilations of genetic cause or related to brain malformation are mostly bilateral whereas cases with cerebral injury mostly show unilateral ventriculomegaly.35 Ventricular dilation is also seen in numerous syndromes in which it involves the frontal horns with a square and sharp shape to the ventricular walls. An apparent mechanism for ventricular dilation is not always found in utero. The prognosis for ventriculomegaly in the fetus is variable. Findings indicative of a more favorable outcome include late diagnosis in the third trimester, slow evolution, a ventricle–hemisphere ratio of no more than 50 percent of normal, and isolated ventriculomegaly. Isolated mild ventriculomegaly (whether unilateral or bilateral) is highly challenging because developmental delay ranges from 0 percent to 36 percent. The underlying mechanism of isolated ventriculomegaly can be related to fetal hypoxia (7 percent),

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early stages of benign external hydrocephalus (16 percent)40 and possible subtle changes of the white matter that are undetectable by conventional MRI. Hydrocephalus, in terms of increased intracranial pressure, may be difficult to identify, because no criteria are available compared to the postnatal period. Indeed, hydrocephalus may be encountered with normal head circumference and visibility of the subarachnoid spaces because of the high water content of the white matter that is malleable, resulting in enlarged ventricles at the expense of the white matter, and because of the counterpressure from amniotic fluid that impedes expansion of the cranial vault. Prolonged hydrocephalus may then lead to axonal degeneration, neuronal loss, gliosis and edema of cerebral tissue. Malformations responsible for ventricular dilation and macrocrania are mainly Chiari II, Dandy–Walker, and aqueductal stenosis. Other malformations also encountered in ventricular dilation, usually not associated with macrocrania, are CCA, holoprosencephalies and arachnoid cyst.53 Inborn errors of metabolism Inborn errors of metabolism manifesting in utero are rare and extremely challenging in terms of diagnosis and prognostic significance. Metabolic diseases can be suspected because of fetal hydrops, IUGR, polyhydramnios, and brain malformation. In utero death, fetal akinesia and arthrogryposis are also part of prenatal manifestations. Nonimmune hydrops fetalis is encountered in lysosomal storage diseases such as mucopolysaccharidosis and sialic acid storage disorders.54,55 CCA can be seen in pyruvate dehydrogenase deficiency, heterotopia in mitochondrial respiratory chain deficiency, and cortical abnormalities in Zellweger disease. Subependymal cysts Subependymal cysts are encountered in numerous diseases. When congenital, they may be the result of hemorrhage, hypoxic-ischemic damage or neurotropic infection. They have been reported in association with congenital viral infections (mainly cytomegalovirus and rubella), metabolic disorders (especially Zellweger syndrome),56 chromosomal abnormalities57 and maternal cocaine consumption. However, subependymal cysts may be an iso-

lated finding in otherwise healthy newborns.58 The etiopathogenesis of subependymal cysts is still unknown. Brain injury Brain injury can also be encountered in genetic syndromes, especially intracranial hemorrhage as a complication of fetal thrombocytopenia.59 Nonimmune causes for fetal thrombocytopenia include severe IUGR, congenital viral infections (especially cytomegalovirus, parvovirus B19), bacterial infections, and genetic syndromes such as thrombocytopenia, absent radius syndrome, trisomies, Wiskott–Aldrich, Kasabach–Meritt, megakaryocytosis and Bernard–Soulier syndrome (thrombasthenia caused by congenital glycoprotein Ib/V/ IX deficiency resulting in thrombocytopenia and giant platelets). Immune causes for fetal thrombocytopenia include fetal and neonatal alloimmune thrombocytopenia and RhD alloimmunized pregnancies with hydropic anemic fetuses. Intracranial hemorrhage is often visualized at the chronic stage as foci of low signal on T2 WI within the cerebral parenchyma and/or the ependymal area (Figure 27.3C). The acute stage of hemorrhage displays a bright signal on T1 WI (Figure 27.3D). In utero second-trimester fetal cerebral infarction can result in cerebral porencephaly. Etiologies consist of vascular occlusive disease (due to a congenital maldevelopment or prothrombotic disorder), infections, trauma, arteriovenous malformation with vascular steal, twin–twin transfusion, and fetal–maternal bleeding. In some cases the cause is never found. Fetal MRI shows loss of cortex and adjacent white matter within a vascular territory, usually with underlying expansion of the adjacent lateral ventricle. Cases are mostly visualized at the porencephalic stage, representing an old injury, and not at the acute stage. However, the utilization of diffusion imaging gives the potential to visualize in utero the acute infarction as a restriction of the motion of water molecules related to cytotoxic edema. In hypoxic-ischemic cases and related disorders, MRI is very often performed at a chronic stage that displays ventriculomegaly with irregular germinal matrix or ventricular walls (due to destruction of the ependyma) and white matter gliosis. This latter damage is not currently identified by MRI. DTI and proton

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Prenatal Diagnosis by Fetal Magnetic Resonance Imaging 921

spectroscopy offer the potential to depict such abnormalities. Congenital infections (especially cytomegalovirus and toxoplasmosis) are also responsible for malformations of the cortex as the result of the chronic response of the fetal brain.20,22,60

Magnetic resonance imaging of non-central nervous system fetal systems Magnetic resonance imaging is an alternative modality to US, which has excellent tissue contrast and a large field of view, is not limited by obesity, overlying bone or oligohydramnios, and can image the fetus in multiple planes. Fetal MRI is useful to appreciate the extent of cervical masses, to differentiate chest masses such as diaphragmatic hernia, cystic adenomatoid malformation and sequestration, to analyze complex genitourinary malformations, and to detect intestinal abnormalities.61 Fetal MRI can be performed from 18 weeks on, depending on the severity of the disease. MRI must be done in an imaging department with fetal and neonatal expertise. Technical issues The basic MRI protocol consists of T2 WI following the three planes of the fetal body: HASTE (Half Fourier Acquisition Single Shot Turbo Spin Echo) and/or True-FISP (Fast Imaging with Steady Precession, bFFE, bTFE)) associated with T1 gradient echo WI (FLASH 2D) sequence in the coronal and sagittal planes.62–65 All sequences are performed with a breath-hold technique. The entire examination time does not exceed 20 minutes. Additional sequences such as diffusion-weighted MRI, hydrography and proton MRS are used in some indications.66–71 Diffusion-weighted sequence is a spin echo echo-planar imaging (SE EPI) single shot sequence that can be performed with a free breathing technique. Recently, normal values of fetal kidney apparent diffusion coefficient (ADC)67,70,72 (and normal values of pediatric kidney ADC) have been reported.73 Few publications have depicted fetal lung diffusion.66–69 Evaluation of the ADC value of fetal kidneys is feasible and, in addition to morphologic exploration, may be a noninvasive means to further explore the fetal kidney. Correla-

tion between ADC values (lung and/or kidney) and gestational age is controversial because of the large variability of ADC values seen at each gestational age. Fetal hydrography is a thick slab RARE sequence74,75 that displays an overall estimation of the tracheobronchial tract, the upper GI tract and the urinary tract. It is also useful to appreciate the amniotic fluid volume. Dynamic cine 2D sequence71 is also used to explore esophagus and bowel peristalsis. More recently, this technique has been shown to offer interesting evaluation of fetal cardiac pathologies.76 Proton MRS can also be performed in utero for lung, liver or amniotic fluid analysis, but is not yet employed in a routine clinical protocol.68–77 Fetal neck Ultrasound can assess most fetal neck abnormalities such as cystic hygroma that can be associated with a chromosomal or other defect (Turner syndrome, Noonan syndrome). MRI examination can be used in some cases of meningoencephalocele to achieve precise definition of CNS anomalies. Lateral masses include mainly cystic lymphangioma, teratoma and branchial cleft cysts, whereas teratoma and lymphangioma are mainly encountered within the anterior cervical area. US with color Doppler is more precise than MRI for analysis of the composition of the mass (septations, calcifications, vascularization). Lymphangioma, the most common mass of the neck, is generally a cystic septated mass with vascularization within the septa. Teratoma is more commonly a solid and calcified mass. Branchial cysts are rarely septated. The MRI contribution is to determine the exact location and the extent of large cervical masses (Figure 27.4A). Note that normal thyroid tissue appears bright on T1 WI, and is considered a landmark for the assessment of neck abnormalities. Fetal chest Fetal MRI is useful in all thoracic abnormalities and can clearly demonstrate the anatomic relationship between the lesion and adjacent organs. Fetal MRI allows correct diagnosis of congenital diaphragmatic hernia (CDH) and evaluation of the consequences on pulmonary growth. Other

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Figure 27.4 A, 23 weeks, sagittal T2 WI: teratoma of the tongue responsible for upper airway obstruction (black arrow). B, 32 weeks, coronal T2 WI: left congenital diaphragmatic hernia containing bowel (star) associated with duodenal obstruction (white arrow). C, 35 weeks,

sagittal T2 WI: suprarenal cystic mass with septations (black arrow) suggesting neuroblastoma. D, 19 weeks, coronal T2 WI: anamnios, lung hypoplasia, major enlargement of the kidneys with bright signal in a fetus presenting Meckel–Grubber syndrome.

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pulmonary malformations, such as cystic adenomatoid malformation, sequestration and bronchogenic cysts, can also be easily identified.78 Pulmonary maturation is more difficult to appreciate but some advances with functional sequences (diffusion WI and MRS) are of interest.68,77,79

Congenital diaphragmatic hernia The most frequent diaphragmatic anomaly is the Bochdalek hernia (90 percent of cases) characterized by a posterolateral diaphragmatic defect, frequently unilateral and left-sided (80 percent). CDH occurs in 1/2,200 to 1/5,500 live births. It is usually isolated and sporadic but karyotype analysis is indicated. This malformation can also be part of a syndrome (e.g. Fryns, Cornelia de Lange, Beckwith–Wiedemann).80 Fetal MRI is useful in cases of CDH to confirm the diagnosis, precisely define the herniated viscera (liver, stomach), search for associated malformations (Figure 27.4B), determine the severity or complete aplasia of the diaphragm, and measure lung volume that is a prognostic factor in CDH.81,82 A lung volume below 25 percent of the normal expected volume correlates with a significant decrease in postnatal survival. It is also possible to appreciate lung maturation through lung signal intensity.66,69

Bronchopulmonary airway malformations Pulmonary malformations are part of a continuum of abnormalities and are often interrelated and well analyzed by US. Cystic adenomatoid malformations are the main lung malformations: these hyperechoic masses appear bright on T2 WI and their size can decrease during pregnancy. Sequestrations are also bright on T2 WI so that color Doppler is mandatory to show the systemic arterial blood supply that is often missed on MRI. However, the differential diagnosis between cystic adenomatoid malformation and sequestration can be difficult. Bronchogenic cysts are generally located along the tracheobronchial tree and can be compressive, resulting in lobar lung emphysema. Bronchogenic cysts appear as unilocular cystic structures. Tracheal, laryngeal and bronchial atresia are extremely rare and often lethal. The most severe form is the CHAOS syndrome (Congenital High Airway Obstruction Syndrome) that manifests as an upper airway obstruction (atresia or stenosis)

with hyperechoic enlarged lungs and inverted diaphragm.83 The contribution of MRI is to identify clearly the upper airway obstruction compared to US. Congenital lobar emphysema is rare and MRI easily confirms the lobar distribution of the lesion that appears bright on T2 WI. Therefore MRI is useful for the assessment of bronchopulmonary airway malformations to identify the exact location of the lesion, achieve a precise diagnosis, determine bilateral involvement, search for associated malformations, and to exclude the CHAOS syndrome.

