Genetic disorders and the fetus : diagnosis, prevention, and treatment [Eighth ed.] 9781119676935, 1119676932

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Genetic Disorders and the Fetus

Dedicated to

Laura and Francia For their love, support, and understanding

“Make assurance double sure.” Shakespeare, Macbeth

Genetic Disorders and the Fetus Diagnosis, Prevention, and Treatment EIGHTH EDITION

EDITED BY

Aubrey Milunsky, MB BCh, DSc, FRCP, FACMG, DCH Professor of Obstetrics and Gynecology Tufts University School of Medicine Formerly Professor of Human Genetics, Pediatrics, Obstetrics and Gynecology Boston University School of Medicine Founder and Co-Director, Center for Human Genetics Cambridge, MA, USA

Jeff M. Milunsky, MD, FACMG Professor of Obstetrics and Gynecology Tufts University School of Medicine Formerly Professor of Genetics and Genomics, and Pediatrics Boston University School of Medicine Co-Director, Center for Human Genetics Director, Clinical and Molecular Genetics Cambridge, MA, USA

This eighth edition first published 2021 © 2021 Aubrey Milunsky and Jeff M. Milunsky Edition History Previous editions: © 2016, 2010 Aubrey Milunsky and Jeff M. Milunsky; © 2004, 1998, 1992, 1986, 1979 Aubrey Milunsky 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 law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Aubrey Milunsky and Jeff M. Milunsky to be identified as the authors of the editorial material in this work has been asserted in accordance with law. Registered Offices John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial Office 9600 Garsington Road, Oxford, OX4 2DQ, UK For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print-on-demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty 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 scientific method, diagnosis, or treatment by physicians for any particular patient. 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. While the publisher and authors have used their best efforts in preparing this work, they 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 merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Library of Congress Cataloging-in-Publication Data Names: Milunsky, Aubrey, editor. | Milunsky, Jeff M., editor. Title: Genetic disorders and the fetus : diagnosis, prevention, and treatment / edited by Aubrey Milunsky, MB BCh, DSc, FRCP, FACMG, DCH and Jeff M. Milunsky, MD, FACMG. Description: Eighth edition. | Hoboken, NJ : Wiley-Blackwell, 2021. Identifiers: LCCN 2021058739 (print) | LCCN 2021058740 (ebook) | ISBN 9781119676935 (hardback) | ISBN 9781119676973 (adobe pdf) | ISBN 9781119676959 (epub) Subjects: LCSH: Prenatal diagnosis. | Fetus–Diseases–Genetic aspects. | Fetus–Abnormalities–Genetic aspects. Classification: LCC RG628 .G46 2021 (print) | LCC RG628 (ebook) | DDC 618.3/2075–dc23 LC record available at https://lccn.loc.gov/2021058739 LC ebook record available at https://lccn.loc.gov/2021058740 Cover Design: Wiley Cover Image: © (Left column): Rasi Bhadramani/iStock/Getty Images. (Top row)(left) Roland Axt-Fliedner and Aline Wolter; (center) Liesbeth van Leeuwen, Malou A. Lugthart, and Eva Pajkrt; (right) Liesbeth van Leeuwen, Malou A. Lugthart, and Eva Pajkrt. (Middle row)(left) Nadine Girard and Kathia Chaumoitre; (center) Nadine Girard and Kathia Chaumoitre; (right) Michael A. Belfort and Alireza A. Shamshirsaz. (Bottom row)(left) Anver Kuliev and Svetlana Rechitsky. Set in 9.5/12pt MinionPro by SPi Global, Chennai, India

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Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv 1 Genetic Counseling: Preconception, Prenatal, and Perinatal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Aubrey Milunsky and Jeff M. Milunsky 2 Preimplantation Genetic Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Anver Kuliev and Svetlana Rechitsky 3 Amniotic Fluid Constituents, Cell Culture, and Neural Tube Defects . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Daniel L. Van Dyke and Aubrey Milunsky 4 Molecular Aspects of Placental Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Wendy P. Robinson and Deborah E. McFadden 5 Fetal Origins of Adult Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 Michael G. Ross and Mina Desai 6 Maternal Serum Screening for Chromosomal Abnormalities and Neural Tube Defects . . . . . . . . . . 240 Howard Cuckle 7 Noninvasive Screening for Aneuploidy Using Cell-Free Placental DNA . . . . . . . . . . . . . . . . . . . . . . . . 301 Lorraine Dugoff 8 Noninvasive Prenatal Diagnosis and Screening for Monogenic Disorders Using Cell-Free DNA . . 318 Ignatia B. Van den Veyver, Natalie Chandler and Lyn S. Chitty 9 Amniocentesis, Chorionic Villus Sampling, and Fetal Blood Sampling . . . . . . . . . . . . . . . . . . . . . . . . . 346 Anthony O. Odibo 10 Prenatal Diagnosis of Neural Tube Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Aubrey Milunsky 11 Prenatal Diagnosis of Chromosomal Abnormalities through Chorionic Villus Sampling and Amniocentesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404 Peter A. Benn 12 Prenatal Diagnosis of Sex Chromosome Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Jeff M. Milunsky 13 Prenatal Diagnosis of Chromosomal Abnormalities: From Karyotype to Microarray . . . . . . . . . . . . 547 Brynn Levy

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Contents

14 Molecular Genetics and Prenatal Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572 Jeff M. Milunsky 15 Prenatal Diagnosis of Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611 Wayne W. Grody 16 Prenatal Diagnosis and the Spectrum of Involvement from Fragile X Mutations . . . . . . . . . . . . . . . . 629 Randi J. Hagerman and Paul J. Hagerman 17 Prenatal Diagnosis of Fetal Malformations by Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647 Liesbeth van Leeuwen, Malou A. Lugthart and Eva Pajkrt 18 Prenatal Diagnosis and Management of Abnormal Fetal Development in the Third Trimester of Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701 Roland Axt-Fliedner and Aline Wolter 19 Prenatal Diagnosis by Fetal Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760 Nadine Girard and Kathia Chaumoitre 20 Prenatal Diagnosis of Skeletal Dysplasias and Connective Tissue Disorders . . . . . . . . . . . . . . . . . . . . 783 Andrea Superti-Furga and Sheila Unger 21 Prenatal Diagnosis of Disorders of Carbohydrate Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803 Deeksha Sarihyan Bali, Stephanie L. Austin and Priya S. Kishnani 22 Disorders of Metabolism of Amino Acids and Related Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . 831 Andrea Gropman, Georgianne Arnold and Jerry Vockley 23 The Mucopolysaccharidoses: Prenatal Diagnosis, Neonatal Screening and Emerging Therapies . . . 868 Lorne A. Clarke 24 Prenatal Diagnosis of the Peroxisomal and Mitochondrial Fatty Acid Oxidation Deficiencies . . . . 890 Ronald J.A. Wanders and Hans R. Waterham 25 Prenatal Diagnosis of Disorders of Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 910 James E. Davison, Steven E. Humphries, Bryan G. Winchester and Sara E. Mole 26 Prenatal Diagnosis of Primary Immunodeficiency Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 982 Jennifer M. Puck 27 Prenatal Diagnosis of the Hemoglobinopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002 John M. Old and Jan Traeger-Synodinos 28 Prenatal Diagnosis of Inherited Disorders of Folate and Cobalamin Metabolism . . . . . . . . . . . . . . . 1035 David S. Rosenblatt and David Watkins 29 Fetal Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1050 Michael A. Belfort and Alireza A. Shamshirsaz 30 In Utero Stem Cell Transplantation, Enzyme Replacement, and Gene Therapy . . . . . . . . . . . . . . . . . 1105 Tippi C. MacKenzie and Marisa E. Schwab 31 Maternal Genetic Disorders That Affect Fetal Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1120 Karin J. Blakemore 32 Pregnancy Termination for Genetic Disorders: Indications and Complications . . . . . . . . . . . . . . . . 1158 Lee P. Shulman

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33 Providing Supportive Psychosocial Care to Parents after Perinatal Loss . . . . . . . . . . . . . . . . . . . . . . . 1179 Marianne H. Hutti 34 Prenatal Diagnosis of Fetal Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1197 Marianne Leruez-Ville, Valentine Faure-Bardon and Yves G. Ville 35 Medicolegal Aspects of Prenatal Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1236 Ellen Wright Clayton 36 Prenatal and Preimplantation Diagnosis: International Policy Perspectives . . . . . . . . . . . . . . . . . . . . 1250 Minh Thu Minh Nguyen and Bartha Maria Knoppers 37 Ethical Issues in the Diagnosis and Management of Genetic Disorders in the Fetus . . . . . . . . . . . . 1267 Frank A. Chervenak and Laurence B. McCullough Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1294

Preface

Certainty and trust are the hallmarks of prenatal diagnosis where doubt and ambiguity are outlaws. At no time was that more apparent than when AM made his first prenatal genetic diagnosis over 50 years ago. “Are you sure?” were that patient’s first words, intoned with deep anxiety. Those words underscored the still cogent need for accuracy in prenatal diagnosis, where mostly a single report would be final. That was a time when prenatal diagnosis depended on amniocentesis-based study, yielding an accuracy rate exceeding 99 percent. Few laboratory tests, then or now, concerned with profoundly important decision-making equal this enviable accuracy. Variants of uncertain significance had not yet entered the genetic lexicon. Today, however, with the newer, beguiling, noninvasive technology, amniocentesis and chorionic villus sampling (CVS) use has declined dramatically. On the altars of convenience and expediency, women are choosing or simply encouraged to have noninvasive prenatal testing, often blissfully unaware that about half of all chromosomal abnormalities detectable through amniocentesis or CVS and authoritatively discussed, would be missed. There is room for concern given the current consensus that all women should be offered either of these two procedures. Similarly, all women with risks of having offspring with a monogenic disorder and a known pathogenic variant should be informed about and offered the option of preimplantation genetic testing (PGT). The extensive and long-established experience of the authors with PGT enshrine this recommendation. Expanded carrier screening should make this a more frequently addressed option. Couples should, however, be carefully counseled about the limitations of such screening. All the facts, figures, guidelines, and recommendations have been updated in this edition, and will

assist the expanded healthcare team in providing optimal care to all patients. Coupled with remarkable advances in technology, prenatal diagnosis has undergone a revolution. Thus far, and continually increasing, more than 4,331 culprit genes and 6,739 associated phenotypes for an extensive array of genetic disorders have been identified. Consequently, since physicians in all medical specialties encounter genetic disorders for which a molecular diagnosis is available, awareness of the options for prenatal diagnosis or PGT has become especially important. Pari passu with these technical advances has come the opportunity to avoid and prevent the occurrence of many lethal and seriously disabling genetic diseases. This progress means that physicians in all specialties incur responsibility (and inevitable liability) to acquire new knowledge of genetic disorders and offer appropriate tests or refer patients for evaluation and genetic counseling. More than 60 million people worldwide are estimated to have DNA studies in the next 5 years. Expanded carrier testing, maternal cell-free plasma DNA testing, whole-exome sequencing, next-generation sequencing, single-cell molecular diagnosis, and advanced fetal imaging, all fully presented in this volume, now complement the well-established procedure for prenatal diagnosis and PGT. Whole-genome analysis for prenatal diagnosis (even if advisable) simply awaits further technical advances. Chromosomal microarrays and whole-exome sequencing have propelled an even greater prenatal diagnostic reach, especially in the face of fetal structural, including skeletal, abnormalities, given the advances in ultrasound and magnetic resonance imaging, so expertly covered in this volume by authors from three European countries. The fetal anomaly scan has to an important extent superseded amniotic fluid 𝛼-fetoprotein for ix