Lung hypoplasia Unilateral lung hypoplasia is rare. Bilateral lung hypoplasia occurs more frequently related to oligohydramnios (premature membrane rupture, bilateral renal impairment, IUGR). Fetal MRI is useful to measure lung volume84 and to appreciate lung maturation.66,69

Fetal heart and mediastinum Ultrasound with color Doppler, 3D and 4D realtime techniques are the primary tools used to assess the fetal heart. Fetal cardiac MRI is currently used in research protocols and is not yet employed clinically. Dynamic cine 2D sequences are also available71,76 but are still difficult to perform in routine practice. MRI is useful for assessment of mediastinal masses to precisely define the tumor (lymphangioma, teratoma). It is also of value in cases of possible esophageal atresia (polyhydramnios, small stomach) (Figure 27.4D) because it is possible to examine fetal deglutition and to verify the entire course of the esophagus.74 Fetal abdomen and pelvis Fetal MRI can accurately diagnose a wide variety of urinary tract disorders and must be seen as a valuable complementary tool to US in the assessment of the urinary system, particularly in cases of inconclusive ultrasound findings with bilateral anomalies.63,85 Prenatal MRI can help to further characterize bowel obstruction, abdominal masses and genital abnormalities.86

Liver and biliary pathologies Fetal hemochromatosis can be diagnosed easily by MRI because of a low signal of the liver on T2*

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Genetic Disorders and the Fetus

WI.87 Numerous genetic syndromes are associated with biliary anomalies, which are precisely identified on MRI, including gallbladder ectopia or agenesis, and choledochal cysts. Liver tumors manifesting prenatally are not common. However, fetal MRI is necessary to identify the location of the tumor and to depict the signal characteristics on T1, T2 and diffusion WI in order to differentiate the three major liver tumors (hamartoma, hemangioma or hepatoblastoma).

Abdominal masses Magnetic resonance imaging is necessary to explore abdominal masses because of its good tissue characterization and the precise anatomic information obtained. MRI enables recognition of a bright signal on T1 WI consistent with a hemorrhagic lesion such as an adrenal hematoma (evaluation of a suprarenal mass being a frequent clinical condition), and also septations in a cystic lesion such as neuroblastoma (Figure 27.4C) through diffusion WI. MRI is also helpful in the assessment of sacrococcygeal teratoma to delineate pelvic and spinal canal extension, and to outline the solid and cystic components. On the other hand, MRI is not as accurate as US in the evaluation of an isolated cystic mass because it cannot differentiate an ovarian cyst from an intestinal duplication.

Bowel obstructions, anorectal malformations Fetal MRI is informative in gastrointestinal abnormalities because of the easy differentiation between the colon (bright signal on T1 WI and low signal on T2 WI) and the small bowel (bright signal on T2 WI). US is generally sufficient for the diagnosis of duodenal atresia that is often associated with trisomy 21. In contrast, fetal MRI is useful to identify the location of other bowel obstructions, detect microcolon that might imply a complete obstruction and analyze the rectal location.86,88,89 MRI is also performed to analyze the bowel complications of omphalocele and gastroschisis.90 Hirschsprung disease cannot be diagnosed prenatally because functional abnormalities only appear in the neonatal period. Microcolon is easily identified on MRI, permitting the diagnosis of megacystis-microcolon-intestinal hypoperistalsis syndrome.89 Anorectal malformations are difficult to identify by US. Fetal MRI can detect such malformations when

urinary fistula is seen through an abnormal bright signal on T2 WI within the rectum, and/or when the rectal cul-de-sac is above the bladder neck (supralevator lesion). This type of malformation can be encountered either alone or as part of the VACTERL association (vertebral, anal, cardiac, tracheal, esophageal, renal and limb abnormalities). Cloacal malformation is the most severe anorectal abnormality and difficult to diagnose by US. MRI is the best imaging technique to evaluate this rare malformation, almost always found in females, characterized by a common single perineal orifice. Urinary tract pathologies, kidney diseases and genital malformations Urinary tract malformations are frequent and generally detected and well explored by US. Fetal MRI can be an additional tool in cases of oligohydramnios that is often associated with severe urinary tract malformation.63 MRI is not justified in cases of unilateral abnormality. In contrast, it is useful in cases with bilateral dilation to examine the bladder neck and possible urethral valve, identify a complex bilateral malformation (duplex system, ureterocele) and exclude a microcolon. MRI is also informative in detecting microcysts and/or an abnormal bright signal on T2 WI in cases of hyperechoic kidneys and/or enlarged kidneys (Figure 27.4D). It is therefore a contributive technique in the assessment of the numerous fetal nephropathies (e.g. polycystic kidney disease, Bardet–Biedl syndrome, Meckel–Gruber syndrome). Note that renal function can be approached by diffusion WI.67,70,72 MRI is also useful to assess genital malformations, especially the internal genital organs that are difficult to delineate by US. Skeletal malformations The development of 3D US has improved the diagnosis of bone malformations. These may be isolated defects, associated with numerous syndromes (i.e. VACTERL), or may represent one of the osteochondrodysplasias. MRI can be used for vertebral examination with gradient echo T2 WI. However, the 3D CT scanner is currently the best imaging tool for the diagnosis of fetal skeletal abnormalities.91

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Conclusion Fetal MRI for CNS and non-CNS structures is a valuable imaging technique, especially in cerebral and thoracic pathologies. Compared to US scan, MRI offers high tissue contrast, large field of view, and functional information (diffusion WI, MRS). These factors explain the significant development of fetal MRI in the last decade. However, fetal MRI remains a complementary imaging technique that is performed in a radiologic unit at a tertiary care facility after US performed by a dedicated sonographer.

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27. Righini A, Bianchini E, Parazzini C, et al. Apparent diffusion coefficient determination in normal fetal brain: a prenatal MR imaging study. Am J Neuroradiol 2003;24:799. 28. Righini A, Zirpoli S, Mrakic F, et al. Early prenatal MR imaging diagnosis of polymicrogyria. Am J Neuroradiol 2004;25:343. 29. Schneider JF, Confort-Gouny S, Le Fur Y, et al. Diffusion-weighted imaging in normal fetal brain maturation. Eur Radiol 2007;17(9):2422. 30. Girard N, Fogliarini C, Viola A, et al. MRS of normal and impaired fetal brain development. Eur J Radiol 2006;57:217. 31. Girard N, Gouny SC, Viola A, et al. Assessment of normal fetal brain maturation in utero by proton magnetic resonance spectroscopy. Magn Reson Med 2006;56:768. 32. Coakley FV, Glenn OA, Qayyum A, et al. Fetal MRI: a developing technique for the developing patient. Am J Roentgenol 2004;182:243. 33. Patel SJ, Reede DL, Katz DS, et al. Imaging the pregnant patient for nonobstetric conditions: algorithms and radiation dose considerations. Radiographics 2007;27:1705. 34. Girard N, Raybaud C, Gambarelli D. Fetal MR imaging. In: Demaerel P, ed. Recent advances in diagnostic neuroradiology. Berlin: Springer-Verlag, 2001:373. 35. Girard N, Ozanne A, Chaumoitre K, et al. [MRI and in utero ventriculomegaly]. J Radiol 2003;84:1933. 36. Malinger G, Ben-Sira L, Lev D, et al. Fetal brain imaging: a comparison between magnetic resonance imaging and dedicated neurosonography. Ultrasound Obstet Gynecol 2004;23:333. 37. Malinger G, Lerman-Sagie T, Watemberg N, et al. A normal second-trimester ultrasound does not exclude intracranial structural pathology. Ultrasound Obstet Gynecol 2002;20:51. 38. Gilles FH, Gomez IG. Developmental neuropathology of the second half of gestation. Early Hum Dev 2005;81:245. 39. Girard N. Imaging brain maturation. In: Carty H, Brunelle F, Sringer D, Kao S, eds. Imaging children, 2nd edn. Edinburgh: Elsevier, 2005:1711. 40. Girard NJ, Raybaud CA. Ventriculomegaly and pericerebral CSF collection in the fetus: early stage of benign external hydrocephalus? Childs Nerv Syst 2001;4–5:239. 41. Norman MG, McGillivray B, Kalousek DK, et al. Congenital malformations of the brain: pathological, embryological, clinical, radiological and genetic aspects. New York: Oxford University Press, 1995.

42. Raybaud C, Levrier O, Brunel H, et al. MR imaging of fetal brain malformations. Childs Nerv Syst 2003;19:455. 43. Sutton LN, Sun P, Adzick NS. Fetal neurosurgery. Neurosurgery 2001;48:124. 44. Simon EM, Hevner RF, Pinter JD, et al. The middle interhemispheric variant of holoprosencephaly. Am J Neuroradiol 2002;23:151. 45. Philip N. Screening for genetic disorders. Childs Nerv Syst 2003;19:436. 46. Chabrol B, Girard N, N’Guyen K, et al. Delineation of the clinical phenotype associated with OPHN1 mutations based on the clinical and neuropsychological evaluation of three families. Am J Med Genet A 2005;138:314. 47. Barkovich AJ, Raybaud CA. Malformations of cortical development. Neuroimaging Clin North Am 2004; 14:104. 48. Sonigo PC, Rypens FF, Carteret M, et al. MR imaging of fetal cerebral anomalies. Pediatr Radiol 1998;28: 212. 49. Raybaud C, Girard N, Levrier O, et al. Schizencephaly: correlation between the lobar topography of the cleft(s) and absence of the septum pellucidum. Childs Nerv Syst 2001;17:217. 50. Kannu P, Aftimos S. FGFR3 mutations and medial temporal lobe dysgenesis. J Child Neurol 2007;22:211. 51. Horton WA, Hall JG, Hecht JT. Achondroplasia. Lancet 2007;370:162. 52. Arnaud-Lopez L, Fragoso R, Mantilla-Capacho J, et al. Crouzon with acanthosis nigricans. Further delineation of the syndrome. Clin Genet 2007;72:405. 53. Zimmerman RA, Bilaniuk LT. Magnetic resonance evaluation of fetal ventriculomegaly-associated congenital malformations and lesions. Semin Fetal Neonatal Med 2005;10:429. 54. den Hollander NS, Kleijer WJ, Schoonderwaldt EM, et al. In-utero diagnosis of mucopolysaccharidosis type VII in a fetus with an enlarged nuchal translucency. Ultrasound Obstet Gynecol 2000;16:87. 55. Froissart R, Cheillan D, Bouvier R, et al. Clinical, morphological, and molecular aspects of sialic acid storage disease manifesting in utero. J Med Genet 2005;42:829. 56. Cuillier F, Cartault F, Lemaire P, et al. [Subependymal pseudocysts in the fetal brain revealing Zellweger syndrome]. J Gynecol Obstet Biol Reprod (Paris) 2004;33:325. 57. Epelman M, Daneman A, Blaser SI, et al. Differential diagnosis of intracranial cystic lesions at head US: correlation with CT and MR imaging. Radiographics 2006;26:173.

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58. Malinger G, Lev D, Ben Sira L, et al. Congenital periventricular pseudocysts: prenatal sonographic appearance and clinical implications. Ultrasound Obstet Gynecol 2002;20:447. 59. Porcelijn L, van den Akker ES, Oepkes D. Fetal thrombocytopenia. Semin Fetal Neonatal Med 2008;13:223. 60. Barkovich AJ, Girard N. Fetal brain infections. Childs Nerv Syst 2003;19:501. 61. Sandrasegaran K, Lall CG, Aisen AA. Fetal magnetic resonance imaging. Curr Opin Obstet Gynecol 2006;18:605. 62. Breysem L, Bosmans H, Dymarkowski S, et al. The value of fast MR imaging as an adjunct to ultrasound in prenatal diagnosis. Eur Radiol 2003;13:1538. 63. Cassart M, Massez A, Metens T, et al. Complementary role of MRI after sonography in assessing bilateral urinary tract anomalies in the fetus. Am J Roentgenol 2004;182:689. 64. Ertl-Wagner B, Lienemann A, Strauss A, et al. Fetal magnetic resonance imaging: indications, technique, anatomical considerations and a review of fetal abnormalities. Eur Radiol 2002;12:1931. 65. Poutamo J, Vanninen R, Partanen K, et al. Diagnosing fetal urinary tract abnormalities: benefits of MRI compared to ultrasonography. Acta Obstet Gynecol Scand 2000;79:65. 66. Balassy C, Kasprian G, Brugger PC, et al. Diffusionweighted MR imaging of the normal fetal lung. Eur Radiol 2007;18:700. 67. Chaumoitre K, Colavolpe N, Shojai R, et al. Diffusionweighted magnetic resonance imaging with apparent diffusion coefficient (ADC) determination in normal and pathological fetal kidneys. Ultrasound Obstet Gynecol 2007;29:22. 68. Fenton BW, Lin CS, Macedonia C, et al. The fetus at term: in utero volume-selected proton MR spectroscopy with a breath-hold technique – a feasibility study. Radiology 2001;219:563. 69. Moore RJ, Strachan B, Tyler DJ, et al. In vivo diffusion measurements as an indication of fetal lung maturation using echo planar imaging at 0.5T. Magn Reson Med 2001;45:247. 70. Savelli S, di Maurizio M, Perrone A, et al. MRI with diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) assessment in the evaluation of normal and abnormal fetal kidneys: preliminary experience. Prenat Diagn 2007;27:1104. 71. Shen SH, Guo WY, Hung JH. Two-dimensional fast imaging employing steady-state acquisition (FIESTA) cine acquisition of fetal non-central nervous system abnormalities. J Magn Reson Imaging 2007; 26:672.

72. Witzani L, Brugger PC, Hormann M, et al. Normal renal development investigated with fetal MRI. Eur J Radiol 2006;57:294. 73. Jones RA, Grattan-Smith JD. Age dependence of the renal apparent diffusion coefficient in children. Pediatr Radiol 2003;33:850. 74. Chaumoitre K, Wikberg E, Shojai R, et al. Fetal magnetic resonance hydrography: evaluation of a singleshot thick-slab RARE (rapid acquisition with relaxation enhancement) sequence in fetal thoracoabdominal pathology. Ultrasound Obstet Gynecol 2006;27:537. 75. Kline-Fath BM, Calvo-Garcia MA, O’Hara SM, et al. Water imaging (hydrography) in the fetus: the value of a heavily T2-weighted sequence. Pediatr Radiol 2007;37:133. 76. Saleem SN. Feasibility of MRI of the fetal heart with balanced steady-state free precession sequence along fetal body and cardiac planes. Am J Roentgenol 2008;191:1208. 77. Clifton MS, Joe BN, Zektzer AS, et al. Feasibility of magnetic resonance spectroscopy for evaluating fetal lung maturity. J Pediatr Surg 2006;41:768. 78. Cannie M, Jani J, de Keyzer F, et al. Magnetic resonance imaging of the fetal lung: a pictorial essay. Eur Radiol 2008;18:1364. 79. Brewerton LJ, Chari RS, Liang Y, et al. Fetal lung-toliver signal intensity ratio at MR imaging: development of a normal scale and possible role in predicting pulmonary hypoplasia in utero. Radiology 2005;235:1005. 80. Enns GM, Cox VA, Goldstein RB, et al. Congenital diaphragmatic defects and associated syndromes, malformations, and chromosome anomalies: a retrospective study of 60 patients and literature. Am J Med Genet 1998;79:215. 81. Gorincour G, Bouvenot J, Mourot MG, et al. Prenatal diagnosis of congenital diaphragmatic hernia using magnetic resonance imaging measurement of fetal lung volume. Ultrasound Obstet Gynecol 2005;26:738. 82. Jani J, Cannie M, Sonigo P, et al. Value of prenatal magnetic resonance imaging in the prediction of postnatal outcome in fetuses with diaphragmatic hernia. Ultrasound Obstet Gynecol 2008;32:793. 83. Mong A, Johnson AM, Kramer SS, et al. Congenital high airway obstruction syndrome: MR/US findings, effect on management, and outcome. Pediatr Radiol 2008;38:1171. 84. Rypens F, Metens T, Rocourt N, et al. Fetal lung volume: estimation at MR imaging – initial results. Radiology 2001;219:236. 85. Yamashita Y, Namimoto T, Abe Y, et al. MR imaging of the fetus by a HASTE sequence. Am J Roentgenol 1997;168:513.

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86. Saguintaah M, Couture A, Veyrac C, et al. MRI of the fetal gastrointestinal tract. Pediatr Radiol 2002;32:395. 87. Brugger PC, Prayer D. Fetal abdominal magnetic resonance imaging. Eur J Radiol 2006;57:278. 88. Benachi A, Sonigo P, Jouannic JM, et al. Determination of the anatomical location of an antenatal intestinal occlusion by magnetic resonance imaging. Ultrasound Obstet Gynecol 2001;18:163.

89. Veyrac C, Couture A, Saguintaah M, et al. MRI of fetal GI tract abnormalities. Abdom Imaging 2004;29:411. 90. Shinmoto H, Kuribayashi S. MRI of fetal abdominal abnormalities. Abdom Imaging 2003;28:877. 91. Cassart M, Massez A, Cos T, et al. Contribution of three-dimensional computed tomography in the assessment of fetal skeletal dysplasia. Ultrasound Obstet Gynecol 2007;29:537.

28

Induced Abortion for Genetic Indications: Techniques and Complications Lee P. Shulman Feinberg School of Medicine, Northwestern University, and University of Illinois at Chicago College of Pharmacy, Chicago, IL, USA

Women who undergo prenatal genetic screening or diagnosis during the first and second trimesters and are found to be carrying fetuses with abnormalities may choose to continue or terminate their pregnancies. Most women found to be carrying fetuses with autosomal trisomies and severe structural abnormalities ultimately choose to terminate their pregnancies, although this is less true for sex chromosome polysomy.1–4 Indeed, Quadrelli and colleagues found this to be true even where legal abortion is not available (Uruguay).5 Studies in Europe,6 Australia,7 and Canada8 have demonstrated that the expansion of prenatal diagnosis and the decision to terminate abnormal fetuses have made for a considerable reduction in infant mortality rates. Indeed, the study by van der Pal-de Bruin et al. demonstrated that differences in the practice of prenatal screening and diagnosis and termination of pregnancies characterized by congenital anomalies contributed to the reductions in overall perinatal mortality rates observed in various European regions.6 Many safe techniques for terminating pregnancies during the first and second trimesters are available; the decision concerning which technique to use is based primarily on fetal gestational age and the experience of the obstetrician and, in some situations, the wishes of the woman. This chapter

Genetic Disorders and the Fetus, 6th edition. Edited by A. Milunsky & J. Milunsky. © 2010 Blackwell Publishing. ISBN: 978-1-4051-9087-9

focuses on the techniques, complications, and risks of abortion performed during the first and second trimesters of pregnancy. Readers desiring more surgically oriented descriptions of the various techniques of pregnancy termination should seek other sources.9–13

First-trimester techniques The expanding use of techniques such as chorionic villus sampling (CVS) (see Chapter 5) and firsttrimester endovaginal ultrasonography now enables many women to undergo safe and reliable first-trimester prenatal screening and diagnosis. Detecting fetal disorders in the first trimester permits women to undergo first-trimester pregnancy termination, a procedure that is safer and less emotionally traumatic than termination performed during the second trimester. Suction curettage Suction curettage remains the most common method for pregnancy termination in the United States.14–16 The procedure is usually performed between 7 and 13 weeks of gestation and does not require hospitalization, except in high-risk cases (e.g. a patient with a bleeding disorder or severe maternal cardiovascular disease). Although there is a burgeoning literature regarding the application and safety of medical abortion, most terminations for fetal abnormalities occur later in the first trimester when the applicability of medical procedures is considerably more limited; accordingly,

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Genetic Disorders and the Fetus

most pregnancy terminations for first-trimester prenatal diagnoses are still accomplished by surgical techniques.

Technique Determination of gestational age must be performed before suction curettage. If pelvic examination demonstrates uterine size to be appreciably different from the reported gestational age, ultrasound should be used to determine fetal viability, the correct fetal number and gestational age. An ultrasound examination before all termination procedures is routine to assess fetal number and gestational age. This practice has been shown to be cost-effective17 and has reduced the frequency of failed evacuation procedures in the first trimester and eliminated the performance of termination procedures in advanced gestational age pregnancies. Goldstein et al.18 found that preprocedure ultrasound and postprocedure examination of products of conception (POC) considerably decreased procedure-related morbidity. A randomized controlled trial from the Whittington Hospital in London, UK, showed that ultrasound guidance of first-trimester suction curettage was associated with a significantly lower complication rate.19 Suction curettage for first-trimester pregnancy termination usually requires cervical dilation.16 The endocervical canal can be manually dilated using instruments having progressively increasing diameters (e.g. Pratt dilators, Hegar dilators). Alternatively, synthetic dilators (e.g. Dilapan (polyacrylonitrile), Lamicel (magnesium sulfate sponge)) or the seaweed Laminaria japonicum are often used. These osmotic dilators serve to dilate the endocervical canal by absorbing cervical moisture. This uptake in water and the resulting expansion of the dilator produces both a softening of the cervix and dilation of the endocervical canal to 2–3 times the original diameter. Schulz et al.20 showed that procedures using L. japonicum resulted in a fivefold reduction in cervical lacerations compared with manual dilation; however, optimal results with L. japonicum require several hours, whereas manual techniques can be applied for immediate dilation. Our clinical experience indicates similar dilating efficiency between Lamicel and L. japonicum, with Lamicel tents resulting in adequate dila-

tion in a shorter interval (4–6 hours) than Laminaria tents (12–14 hours). Hern21 reported that although Laminaria and Dilapan demonstrated similar efficacy for cervical dilation, the Dilapan dilator was more likely to disintegrate, retract or present minor problems associated with poor dilation (e.g. dilator stuck in cervical canal). Endogenous prostaglandins released as a result of cervical manipulation and dilation may also cause cervical softening; administration of certain prostaglandin analogs are known to result in cervical softening22 and make for a more facile cervical dilation. Oral misoprostol (a prostaglandin E1 analog) appears effective and safe for facilitating cervical dilation before first-trimester suction curettage.23 Vaginal and oral misoprostol have provided safe and effective preoperative dilation.24 MacIsaac et al.25 demonstrated that vaginal misoprostol (400 mg) was superior to oral misoprostol (400 mg) with regard to mean dilation and caused less discomfort than Laminaria tents in a randomized trial of women undergoing surgical abortion. Antiprogesterone compounds such as mifepristone (RU486) have also been shown to be effective in softening the cervix and facilitating cervical dilation.26 Carbonne et al.27 compared vaginal gemeprost (a prostaglandin E1 analog) with oral mifepristone and found that although both products reduced the time for cervical dilation before first-trimester suction curettage, cervical dilation was easier with a 48-hour regimen of oral mifepristone than with vaginal gemeprost. The efficacy of mifepristone for cervical ripening before firsttrimester uterine evacuation has been demonstrated by a study sponsored by the World Health Organization.28 Platz-Christensen et al.29 found mifepristone to be equal to misoprostol for cervical ripening and dilation before first-trimester pregnancy termination, with misoprostol being less expensive and easier to administer than mifepristone. Regardless of the mechanical or chemical technique used, cervical softening can be accomplished before dilation and will facilitate the dilation procedure, shorten the overall operative time, and reduce the morbidity associated with the procedure.30 The Society of Family Planning has published a guideline stating that while the available literature did not support the need for cervical priming to routinely reduce the risk of complica-