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the diagnosis of neural tube defects. Nevertheless, the significant worldwide prevalence of neural tube defects makes maternal serum 𝛼-fetoprotein screening a continuing vital test, critically evaluated here by a pioneer. The diagnosis of neural tube defects and their consequences, as well as the frequently failed efforts at prevention, are thoroughly detailed. Knowledge of the common sex chromosome aneuploidies and some of the rarer variants continues to expand. Up-to-date summaries of all these disorders are presented with specific recommendations for genetic counseling. Molecular prenatal diagnosis has now become routine and the multiple technologies utilized, with their benefits and limitations along with clinically based caveats and considerations, are presented in a significantly updated chapter. The advent of next-generation sequencing has, by targeting panels or whole-exome approaches, resulted in more opportunities for avoidance and prevention through prenatal diagnosis. These widely available technologies not only address previously diagnosed childhood-onset disorders, but also those of adult onset, including cardiomyopathies, malignancies, and neurologic disorders. Progress and refinement in the diagnosis and management of a wide range of metabolic disorders are fully and authoritatively updated, and equally complemented by the detailed in-depth advances in molecular diagnostics that include the hemoglobinopathies, fragile X syndrome, cystic fibrosis, disorders of folate metabolism, and the immunodeficiency diseases. Pregnancy termination is a sad but fortunately uncommon consideration following prenatal diagnostic studies. The techniques and complications are fully discussed and complemented by insightful senior experience with the management of grief after pregnancy and perinatal loss. Discussion about the care and management of mothers with genetic disorders that affect fetal health and those who transmit infection to the fetus is sharply focused on diagnosis, prevention, avoidance, and treatment. Fetal health is doubly important given the known fetal origins of adult disease that go far beyond hypertension, obesity, and diabetes, to include the epigenetic phenomena induced by the maternal pregnancy environment. A thorough

exposition on the importance of placental development, structure, function, genetics, and pathology on fetal growth and development is expertly presented in this edition. Steadily, but surely, fetal gene therapy via hematopoietic stem cell transplantation is taking root, while a remarkable chapter on fetal surgery by a leader in the field points to new brave surgical remedies. While all authors acknowledge continuing progress in molecular genetics, inconclusive results due to variants of uncertain significance are not infrequent. Laboratory conclusions can be further compounded by a host of issues that potentially befuddle interpretation. Commonly encountered issues include delineation of normal variation or polymorphisms, difficulty determining the pathogenicity of variants, depth of sequencing coverage, regions of high GC content, mosaicism, DNA contamination, digenic inheritance, locus heterogeneity, and false-positive and false-negative results. The concurrence of an incidental (secondary) finding on fetal DNA analysis will predictably arouse great parental anxiety. Current laws and public policy regarding prenatal diagnosis and PGT in 16 countries are examined in detail regarding international differences, with special reference to the guidelines relevant to the emerging technologies. A senior physician-lawyer, in reviewing the important principles in the torts of wrongful birth and wrongful life, focused on the potential liability of those involved in reproductive medicine. Professional ethics in obstetrics, with emphasis on the ethical principles of beneficence and autonomy and the ethical concept of the fetus as a patient, receive in-depth discussion by doyens in this field. This volume is a major repository of facts about prenatal diagnosis and provides a critical analysis and synthesis of established and new knowledge based on the long experience of the senior contributing authorities in their respective fields. In addition, a broad international perspective is presented with contributions from recognized international experts from nine countries. The guidance provided and the insights and perspectives of these authors make this volume a valuable and indispensable resource for all those whose focus is securing fetal health through prenatal diagnosis or PGT.

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This text is very well referenced and replete with evidence-based guidance and reflective of the lifetime experience and wisdom of distinguished senior authors. This edition encompasses 152 tables, 167 figures, and over 10,000 references. A valuable index will facilitate the reader’s search for specific information. The major technologic advances in genetics have made the requirement for preconception, prenatal, and perinatal genetic counseling of paramount importance. Even though the underlying principles

and prerequisites are well established, the many advances have introduced a panoply of new challenges discussed in detail in a comprehensive, heavily referenced opening chapter. We are in the golden era of human genetics, and through new discoveries and insights have increased opportunities for the diagnosis, prevention, and treatment of many serious and lethal genetic disorders. Aubrey Milunsky and Jeff M. Milunsky Cambridge

Acknowledgments

This eighth edition marks the 42nd year of this text and reflects the continuing remarkable advances made in achieving accurate prenatal and preimplantation diagnoses. The first book on this subject (The Prenatal Diagnosis of Hereditary Disease) was published some 48 years ago (by AM). The distillation of accrued biological, technological, ethical, and legal knowledge has graced these pages and enriched the reference value of these editions. The wisdom, insight, perspective, expertise, experience, and knowledge of these senior contributing authors has made these volumes a valuable and authoritative text. Moreover, these authors have again provided an international perspective, this edition having contributions from nine countries. Expert care in human genetics, maternal–fetal medicine, and perinatal medicine has demanded knowledge, up-to-date information, guidance, and expertise in each volume. This has been achieved only by the willingness of the internationally recognized authoritative authors who have taken the time to share their knowledge, experience, and wisdom. For this we are most appreciative. We are also grateful and indebted to our friends and colleagues who have died and who were

expert contributing authors to earlier editions. We remember them with pride and sadness: Bruno Brambati, MD, David J.H. Brock, PhD, Jacob A. Canick, PhD, Louis Dalliaire, MD, PhD, Sherman Elias, MD, H.J. Evans, PhD, FRSE, John C. Fletcher, PhD, Fredric Frigoletto, MD, Albert B. Gerbie, MD, Leonard A. Herzenberg, PhD, George Hug, MD, Lillian Y.F. Hsu, MD, Mary Z. Pelias, PhD, JD, Arthur Robinson, MD, Richard H. Schwarz, MD, Margery W. Shaw, MD, JD, Irving Umansky, MD, Yury Verlinsky, PhD, and Dorothy C. Wertz, PhD. It is possible that we are unaware of the passing of a few authors and regret any omission. We remain eternally grateful to all these remarkable physicians and scientists. AM deeply appreciates the exemplary work and dedication to perfection of his assistant Marina Nguyen. JM is very grateful for the precision, accuracy, and patience of his assistant Emma Rebholz during the writing and editing of this text.

Aubrey Milunsky Jeff M. Milunsky

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Contributors

Georgianne L. Arnold,

MD Professor of Pediatrics University of Pittsburgh School of Medicine Clinical Director, Division of Medical Genetics Children’s Hospital Pittsburgh Pittsburgh, PA, USA

Stephanie L. Austin,

MS, MA, CGC

Genetic Counsellor Senior Research Program Leader Division of Medical Genetics Duke University Durham, NC, USA

Roland M. Axt-Fliedner,

MD, PhD Professor of Obstetrics and Gynecology Justus-Liebig-University Head Division of Prenatal Medicine and Fetal Therapy University Hospital Giessen, Germany

Deeksha Sarihyan Bali,

PhD, FACMGG Professor of Pediatrics Laboratory Director Biochemical Genetics Laboratory Division of Medical Genetics Duke Health Durham, NC, USA

Michael A. Belfort, MBBCH, D. Mid. COG(SA), D.A (SA), MD, PhD, FRCOG, FRCSC, FACOG Professor and Ernst Bertner Chair, Department of Obstetrics and Gynecology Secondary appointments: Departments of Anesthesiology, Surgery, Neurosurgery Baylor College of Medicine Obstetrician and Gynecologist-in-Chief FB McGuyer and Family Chair in Fetal Surgery Medical Director, Texas Children’s Fetal Center

Texas Children’s Hospital Houston, TX, USA

Peter A. Benn, BSc, MSc, PhD, FACMG, DSc Professor Emeritus Department of Genetics and Genome Sciences University of Connecticut Health Center Farmington, CT, USA Karin J. Blakemore,

MD Professor of Gynecology and Obstetrics, Oncology, and Medical Genetics Director, Prenatal Genetics Center Division of Maternal-Fetal Medicine Department of Gynecology and Obstetrics Johns Hopkins University School of Medicine Baltimore, MD, USA

Natalie Chandler, FRCPath Senior Clinical Scientist North Thames Genomic Laboratory Hub Great Ormond Street Hospital for Children NHS Foundation Trust London, UK Kathia Chaumoitre,

MD, PhD

Professor of Paediatric Radiology Imaging Department North Hospital Aix-Marseille University Marseille, France

Frank A. Chervenak, MD, MMM Professor and Chair Department of Obstetrics and Gynecology Associate Dean, International Medicine Zucker School of Medicine at Hofstra/Northwell Lenox Hill Hospital New York, NY, USA

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Contributors

Lyn S. Chitty, PhD, MB, BS, MRCOG Professor of Genetics and Fetal Medicine Medical Director North Thames Genomic Laboratory Hub Great Ormond Street Hospital for Children Genetics and Genomic Medicine, UCL Institute of Child Health London, UK Lorne A. Clarke,

MDCM, FRCPC, FCCMG Professor of Medical Genetics University of British Columbia Department of Medical Genetics British Columbia Children’s Hospital Research Institute Vancouver, British Columbia, Canada

Howard Cuckle,

MSc, DPhil Professor Faculty of Medicine, Tel Aviv University Tel Aviv, Israel

James E. Davison,

MBChB, PhD Consultant in Paediatric Metabolic Medicine Department of Metabolic Medicine Great Ormond Street Hospital for Children NHS Foundation Trust London, UK

Mina Desai, PhD Associate Professor of Obstetrics & Gynecology David Geffen School of Medicine at UCLA Los Angeles, CA, USA Director of Perinatal Research The Lundquist Institute at Harbor-UCLA Medical Center Torrance, CA, 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 Université de La Mediterranée Head of Neuroradiology Timone Hospital Aix-Marseille University Marseille, France

Wayne W. Grody, MD, PhD Professor Divisions of Medical Genetics and Molecular Diagnostics Departments of Pathology and Laboratory Medicine, Pediatrics, and Human Genetics University of California Los Angeles School of Medicine UCLA Institute for Society and Genetics Director Molecular Diagnostic Laboratories and Clinical Genomics Center UCLA Medical Center Los Angeles, CA, USA Andrea Gropman,

MD, FAAP, FACMG Professor of Neurology and Pediatrics Chief, Neurogenetics and Neurodevelopmental Disabilities Children’s National Medical Center and the George Washington University School of Medicine Washington DC, USA

Paul J. Hagerman,

Lorraine Dugoff,

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

Valentine Faure-Bardon

Randi J. Hagerman, MD Distinguished Professor of Pediatrics Endowed Chair in Fragile X Research Medical Director MIND Institute University of California, Davis Health System Sacramento, CA, USA

MD Professor of Obstetrics and Gynecology University of Pennsylvania School of Medicine Chief, Reproductive Genetics Division Department of Obstetrics and Gynecology Hospital of the University of Pennsylvania Philadelphia, PA, USA

Lecturer in Obstetrics and Fetal Medicine Department of Obstetrics and Fetal Medicine Hôpital Necker-Enfants Malades, GH Paris-Centre Université de Paris Paris, France

Steven E. Humphries,

PhD Emeritus Professor of Cardiovascular Genetics Institute of Cardiovascular Science University College London London, UK

Contributors xvii

Marianne H. Hutti, PhD, WHNP-BC, FAANP, FAAN Professor College of Nursing Senior Nurse Scientist University of Kentucky Healthcare – Ambulatory Services University of Kentucky Lexington, KY, USA

Malou A. Lugthart, MD Research Fellow in Fetal Medicine Department of Obstetrics Amsterdam Reproduction and Development Research Institute Amsterdam University Medical Centers University of Amsterdam Amsterdam, The Netherlands

Priya S. Kishnani, MD, FACMGG C.L. and Su Chen Professor of Pediatrics and Professor of Molecular Genetics and Microbiology Duke University School of Medicine Medical Director, YT and Alice Chen Pediatrics Genetics and Genomics Center Division Chief, Medical Genetics Duke Health Durham, NC, USA

Tippi C. MacKenzie,

Bartha Maria Knoppers,

PhD, OC, OQ Canada Research Chair in Law and Medicine Director, Centre of Genomics and Policy Department of Human Genetics Faculty of Medicine McGill University Montreal, Quebec, Canada

Anver Kuliev, MD, PhD Clinical Professor Herbert Wertheim College of Medicine Florida International University Miami, FL Director of Research Reproductive Genetics Innovations Northbrook, IL, USA Marianne Leruez-Ville, MD, PhD Consultant in Medical Virology National Reference Laboratory for Congenital Cytomegalovirus Infections Hôpital Necker-Enfants-Malades, GH Paris Centre Université de Paris Paris, France Brynn Levy,

MSc(Med), PhD, FACMG Professor of Pathology and Cell Biology, Columbia University Medical Center Medical Director, Clinical Cytogenetics Laboratory Co-Director, Laboratory of Personalized Genomic Medicine Vagelos College of Physicians and Surgeons Columbia University Irving Medical Center & the New York Presbyterian Hospital New York, NY, USA

MD Professor of Surgery Co-Director, Center for Maternal-Fetal Precision Medicine University of California, San Francisco San Francisco, CA, USA

Laurence B. McCullough,

PhD Professor Department of Obstetrics and Gynecology Zucker School of Medicine at Hofstra/Northwell Ethics Scholar Department of Obstetrics and Gynecology Lenox Hill Hospital New York, NY, USA

Deborah E. McFadden, MD, FRCPC Clinical Professor Department of Pathology and Laboratory Medicine University of British Columbia Head and Medical Director Department of Pathology and Laboratory Medicine Children’s & Women’s Hospitals of British Columbia Vancouver, BC, Canada Aubrey Milunsky, MD, DSc, FRCP, FACMG, DCH Professor of Obstetrics and Gynecology Tufts University School of Medicine, Boston, MA, USA Formerly Professor of Human Genetics, Pediatrics, Obstetrics and Gynecology, and Pathology Boston University School of Medicine Founder and Co-Director, Center for Human Genetics Cambridge, MA, USA Jeff M. Milunsky,

MD, FACMG Professor of Obstetrics and Gynecology Tufts University School of Medicine Formerly Professor of Genetics and Genomics, and Pediatrics Boston University School of Medicine Co-Director, Center for Human Genetics Director, Clinical Genetics and Molecular Genetics Cambridge, MA, USA