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tions associated with first-trimester suction curettage, there was no evidence that osmotic dilators were more effective than pharmacologic agents and that pharmacologic agents were more easy to use.31 If manual dilation is required to dilate the cervix, placement of a paracervical block is appropriate before the procedure; xylocaine without epinephrine is one agent commonly used. If synthetic dilators or L. japonicum are used, a paracervical block can be deferred until their removal. Some operators add synthetic vasopressin (Pitressin) or other vasoactive substances to the injectable anesthetic,32 although the safety and efficacy of this practice are undetermined. Vasovagal syncope, or “cervical shock,” can occur after administration of a paracervical block. Although the patient may appear to have had a seizure, vasovagal syncope is self-limited and is differentiated from seizure activity by bradycardia, rapid recovery, and a lack of postictal state. Use of atropine in the administered anesthetic agent can prevent vasovagal syncope in women who have demonstrated such activity in the past.10 Once endocervical dilation has been achieved, a suction curette (without suction having been started) is inserted into the uterine cavity. The choice of suction curette size is dependent on gestational age. The size of the suction curette usually equals the gestational age (in weeks) of the pregnancy. For example, a number 9 suction curette (9 mm diameter) would be used to evacuate a 9 week-sized uterus. Transparent polyethylene tubing is connected to the curette once the curette is within the uterine cavity. In turn, the other end of the tubing is connected to the collection vessel. Suction is then applied using an aspiration device (e.g. Model VH-II Aspiration Machine, Berkeley Bio-Medical Engineering, Berkeley, CA). The curette is rotated on its axis with little motion along the longitudinal axis of insertion, aspirating uterine contents. When no additional tissue can be aspirated, the curette is withdrawn, with suction being maintained. A metal curette can be used to verify that all products of conception have been removed; however, experienced clinicians will frequently not perform sharp curettage after suction curettage because too vigorous curettage can markedly increase the risk for postabortion intrauterine adhesion formation. When performed, the

931

sharp metal curette used for this purpose should be the largest that easily passes into the uterus. If products remain within the cavity after sharp curettage, suction curettage is repeated. After the procedure, patients are monitored for 30 minutes for hemorrhage or changes in vital signs. Women who are Rh negative and unsensitized receive 300 mg of Rh-immune globulin. Prophylactic antibiotics (e.g. 5-day regimen of tetracycline or doxycycline) are effective in preventing infection.33,34 Methylergonovine maleate (Methergine) is effective for decreasing the risk of postabortion bleeding from uterine atony and helps prevent development of hematometra35 (see below). Accordingly, patients are given a five-dose regimen of 0.2 mg Methergine to be taken orally every 4 hours, beginning immediately after completion of the procedure. In all cases, POC should be examined carefully by the clinician to assess completion of the procedure and gross placental or fetal abnormalities.18

Morbidity While unsafe abortion remains a prominent cause of morbidity and mortality in the developing world,36 legal first-trimester suction curettage remains a safe and effective method for pregnancy termination in developed nations.37–39 In a series of 170,000 consecutive suction curettage procedures performed between 5 and 14 weeks of gestation, Hakim-Elahi et al.38 reported that only one in 1,405 cases (0.07 percent) required hospitalization because of incomplete abortion, sepsis, uterine perforation, hemorrhage, inability to complete the procedure, or combined (intrauterine and tubal) pregnancy. Minor complications such as mild infection, incomplete abortion requiring repeat suction, cervical stenosis or laceration, or seizure resulting from the administration of local anesthetic occurred in one of 118 cases (0.84 percent).38 The safety reported in earlier studies39–42 has been confirmed with comparable safety in programs located in historically underserved areas of the world.43–45 Complication rates for suction curettage performed before the 7th week are higher than for procedures done between weeks 8 and 10.14,46 This likely reflects difficulty in completing uterine evacuation as a result of the small size of the conceptus

932

Genetic Disorders and the Fetus

at this stage of pregnancy, a problem frequently encountered with menstrual extraction (see below). Suction curettage performed after the 13th week is technically more difficult and results in a higher rate of complications because of the larger size of the conceptus. First-trimester suction curettage procedures performed in an outpatient setting result in low rates of morbidity and mortality, comparable to procedures performed within a hospital setting.14 Pregnancy outcomes examined in Danish women who had undergone surgical abortions revealed an increased risk of subsequent preterm and post-term deliveries.47 There was no increased risk for any pregnancy-related complication compared with those of similar parity and age among those women.48 Although there were initial concerns regarding a possible increased risk for breast cancer among women undergoing pregnancy termination an extensive and robust evaluation of international data shows no increased risk.49

Immediate complications Complications resulting from suction curettage can be either immediate or delayed. Immediate complications include hemorrhage and uterine perforation. Postabortion hemorrhage usually results from cervical laceration, uterine perforation or uterine atony. The risk of cervical laceration can be decreased by either careful manual dilation or use of cervical osmotic dilators (e.g. Laminaria, Lamicel).50 The location of uterine perforation, a complication more significant in the pregnant than in the nonpregnant state, determines the amount of bleeding and expression of symptoms. A fundal perforation may go undetected because there is likely to be neither excess bleeding nor other symptoms. However, a lateral uterine perforation may lacerate the uterine artery or uterine vein, resulting in immediate and profuse bleeding per vagina. A broad ligament hematoma may also develop as a result of a lateral perforation and present as a delayed complication manifest by diffuse lower abdominal pain, pelvic mass or maternal fever. Use of general anesthesia has been associated with an increased risk for cervical laceration and uterine perforation,51 as well as uterine atony. However, Hakim-Elahi et al.38 found no increase in morbidity in women undergoing

suction curettage with general anesthesia using methohexital. Immediate postoperative pain without overt bleeding per vagina may indicate development of hematometra. Hematometra (also known as uterine distension syndrome or postabortion syndrome) usually presents with dull, aching lower abdominal pain, possibly accompanied by tachycardia, diaphoresis or nausea. The onset is usually within the first hour after completion of the procedure. Pelvic examination reveals a large globular uterus that is tense and tender. Treatment requires immediate uterine evacuation, allowing the uterus to contract to a normal postprocedure size. Administration of intramuscular Methergine (0.2 mg) is then given to ensure continued contraction of the uterus. Overall, hemorrhage, cervical laceration, and uterine perforation occurred in 1.1 percent of 42,598 suction curettage procedures performed at 8 weeks of gestation.35 These complications were even less frequent (0.06 percent) in another series of 170,000 consecutive cases.38 The marked difference in complication rates between the two studies may reflect operator experience. Tietze and Lewit46 published their report at a time when legal abortion was just beginning to become available in the United States; relatively few physicians were experienced in suction curettage. Hakim-Elahi et al.38 described the ongoing experience of three large Planned Parenthood abortion clinics in New York City from 1971 (when abortion became legal in the state of New York) through 1987; most of the procedures were performed by experienced obstetricians.

Delayed complications Delayed complications of suction curettage may be defined as those occurring more than 72 hours after the procedure. These occur in 1–2 percent of cases and include fever, infection, hemorrhage, and retained POC (usually occurring in combination).38,46 Retained POC may present as postabortion bleeding, fever, midline pelvic mass or pelvic/ abdominal pain. Ultrasound can be helpful in arriving at a diagnosis for delayed postabortion complications; however, any evidence of retained POC (e.g. enlarged uterus) should prompt the physician to repeat the suction curettage.

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Many delayed complications should, in theory, be preventable. Careful examination of the tissue obtained by suction curettage should detect an unsuccessful termination of a singleton pregnancy due to either an ectopic pregnancy or technical difficulties in completely evacuating the uterus. Failure to obtain chorionic villi necessitates an ultrasound examination; if an intrauterine pregnancy is visualized, ultrasonography can be used to assist in locating the POC for suction curettage. Women with suspected ectopic pregnancies should be carefully monitored with serial human chorionic gonadotropin (hCG) levels. Although surgical interventions (e.g. salpingectomy, salpingostomy) have traditionally been used to treat ectopic pregnancies, nonsurgical regimens using methotrexate are now commonly used.52,53

Mortality First-trimester suction curettage has the lowest maternal mortality rate of any surgical method of pregnancy termination.16 The reported death rate is far less than the national maternal mortality rate of 9 per 100,000 livebirths.16,38,54 Hakim-Elahi et al.38 reported no maternal deaths in 170,000 consecutive first-trimester suction curettage procedures. The above-cited studies, as well as studies by other investigators,30–32 all indicate that firsttrimester suction curettage is the safest method for surgical pregnancy termination (Table 28.1); second-trimester techniques of dilation and evacuation, intra-amniotic instillation of abortifacients, and hysterotomy or hysterectomy all carry higher mortality rates (Table 28.2). Despite the differences in morbidity and mortality in first- and

933

second-trimester procedures, mortality rates after the Roe v. Wade US Supreme Court ruling legalizing abortion were considerably lower for all induced abortion compared with other types of abortion (e.g. spontaneous).55 Manual vacuum aspiration (MVA) Meyer57 reported on the transition from menstrual extraction, typically performed at or before 42 days gestation, to manual vacuum aspiration (MVA). The use of pregnancy tests, pathology evaluation, and diagnostic assessment in cases of failure to obtain POC was detailed in a successful in-office use of MVA for early pregnancy termination.56 MVA is performed in a somewhat different fashion from menstrual extraction and is facilitated with current pregnancy diagnostic methods. It is important to recognize that such technqiues that are applicable to early pregnancy termination are

Table 28.1 Mortality rates associated with first-trimester suction curettage Study

Years of study

Number

Number

of deaths

of cases

Nathanson40

1970–1971

0

26,000

Hodgson and

1972–1973

0

10,453

Hodgson42

1972–1973

0

Atrash et al.54

1972–1982

0.8*

100,000*

Hakim-Elahi et al.38

1971–1987

0

170,000

Portmann41 20,248

* Mortality rate calculated from Centers for Disease Control Annual Abortion Surveillance Reports.

Table 28.2 Death rate from legal abortion in the United States, 1972–1982 Procedure

Gestational age (weeks) 8

9–10 1.0

11–12

Suction curettage

0.5

D and E

NA

NA

NA

1.8

Instillation

NA

NA

Hysterectomy/hysterotomy

NA

48.2

Total

0.5

1.1

13–15

16–20

21

NA

NA

NA

3.6

9.5

NA

5.0

33.1

62.6

1.8

4.3

Source: Adapted from Atrash et al.,55 Table II. Note: Death rate is the number of maternal deaths per 100,000 procedures.

Total 0.8

10.4

5.1

10.9

11.7

10.1

80.9

115.1

44.8

11.1

11.8

1.6

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Genetic Disorders and the Fetus

unlikely to currently be of value to clinicians or women considering pregnancy management options for embryos or fetuses characterized by congenital or acquired conditions. As with suction curettage (see below), assessment of gestational age and maternal anatomic findings that could complicate the procedure is carried out before starting the procedure. MVA is usually performed in the office and, unlike menstrual extraction, may involve minimal dilation (osmotic dilators, active dilators, prostaglandin) before insertion of a curette. Once inserted, the curette is attached to a manual aspirator and several aspirations are performed until the operator determines that the uterus is emptied. Tissue obtained at the time of aspiration is sent to the pathology laboratory for confirmation of POC. The procedure is usually performed from 6 to 10 completed weeks of gestation.57,58 Kulier et al.59 found no differences regarding the efficacy and safety of MVA and dilation and suction curettage; cost and time to completion were considerably reduced in the MVA procedures. Mulayim et al.60 recently found that administration of sublingual misoprostol (400 µg) after MVA significantly reduced postoperative bleeding compared to those who did not receive misoprostol. The increasing worldwide use of medical pregnancy termination algorithms will likely reduce the use of MVA and other surgical pregnancy termination procedures. Although most first-trimester termination procedures for fetal abnormalities will be performed after 10 completed weeks, this procedure holds great promise for its safety, efficacy, and cost effectiveness. Medical abortion The past two decades have witnessed the development of medical, or pharmacologic, regimens that are now used in the US and worldwide to induce first- and second-trimester abortions. Used to induce approximately 6 percent of all abortions in the US in 2003,61 medical abortion is primarily used by women seeking pregnancy termination up to 63 days’ gestation.62 Accordingly, it is not a procedure that can be chosen by women seeking pregnancy termination for fetal abnormalities. Nonsurgical methods for pregnancy termination are used by women seeking pregnancy termination

in the second trimester and developments in firsttrimester algorithms may, in the future, be amenable for the termination of first-trimester pregnancies characterized by fetal anomalies. Progesterone plays an integral role in implantation and early development of the conceptus; its deficiency is associated with pregnancy loss.63 Progesterone antagonists are effective in interrupting early pregnancies, which are dependent on progesterone for normal development and maintenance. This offers a nonsurgical method for early pregnancy interruption. The most commonly used antiprogesterone analog for early pregnancy interruption is RU486, or mifepristone. RU486 has high affinity for progesterone receptors, blocking normal progesterone binding and function. This compound has been shown to be an effective and safe abortifacient in early gestation. In a series of 100 very early pregnancies (within 10 days of a missed period), RU486 was effective in causing complete abortion in 85 of the 100 women.64 Baird et al.65 showed that a single oral dose (400–600 mg) of RU486 induced bleeding per vagina in most patients; however, the frequency of incomplete abortion in women using only a single dose of RU486 was approximately 20 percent. Use of a prostaglandin analog (gemeprost suppository per vagina or sulprostone intramuscular injection) as an adjunct to RU486 resulted in complete abortion (without need for further surgical evacuation) in 95 of 100 women whose pregnancies were 42 days or less after their last menstrual period.65 Similar success rates were reported by others.66–71 Misoprostol, a prostaglandin E1 analog, is also effective as a co-inductor of first-trimester abortion. Vaginal misoprostol appears far more effective and better tolerated than oral misoprostol for the induction of first-trimester abortion with mifepristone.72 The optimal drug dosages for the combination of RU486 and prostaglandin analogs and the most efficacious time during pregnancy for these drugs to be administered have been extensively studied.62 The US FDA approved the protocol of mifepristone, 600 mg orally, followed approximately 48 hours later by misoprostol, 400 µg orally. There has been considerable discussion concerning the safety and efficacy of vaginal misoprostol, with several case reports of sepsis associated with vaginal use.