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Contributors

Sara E. Mole, PhD Professor in Molecular Cell Biology UCL Great Ormond Street Institute of Child Health MRC Laboratory of Molecular Cell Biology Genetics and Genomics Medicine Research & Teaching Department Inborn Errors of Metabolism Section University College London London, UK Minh Thu Minh Nguyen, LLM, LLB, BSc Academic Associate Centre of Genomics and Policy McGill University Montreal, Quebec, Canada

Anthony O. Odibo,

MD, MSCE Professor Maternal Fetal Medicine Department of Obstetrics and Gynecology University of South Florida Tampa, FL, USA

John M. Old,

PhD, FRCPath Clinical Scientist Emeritus Reader in Hematology University of Oxford, Oxford, UK

Eva Pajkrt,

MD, PhD Professor of Obstetrics Head of Obstetrics and Fetal Medicine Department of Obstetrics Amsterdam Reproduction and Development Research Institute Amsterdam University Medical Centers University of Amsterdam Amsterdam, The Netherlands

Jennifer M. Puck, MD Professor of Pediatrics Division of Allergy/Immunology and Blood and Marrow Transplantation University of California San Francisco Department of Pediatrics San Francisco, CA, USA Svetlana Rechitsky, PhD Clinical Associate Professor Herbert Wertheim College of Medicine Florida International University Miami, FL, USA President Reproductive Genetic Innovations Northbrook, IL, USA

Wendy P. Robinson, PhD Professor, Department of Medical Genetics University of British Columbia Senior Scientist Child and Family Research Institute Vancouver, BC, Canada David S. Rosenblatt,

MDCM, FCCMG, FRCPSC, FAAP, FACMG Dodd Q. Chu and Family Chair in Medical Genetics Professor Departments of Human Genetics Medicine, Pediatrics, and Biology Faculties of Medicine and Science McGill University Montreal, Quebec, Canada

Michael G. Ross, MD, MPH Distinguished Professor of Obstetrics and Gynecology and Public Health Geffen School of Medicine at University of California Los Angeles Fielding School of Public Health at UCLA Los Angeles, CA, USA Co-Director Institute for Women’ and Children’s Health The Lundquist Institute at Harbor-UCLA Medical Center Torrance, CA, USA Marisa E. Schwab, MD Surgery Resident The Center for Maternal-Fetal Precision Medicine and Department of Surgery University of California, San Francisco San Francisco, CA, USA Alireza A. Shamshirsaz Associate Professor of Surgery, Obstetrics and Gynecology Departments of Obstetrics and Gynecology and Surgery Baylor College of Medicine Chief, Division of Fetal Therapy and Surgery Co-Chief, Maternal Fetal Surgery Section Texas Children’s Fetal Center Texas Children’s Hospital Houston, TX, USA

Lee P. Shulman,

MD, FACMG, FACOG The Anna Ross Lapham Professor in Obstetrics and Gynecology Feinberg School of Medicine of Northwestern University Medical Director, Insight Medical Genetics Medical Director, Reproductive Genetic Innovations Chicago, IL, USA

Contributors xix

Andrea Superti-Furga,

MD

Professor of Pediatrics University of Lausanne Head, Division of Genetic Medicine Lausanne University Hospital Lausanne, Switzerland

Jan Traeger-Synodinos,

DPhil (Oxon), ErCLG Professor of Genetics Director of the Laboratory of Medical Genetics National and Kapodistrian University of Athens, Greece

Sheila Unger,

MD, FRCPC Chief MER1 Division of Genetic Medicine Lausanne University Hospital Lausanne, Switzerland

Ignatia B. Van den Veyver,

MD Professor of Obstetrics and Gynecology and Molecular and Human Genetics Baylor College of Medicine; Director of Clinical Prenatal Genetics Texas Children’s Hospital Houston, TX, USA

Daniel L. Van Dyke,

PhD Professor of Laboratory Medicine and Pathology Mayo Medical School and Mayo Clinic Cytogenetics Laboratory Rochester, MN, USA

Liesbeth van Leeuwen,

MD, PhD Associate Professor Department of Obstetrics Amsterdam Reproduction and Development Research Institute Amsterdam University Medical Centers University of Amsterdam Amsterdam, The Netherlands

Yves G. Ville,

MD Professor of Obstetrics and Fetal Medicine Head of Department of Obstetrics and Fetal Medicine Hôpital Necker-Enfants Malades, GH Paris-Centre Université de Paris Paris, France

Gerard Vockley,

MD, PhD Professor of Pediatrics and Human Genetics Cleveland Family Endowed Professor of Pediatric Research Department of Pediatrics University of Pittsburgh School of Medicine and Graduate School of Public Health Chief, Division of Medical Genetics Director, Center for Rare Disease Therapy Children’s Hospital of Pittsburgh Pittsburgh, PA, USA

Ronald J.A. Wanders University of Amsterdam, Academic Medical Center Departments of Clinical Chemistry and Pediatrics Emma Children’s Hospital Laboratory of Genetic Metabolic Diseases Amsterdam, The Netherlands

Hans R. Waterham,

PhD Professor, Functional Genetics of Metabolic Diseases Departments of Laboratory Medicine and Pediatrics University of Amsterdam Academic Medical Center Laboratory for Genetic Metabolic Diseases Amsterdam, The Netherlands

David Watkins,

PhD Research Associate Department of Human Genetics McGill University Scientist Division of Medical Genetics, Department of Specialized Medicine McGill University Health Centre Montreal, Quebec, Canada

Bryan G. Winchester,

MA, PhD Emeritus Professor of Biochemistry ULC Great Ormond Street Institute of Child Health University College London London, UK

Aline Wolter, MD Justus-Liebig-University, Giessen Department of Obstetrics and Gynecology Division of Prenatal Medicine and Fetal Therapy University Hospital Giessen, Germany Ellen Wright Clayton,

MD, JD Craig-Weaver Professor of Pediatrics Professor of Law Center for Biomedical Ethics and Society Vanderbilt University Nashville, TN, USA

1

Genetic Counseling: Preconception, Prenatal, and Perinatal Aubrey Milunsky 1,2 and Jeff M. Milunsky 1,2 1 2

Center for Human Genetics, Cambridge, MA, USA Tufts University School of Medicine, Boston, MA, USA

The time is fast approaching when virtually all the culprit genes and their mutations for >7,000 rare monogenic disorders1 will be known. Thus far, causal single genes and their mutations have been determined for 5,673 genetic disorders,2 enabling preimplantation genetic testing or prenatal genetic diagnosis. These advances using chromosomal microarrays, whole-exome sequencing and even whole-genome sequencing together with fetal imaging and noninvasive prenatal testing, expand the era in which all couples have the option of avoiding or preventing having children with irreversible, irremediable, crippling, or lethal monogenic disorders. Primary care physicians, and those in all medical specialties, will need to inform their patients of this key option. This imperative is already partly in current practice. Missing is the requirement of physicians to request and obtain the precise name of the genetic disorder in question or an existing DNA report on a family member, for prospective parents to benefit from available options. Increasingly, couples are seeking prenatal diagnosis for adult-onset genetic disorders in which mutations have been determined. Huntington disease prenatal diagnosis has been in the vanguard for many years, but now there are requests for adult-onset dominantly transmissible disorders including breast and other malignancies,

frontotemporal dementia, neurodegenerative disorders, and cardiomyopathies. The remarkable advances in genetics provide a cogent need to confer and refer. Physicians should not invite legal purview for a failure to inform, offer, refer, or provide genetic testing. In context, couples at risk for having progeny with abnormalities expect to be informed about their risks and options, optimally during preconception counseling. Their concerns are serious, given the significant contribution of genetic disorders to morbidity and mortality in children and adults.

The burden of genetic disorders and congenital malformations A conservative estimate for the world population prevalence of rare diseases (71.9–80 percent considered as genetic) is 3.5–5.9 percent, equating to 263–446 million individuals affected at any point in time.3, 4 In India, which has a quarter of the world’s neonatal deaths (an estimated 753,000 in 2013), about 9 percent were due to congenital anomalies.5 An estimated 7.9 million infants worldwide are born each year with a major congenital malformation according to a report in 2013.6 The likelihood of having a child with a congenital malformation varies from 2 to 10 percent7 due to multiple factors

Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, Eighth Edition. Edited by Aubrey Milunsky and Jeff M. Milunsky. © 2021 Aubrey Milunsky and Jeff M. Milunsky. Published 2021 by John Wiley & Sons Ltd.

1

2

Genetic Disorders and the Fetus

that complicate efforts to accurately diagnose and determine the incidence or prevalence of congenital anomalies or genetic disorders. Box 1.1 lists 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. Of the >7,000 rare genetic disorders, about 1 in 12 to 1 in 16 individuals are affected,1 aware or unaware. Given a world population of 7.6 billion, an estimated 473 million are likely to have a rare disease.1

More than 4,331 genes with phenotype-causing mutations have been identified, including 6,739 phenotypes with a known molecular basis.2 Severe intellectual disability is considered to be largely genetic in origin8, 9 with a global prevalence between 0.5 and 1.0 percent.10 Despite continuing progress in the discovery of genes causally related to neurodevelopmental delay,11 in less than 40 percent of cases is there a definitive recognition of cause. The European Organization for Rare Diseases maintained that about 30 percent of all patients with a rare disease died before the age of 5 years.12 In the United States in 2013–2014,

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 and MRI • Accuracy of diagnosis • Age at diagnosis • Case selection, bias, and ascertainment • Congenital hypothyroidism • Consanguinity • Definitions of major and minor congenital anomalies • Diagnostic DNA analysis • Duration of follow-up • Economic level in developed or developing world • Environmental toxins • Family history • Frequency, inclusion, and exclusion of stillbirths, fetal deaths, and elective pregnancy termination • Frequency of certain infectious diseases • Frequency of de novo gene mutations • History of recurrent spontaneous abortion • In vitro fertilization • Incidence and severity of prematurity • Infertility • 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 •

Maternal fever or use of hot tub in the first 6 weeks of pregnancy • Maternal folic acid supplementation • Maternal grandmother’s age • Maternal obesity • Maternal serum screening for chromosome abnormalities • Maternal smoking • Maternal-specific susceptibility genes • Maternal use of medication • Mortality rates decreasing • Multiple pregnancy rate • Necropsy • Noninvasive prenatal testing using cell-free fetal DNA for chromosomal abnormalities and monogenic disorders • Parent with a congenital abnormality or genetic disorder • Paternal age • Previous affected child • Previous maternal immunization/vaccination • Season of the year • Training and expertise in examination of newborns • Use of chromosomal analysis • Use of chromosomal microarray • Use of whole-exome sequencing • Use of whole-genome sequencing • Use of death certificates • Use of registry data •

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Genetic Counseling: Preconception, Prenatal, and Perinatal 3

congenital malformations, deformations, and chromosomal abnormalities accounted for the most infant deaths – 4,746 (20.4 percent) out of 23,215 – in any category of causation.13

Incidence and prevalence of genetic disorders and congenital malformations Estimates of aneuploidy in oocytes and sperm reach 25 percent and 3–4 percent, respectively.14, 15 Estimates, especially for oocytes, vary widely (see Chapter 2). The effect of maternal age, among other factors, is important. At 25 years, early thirties, and >40 years of age, the rate of aneuploidy approximates 5 percent, 10–25 percent, and 50 percent, respectively.15–19 Estimates of aneuploidy and structural chromosomal abnormalities in sperm vary from 7 to 14 percent.20 Not surprisingly, then, about one in 13 conceptions results in a chromosomally abnormal conceptus,21 while about 50 percent of first-trimester spontaneous abortions are associated with chromosomal anomalies.22 One study of blastocysts revealed that 56.6 percent were aneuploid. Moreover, these blastocysts produced in vitro from women of advanced maternal age also revealed mosaicism in 69.2 percent.23 Similar results have been reported by others.24 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 that have implications for reproduction later in life (see Chapters 11 and 13). Between 5.6 and 11.5 percent of stillbirths and neonatal deaths have chromosomal defects.25 Congenital malformations with obvious structural defects are found in about 2 percent of all births.26 This was the figure in Spain among 710,815 livebirths,27 with 2.25 percent in Liberia,28 2.03 percent in India,29 and 2.53 percent among newborn males in Norway.30 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.31 Pooled data from 12 US population-based birth defects surveillance systems, which included 13.5 million livebirths (1999–2007), revealed that American Indians/Alaska natives had a ≥50 percent greater prevalence for seven congenital malformations (including anotia or microtia, cleft lip, trisomy 18, encephalocele, limb-reduction

defect).32 Factors that had an impact on the incidence/prevalence of congenital malformations are discussed later. Over 25,500 entries for genetic disorders and traits have been catalogued.2 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).33 Baseline birth prevalence of rare single-gene disorders for multiple countries are shown in Figure 1.1, which highlights the contribution of consanguinity-associated disorders.34 Polygenic disorders occurred at a rate of 46.4 in 1,000 (Table 1.1). A key study of homozygosity in consanguineous patients with an autosomal recessive disease showed that, on average, 11 percent of their genomes were homozygous.35 Each affected individual had 20 homozygous segments exceeding 3 cM. At least 3–4 percent of all births are associated with a major congenital defect, intellectual disability, or a genetic disorder, a rate that doubles by 7–8 years of age, given later-appearing and/or later-diagnosed genetic disorders.36, 37 If all congenital defects are considered, Baird et al.33 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.38 The bicuspid aortic valve is the most common congenital cardiac malformation and in the final analysis may cause higher mortality and morbidity rates than all other congenital cardiac defects.39 About 27 percent suffer cardiovascular complications requiring surgery.40, 41 Mitral valve prolapse affects 2–3 percent of the general population, involving more than 176 million people worldwide.42 A Canadian study of 107,559 patients with congenital heart disease reported a prevalence of 8.21 per 1,000 livebirths, rising to an overall prevalence of 13.11 per 1,000 in adults.43 The authors concluded that adults now account for some two-thirds of the prevalence of congenital heart disease. Categorical examples of factors associated with an increased risk of congenital heart disease or malformations in the fetus are shown in Box 1.1. A metropolitan Atlanta study (1998–2005) showed an overall

4

Genetic Disorders and the Fetus

22 20 Consang-associated

Rate/1,000 births

18 16

Genetic type unknown

14

Recessive

12 10

X-linked

8 Early onset dominant

6 4 2 0

EMR

AFR

SEAR

EUR

WPR

AMR

World W Europe

Figure 1.1 Total baseline birth prevalence of rare single-gene disorders by World Health Organization (WHO) region, highlighting the important contribution of consanguinity to monogenic disorders. Source: Blencowe et al. 2018.34 Reproduced with permission from Springer.