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However, no causation has been demosntrated, and studies have shown that vaginal administration of misoprostol may be more effective than oral adminstration.73

Morbidity and mortality Most women (90–95 percent) treated with RU486 and prostaglandin analogs report transient abdominal cramping.62 Other side effects include nausea, vomiting, diarrhea, breast tenderness, and bleeding per vagina that is heavier than normal menses. In the WHO study,74 these minor complications occurred in similar frequencies in both the singledose study group and the five-dose study group. More serious complications (e.g. heavy bleeding, infection) are rare. No deaths resulting from the administration of RU486, with or without prostaglandin analogs, were reported in the WHO study74 or in any of the cited studies. Medical protocols may be applicable to women considering pregnancy termination up to 12 weeks of gestation following an abnormal prenatal diagnostic test. A regimen of mifespristone followed 48 hours later by a course of vaginal gemeprost resulted in complete abortion in 24 of 25 women, with 23 of the 24 successful cases requiring no more than two gemeprost vaginal pessaries.75 The sole failure underwent surgical evacuation because of heavy bleeding. In a different study, a regimen of mifepristone followed 36–48 hours later by repeated doses of misoprostol was successful in 458 of 483 (94.8 percent) cases.76

Other systemic abortifacients Drugs and substances used for indications other than abortion can also act as systemic abortifacients. Prostaglandin analogs, currently used for nonabortion indications, have been used without medical approbation in areas where legal abortions are either not available or prohibitively expensive. For example, Coelho et al.77 from Brazil described using misoprostol, a readily available prostaglandin E1 analog commonly prescribed for the treatment of peptic ulcer disease and gastritis resulting from chronic NSAID use. Misoprostol also causes uterine contractions and thus has become a popular abortifacient in Brazil, where abortion is illegal. However, misoprostol has been shown to be effective when used in combination with mife-

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pristone (see above). Misoprostol used alone as an abortifacient may not be as effective as mifepristone but will result in complete abortion in almost two-thirds of cases.78,79 A high-dose regimen of oral misoprostol was highly effective in causing complete abortion, with 91 percent of women having a complete second-trimester abortion within 24 hours.80 Another prostaglandin E1 analog, gemeprost, has also been shown to have similar efficacy when used alone as an abortifacient,81 although it also has been shown to be an effective co-inductor of uterine evacuation when used with mifepristone. Another systemic abortifacient that was used is methotrexate. Before FDA approval for a mifepristone-based regimen and the extensive use of methotrexate to treat ectopic pregnancy,52,53 methotrexate was used in combination with prostaglandin analogs to induce early pregnancy termination. Although initial studies suggest the efficacy of methotrexate combination regimens to be less than mifepristone combination regimens, relatively high rates of completed terminations can be obtained when methotrexate is used with misoprostol or gemeprost.82,83

Second-trimester techniques Despite increasing use of screening modalities incorporating first-trimester assessments, CVS, and endovaginal ultrasonography, the majority of prenatal diagnostic testing is still performed in the late first and second trimesters. Thus, the performance of second-trimester genetic pregnancy terminations remains a necessity for centers providing prenatal diagnosis, despite the fact that only 12.5 percent of abortions performed in 2000 were for women presenting at or after 13 weeks gestation.39 In general, second-trimester pregnancy termination procedures carry morbidity and mortality rates higher than first-trimester techniques. Some centers, including ours, have begun to utilize preoperative or preinduction procedures to ensure the delivery of a demised fetus. Prior to dilation and evacuation (D and E), either a KCl intracardiac injection or cord avulsion can be performed while a KCl intracardiac injection can be performed prior to initiation of systemic pharmacotherapy for a labor induction pregnancy

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termination. Even though many feel that performing such procedures increases the cost and adversely affects the safety of procedures, recent state and federal court rulings can be interpreted in a way that could possibly place operators at risk if such fetocidal procedures are not performed. Clinicians who perform pregnancy terminations are strongly encouraged to review the relevant laws in their specific community and jurisdiction. Dilation and evacuation In the United States, D and E is the most common technique used for second-trimester pregnancy termination.15 D and E has the lowest mortality rate of all second-trimester pregnancy termination procedures (see Table 28.2) and morbidity rates comparable to or lower than other secondtrimester techniques.10,84 No adverse impact has been demonstrated on future child bearing after a second-trimester D and E if preoperative cervical dilation was achieved by the use of Laminaria.85 Women undergoing D and E do not usually require hospitalization, unlike those who undergo labor induction techniques; D and E is therefore less expensive than labor induction techniques.86 The psychologic benefits of a rapid outpatient method have also been documented. Kaltreider et al.87 reported that 30 patients undergoing D and E experienced less postoperative pain, anger, and depression than 20 women undergoing labor induction methods. D and E also requires less time to complete than labor induction methods.88 Although D and E is the most commonly used technique for second-trimester pregnancy termination, labor induction methods (e.g. systemic prostaglandins) are still the most commonly used for genetic pregnancy termination. An informal survey of the seven prenatal diagnostic centers involved in the US Collaborative Chorionic Villus Sampling Study89 showed that six of the seven centers participating in the study used labor induction methods (primarily vaginal prostaglandin suppositories) for second-trimester genetic terminations. There are several reasons why D and E is not commonly performed for second-trimester genetic pregnancy terminations. First, not all obstetrician– gynecologists (OB/gyns) are trained or are willing to perform this procedure. However, most large

medical centers now have personnel trained in this procedure. OB/gyns who perform second-trimester D and E require special training and ongoing experience; lower rates of morbidity and mortality are achieved only when D and E is performed by such physicians.90 A second rationale for not using D and E for pregnancy terminations performed for genetic indications is concern about the ability to confirm the genetic diagnosis. However, considerable success in confirming abnormal prenatal diagnoses by pathologic, cytogenetic or DNA analyses of POC obtained by D and E has been reported.91–93 Specifically, prenatal diagnoses were confirmed in 114 consecutive pregnancies terminated after diagnosis of fetal abnormalities.93 A fetal cytogenetic complement from POC was obtained in all but one of 114 cases studied. Ultrasound-directed retrieval of selected organs confirmed prenatal ultrasound diagnoses of fetal structural abnormalities in 13 cases. Bernick et al.94 were able to obtain a cytogenetic result on approximately 99 percent of their cases of D and E. Diagnostic confirmation is thus possible following D and E in the majority of cases; confirmation of fetal cytogenetic abnormalities should be possible in almost all cases, whereas the diagnosis of structural abnormalities will rely on not only the expertise of the clinician but also the expertise of the pathologist and geneticist who evaluate the particular abnormal pregnancy. However, detection of associated structural defects in cases characterized by multiple anomalies will likely require pathologic evaluation of an intact fetus to have the best chance of identifying a syndrome and thus provide the most accurate counseling for future pregnancies.

Technique As with first-trimester pregnancy termination, assessment of gestational age must be performed before second-trimester abortion procedures. In almost all cases of second-trimester genetic termination, ultrasound has been performed before the decision to terminate the pregnancy. Secondtrimester D and E invariably requires dilation of the cervix. Although careful manual dilation usually allows sufficient cervical dilation to enable uterine evacuation in most cases, this technique carries increased risk for cervical laceration, hemorrhage,

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and unsuccessful uterine evacuation.20 The preferred technique uses cervical dilators that gradually expand within the endocervical canal as a result of absorbing moisture from the cervix (see above). Many obstetricians use Laminaria tents made from the seaweed L. japonicum for second-trimester D and E procedures. Proper use of Laminaria tents requires leaving them in place for 12–18 hours to achieve optimal cervical dilation, usually necessitating a 2-day procedure. Alternatively, synthetic dilating devices (e.g. Lamicel, Dilapan) achieve safe and optimal dilation within 6–8 hours and enable the entire procedure to be completed within 1 day. Older techniques using general anesthesia should be avoided, if possible, because they increase maternal morbidity and mortality by relaxing the uterus.95 However, more current methods of anesthesia, including genreal endotracheal methods and spinal and epidural methods, can be used to provide safe and effective pain management. A paracervical block is administered before uterine evacuation. Some OB/gyns add small amounts of vasopressin or other vasoactive substances to the xylocaine, apparently resulting in significantly less intraoperative blood loss.32 Occasionally patients experience vasovagal syncope following administration of the paracervical block (see above); this usually resolves quickly. Certain maternal cardiac disorders (e.g. cardiac arrhythmias) may be relative contraindications to paracervical analgesia. Prophylactic antibiotics may be given at the time of cervical dilation, and antiemetic and antianxiety medications are provided as needed. Products of conception are evacuated using instruments specifically designed to extract intrauterine contents at this stage of gestation. We prefer either Sopher or Bierer forceps; other available ovum forceps are also listed in Box 28.1. Concurrent ultrasonography is also helpful in facilitating uterine evacuation,96,97 especially when the extraction of intact specific fetal parts is necessary to confirm prenatal diagnoses.92,93 Although ultrasound guidance is not a requirement for safe and successful uterine evacuation, it often facilitates the evacuation procedure, particularly in problematic cases such as when patients have severe uterine anteversion or anteflexion.98 After the POC have been evacuated, suction curettage is performed to remove any remaining

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Box 28.1 Ovum forceps used for second-trimester dilation and evacuation

Barrett Bierer Clemetson Forester Kelly placental forceps Moore Peterson Sanger Sopher Van Lith

tissue. As with first-trimester procedures, the OB/ gyn must examine the specimen to verify that all POC have been removed. For terminations performed because of fetal anatomic abnormalities, POC should also be examined and labeled by individuals with expertise in dysmorphology before preparing specimens for pathologic and other confirmatory laboratory analyses. The confirmatory analyses selected (e.g. cytogenetic, DNA, enzymatic) will depend on the specific prenatal diagnosis. After D and E, patients are observed for excessive vaginal bleeding or changes in vital signs. Patients are instructed to expect some lower abdominal cramping, vaginal bleeding (similar to menstrual flow in volume), and possibly low-grade fever. Severe manifestations of these signs and symptoms may presage serious complications and require immediate evaluation by a physician. To prevent uterine atony, intramuscular Methergine (0.2 mg) is given immediately on completion of the procedure, followed by an oral regimen of 0.2 mg Methergine every 4 hours for five doses. Rhimmune globulin (300 µg) is administered to unsensitized Rh-negative patients. It is prudent to contact patients by telephone 24–48 hours after the procedure and arrange postoperative visits no later than 10 days to 2 weeks later.