Table 1.1 The frequencies of genetic disorders in 1,169,873 births, 1952–198334 . Rate per million

Total births

livebirths

(percent)

Dominant

1,395.4

0.14

Recessive

1,665.3

0.17

X-linked

532.4

0.05

Chromosomal

1,845.4

0.18

Multifactorial

46,582.6

4.64

Category A

Genetic unknown

1,164.2

0.12

Total

53,175.3

5.32 a

B All congenital anomalies 740–759 b

52,808.2

5.28

Congenital anomalies with genetic etiology

26,584.2

2.66

79,399.3

7.94

(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.

Source: Blencowe et al. 2018.34 With permission from Elsevier.

prevalence of 81.4 per 10,000 for congenital heart disease among 398,140 livebirths,44 similar to a Belgian study of 111,225 live and stillborn infants ≥26 weeks of gestation with an incidence of 0.83 percent, chromosome abnormalities excluded.45 A EUROCAT registry study found an increasing prevalence of severe congenital heart defects (single ventricle, atrioventricular septal defects, and tetralogy of Fallot) possibly due to increasing

obesity and diabetes.46 In a study of 8,760 patients with autism spectrum disorders and 26,280 controls, a statistically significant increase in the odds of concurrent congenital heart disease (odds ratio [OR] 1.32) was noted.47 Atrial septal defects and ventricular septal defects were most common. Incidence/prevalence rates of congenital defects are directly influenced by when and how diagnoses are made. Highlighting the importance of how

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Genetic Counseling: Preconception, Prenatal, and Perinatal 5

early a diagnosis is made after birth, the use of echocardiography, and the stratification of severity of congenital heart defects, Hoffman and Kaplan48 clarified how different studies reported the incidence of congenital heart defects, varying from 4 in 1,000 to 50 in 1,000 livebirths. They reported an incidence of moderate and severe forms of congenital heart disease in about 6 in 1,000 livebirths, a figure that would rise to at least 19 in 1,000 livebirths if the potentially serious bicuspid aortic valve is included. They noted that if all forms of congenital heart disease (including tiny muscular ventricular septal defects) are considered, the incidence increases to 75 in 1,000 livebirths. The newer genetic technologies, including chromosomal microarray, whole-exome sequencing, next-generation sequencing, and whole-genome sequencing, have helped unravel the causes of an increasing number of isolated or syndromic congenital heart defects.49, 50 Identified genetic causes include monogenic disorders in 3–5 percent of cases, chromosomal abnormalities in 8–10 percent, and copy number variants in 3–25 percent of syndromic and 3–10 percent of isolated congenital heart defects.49, 51 A next-generation sequencing study indicated that 8 percent and 2 percent of cases were due to de novo autosomal dominant and autosomal recessive pathogenic variants, respectively.52 Pregestational diabetes in 775 of 31,007 women was statistically significantly associated with sacral agenesis (OR 80.2), holoprosencephaly (OR 13.1), limb reduction defects (OR 10.1), heterotaxy (12.3), and severe congenital heart defects (OR 10.5–14.9).53 Maternal obesity is associated with an increased risk of congenital malformations.54–65 The greater the maternal body mass index (BMI), the higher the risk, especially for congenital heart defects,59, 60, 62, 65 with significant odds ratios between 2.06 and 3.5. In a population-based case–control study, excluding women with preexisting diabetes, Block et al.66 compared the risks of selected congenital defects among obese women with those of average-weight women. They noted significant odds ratios for spina bifida (3.5), omphalocele (3.3), heart defects (2.0), and multiple anomalies (2.0). A Swedish study focused on 1,243,957 liveborn singletons and noted 3.5 percent with at least one major congenital abnormality.64

These authors used maternal BMI to estimate risks by weight. The risk of having a child with a congenital malformation rose steadily with increasing BMI from 3.5 percent (overweight) to 4.7 percent (BMI ≥40). Our own67, 68 and other studies69 have implicated the prediabetic state or gestational diabetes as contributing to or causing the congenital anomalies in the offspring of obese women. In this context, preconception bariatric surgery seems not to reduce the risks of congenital anomalies.61, 70–72 It appears that folic acid supplementation attenuates but does not eliminate the risk of spina bifida when associated with diabetes mellitus73 or obesity74 (see Chapter 10). In contrast, markedly underweight women reportedly have a 3.2-fold increased risk of having offspring with gastroschisis,74 in all likelihood due to smoking and other acquired exposures.75, 76 Indeed, a study of 173,687 malformed infants and 11.7 million unaffected controls, when focused on maternal smoking, yielded significant odds ratios up to 1.5 for a wide range of major congenital malformations in the offspring of smoking mothers.77 Young nulliparous women have an increased risk of bearing a child with gastroschisis, those between 12 and 15 years of age having a more than fourfold increased risk.78 A Californian population-based study (1995–2012) recorded a prevalence for gastroschisis of 2.7 cases per 10,000 livebirths.75 The surveillance system of the National Network of Congenital Anomalies of Argentina reported a 2009–2016 study of 1,663,610 births with 702 born with limb reduction defects.79 The prevalence was 4.22/10,000 births. In 15,094 stillbirths, the prevalence rose to 30.80/10,000. A Chinese study of 223 newborn deaths in a neonatal intensive care unit noted that 44 (19.7 percent) had a confirmed genetic disorder.80 The National Perinatal Epidemiology Centre in Ireland in a study of fatal fetal anomalies recorded 2,638 perinatal deaths, 939 (36 percent) having a congenital anomaly, 43 percent of which were chromosomal.81 More than a single anomaly was noted in 36 percent (333 of 938) of their cases. These numbers led to a significant genetic disease burden and have accounted for 28–40 percent of hospital admissions in North America, Canada, and England.82–84 Notwithstanding their frequency, the causes of about 60 percent of congenital malformations remain obscure.85, 86

6

Genetic Disorders and the Fetus

The effect of folic acid supplementation, via tablet or food fortification, on the prevalence of neural tube defects (NTDs), is now well known to reduce the frequency of NTDs by up to 70 percent87, 88 (see Chapter 10). 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.89 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 (16

Immune system Susceptibility to infection

100

Juvenile rheumatoid-like arthritis

1.2

Gastrointestinal Congenital defects of the gastrointestinal tract

6

Celiac disease

5.4

Dysphagia

55

Endocrine/metabolic Overweight/obesity

23–70

Hypothyroidism

50

Diabetes mellitus

1.4–10.6

Hyperthyroidism

1–3

Ophthalmologic Eye disorders a

80

Cataract

17–29

Keratoconus

8–10

Hematologic/oncologic Leukemia

2–3 (>20-fold excess)

Testicular cancer

Standardized incidence ratio of 2.9

Transient myeloproliferative disorder

50 years of age had associated risks of fetal death almost twice that of younger fathers.426 A Swiss population study found that the proportion of younger fathers was uniformly different between those with and without Down syndrome offspring. Young fathers had an almost twofold increased odds for siring a child with trisomy 21.427 The authors stated the need for confirmation of their findings. Paternal age should garner more attention during genetic counseling,428 especially with the availability of molecular analysis of multiple genes susceptible to de novo mutations in both noninvasive prenatal testing (see Chapter 8) and prenatal diagnosis (see Chapter 14).

A previous fetus or child with a genetic disorder A genetic evaluation and counseling are usually indicated when a previous fetus or child has or had a genetic disorder, unless the matter is straightforward (e.g. previous trisomy 21) and the obstetrician is well informed. Careful inquiry should be made about the health status of a previous child. Failure or delay in the diagnosis of a monogenic disorder leaves the parents without the option of prenatal diagnosis in a subsequent pregnancy. In addition, it deprives them of the option of preimplantation genetic testing for those disorders with known mutations. Failure to make an early diagnosis of a genetic disorder during the first 5 years of life is common. For example, the Rotterdam Clinical Genetics Group reported that 50 percent of children affected by neurofibromatosis had been treated for related symptoms before a specific diagnosis had been made.429 Such delay has become

problematic given that the NF1 gene and genes for many other monogenic disorders are routinely sequenced for a precise diagnosis. Frequently, distressed parents will select a different physician for a subsequent pregnancy and a new or more recent insight may shed light on the cause of the previous disorder. For example, confined placental mosaicism (see Chapter 4) may now serve to explain the discrepancy between reported chromosomal findings at the time of CVS and fetal tissues obtained at elective abortion. Confined placental mosaicism may also be associated with intrauterine growth restriction (see Chapter 4), requiring serial ultrasounds during the pregnancy. Given the heterogeneous nature of genetic disease, being alert to alternative mechanisms of causation will on occasion be rewarding. For example, during a consultation with a patient who had previously delivered a child with the autosomal recessive Meckel–Gruber syndrome, preparatory discussions about establishing the specific mutation from each parent could reveal that the father is not a carrier of a mutation in the culprit gene. Although nonpaternity is more likely, a judicious approach would also include consideration of uniparental disomy.430, 431 This mode of inheritance, in which an offspring can inherit two copies – part or all of a chromosome from one parent and no copy from the other parent – has been seen in a number of disorders, including Prader–Willi syndrome and Angelman syndrome (see discussion later and Chapter 14). About 25 percent of cases of Prader–Willi syndrome are caused by maternal uniparental disomy.432 Involvement of chromosomes 7, 11, 14, and 15 have been notable. Uniparental disomy is caused primarily by meiotic nondisjunction events and followed by trisomy or monosomy “rescue.” Most cases described have been associated with advanced maternal age and have been detected primarily in the process of prenatal genetic studies.433, 434 Recognition of the molecular basis of a disorder from which a previous child died may provide a couple with an opportunity for prenatal diagnosis in a subsequent planned pregnancy. A caveat would be the availability of analyzable tissue from the deceased child. In the recent past this was mostly not done, but with the escalation of new discoveries in genetics, tissues should now be frozen for potential future DNA analysis. The establishment

CHAPTER 1

Genetic Counseling: Preconception, Prenatal, and Perinatal 29

of the molecular basis of recognized syndromes, previously undetectable prenatally, now provides new opportunities for couples seeking prenatal diagnosis. Examples abound and include some of the craniosynostosis syndromes, certain skeletal dysplasias, and many other disorders. In one of our cases, a father with metaphyseal dysplasia of Schmid, troubled by the indignities and hurts of growing up with severe short stature, elected prenatal diagnosis at a preconception visit. Subsequent mutation analysis of conceived twins yielded a normal prenatal diagnosis result confirmed postnatally.435 Heterogeneity and pleiotropism also require consideration in the context of a previous child’s disorder and anticipation of future prenatal diagnosis. For example, a previous child with tuberous sclerosis or a fetus with a cardiac rhabdomyoma would prompt molecular analysis of the TSC1 and TSC2 genes for more precise future prenatal diagnosis.436

A parent with a genetic disorder Physicians are now advised to determine whether a culprit gene has been found for a specific genetic disorder under discussion, since prenatal diagnosis would then be available for that couple or their children. Adult-onset genetic disorders (breast/ovarian cancer, colon cancer, hypertrophic cardiomyopathy, long QT syndrome) serve as examples where prenatal diagnosis is an option. The long-established prenatal diagnoses for both presymptomatic and symptomatic neurodegenerative disorders437 continue to be expanded to include disorders such as amyotrophic lateral sclerosis and frontotemporal dementia by analysis of the C9orf72 gene.438 In prenatal diagnosis discussions for all adult-onset disorders, there is a natural focus on the tortured questions of personal existence and self-extinction. One example is that of a young father with CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) who, faced with our prenatal diagnosis of this disorder, by mutation analysis of the Notch3 gene, with his wife, elected termination.439 Mutation analysis in a subsequent pregnancy assured an unaffected fetus.440 These consultations may invoke deep personal emotional conflict, especially when pleiomorphic genes are concerned. For example, a parent with tuberous sclerosis and normal intelligence could

not be certain that an affected child would not have intellectual disability. This was especially evident in our series of 50 couples having prenatal diagnosis for tuberous sclerosis.436 Discovery of fetal cardiac rhabdomyoma led to sequencing of both the TSC1 and TSC2 genes in the fetus and diagnosis in one of the asymptomatic parents. Parental decisions are neither simple nor predictable. In a UK study441 of 644 deaf individuals and 143 with hearing impairment, 2 percent opined that they would prefer to have deaf children and would consider an elective abortion if the fetus was found to be hearing! Prospective mothers with insulin-dependent diabetes mellitus (IDDM) could find their disorder harder to control during pregnancy. Diabetes should be well controlled before pregnancy. The better the control, the lower the risk of having a child with congenital defects.442, 443 An Australian study noted that with good preconception care of type 1 IDDM, the major congenital malformation rate decreased from a high of 14 percent to 2.2 percent.444 Notwithstanding extant knowledge about IDDM and pregnancy, a report of 273 women noted rates of stillbirth (1.85 percent), perinatal mortality (2.78 percent), and congenital anomalies (6 percent).445 An important Stockholm study of 1,089 stillbirths usefully separated causes in preterm and term/post-term births.446 Infection and intrauterine growth restriction/placental insufficiency accounted for over 44 percent of cases in about equal proportion. The genetics of diabetes is complex with multiple types, both polygenic, multifactorial, syndromic, and monogenic in origin. The polygenic type 1 diabetes (T1DM) and type 2 diabetes (T2DM) have over 40 and 90 genes implicated, respectively. Between 1 and 5 percent of diabetes is monogenic and symptoms overlap with T1DM and T2DM diabetes.447, 448 Affected monogenic type patients mostly do not have islet autoantibodies, often have endogenous insulin production, and are frequently misdiagnosed.449, 450 Both T2DM and monogenic diabetes are often not insulin-dependent, have a family history of diabetes, and can occur in the young. Usually, insulin resistance does not occur, nor does acanthosis nigricans in monogenic diabetics, who are mostly not obese.449 Diabetes diagnosed in the first year of life is monogenic and due to KATP channel mutations.451 There are multiple types of monogenic autosomal

30 Genetic Disorders and the Fetus

dominant maturity-onset diabetes of the young (MODY), four subtypes predominating with mutations in HNFIA (52 percent), GCK (32 percent), HNF4A (10 percent), and HNF1B (6 percent).452 A precise preconception molecular diagnosis is important so as to direct appropriate treatment. No pharmacologic treatment is indicated for the GCK-MODY type, low dose sulfonylureas are prescribed for HNF1A-MODY and HNF4A-MODY, with high-dose sulfonylureas for KATP channel-related diabetes.451 Pregestational T1DM and T2DM are associated with poorer pregnancy outcomes, including up to a fourfold higher rate of perinatal mortality.453 The poorer glycemic control at the time of conception and the first trimester, the higher the frequency of stillbirths, congenital abnormalities, perinatal morbidity and mortality, macrosomia, dystocia in labor, and maternal mortality.454–458 Obesity, with its burden of obstetric complications and congenital anomalies457 (as discussed earlier), compounds all the problems in the diabetic mother. Pregnant women with the chronic multifactorial autoimmune disease systemic lupus erythematosus (SLE) face a host of complications. This disorder, with its predilection for women of childbearing age, is more prevalent in non-white populations and is characterized by involvement that includes renal, cardiovascular, musculoskeletal, neurological, rheumatological, and cutaneous systems.459 Adverse pregnancy outcomes include fetal death, preterm births, intrauterine growth restriction, and neonatal lupus.460 Women with anti-Ro/anti-La antibodies, the latter being specific for the diagnosis of SLE and Sjögren syndrome,461 can be asymptomatic. Anti-Ro antibodies may precede the clinical manifestations of SLE by an average of 3.6 years.462 Note, however, these antibodies are found in up to 3 percent of the general population.463 The prime consequences of having anti-Ro antibodies is the risk of fetal/neonatal heart block and neonatal lupus. In a study of 325 children with second- or third-degree heart block, the overall mortality rate was 17.5 percent. Death in utero occurred in 6 percent.464 The risk of offspring being born with congenital heart block to a mother with anti-Ro antibodies is between 0.2 and 2.0 percent, but 15–20 percent if there has been a previously affected fetus or neonate.464, 465 After

two affected pregnancies, the subsequent pregnancy risk is 50 percent.466 Complex therapeutic considerations include fluorinated glucocorticoids (dexamethasome and betamethasome) and maternal fetal echocardiography monitoring.467, 468 Neonatal lupus with congenital heart block will usually require pacemaker implantation.469, 470 For mothers with a previous affected pregnancy, hydroxychloroquine has been recommended as a pre-emptive treatment.471, 472 Fortunately, only a third of mothers carrying fetuses with complete heart block have an identified autoimmune disorder such as lupus or Sjögren disease.473 Certain genetic disorders may threaten maternal and fetal health in pregnancy and are discussed in detail in Chapter 31.

A history of infertility Beyond the issues of paternal age discussed earlier, there is the evidence that structural chromosomal abnormalities, which occur in 0.25 percent of births, more frequently have their origin in paternal chromosomes. In a 2006 report, 72 percent of de novo unbalanced chromosomal rearrangements were of paternal origin.474 The likelihood of having a translocation doubled every 10 years after the age of 25.475 An American Cancer Society Study of 2,532 cases of hematological cancers noted that men over 35 had a 63 percent higher risk of having affected offspring when compared with those under 25.476 A small, but statistically significant increased risk of nonchromosomal congenital malformations associated with advanced paternal age was reported by the National Birth Defects Prevention Study.477 Malformations included were cleft lip, diaphragmatic hernia, right ventricular outflow tract obstruction, and pulmonary stenosis. About 10 percent of couples have infertility. A World Health Organization multicenter study concluded that the problem appeared predominantly in males in 20 percent of cases, predominantly in females in 38 percent, and in both partners in 27 percent. In the remaining 15 percent of cases, no definitive cause for the infertility was identified.478 Care should be exercised in the preconception counseling of a couple with a history of infertility. In the absence of a recognizable cause, karyotyping of both is recommended. Unrecognized spontaneous abortions may have occurred without the patient’s awareness, caused by overt structural

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Genetic Counseling: Preconception, Prenatal, and Perinatal 31

chromosome rearrangements or microdeletions or duplications (see Chapters 11 and 13). Microarrays performed after routine cytogenetics on products of conception in 2,389 cases revealed significant copy number changes or whole-genome uniparental disomy in 1.6 percent and 0.4 percent of cases, respectively.479 A study of 1,300 infertile men revealed chromosomal abnormalities in 10.6 percent and Y-microdeletions in 4.0 percent.480 Recognized habitual abortion due to the same causes would also require cytogenetic analysis. Such studies may reveal a parent (rarely both) with a chromosomal rearrangement with significant risks for bearing a child with intellectual disability and/or malformations, who could benefit from prenatal or preimplantation diagnosis. Other examples of disorders characteristically associated with recurrent pregnancy loss or infertility include premature ovarian failure in fragile X syndrome carriers (see Chapter 16), and the X-linked disorders steroid sulfatase deficiency481 and incontinentia pigmenti.482 Thrombophilia as a significant cause remains uncertain.483, 484 In about 8 percent of women experiencing recurrent abortion a mutation in the SYCP3 gene (which encodes an essential component of the synaptonemal complex, key to the interaction between homologous chromosomes) was noted.485 An extensive list of genes related to premature ovarian failure have been recognized,486 especially noteworthy in a highly consanguineous population.487 Consequently, next-generation sequencing488 or whole-exome sequencing,489–492 cost issues aside, would be indicated. Although the investigation to determine the cause of male or female infertility can be extensive, several observations are pertinent here. We recognized that congenital bilateral absence of the vas deferens (CBAVD),493 which occurs in 1–2 percent of infertile males, is primarily a genital form of CF (see Chapter 15). Men with CBAVD494 should have CF gene analysis (sequencing, poly T variant analysis, deletion analysis). A meta-analysis concluded that among CBAVD patients, 78 percent had one recognizable CFTR gene mutation whereas 46 percent were noted to have two mutations.495 The mutation detection rate is likely to exceed 92 percent including large gene rearrangements.496 Of interest is the observation of Traystman et al.497 that CF carriers may be at higher risk for infertility

than the population at large. Men who test negative for a CFTR mutation should have the ADGRG2 gene on the X chromosome sequenced.498, 499 Some patients with CBAVD (21 percent in one study500 ) also have renal malformations. These patients may have a normal sweat test and thus far no recognizable mutations in the CF gene.500, 501 Renal ultrasound studies are recommended in all patients with CBAVD who have normal CFTR analyses. The partner of a male with CBAVD and a recognized mutation(s), after gene analysis, should routinely be offered sequencing and deletion analysis of the CFTR gene. Such couples frequently consider epididymal sperm aspiration,502, 503 with pregnancy induced by IVF. Precise prenatal and/or preimplantation genetic testing can be achieved only if specific mutations have been recognized. Significant male infertility is mainly associated with XXY males (see Chapter 12), autosomal translocations, Kallman syndrome, Ymicrodeletions, autosomal inversions, CBAVD, mixed gonadal dysgenesis, and X-linked and autosomal gene mutations.504 We reported a 28-year-old with azoospermia and bilateral congenital cataracts associated with a contiguous deletion including the Nance–Horan gene at Xp23.13 and implicating the SCML1 gene.505 The global prevalence of Yq microdeletions approximates 7.5 percent in infertile males.506 Genes including DAZ (“deleted in azoospermia”), YRRM (Y chromosome RNA recognition motif),507, 508 and others may be deleted singly or together in the region of Yq11.23.509 Couples must be informed that male offspring of men with these interstitial deletions in the Y chromosome will have the same structural chromosome defect. The female partner of the male undergoing intracytoplasmic sperm injection (ICSI) needs explanations about procedures and medications for her that are not risk free. Patients should realize that ICSI followed by IVF is likely to achieve pregnancy rates between 20 and 24 percent,510 a success rate not very different from the approximately 30 percent rate in a single cycle after natural intercourse at the time of ovulation.510 Pregnancy follow-up data from cases culled from 35 different programs reported in a European survey511 and a major American study of 578 newborns showed no increased occurrence of congenital malformations.214 However, a statistically significant increase in sex chromosome