Morbidity When performed by an experienced OB/gyn,90 D and E carries significantly lower morbidity rates

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than do methods requiring labor induction or surgical procedures (i.e. hysterotomy, hysterectomy).54,98–102 Kafrissen et al.101 compared the safety of 9,572 D and E procedures with 2,805 instillation procedures using an instillate composed of prostaglandin F2α and urea. All procedures were performed between 13 and 24 weeks of gestation. Serious complications (fever >38 °C, hemorrhage requiring blood transfusion or performance of unintended surgery as result of an abortion-related incident) occurred in 0.49 percent of patients undergoing D and E procedures compared with 1.03 percent of patients undergoing prostaglandin/ urea procedures. Only uterine perforation occurred more frequently in the D and E group. Among women undergoing abortions through the 15th menstrual week, Robins and Surrago100 found that 400 patients undergoing D and E had a lower frequency of complications (i.e. blood loss requiring transfusion, cervical laceration, retained POC, fever, vomiting, and diarrhea) than 112 patients undergoing labor induction abortions by intravaginal prostaglandin suppositories. Peterson et al.103 reported that the rate of unplanned hospitalizations resulting from D and E at 13 weeks was 0.6 percent, but was 1.4 percent at 20–21 weeks. Schneider et al.104 found that D and E procedures performed from 18 to 22 weeks were characterized by low complication rates of less than 1 percent. Thus, D and E performed later in the second trimester results in morbidity rates no greater, and possibly less, than labor induction techniques. Nonetheless, the other substantive advantages of D and E (e.g. outpatient procedure, less expensive to perform) make this technique more advantageous compared with other second-trimester techniques (i.e. systemic or intra-amniotic abortifacients, hysterotomy or hysterectomy). A major complication of D and E is uterine perforation. The severity of signs and symptoms depends on the location of the uterine perforation. Lateral perforations involving laceration of the uterine artery or vein are most dangerous because of the possibility of profuse hemorrhage. The use of concurrent ultrasound guidance may reduce the incidence of uterine perforation.96,98 Other causes of hemorrhage include cervical or vaginal lacera-

tion, uterine atony, retained POC, and coagulopathy (apparently secondary to release of tissue thromboplastin into the maternal venous system during D and E). Although ultrasound-directed uterine evacuation, postoperative Methergine, and careful inspection of POC will reduce the incidence of intraoperative and postoperative hemorrhage, complications will invariably occur. Operators must be prepared to administer necessary resuscitation maneuvers needed to stabilize such patients and to subsequently manage their complications. Infection is another serious complication that may occur after D and E. Antibiotic prophylaxis is effective in decreasing febrile morbidity in both first- and second-trimester uterine evacuation procedures.16,33,34 Frequently, postoperative infection is the result of retained POC. If there is any evidence of retained POC, suction curettage should be performed to evacuate the uterus. Ultrasonography may be particularly useful in evaluating and treating such patients.

Mortality Overall, D and E is the safest technique for secondtrimester pregnancy termination. D and E is as safe as having a normal pregnancy and delivery,105 which is untrue of other techniques of secondtrimester pregnancy termination. The Joint Program for the Study of Abortion (JPSA III) showed D and E to be associated with the lowest maternal death to case ratio compared with instillation techniques or hysterotomy/hysterectomy (see Tables 28.2 and 28.3).54,102 Maternal mortality rates associated with D and E procedures increase with the gestational age at which the procedure is performed and become similar to that of instillation procedures later in the second trimester (i.e. >16 weeks of gestation) (see Table 28.3).106 In addition to safety, the other benefits of D and E procedures (e.g. low cost, shorter time to complete abortion, less psychologic stress) make D and E the preferred method for almost all second-trimester pregnancy terminations. Systemic abortifacients The primary advantage of systemic abortifacients is ease of use. Their noninvasive application does

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Table 28.3 Mortality rates of second-trimester pregnancy termination procedures Procedure

Gestational age at time of abortion (weeks) 13–15

16–20

21

Total rate

D and E

3.2

9.2

12.0

4.9

All instillation*

5.5

12.0

13.3

9.6 11.6

Saline

1.7

15.2

12.9

Prostaglandin F2α

12.1

6.0

14.2

6.4

Hysterotomy/

64.9

84.5

123.0

47.8

hysterectomy Source: Adapted from Grimes and Schulz.102 Note: All mortality rates are deaths per 100,000 cases. * Refers to all instillation procedures, irrespective of agent(s) used.

not require surgical expertise; therefore, they can be easily used by non-OB/gyns. However, clinicians who perform second-trimester pregnancy terminations with systemic abortifacients must be ready to provide surgical care should there be a failed induction or retained POC, or if such techniques result in the uncommon but life-threatening complication of uterine rupture.106 The most commonly used systemic abortifacients for second-trimester pregnancy termination are prostaglandin analogs that stimulate uterine contractions and result in the expulsion of the POC.16 However, antiprogesterones such as RU486 have been shown to facilitate and expedite secondtrimester pregnancy termination performed by use of systemic abortifacients,107–109 although their use alone in such cases has been shown to yield poor results, with incomplete expulsion of the POC.110 The most frequently used prostaglandin analog in the United States is prostaglandin E2 (PGE2; dinoprostone) in suppository (20 mg) form. The suppository is placed intravaginally on a regular schedule (usually every 3–4 hours until delivery), either directly into the posterior fornix of the vagina or held in place with a diaphragm. Intramuscular injections of 15-methyl prostaglandin F2α are also used for second-trimester pregnancy termination; however, this abortifacient is no longer marketed in the United States.

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Several groups have reported the use of misoprostol for first- and second-trimester pregnancy terminations. In the second trimester, pregnancy terminations using misoprostol are performed with a 200 mcg intravaginal tablet that is repeated every 12 hours until completion. One study found no effect with regard to procedure efficacy when misoprostol was provided in oral form.111 However, Jain and Mishell112 showed that the rate of successful abortion was 81 percent with dinoprostone and 89 percent with misoprostol, with similar times to completion. The main advantage of misoprostol over dinoprostone is considerably fewer gastrointestinal effects and less hyperpyrexia. Other studies113,114 demonstrated that PGE1 analogs are as effective as PGE2 analogs for inducing secondtrimester pregnancy termination, but with considerably fewer and less severe side effects; these findings are comparable to a study by Hamoda et al.76 showing a 91 percent success rate for secondtrimester pregnancy termination using a high-dose misoprostol protocol. Autry et al.84 found the use of misoprostol to be the safest method for medical induction for second-trimester pregnancy termination. Surrago and Robins115 reported that the mean time from drug administration to abortion was 13.4 hours using 20 mg PGE2 vaginal suppositories (one every 3 hours); 90 percent of women aborted within 24 hours. Robins and Mann116 reported that the mean time from drug administration to abortion was 16 hours using 15-methyl prostaglandin F2α (250 mcg intramuscular every 2 hours), with 80 percent of women completing the abortion after 24 hours. Borgida et al.117 found intravaginal PGE2 to be a more effective and a more rapid abortifacient than intramuscular 15methyl prostaglandin F2α. Concurrent intravenous oxytocin augmentation is used in some centers, although it is unclear whether this significantly decreases the interval to expulsion.115 Owen and Hauth118 found that concurrent oxytocin expedited pregnancy termination when vaginal PGE2 suppositories were used. Pretreatment with Laminaria or synthetic dilators definitely shortens the time to abortion, thereby reducing the amount of prostaglandin administered and, accordingly, the side effects experienced.119,120

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Systemic abortifacients result in an intact abortus which, in some cases, may be necessary for diagnostic confirmation. However, D and E usually provides tissues adequate for most diagnostic confirmations.91–93 This is true even in cases of structural anomalies, and certainly for cytogenetic studies. Thus, there may be little or no diagnostic or obstetric advantage in inducing labor by systemic abortifacients compared with using D and E, except for situations in which personnel trained in D and E are not available.

Morbidity and mortality Maternal systemic effects of PGE2 vaginal suppositories include nausea, vomiting, diarrhea, hypotension, and tachycardia. Surrago and Robins115 reported vomiting in 37 percent and diarrhea in 31 percent of 112 patients undergoing pregnancy termination by PGE2 vaginal suppositories; 29 percent of the women had fever >38 °C during the termination procedure. Gastrointestinal side effects are more common when intramuscular 15methyl prostaglandin F2α is used: 83 percent reported vomiting and 71 percent had diarrhea in another series.116 However, Jain and Mishell112 reported significantly fewer side effects with misoprostol, with efficacy similar to that of dinoprostone. Because of the relatively high incidence of incomplete abortion, complications such as blood loss requiring transfusion and sepsis can occur after the administration of systemic prostaglandins. Although systemic prostaglandins are relatively safe and easy to use, maternal mortality can still occur as a result of failure to recognize complications in a timely fashion or from intraoperative complications (e.g. uterine perforation, hemorrhage) secondary to surgical procedures required for incomplete abortions. Intra-amniotic abortifacients and hysterotomy/hysterectomy Intra-amniotic techniques for pregnancy termination, while once very popular, are essentially no longer used because of the morbidity and mortality associated with the techniques as well as the development of effective pharmacologic agents to effectively induce uterine contractions. Hysterotomy is

warranted only in situations in which systemic or intra-amniotic methods of termination have failed and no trained personnel experienced in performing D and E are available. Hysterectomy may be justified in very rare instances when the need for termination is accompanied by uterine pathology (e.g. cancer). We have not encountered such a need in over 25 years of offering prenatal diagnostic services. Counseling patients about secondtrimester procedures How should patients be counseled concerning second-trimester pregnancy termination procedures? If alternatives exist, that will primarily depend on the wishes of the patient, the fetal diagnosis, and the potential need for further pathologic assessment. With regard to safety, D and E is the method of choice if trained personnel are available and if the pregnancy is 17 weeks of gestation) is less clear. D and E carries similar morbidity and mortality rates as systemic abortifacient procedures when performed at this stage of pregnancy, although Schneider et al.104 found that D and E was as safe as induction methods when performed between 18 and 22 weeks. When considering other benefits of D and E (e.g. rapid procedure, lower costs, improved psychologic well-being), it may be the preferred method for second-trimester pregnancy termination after 17 weeks of gestation, except when an intact fetus is needed for diagnostic confirmation. Few data exist concerning the safety of even later pregnancy termination procedures (>20 weeks of gestation). Many states preclude pregnancy termination after 24 weeks of gestation except to save the life of the mother. If necessary, however, either intra-amniotic agents or systemic prostaglandins should be used because the size of the conceptus at this stage of pregnancy makes routine D and E technically problematic. Ongoing federal legislation in the United States may have further negative implications for performing D and E in the later portion of the second trimester.121,122

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Selective abortion and fetal reduction in multiple gestations Improved ultrasound technology and invasive prenatal diagnostic procedures (e.g. amniocentesis, CVS) have enabled earlier and more accurate detection of fetal abnormalities in multiple gestations (see Chapters 5, 6 and 25). Occasionally, this leads to the dilemma of detecting discordance in a multiple gestation involving normal and abnormal fetuses. In such cases, selective abortion is used, with the objective of causing death of the abnormal fetus(es) with continued gestation of the normal fetus(es). Despite changes in the practice of infertility intervention that have considerably reduced the number of higher-order multifetal pregnancies,123 assisted reproductive technologies along with advanced maternal age create the need for consideration of selective abortion in women presenting for prenatal diagnosis. Pregnancies with three or more fetuses have significantly higher spontaneous abortion rates than singleton or twin pregnancies,124,125 and infants of multiple birth have significantly higher morbidity and mortality rates than singletons.126 A variation of selective abortion applied to multifetal pregnancies (i.e. three or more fetuses) is to arbitrarily “reduce” the number of fetuses (usually to two or three) to reduce the risks of mortality and morbidity of the remaining fetuses. In this section, we discuss the surgical aspects of selective abortion and fetal reduction. Detailed ethical discussion of such decisions is provided elsewhere,127–129 and in Chapter 35. Second-trimester selective abortion Selective abortion of abnormal fetuses was initially performed in the second trimester. Aberg et al.130 reported the birth of a normal infant at 33 weeks after selective termination at 20 weeks of gestation of a co-twin affected with Hurler syndrome. Selective termination in this case was performed by fetal exsanguination by ultrasound-directed needle cardiac puncture. In the second report of a successful selective birth, Beck et al.131 used hysterotomy at 22 weeks of gestation to remove an abnormal twin. Subsequent successes were also reported by Rodeck et al.,132 who used fetoscopic-guided air embolization into the umbilical vein to selectively

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abort abnormal fetuses. Antsaklis et al.133 used a fetoscopic-guided intracardiac injection of calcium gluconate.

Technique Most centers in the United States that perform second-trimester selective abortion currently use the intracardiac potassium chloride (KCl) injection technique.134,135 Detailed descriptions of the other techniques can be found elsewhere.136 Ultrasound is required to locate and identify the normal and abnormal fetuses. In the case of a fetus with a structural abnormality, ultrasonographic visualization of the fetal defect at the time of selective abortion is sufficient. However, for cases in which fetuses have diagnosed abnormalities with no discriminating ultrasonographic findings (e.g. a fetus with Duchenne muscular dystrophy diagnosed by DNA analysis of amniotic fluid cells), careful documentation of fetal positions at the time of amniocentesis is necessary for determining the normal and abnormal fetuses at the time of selective abortion. An ultrasound examination is initially performed to confirm fetal number, viability, gestational age, placental location, and positions of the normal and abnormal fetuses. Choice of needle insertion site is based on ultrasound determination of the easiest access to the fetus(es) to be terminated. Before needle insertion, the patient may be premedicated for sedation and to decrease fetal movements (e.g. intravenous meperidine 50 mg; prochlorperazine 10 mg; and diazepam 5–10 mg) and to inhibit uterine contractions (ritodrine hydrochloride 3 mg).137 Under continuous ultrasound guidance and through an aseptic field, a 20 or 22 gauge needle is inserted transabdominally into the amniotic sac of the abnormal fetus. The needle stylet is removed and a 5 mL syringe is attached to the hub of the needle to withdraw amniotic fluid for confirmatory studies (if applicable). The tip of the needle is then passed into the fetal thorax and heart. Correct placement of the needle is confirmed by observation of negative pressure within the 5 ml syringe. Sterile KCl (2 mEq/mL) is injected in 2 mL increments until asystole is ultrasonographically demonstrated136; in the series reported by Golbus et al.,136 the volume of KCl needed to cause asystole ranged from 2 to

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Genetic Disorders and the Fetus

7 mL. Intracardiac instillation of KCl may not always result in permanent asystole of the affected fetus; several instillations may be required to complete the procedure. The overall incidence of multiple instillations has not been determined, although most procedures are apparently completed after a single instillation.136,137 After the procedure, ultrasound examination should be repeated every 30 minutes for 1–2 hours to verify absence of fetal heart activity. If fetal heart activity is still present, the procedure must be repeated. Unsensitized Rh-negative patients should receive Rh-immune globulin. The benefit of prophylactic antibiotics is as yet undetermined. Golbus et al.136 administered antibiotic prophylaxis for selected cases requiring a greater degree of manipulation. Chitkara et al.137 gave all patients intravenous antibiotic prophylaxis (cefazolin sodium 1 g) before selective abortion. Serial ultrasound examinations should be scheduled to monitor the surviving fetus(es).

Morbidity and mortality Selective pregnancy reduction carries several predictable risks: inadvertent loss of remaining fetuses, premature labor, premature delivery, disseminated intravascular coagulopathy (DIC), infection, and psychologic problems.127 Pregnancy loss rates after DIC selective abortion have ranged widely. Golbus et al.136 reported the outcomes of 18 patients undergoing selective abortions in twin pregnancies during the second trimester by one of the following methods: intracardiac KCl (n = 57), cardiac puncture-air embolus (n = 57), hysterotomy (n = 52), fetal exsanguination (n = 51), and cardiac tamponade using intracardiac saline (n = 51). Fourteen women were delivered of normal infants and four women lost their entire pregnancy. All four complete pregnancy losses involved monochorionic twins. Transabdominal intracardiac instillation of KCl was considered to be the procedure of choice because of its ease of performance.136 Chitkara et al.137 reported a series of 17 secondtrimester twin selective abortions in which a cotwin was found to have a cytogenetic or structural abnormality. Intracardiac instillation of KCl was used in 10 cases: fetal exsanguination and air embolus (2); air embolus and saline intracardiac injection (2); air embolus and fetal exsanguination

(2). Exsanguination and saline intracardiac injection was used in one case. In each of the first six consecutive cases, the entire pregnancy was lost; however, in the last 11 cases (10 of which were performed by intracardiac instillation of KCl), all women were delivered of healthy infants. Centers with considerable overall experience report a procedure-related pregnancy loss rate of approximately 5 percent.134,138–140 To date, no reported cases of second-trimester selective abortion have resulted in maternal mortality. Greater experience using KCl has demonstrated a decreasing morbidity with second-trimester selective reduction.141 First-trimester fetal reduction First-trimester fetal reduction is most commonly used to reduce the number of fetuses in multifetal pregnancies (three or more fetuses) to decrease the risk of preterm delivery. CVS and endovaginal ultrasonography have permitted detection of fetal abnormalities in the first trimester, making this technique applicable for selective abortion of abnormal fetuses. Counseling women carrying a multifetal gestation with one or more affected fetuses should be nondirective and include the fetal and maternal implications of selective reduction and pregnancy continuation.142 Mulcahy et al.143 first reported the selective termination of a male co-twin at risk for hemophilia A; fetal sex was determined by CVS and selective abortion was performed.

Technique Most centers in the United States currently use transabdominal intracardiac instillation of KCl to cause fetal death.144–147 The technique for first-trimester transabdominal intracardiac instillation of KCl is the same as the technique used for secondtrimester intracardiac instillation of KCl (see above), except that only 1–2 mEq KCl are usually needed to cause asystole.141,142 The postoperative protocol is essentially the same for both secondand first-trimester procedures, although confirmation of fetal asystole can be accomplished in 15–30 minutes after the procedure. Timor-Tritsch et al.148,149 reported the successful use of “transvaginal puncture” for first-trimester reduction with salutary outcomes similar to those for transabdominal procedures. This transvaginal

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approach may be the only option available if this approach provides the only access to the abnormal fetus(es). However, this procedure is essentially no longer performed because of potential safety issues as well as the increasing international experience with transabdominal KCl instillation. The optimal gestational age to perform selective pregnancy reduction remains uncertain. Those who perform transabdominal procedures generally advocate 10–11 weeks,128,143,145,146 whereas those who use a transcervical aspiration approach recommend performing selective reduction at 6–9 weeks of gestation.147 First-trimester fetal reduction precludes subsequent second-trimester maternal serum AFP (MSAFP) screening and amniotic fluid AFP (AFAFP) analysis. Grau et al.150 reviewed MSAFP and AFAFP analyses of 40 women who underwent fetal reduction procedures at approximately 12 weeks of gestation. Twenty-one of the 22 women (95.5 percent) who elected to undergo MSAFP screening during the second trimester were found to have elevated MSAFP levels. Among 53 amniotic fluid specimens analyzed from the women carrying multiple gestations and obtained during the second trimester, 13 (24.5 percent) were found to have abnormally elevated AFAFP levels (>2.0 MoM), and one specimen (1.9 percent) was positive for acetylcholinesterase. None of the abnormal MSAFP or AFAFP levels or the single case with a positive acetylcholinesterase were associated with fetal abnormalities. The effect of first-trimester reduction procedures on the maternal serum analytes AFP, unconjugated estriol, and hCG have also been studied.151 We confirmed the elevation of second-trimester MSAFP levels were confirmed and levels of hCG and unconjugated estriol remained unaltered. As such, MSAFP screening and AFAFP analysis after selective abortion does not provide useful clinical information regarding the presence of fetal neural tube defects or other structural defects associated with elevated AFP levels. The efficacy of multianalyte screening for fetal chromosome abnormalities (see Chapter 24) in these cases remains undetermined; however, the lack of utility of MSAFP strongly suggests that multianalyte algorithms are likely not amenable for prenatal screening after multifetal pregnancy reduction.

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Morbidity and mortality As with second-trimester selective abortion, fetal reduction may result in inadvertent loss of remaining fetuses, premature labor, premature delivery, DIC, infection, and psychologic problems.127 Multiple instillations may also be required to achieve asystole. Tabsh147 reported that none of the 40 women undergoing selective fetal reduction by intracardiac instillation of KCl lost the entire pregnancy. Evans et al.128 reported that five of 22 patients (22.7 percent) lost their entire pregnancy. Lynch et al.144 reported that in 85 cases of fetal reduction by transabdominal intracardiac instillation of KCl, 45 women were delivered of viable infants, 32 pregnancies were ongoing, and only eight women (9.4 percent) had lost their entire pregnancy. Hemorrhage, maternal infection, premature rupture of the membranes, and premature labor and delivery have predictably occurred, although the frequency of these complications is surprisingly low.152–155 Although DIC is recognized as occurring in cases of spontaneous fetal death in multiple gestations,156 it has thus far not been reported in women undergoing selective fetal reduction. Maternal mortality has not been reported. Reports of collaborative studies of clinical outcomes of multifetal reduction for a variety of indications demonstrate considerable improvement in overall pregnancy loss rates. Most studies report an approximate 5 percent risk of pregnancy loss, with lower loss rates being associated with experienced operators157 and the number of fetuses being reduced.141,142 Antsaklis et al.133 noted that secondtrimester selective abortion has a risk of pregnancy loss (8.3 percent) comparable to procedures performed in the first trimester (5.6 percent; p not significant), a finding echoed by others138 (4.3 percent pregnancy loss in the first trimester and 4.0 percent in the second trimester; p not significant). Stone and colleagues158 reported on their singlecenter experience with multifetal pregnancy reduction and found that overall loss rates had remained stable at 4.7 percent with 95.2 percent delivering after 24 weeks and the lowest loss rate (2.1 percent) occurring in women reducing from a twin pregnancy to a singleton. Monochorionic multifetal pregnancy is a contraindication to first-trimester selective reduction. The reported rate of pregnancy

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loss in cases of monochorionic multifetal pregnancies using conventional instillation techniques approaches 100 percent136,146; however, the use of novel technqiues that occlude and transect the cord of the reduced fetus has been shown to allow for successful reduction of monoamniotic and monochorinic twins.159 CVS is routinely offered before multifetal reduction. In more than 75 cases there have been no pregnancy losses or an inaccurate diagnostic outcome. Eddleman et al.160 reported a 1.4 percent pregnancy loss rate and a “probable” 1.2 percent karyotypic inaccuracy rate among 73 women who underwent CVS for 165 fetuses before multifetal reduction. However, other groups report successful prenatal diagnosis following multifetal reduction.161,162 Ferrara and colleagues163 showed that there was no difference between the loss rates observed in women undergoing multifetal pregnancy reduction who underwent CVS prior to reduction compared to those who did not. Regardless of the beliefs of clinicians as to the “best” approach to prenatal diagnosis and fetal reduction, it seems that the outcomes of both protocols are similar. A small percentage of cases will not permit a particular approach to prenatal diagnosis because of technical or anatomic considerations. However, for the majority of cases in which prereduction CVS or postreduction amniocentesis is feasible, patient choice should play a central role in determining which approach to use for women desiring prenatal diagnosis and multifetal reduction.