32 Genetic Disorders and the Fetus

defects has been observed.512 Prenatal diagnosis is recommended in all pregnancies following ICSI. Even “balanced” reciprocal translocations in males may be associated with the arrest of spermatogenesis and resultant azoospermia.513 In one series of 150 infertile men with oligospermia or azoospermia, an abnormal karyotype was found in 10.6 percent (16/180), 5.3 percent (8/150) had an AZF-c deletion, and 9.3 percent (14/150) had at least a single CF gene mutation.514 This study revealed a genetic abnormality in 36/150 (24 percent) of men with oligospermia or azoospermia. A Turkish study of 1,696 males with primary infertility showed 8.4 percent with a chromosomal abnormality and 2.7 percent with a Y-chromosome microdeletion.515 Rarer disorders may need to be considered in the quest to determine the cause of infertility including, for example, the blepharophimosis, ptosis, epicanthus inversus syndrome, which may respond to treatment.516 In a study of 75,784 women to determine all-cause and cause-specific mortality, those with infertility had a 10 percent increased risk of death from any cause.517 Death from breast cancer was more than doubled. In a major prospective Danish study, 3,356 women who had children born after frozen embryo transfer were compared with 910,291 fertile women. The incidence rate of childhood cancer was 17.5 per 100,000 for children born to fertile women, and 44.4 per 100,000 in children born after the use of frozen embryos.518 The statistically significant increased risk was primarily leukemia and sympathetic nervous system tumors. The cause(s) remain unknown. A US study did not find a significant association, but had a shorter follow-up period (30 percent complained of anxiety, depression, and headaches.533 Between 20 and 30 percent of carriers experience irregular or absent menses due to primary ovarian insufficiency.534 This latter recognition during routine obstetric care often serves as an alert to check fragile X syndrome carrier status. We have also seen instances where recognition of carrier status has led to reversal of a putative diagnosis of parkinsonism or early dementia, instead of an actual diagnosis of the fragile X tremor ataxia syndrome manifesting in a grandfather over 60 years of age (see Chapter 16). Carrier status for women with a family history of hemophilia A or B cannot be excluded by a normal activated partial thromboplastin time or normal factor VIII or factor IX levels.535 A definitive molecular diagnosis combined with linkage analysis where necessary is needed, especially if prenatal or preimplantation diagnosis is sought. Determination of a pathogenic variant in the structurally complex factor VIII gene enables confirmation of carrier status.536, 537 Prenatal diagnosis requests for hemophilia A are uncommon, but have been provided.538–540 Preimplantation genetic testing (see Chapter 2) for hemophilia has also been accomplished.541 Noninvasive prenatal diagnosis of hemophilia A and B in hemophilia carriers using maternal plasma and factor VIII and factor IX sequence variants has been demonstrated542 (see Chapter 8). We all carry a host of deleterious recessive genes (∼100–300)543 and technical advances have enabled routine simultaneous testing of hundreds of autosomal recessive and X-linked disorders which affect about 1 in 300 pregnancies.544 Not well understood by patients is the fact that expanded carrier testing545–555 examines only a few common mutations in each gene analyzed. The net effect is a significant reduction in the risk of being a carrier of the gene tested. Unfortunately, the refrain heard from patients having had expanded carrier testing is “I am not a carrier.” Financial constraints prevent many couples benefitting from the extensive panel of carrier tests, leaving them with the previously required indications of ethnicity, affected offspring,

or family history. This type of limited carrier testing, which includes CF and spinal muscular atrophy, misses about 70 percent of carriers of rare disorders.556 For the most part carriers of autosomal recessive disorders are asymptomatic. An important exception are the carriers of the sickle cell disease gene mutation p.Glu6-Val in the β-globin chain of hemoglobin, who have an increased risk of both venous thromboembolism and chronic renal disease.557 This is an important realization that should lead to care and surveillance, given that about 300 million worldwide have the sickle cell trait. Autosomal recessive disease severity when due to compound heterozygous pathogenic variants will be a consequence of the variable expression of the two alleles (e.g. CF with the p.Phe508del and the p.Arg117His alleles resulting only in CBAVD) (see Chapter 15). Gene modifiers too will affect the phenotype. Variant interpretation remains a challenge as well as increasing the need and time taken for genetic counseling given that over 1,800 autosomal recessive genes are known.543 Clearly, the purpose of expanded carrier screening (see Chapter 14) for healthy couples enables them to benefit from available options that include preimplantation genetic testing, routine prenatal diagnosis, adoption, donor sperm or ova, or surrogacy. This approach has proved acceptable to the American College of Obstetricians and Gynecologists, the American College of Medical Genetics and Genomics, the Society for Maternal-Fetal Medicine, and the National Society of Genetic Counselors.558, 559 The clinical utility and efficacy has been clearly demonstrated.546, 549, 551, 558 Johansen Taber et al.560 reported on the actions and reproductive outcomes of 391 at-risk couples from a tested population of over 270,000 using a panel of 176 genetic disorders. Over 75 percent who had preconception testing, planned or acted to avoid having an affected progeny. More than 50 percent of at-risk couples terminated pregnancies. Relying on a survey study, the authors acknowledge, has limitations so far as memory, response bias, and selection (infertility problems) are concerned. In a smaller study, others549 demonstrated the clear superiority of expanded carrier screening compared with ethnicity-based testing, with over threefold detection. Punj et al. offered preconception next-generation sequencing to determine

34 Genetic Disorders and the Fetus

carriers and found 12/71 couples at risk.548 Eight were carriers of hemochromatosis. These authors analyzed 728 genes in 202 individuals, 78 percent being determined to have at least one positive carrier result. In this exploratory study, which used a 148 gene-panel rather than the ACMG actionable panel of 59 genes, 3.5 percent of participants had a medically actionable variant548 (see Chapter 14). Applying their analysis to the ACMG panel, 2.9 percent had an actionable variant. Ethnicity-based carrier testing (Table 1.5) remains the only option for large swaths of the world’s population. Selective Ashkenazi Jewish mutation carrier testing, for example, for disorders listed in Table 1.5 do provide valuable but limited information, leading to options noted above. A study of 6,805 Jewish patients (Ashkenazi, Sephardi, and Mizrahi) having expanded carrier screening showed that 64.6 percent were identified as a carrier of one or more of 96 disorders562 (Table 1.6). The authors noted that >80 percent of the reported variants would have been missed by standard Ashkenazi Jewish screening protocols. One in 16 couples were identified as joint carriers with a 25 percent risk of having an affected child. A novel, likely pathogenic variant was seen in about 2.5 percent of patients tested. A whole-exome sequencing study of 123,136 cases examined carrier rates in six ethnic groups, focusing on 415 genes associated with severe recessive disorders.563 These authors found that 32.6 percent (East Asian) and 62.9 percent (Ashkenazi Jewish) were variant carriers of at least one of the 415 genes. A pan-ethnic screen using these 415 genes would identify up to 2.52 percent of at-risk couples. However, the limitations of ethnic-based carrier testing were revealed by a genetic ancestry analysis of >93,000 individuals having expanded carrier testing using a 96-gene panel.565 Nine percent of those tested had an ancestry from a lineage inconsistent with self-reported ethnicity. Multiple published reports on preconception or prenatal expanded carrier screening using large but variable-sized gene panels overwhelmingly support this approach above ethnicity-based testing.545, 549, 566–572 Although not currently required in preconception carrier screening, testing for hereditary cancer risk should be considered. A personal or family history of cancer as well as ethnicity currently

serves as an indication for screening. Autosomal dominant disorders are otherwise not usually subject to screening. In a study of 26,906 individuals in the Healthy Nevada Project screened for BRCA-related breast and ovarian cancer, Lynch syndrome, and familial hypercholesterolemia, 1.33 percent were found to be carriers of pathogenic or likely pathogenic variants.573 Moreover 90 percent of carriers had not been identified previously, and only 25.2 percent had a relevant family history. These three disorders determined by screening (not family history) are not usually considered for prenatal diagnosis or preimplantation genetic testing. However, other autosomal dominant disorders with manifestations in childhood (e.g. multiple endocrine neoplasia type 2B, familial adenomatous polyposis, long QT syndrome, cardiomyopathy) do qualify for preconception, preimplantation, and prenatal testing. A study of 23,179 individuals with a family history of cancer had next-generation sequencing using a 30-gene panel.574 A total of 2,811 pathogenic variants were found in 2,698 individuals for an overall pathogenic frequency of 11.6 percent. For those of Ashkenazi Jewish descent three-quarters of the pathogenic variants in the BRCA1 and BRCA2 genes would have been missed if only the routine three common founder mutations were tested. Geneticists and genetic counselors will attest to the frequent challenges they encounter faced by their patients’ difficulty comprehending genetic test results, implications, and options. On the heels of the technologic advances in genetics have come commercialization in the form of direct-to-consumer (DTC) testing. Few patients are cognizant of the commercialization realities that include selling of their data, receiving misleading results, being faced with incorrect, false-positive or false-negative results, a lack of informed consent, confidentiality, and privacy.575–580 There is a wide spectrum of laws that govern genetic testing in most countries, with special reference to laboratory accreditation, staff certification, genetic counseling requirements, and informed consent. In one study of identical twins there was a lack of concordance between laboratories.581 In an illustrative case, the result provided was actionable, but no action was taken by the recipient of the DTC communication.582 Ethical breaches, including testing of children, further complicate DTC practices.583

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Genetic Counseling: Preconception, Prenatal, and Perinatal 35

Table 1.5 Genetic disorders in various ethnic groups. Ethnic group

Genetic disorder

Africans (black)

Sickle cell disease and other disorders of hemoglobin α- and β-thalassemia Glucose-6-phosphate dehydrogenase deficiency Benign familial leucopenia High blood pressure (in females)

Afrikaners (white South Africans)

Variegate porphyria Fanconi anemia

American Indians (of British Columbia)

Cleft lip or palate (or both)

Amish/Mennonites

Ellis–Van Creveld syndrome Pyruvate kinase deficiency Hemophilia B

Armenians

Familial Mediterranean fever

Ashkenazi Jews

A-β-lipoproteinemia Bloom syndrome Breast cancer Canavan disease Colon cancer Congenital adrenal hyperplasia Dysferlinopathy (limb girdle muscular dystrophy 2B) Dystonia musculorum deformans Factor XI (PTA) deficiency Familial dysautonomia Familial hyperinsulinism Fanconi anemia (type C) Galactosemia Gaucher disease (adult form) Iminoglycinuria Joubert syndrome Maple syrup urine disease Meckel syndrome Niemann–Pick disease Pentosuria Retinitis pigmentosa 590 Tay–Sachs disease Warsaw Breakage syndrome 561

Chinese

Thalassemia (𝛼) Glucose-6-phosphate dehydrogenase deficiency (Chinese type) Adult lactase deficiency

36 Genetic Disorders and the Fetus

Table 1.5 (Continued) Ethnic group

Genetic disorder

Eskimos

E1 pseudocholinesterase deficiency Congenital adrenal hyperplasia

Finns

Aspartylglucosaminuria Congenital nephrosis

French Canadians

Neural tube defects Tay–Sachs disease

Irish

Neural tube defects Phenylketonuria Schizophrenia

Italians (northern)

Fucosidosis

Japanese and Koreans

Acatalasia Dyschromatosis universalis hereditaria Oguchi disease

Maori (Polynesians)

Clubfoot

Mediterranean peoples (Italians,

Familial Mediterranean fever

Greeks, Sephardic Jews, Armenians,

Glucose-6-phosphate dehydrogenase deficiency

Turks, Spaniards, Cypriots)

(Mediterranean type) Glycogen storage disease (type III) Thalassemia (mainly β)

Norwegians

Cholestasis-lymphedema Phenylketonuria

Yugoslavs (of the Istrian Peninsula)

Schizophrenia

Table 1.6 Residual risk values for diseases in Ashkenazi Jewish populations. 100% Ashkenazi

Probability of

Jewish carrier Disease

frequency

Detectability

Residual

affected fetus if

risk

parents pos/nega

Gaucher disease

1 in 15

0.95

1 in 281

1 in 1,124

Cystic fibrosis

1 in 23

0.94

1 in 368

1 in 1,472

Tay–Sachs disease

1 in 27

0.98

1 in 1,301

1 in 5,204

Familial dysautonomia

1 in 31

>0.99

1 in 3,001

1 in 12,004

Canavan disease

1 in 55

>0.97

1 in 1,801

1 in 7,204

Glycogen storage disease type 1a

1 in 64

0.95

1 in 1,261

1 in 5,044

Hyperinsulinemic hypoglycemia

1 in 68

0.90

1 in 671

1 in 2,684

Mucolipidosis IV

1 in 89

0.95

1 in 1,761

1 in 7,044

Maple syrup urine disease

1 in 97

0.95

1 in 1,921

1 in 7,684

Fanconi anemia

1 in 100

0.99

1 in 9,901

1 in 39,604

Dihydrolipoamide dehydrogenase deficiency

1 in 107

>0.95

1 in 2,121

1 in 8,484

Niemann–Pick disease type A

1 in 115

0.97

1 in 3,801

1 in 15,204 1 in 9,524

Usher syndrome type 3

1 in 120

>0.95

1 in 2,381

Bloom syndrome

1 in 134

0.99

1 in 13,301

1 in 53,204

Usher syndrome type 1F

1 in 147

≥0.75

1 in 585

1 in 2,340

Nemaline myopathy

1 in 168

>0.95

1 in 3,341

1 in 13,364

a One

parent is positive and one parent is negative by carrier screening.