References 1. Verp MS, Bombard AT, Simpson JL, et al. Parental decision following prenatal diagnosis of fetal chromosome abnormality. Am J Med Genet 1988;29:613. 2. Grevengood C, Shulman LP, Dungan JS, et al. Severity of abnormality influences decision to terminate pregnancies affected with fetal neural tube defects. Fetal Diagn Ther 1994;9:273. 3. Zlotogora J. Parental decisions to abort or continue a pregnancy with an abnormal finding after an invasive prenatal test. Prenat Diagn 2002;22:1102. 4. Shaffer BL, Caughey AB, Norton ME. Variation in the decision to terminate pregnancy in the setting of fetal aneuploidy. Prenat Diagn 2006;26:667.

5. Quadrelli R, Quadrelli A, Mechoso B, et al. Parental decisions to abort or continue a pregnancy following prenatal diagnosis of chromosomal abnormalities in a setting where termination of pregnancy is not legally available. Prenat Diagn 2007;27:228. 6. Van der Pal-de Bruin KM, Graafmans W, Biermans MC, et al. The influence of prenatal screening and termination of pregnancy on perinatal mortality rates. Prenat Diagn 2002;22:966. 7. Davidson N, Halliday J, Riley M, King J. Influence of prenatal diagnosis and pregnancy termination of fetuses with birth defects on the perinatal mortality rate in Victoria, Australia. Paediatr Perinat Epidemiol 2005;19:50. 8. Liu S, Joseph KS, Kramer MS. Relationship of prenatal diagnosis and pregnancy termination to overall infant mortality in Canada. JAMA 2002;287:1561. 9. Hern WM. First and second trimester abortion techniques. In: Leventhal JM, ed. Current problems in obstetrics and gynecology. Chicago: Year Book Medical Publishers, 1983:5. 10. Stubblefield PG. Pregnancy termination. In: Gabbe SG, Niebyl JR, Simpson JL, eds. Obstetrics: normal and problem pregnancies. New York: Churchill Livingstone, 1991:1303. 11. Shulman LP, Ling FW. Surgical termination of pregnancy. In: Mann WJ, Stovall TG, eds. Gynecologic surgery. New York: Churchill Livingstone, 1996:795. 12. Shulman LP, Lipscomb GH, Ling FW. Management of abnormal pregnancies. In: Paul, M, Lichtenberg, S, Borgatta L, Grimes D Stubblefield P, eds. A clinician’s guide to medical and surgical abortion. New York: Churchill Livingstone, 1999:482. 13. Lohr PA. Surgical abortion in the second trimester. Reprod Health Matters 2008;16:151. 14. Castodot RG. Pregnancy termination: techniques, risks and complications and their management. Fertil Steril 1986;45:5. 15. American College of Obstetricians and Gynecologists. Methods of midtrimester abortion. ACOG Technical Bulletin 109. Washington, DC: American College of Obstetricians and Gynecologists, 1987. 16. Stubblefield PG, Carr-Ellis S, Borgatta L. Methods for induced abortion. Obstet Gynecol 2004;104:174. 17. Singh M, Porter C, Johnson L. Role of routine ultrasound scan in pre-termination of pregnancy assessment in community setting. J Obstet Gynaecol 2008; 28:508. 18. Goldstein SR, Danon M, Watson C. An updated protocol for abortion surveillance with ultrasound and immediate pathology. Obstet Gynecol 1994;83:797. 19. Acharya G, Morgan H, Paramanantham L, Fernando R. A randomized controlled trial comparing surgical

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termination of pregnancy with and without continuous ultrasound guidance. Eur J Obstet Gynecol reprod Bio 2004;114:69. Schulz KF, Grimes DA, Cates W Jr, et al. Measures to prevent cervical laceration during suction curettage abortion. Lancet 1983;1:1182. Hern WM. Laminaria versus Dilapan osmotic cervical dilators for outpatient dilation and evacuation abortion: randomized cohort comparison of 1,001 patients. Am J Obstet Gynecol 1994;171:1324. Uldberg N, Ulmsten U. The physiology of cervical ripening and cervical dilatation and the effect of abortifacient drugs. Baillière’s Clin Obstet Gynecol 1990; 4:263. Ngai SW, Tang OS, Lao T, et al. Oral misoprostol versus placebo for cervical dilatation before vacuum aspiration in first trimester pregnancy. Hum Reprod 1995;10:1220. Singh K, Fong YF. Preparation of the cervix for surgical termination of pregnancy in the first trimester. Hum Reprod Update 2000;6:442. MacIsaac L, Grossman D, Balisteri E, et al. A randomized controlled trial of laminaria, oral misoprostol and vaginal misoprostol before abortion. Obstet Gynecol 1999;93:766. Lefebvre Y, Proulx L, Elie R, et al. The effects of RU-486 on cervical ripening: clinical studies. Am J Obstet Gynecol 1990;162:61. Carbonne B, Brennand JE, Maria B, et al. Effects of gemeprost and mifepristone on the mechanical properties of the cervix prior to first trimester termination of pregnancy. Br J Obstet Gynaecol 1995;102:553. World Health Organization. Cervical ripening with mifepristone (RU486) in late first trimester abortion. World Health Organization Task Force on Postovulatory Methods of Fertility Regulation. Contraception 1994;50:461. Platz-Christensen JJ, Nielsen S, Hamberger L. Is misoprostol the choice for induced cervical ripening in early pregnancy termination? Acta Scand Obstet Gynecol 1995;74:809. Darney PD, Dorwand K. Cervical dilation before firsttrimester elective abortion: a controlled comparison of meteneprost, laminaria and hypan. Obstet Gynecol 1987;70:397. Allen RH, Goldberg AB, Board of Society of Family Planning. Cervical dilation before first-trimester surgical abortion ( 1), both of which are reliably detected on ultrasound examination.32–34 These fetuses can expect a good outcome with optimal standard postnatal care.35 However, those fetuses with a low LHR (≤1) and liver herniation into the chest (“liver up”) experience much greater morbidity and mortality, even with the highest quality postnatal care, including extracorporeal membrane oxygenation (ECMO), high-frequency ventilation, surfactant therapy, inhaled nitric oxide therapy, and delayed surgical repair.36–40 For these severely affected fetuses, correction of the defect before birth has the potential to improve outcomes. Strategies for in utero treatment of CDH have been a driving force in research and development in fetal surgery over the past two decades.41,42 The evolution in techniques and overall treatment strategy for fetal CDH mirrors a global trend in fetal surgery: there has been a transition from open hysterotomy to minimally invasive fetoscopic and percutaneous approaches; a move away from postnatal repair to a direct assault on the fetal pathophysiologic defect19,23; and a push for validation of new methods via proper randomized controlled trials43 rather than reliance on anecdotal case reports.44 Almost 20 years have passed since the first successful open fetal surgery for severe CDH was performed at the UCSF.45 The procedure, reserved for

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Figure 31.2 Schematic drawing of the EXIT procedure.

C H APTER 31

fetuses diagnosed before 25 weeks’ gestation and without evidence of liver herniation, involved complete anatomic repair of the diaphragmatic defect after a maternal hysterotomy and partial removal of the fetus. Although complete repair before birth was feasible, it did not improve outcome over controls and the approach has long since been abandoned.46 Though the initial results of in utero therapy for CDH were disappointing, further research and experimental work in animals led to the development of temporary tracheal occlusion or PLUG (Plug the Lung Until it Grows).47–53 This strategy takes advantage of the natural dynamics of fetal breathing and lung development in order to compensate for the abnormal circumstances caused by the malformed diaphragm. During fetal development, the lung produces a continuous flow of lung fluid that exits through the trachea into the AF. Blocking the egress of this fluid by plugging the trachea, however, allows fluid to build up in the lung, causing pulmonary hyperplasia and creating enough pressure in the lung to counteract the compressive force of the herniated viscera. Lung growth is thus enabled and the growing lungs can begin to push the herniated viscera out of the chest and back into the abdomen.42 Initially, clinical fetal tracheal occlusion was achieved using open fetal surgical techniques that required a hysterotomy. However, this considera-

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ble manipulation of the uterus and fetus incited vigorous postoperative preterm labor, often leading to PROM and preterm delivery.54 With the belief that several small (3–5 mm) uterine incisions might incite less uterine irritability and preterm labor than would one long hysterotomy, minimally invasive techniques were developed (Figure 31.3). Promising initial results led to a prospective, randomized NIH trial at UCSF comparing fetoscopic balloon tracheal occlusion to standard postnatal care for fetuses diagnosed with severe CDH. However, the experimental group failed to show any benefit compared to the control group (73 percent survival rate versus 77 percent, respectively), and the trial was halted at 24 patients.55 All fetuses that underwent tracheal occlusion were born prematurely due to preterm labor, compared to only four out of 13 in the control group. Most likely, the potential benefits of lung growth from tracheal occlusion were offset by the adverse effects of prematurity, caused by the fetal surgery. An additional factor may have been that occlusion was not reversed in utero: increasing data have shown that reversing tracheal occlusion before birth may optimize the combination of lung growth and development of type II pneumocytes that produce surfactant protein, important in postnatal gas exchange.56 After the UCSF NIH tracheal occlusion trial, the European FETO Task Group began performing

Perfusion scope

Ultrasound

Figure 31.3 Percutaneous balloon tracheal occlusion. Under sonographic and endoscopic guidance, the fetal trachea is cannulated with the telescope. After inflation, the balloon is detached 2 cm proximally to the carina (inset).

Balloon inflated Balloon detached

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percutaneous fetal endoscopic tracheal occlusion. Using further miniaturized endoscopes available in Europe, these investigators were able to subsequently reverse occlusion in utero with a second fetoscopic procedure, allowing mothers the possibility of a vaginal delivery at their referring tertiary center. Fetuses with left-sided CDH, liver herniation into the chest, and LHR < 1.0 on ultrasound were considered candidates for the treatment. In their study, survival to discharge was 55 percent, compared with 0.01, asthenia (31.4 percent and 11.4 percent, p < 0,001), myalgia (21.5 percent and 6.7 percent, p < 0,001), rhinopharyngo-tracheo-bronchitis (42.1 percent and 29.5 percent, p = 0,089) and flu-like syndrome defined as the simultaneous occurrence of fever and at least one of these signs (24.5 percent and 9.5 percent, p < 0,001), lymphocytosis ≥40 percent (39.2 percent and 5.7 percent, p < 0,001), increased aminotransferases blood levels (one or both >40 iu/L) (35.3 percent and 3.9 percent, p < 0,001). Platelet count was significantly lower in primary infection but within normal range.55 A careful medical interview is useful because symptoms can be recalled, allowing quite precise dating of the onset of infection. Serology Diagnosis of primary infection can be achieved in two circumstances. 1. The presence of maternal clinical manifestations already described, leading to the serologic diagnosis. 2. Screening for seroconversion on a seronegative mother, usually when there is a risk of contamination (e.g. parents of infants in day care centers, workers in day care centers). Nevertheless, this systematic screening program is not recommended by most public health authorities and can be called “wild screening.” This has been estimated to be around 20 percent in France. If performed, the determination of the maternal serologic status should be done as early as possible at the beginning of pregnancy, or before if possible. If there is no virus-specific IgG in the serum, the pregnant woman is seronegative. Adequate counseling can be done in an attempt to prevent primary infection during pregnancy.56 If IgG and IgM are positive, primary infection can be suspected because it is consistently associated with a virus-specific IgM antibody response. Nevertheless, the presence of specific IgM is not sufficient to diagnose CMV primary infection.57 In this situation, IgG avidity assay can help to identify recent infection.57–59 This assay is based on the observation that virus-specific IgG of low avidity is

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produced during the first months after primary infection, whereas a maturation process leads to the production of IgG of higher avidity. The presence of high IgM levels and a low IgG avidity index (which depends upon each laboratory technique) (3 months before. It is of major importance that serologic testing can be done as early as possible during pregnancy, because after 3 months, even a high-avidity index does not exclude infection around the onset of the pregnancy (periconceptional infections).60 An intermediate index (>30 percent and