Source: Modified from Scott et al.564

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Genetic Counseling: Preconception, Prenatal, and Perinatal 37

Professional organizations, aware of all these issues, have discouraged the use of DTC genetic testing. Position statements have accordingly been issued by the American College of Obstetricians and Gynecologists,584 the American College of Medical Genetics and Genomics,585 the Joint Society of Obstetricians and Gynecologists, and the Canadian College of Medical Genetics.586 A range of laws exist in Europe, with France and Germany banning DTC genetic testing.587 Serious concern has been expressed about the ethical, legal, and regulatory challenges of DTC testing in Ireland588 and Europe.589

A family history of a genetic disorder The explicit naming of a specific genetic disorder when the family history is being discussed facilitates evaluation and any possible testing. Difficulties are introduced when neither family nor previous physicians have recognized a genetic disorder within the family, sometimes revealed by expanded carrier screening591 or whole-exome sequencing.592 Such a disorder may be common (e.g. factor V Leiden deficiency) but nevertheless unrecognized. Clinical clues would include individuals in the family with deep-vein thrombosis, sudden death possibly due to a pulmonary embolus, and yet other individuals with recurrent pregnancy loss.593 Venous thromboembolism is the third leading cause of cardiovascular death in the United States, and provides additional insights into the genetic basis of unprovoked pulmonary embolism. Using whole-exome sequencing in 393 affected individuals and 6,114 controls, Desch et al.594 identified four genes (PROS1, STAB2, PROC, SERPINC1) with pathogenic variants, expanding the need for genetic testing given the history of thromboembolism. For some families, individuals with quite different apparent clinical features may, in fact, have the same disorder. Seventeen cancers in different organs in family members may not be recognized as manifestations of the same common mutation. In hereditary nonpolyposis colon/rectal cancer, various family members may suffer from other cancers including the uterus, ovary, breast, stomach, small bowel, ureter, melanoma, or salivary glands. Analysis of the five culprit genes in the proband would enable detection of the mutation, which could then be assayed in other family members at

risk. In another example, there may be two or more deceased family members who died from “kidney failure,” and another one or two who died from a cerebral aneurysm or a sudden brain hemorrhage. Adult polycystic kidney disease (APKD) may be the diagnosis, which will require further investigation by both ultrasound and DNA analysis. Moreover, two different genes for APKD have been identified (about 85 percent of cases due to APKD1 and close to 15 percent due to APKD2),595 and a rare third locus is known. In yet other families, a history of hearing impairment/deafness in some members and sudden death in others may translate to the autosomal recessive Jervell and Lange–Nielsen syndrome.596 This disorder is characterized by severe congenital deafness, a long QT interval, and large T waves, together with a tendency for syncope and sudden death due to ventricular fibrillation. Given that a number of genetic cardiac conduction defects have been recognized, a history of an unexplained sudden death in a family should lead to a routine electrocardiogram at the first preconception visit and possibly mutation analysis of at least 15 long QT syndrome genes.597 Other disorders in which sudden death due to a conduction defect might have occurred, with or without a family history of cataract or muscle weakness, should raise the suspicion of myotonic muscular dystrophy (see Chapter 31). Rare named disorders in a pedigree should automatically raise the question of the need for genetic counseling. We have seen instances (e.g. pancreatitis) in which, in view of its frequency, the disorder was simply ascribed to alcohol or idiopathic categories. Hereditary pancreatitis, although rare, is an autosomal dominant disorder for which several genes are known.598 Awareness of the clinical manifestations in carrier females of X-linked disorders is important given health and risk implications (Table 1.7). The pattern of inheritance of an unnamed disorder may signal a specific monogenic form of disease. For example, unexplained intellectual disability on either side of the female partner’s family calls for fragile X DNA carrier testing. Moreover, unexpected segregation of a maternal premutation may have unpredicted consequences, including reversion of the triplet repeat number to the normal range.671 Genetic counseling may be valuable, more especially because the phenomena

38 Genetic Disorders and the Fetus

Table 1.7 Signs in females who are carriers of selected X-linked recessive disease pertinent to prenatal diagnosis. Selected disorders

Key feature(s) that may occur

Selected references

Aarskog–Scott syndrome allelic with

Widow’s peak or short stature

599

Achromatopsia

Decreased visual acuity and myopia

600

Adrenoleukodystrophy

Neurologic and adrenal dysfunction

601, 602

Alport syndrome

Microscopic hematuria and hearing impairment

603

Ameliogenesis imperfecta,

Mottled enamel vertically arranged

604

Arthrogryposis multiplex congenita

Club foot, contractures, hyperkyphosis

605

ATRX syndrome 𝛼-thalessemia/ID

Mild intellectual disability, hemoglobin H inclusions

599, 606

Tapered fingers, short, widely spaced, flexed toes,

607

XLMR 16

hypomaturation type

syndrome Borjeson–Forssman–Lehmann syndrome

mild mental retardation

Choroideremia a

Chorioretinal dystrophy

608

Chondrodysplasia punctata 1

Mild intellectual disability, possible bone defects

599

Chronic granulomatous disease

Cutaneous and mucocutaneous lesions

609–611

Cleft palate

Bifid uvula

612

Conductive deafness with stapes

Mild hearing loss

613

Mild high-pitch hearing loss

599

Dilated cardiomyopathy

Cardiac failure

614

Duchenne/Becker muscular

Pseudohypertrophy, muscle weakness,

615–618

and short stature

fixation Deafness X-linked 1 allelic with Charcot-Marie-Tooth 5

dystrophy

cardiomyopathy/conduction defects

Dyskeratosis congenita

Retinal pigmentation

619

Ectodermal dysplasia

Variable severity of skin, hair, nails, and teeth

599

Emery–Dreifuss muscular dystrophy

Cardiomyopathy/conduction defects

620–622

Fabry disease

Angiokeratomas, corneal dystrophy, "burning"

623, 624

hands and feet, rhabdomyolysis FG syndrome

Anterior displaced anus, facial dysmorphism

625

Fragile X syndrome

Mild-to-moderate intellectual disability, behavioral

626–628

aberrations, schizoaffective disorder, premature ovarian failure, fragile X tremor ataxia syndrome, women and men premutation carriers G6PD deficiency

Hemolytic crises, neonatal hyperbilirubinemia

629

Hemophilia A and B

Bleeding tendency

630

Hypohydrotic ectodermal dysplasia

Sparse hair, decreased sweating

631, 632

Ichthyosis

Ichthyosis

633

KDM5C gene disease

Intellectual disability

634

Lissencephaly and agenesis of the

Epilepsy with subcortical band heterotopia

599

Lenticular cataracts

635

MASA syndrome/SPG1

Mild intellectual disability, abducted thumbs

599

McLeod neuroacanthocytosis

Chorea, late-onset cognitive decline

636

Menkes disease

Patchy kinky hair, hypopigmentation

637, 638

Myopia

Mild myopia

639

Nance–Horan syndrome b

Posterior Y-sutural cataracts and dental anomalies

640

Norrie disease

Retinal malformations

641

Ocular albinism type 1

Retinal/fundal pigmentary changes

642

corpus callosum Lowe syndrome

syndrome

(see Chapter 16)

CHAPTER 1

Genetic Counseling: Preconception, Prenatal, and Perinatal 39

Table 1.7 (Continued) Selected disorders

Key feature(s) that may occur

Selected references

Oculofaciodigital syndrome (OFD1)

Facial dysmorphism, abnormal digits, and polycystic

599

allelic with

kidneys

Simson–Galabia–Beheld syndrome 2 and Joubert syndrome Oligodontia

Hypodontia

643

Opitz G/BBB syndrome

Hypertelorism

644

Opitz–Kaveggia syndrome

Mild intellectual disability, hypertelorism

599

Ornithine transcarbamylase

Hyperammonemia, psychiatric/neurologic

645, 646

deficiency

manifestations

Ovarian cancer

Ovarian cancer

647

Pelizaeus–Merzbacher

Possible mild spasticity

648

Retinoschisis

Peripheral retinal changes

649

Retinitis pigmentosa

Night blindness, concentric reduction of visual field,

650, 651

pigmentary fundal degeneration, extinction of electroretinogram, cone disruption, vision loss MECP2-duplication syndrome

Intellectual disability, neuropsychiatric features,

652

endocrine abnormalities 653, 654

Simpson–Golabi–Behmel syndrome

Extra lumbar/thoracic vertebrae, accessory nipples,

Spinal and bulbar muscular atrophy

Muscle weakness and cramps

655

Split-hand/split-foot anomaly

Mild split-hand/split-foot anomaly

656

Spondyloepiphyseal dysplasia, late

Arthritis

657

Slight hypoplasia of ulnar side of hand and mild

658

facial dysmorphism

onset Ulnar hypoplasia with lobster-claw deficiency of feet

syndactyly of toes

Wiskott–Aldrich syndrome a

Abnormal platelets and lymphocytes

659, 660

X-linked intellectual disability

Mostly intellectual disability (many genes),

661–663

occasional short stature, hypertension, psychiatric symptoms X-linked mental retardation

Short stature, hypertelorism

599, 664, 665

X-linked mental retardation

Cerebellar hypoplasia, distinctive facies

666, 667

X-linked myotubular myopathy

Weakness, respiratory problems

668

X-linked protoporphyria

Life-long photosensitivity; liver disease

669

X-linked retinitis pigmentosa

Retinal changes

670

(OPHN1)

a Uncertain. b May

be same disorder.

of pleiotropism (several different effects from a single gene) and heterogeneity (a specific effect from several genes) may confound interpretation in any of these families. History of a previous child with intellectual disability with a diagnosis deemed “idiopathic” or of unknown cause after chromosomal, fragile X and biochemical analyses, is no longer tenable without whole-exome sequencing672, 673 (see Chapter 14). Over 700 genes involved in intellectual disability of monogenic origin have been recognized.674, 675

In a meta-analysis of 3,350 individuals with neurodevelopmental disorders676–678 the diagnostic yield was 36 percent using whole-exome sequencing. More recently, whole-exome sequencing for patients sent for a chromosomal microarray yielded diagnoses in about 27 percent of intellectual disability cases.676

Consanguinity A wide swath of the world’s population have high rates of consanguinity (50–70 percent of births to

40 Genetic Disorders and the Fetus

consanguineous parents). This especially applies to India, Pakistan, Bangladesh, the Middle East, and Africa. Medical literature is replete with examples of rare severe autosomal recessive disease in these populations. Where family history does not reveal unknown or hidden consanguinity, purposeful or incidental, significant runs of homozygosity seen on a chromosomal microarray (see Chapter 13) frequently will. In those instances, recognition of a shared gene and its mutation within a shared region may unexpectedly lead to a rare diagnosis. Not as well known, perhaps, is that shared variant homozygosity markedly reduces the fertility rate of close consanguineous couples.679 Consanguineous couples face increased risks of having children with autosomal recessive disorders; the closer the relationship, the higher the risks. A study in the United Arab Emirates of 2,200 women ≥15 years of age (with a consanguinity rate of 25–70 percent) concluded that the occurrence of malignancies, congenital abnormalities, intellectual disability, and physical handicap was significantly higher in the offspring of consanguineous couples.680, 681 The pooled incidence of all genetic defects, regardless of the degree of consanguinity, was 5.8 percent, in contrast with a nonconsanguineous rate of 1.2 percent, similar to an earlier study.681, 682 A Jordanian study also noted significantly higher rates of infant mortality, stillbirths, and congenital malformations among the offspring of consanguineous couples.683 A Norwegian study of first-cousin Pakistani parents yielded a relative risk for birth defects of about twofold.684 In that study, 28 percent of all birth defects were attributed to consanguinity. An observational study of 5,776 Indian newborns noted a birth defect prevalence of 11.4 per 1,000 births with a consanguinity rate of 44.74 percent.685 A study from Saudi Arabia, where the consanguinity rate exceeds 50 percent, focused on whole-exome sequencing of 2,219 families who had or had lost an affected fetus or child. The study group was constituted by 1,653 individual samples, 127 twosomes, 370 trios, 58 quads, and 11 others.686 They resolved many cases by determining known causal recessive genes and their mutations, but also discovering multiple previously unknown pathogenic variants. In addition, they recognized some genes that also had a dominant rather than recessive mode of inheritance. Their

prenatal diagnostic detection rate was 46.2 percent (30/65 cases), 87 percent of which were autosomal recessive. Whole-exome sequencing following discovery of a fetal anomaly not resolved by karyotyping or chromosomal microarray may well provide a precise diagnosis. In a study of 102 anomalous fetuses, a definitive or probable diagnosis was made in 21 (20.6 percent).687 A similar small study of 19 families with fetal anomalies yielded candidate variants in 12 (63 percent).688 A systematic evidence-based review of exome and genome sequencing for congenital anomalies or intellectual disability on behalf of the ACMG concluded that a change in patient management was observed in nearly all studies, including an impact on reproductive outcomes.689 The occurrence of rare, unusual or unique syndromes invariably raises questions about potential consanguinity and common ancestral origins. Clinical geneticists will frequently be cautious in these situations, providing potential recurrence risks of 25 percent. Consanguineous couples may opt for the entire gamut of prenatal tests to diminish even their background risks, with special focus on their ethnic-specific risks.690 Abnormal or concerning prenatal ultrasound observations in pregnancies by consanguineous couples may prompt prenatal whole-exome sequencing.691

Environmental exposures that threaten fetal health Concerns about normal fetal development after exposure to medications, alcohol, illicit drugs, chemical, infectious or physical agents, and/or maternal illness are among the most common reasons for genetic counseling during pregnancy. Many of these anxieties and frequently real risks could be avoided through preconception care. Public health authorities, vested with the care of the underprivileged in particular, need to focus their scarce resources on preconception and prenatal care and on the necessary public education regarding infectious diseases, immunization, nutrition, and genetic disorders. In preconception planning, careful attention to broadly interpreted fetal “toxins” is necessary, and avoidance should be emphasized. Alcohol, smoking, illegal drug use, certain medications, and X-ray exposure require discussion. Estimates of the prevalence of the fetal alcohol spectrum disorder

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approximate 2 per 1,000 livebirths692 in the United States but in certain regions and countries rates reach as high as 10 percent.693–695 There is a limited list of known and proven human drug teratogens696 (see Chapter 3). Maternal use of specific teratogenic medications,697 such as isotretinoin, may be missed, unless the physician expressly inquires about them. Preconception advice to avoid heat exposure in early pregnancy is appropriate. Our observations showed a 2.9 relative risk for having a child with a NTD in mothers who used a hot tub during the first 6 weeks of pregnancy.698 High fever in the very early weeks of pregnancy is a potential teratogen698 and should be avoided and treated promptly. Animal studies show that commonly used drugs enter the fetal brain.699 A report from the Spanish Collaborative Study of Congenital Malformations noted a 2.8-fold increased risk of Down syndrome in the offspring of women ≥35 years of age and who were taking oral contraceptives when they became pregnant.700 Identification of preconception options

The time to deal with unwanted risks is not during the second trimester of pregnancy, as is so often the case in practice. Preconception counseling will identify specific risks and attendant options, which include the following: • Knowledge of family history • Attention to maternal health (e.g. diabetes control,701, 702 confirm cardiac and vascular normality) • Decision not to have children (includes consideration of vasectomy or tubal ligation) • Adoption • In vitro fertilization • Gamete intrafallopian tube transfer or allied techniques • Artificial insemination by donor • Ovum donation (includes surrogacy) • Intracytoplasmic sperm injection • Carrier detection tests • Noninvasive prenatal screening by fetal DNA in the maternal circulation • Naternal serum 𝛼-fetoprotein screening for NTDs • Prenatal diagnosis (CVS, amniocentesis, cordocentesis, ultrasound, MRI) • Preimplantation genetic testing

Fetal treatment or surgery for selected disorders Folic acid supplementation in periconceptional period (see Chapter 10) • Noninvasive prenatal testing (aneuploidy; monogenic disorders) • Selective abortion • •

Genetic counseling as a prelude to prenatal diagnosis The assumption that noninvasive prenatal testing for common chromosomal abnormalities (see Chapter 7) is a screening and not a diagnostic test, is unfortunately common. Many women receiving a normal report opt to avoid an amniocentesis. The vast majority will be vindicated, but some will complete pregnancy with a child having a disorder that could have been diagnosed in early gestation. Physicians and counselors are advised to remind women of this limitation, given that about half of all chromosomal abnormalities will be missed by the noninvasive screen.703 Prospective parents should understand their specific indication for prenatal tests and the limitations of such studies. Frequently, one or both members of a couple fail to appreciate how focused the prenatal diagnostic study will be. Either or both may have the idea that all causes of intellectual disability or congenital defects will be detected or excluded. It is judicious for the physician to urge that both members of a couple come for the consultation before CVS or amniocentesis. Major advantages that flow from this arrangement include a clearer perception by the partner regarding risks and limitations, a more accurate insight into his family history, and an opportunity to detect an obvious (although unreported or undiagnosed) genetic disorder of importance (e.g. Treacher–Collins syndrome, facioscapulohumeral dystrophy or one of the orofacial–digital syndromes). Women making an appointment for genetic counseling should be informed about the importance of having their partner with them for the consultation, avoiding subsequent misunderstanding about risks, options, and limitations. Before prenatal genetic studies are performed, a couple should understand the inherent limitations both of the laboratory studies and, when relevant, of ultrasound. For detection of chromosomal

42 Genetic Disorders and the Fetus

disorders, they should be aware of potential maternal cell admixture and mosaicism (see Chapter 11). When faced with potential X-linked hydrocephalus, microcephaly, or other serious X-linked disorders, and the realization of less than 100 percent certainty of diagnosis, couples may elect fetal sex determination as the basis for their decision to keep or terminate a pregnancy at risk. For some, neither chromosomal microarrays, biochemical assays, nor DNA analyses will provide results with 100 percent certainty. The time taken to determine the fetal karyotype or other biochemical parameters should be understood before amniocentesis. The known anxiety of this period can be appreciably aggravated by a long, unexpected wait for a result. The need for a second amniocentesis is rarer nowadays but, in some circumstances, fetal blood sampling remains an additional option that may need discussion. Despite the very unlikely eventuality that no result may be obtained because of failed cell culture or contamination, this issue should be mentioned. The potential possibility for false-positive or false-negative results should be carefully discussed when applicable. Any quandary stemming from the results of prenatal studies is best shared immediately with the couple. The role of the physician in these situations is not to cushion unexpected blows or to protect couples from information that may be difficult to interpret. All information available should be communicated, including the inability to accurately interpret the observations made. This is especially so with the use of the chromosomal microarray (see Chapter 13) and whole-exome sequencing (see Chapter 14). Cautions are appropriate with special reference to VOUS (see Chapter 14), that require in addition, parental samples to determine inherited or de novo changes. Other key issues to be considered by the genetic counselor and discussed when appropriate with the consultand follow. Informed consent

Consent for minor procedures including amniocentesis and CVS has been a requirement for decades and needs no repetition. However, the advent of chromosomal microarrays (see Chapter 13) and whole-exome sequencing for prenatal diagnosis (see Chapter 14) requires additional explanations and caveats. Informed consent for

these two technologies is focused on the potential results, not sampling risks and procedures. The specific issues primarily involve the interpretation of results, their significance, the small possibility of uncertain findings, test limitations, and incidental results. Chromosomal microarray testing adds up to 6–10 percent to a prenatal diagnosis result (see Chapter 13) beyond the 8–10 percent for routine karyotyping, and whole-exome sequencing when done after the ultrasound discovery of fetal structural abnormality adds an additional 6.2–80 percent.691, 704–708 This absurd range reflects very small case series, varying indications, and the presence of single or multiple fetal abnormalities. A more likely detection range would be between 8.5 and 32 percent.707, 708 Prenatal diagnosis using whole-exome sequencing (see Chapter 14) is primarily focused on pregnancies in which fetal structural abnormality has been observed. A much less frequent indication would be a recent or late diagnosis of a parent with a likely monogenic disorder characterized by genetic heterogeneity. No matter the indication, the informed consent obtained incorporates and extends current practice for chromosomal microarray tests. The decision to offer whole-exome sequencing will almost inevitably come on the heels of the detection of fetal abnormality and in an atmosphere of tension and anxiety. Any center offering whole-exome sequencing will have, of necessity, established their informed consent procedure. The following list of pointers are likely to find common ground: 1. Pre- and postgenetic counseling by a geneticist or genetic counselor is a prerequisite, with strict adherence to ethical standards.709, 710 2. Both parents should be in attendance. 3. Explanations should use simple language, no jargon, and be in the language of the parents (with an interpreter, if needed). 4. The details of the fetal abnormality, effect on a child (pain; disability), a progressive disorder or not, and life expectancy. 5. The use of targeted sequencing, trios, and gene panels will need explanations, including the reason and need for prior or simultaneous chromosomal microarrays. 6. The time needed to obtain a result.

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7. The likely detection rate and the limitations of whole-exome sequencing (e.g. repeat expansion disorder; mosaicism). 8. The occurrence of false positives, false negatives, or error. 9. The unexpected discovery of nonpaternity or consanguinity. 10. The detection of a variant of unknown significance.709 11. A “secondary finding”711–714 unrelated to the original purpose of the analysis. 12. The opportunity for the parents to opt out of receiving “secondary findings”715 which they should understand may have personal important health implications. 13. The choice to refuse testing. Presymptomatic or predictive testing

Presymptomatic or predictive testing is available for a rapidly increasing number of disorders, especially neuromuscular and neurodegenerative (see Chapter 14). Huntington disease is the prototype, and predictive testing using guidelines promulgated by the World Federation of Neurology,716–719 the International Huntington Association, and the European Huntington Disease Network719 are well established. Various programs report that a majority of patients are able to cope when it is found that they are affected,225–230, 720, 721 and, at least after a 1-year follow-up, potential benefit has been shown even in those found to be at increased risk.722 A European collaborative study evaluated 180 known carriers of the Huntington disease gene mutation and 271 noncarriers, all of whom received a predictive test result. Although the follow-up was only 3 years for about half the group, pregnancies followed in 28 percent of noncarriers and only 14 percent of carriers.723 Prenatal diagnosis was elected by about two-thirds of those who were carriers. Genetic counseling for Huntington disease when intermediate alleles with 27–35 CAG repeats are determined, pose significant challenges.724 Intermediate CAG repeats have been associated with behavioral, movement, and cognitive problems.725–727 The concern is the unpredictable likelihood of expansion which might account for 7 percent of new mutations.724 Providing counseling for those with low penetrance alleles (36–39 CAG repeats) is no less challenging. Repeats in

this range are estimated to occur randomly in the general population with a frequency of about 1 in 400.728 For patients with 36–39 repeats considering prenatal diagnosis, many factors will need to be addressed. These include all options discussed earlier and uncertainty, penetrance, anticipation, age of onset, and life expectancy. Experienced geneticists with an established program that includes predictive/presymptomatic testing for Huntington disease should preferably be consulted. As others earlier,729 we remain very concerned about the use of a test that can generate a “no hope” result. Even in sophisticated programs offering Huntington disease tests, fewer than expected at-risk individuals requested testing.730 A multicenter Canadian collaborative study evaluated the uptake, utilization, and outcome of 1,061 predictive tests, 15 prenatal tests, and 626 diagnostic tests from 1987 to 2000. The uptake for predictive testing was about 18 percent (range 12.5–20.7 percent).731 Of the 15 who had prenatal tests, 12 had an increased risk, which led to pregnancy termination in all but one.731 The motivations leading to the very difficult decision to have or not to have a predictive test are being recognized as extremely complex.732 In a Danish study before DNA tests were available, one in 20 individuals at risk for Huntington disease committed suicide, more than double the population rate,733 highlighting earlier reports of high suicide rates734 and emphasizing the erosive effects of uncertainty. However, a worldwide assessment of suicide rates, suicide attempts, or psychiatric hospitalizations after predictive testing did not confirm a high rate of suicide.735 In their worldwide questionnaire study sent to predictive testing centers, the authors noted that 44 individuals (0.97 percent) among 4,527 tested had five suicides, 21 suicide attempts, and 18 hospitalizations for psychiatric reasons. All those who committed suicide had signs of Huntington disease, while 11 (52.4 percent) of the 21 individuals who attempted suicide were symptomatic. Suicidal ideation or attempts remain a devastating reality for Huntington disease, especially given the psychopathology in those affected.736, 737 Depression, anxiety, and bipolar disorder are not infrequent. Suicidal behavior may be about 12 times that in the population at large, reaching an estimated 20 percent.738, 739 Others have written about the psychologic burden

44 Genetic Disorders and the Fetus

created by knowledge of a disabling fatal disease decades before its onset.740–742 Hayden743 warned that it is inappropriate to introduce a predictive test that “has the potential for catastrophic reactions” without a support program, including pretest and post-test counseling and specified standards for laboratory analyses. In one study, 40 percent of individuals tested for Huntington disease and who received DNA results required psychotherapy.744 A 5-year longitudinal study of psychologic distress after predictive testing for Huntington disease focused on 24 carriers and 33 tested noncarriers. Mean distress scores for both carriers and noncarriers were not significantly different but carriers had less positive feelings.745 A subgroup of tested persons were found to have long-lasting psychologic distress. An interview study of 20 who tested negative for Huntington disease revealed reactions that included obvious relief and gratitude, wishes to have (more) children, and life changes that included pursuit of a career and ending an unhappy relationship.746 Negative reactions included survivor guilt with sadness and depression or a feeling of pressure to do something extraordinary with their lives. Homozygotes for Huntington disease are rare747, 748 and reported in one out of 1,007 patients (0.1 percent). Counseling a patient homozygous for Huntington disease about the 100 percent probability of transmitting the disorder to each child is equivalent to providing a nonrequested predictive test,749 while failing to inform the patient of the risks would be regarded as the withholding of critical information. Pretest counseling in such cases would take into consideration a family history on both sides and therefore be able to anticipate the rare homozygous eventuality. On the other hand, an increasing number of examples already exist (see Chapter 14) in which presymptomatic testing is possible and important to either the patient or future offspring or both. Uptake has been high by individuals at risk, especially for various cancer syndromes.750 Use of DNA linkage or mutation analysis for ADPKD751, 752 may lead to the diagnosis of an unsuspected associated intracranial aneurysm in 8 percent of cases (or 16 percent in those with a family history of intracranial aneurysm or subarachnoid hemorrhage753 ) and preemptive surgery, with avoidance of a life-threatening sudden cerebral hemorrhage.

It is worth noting that a subgroup of families has features similar to Marfan syndrome and that haplo-insufficiency of the PKD1 gene influences the transforming growth factor-β (TGFβ) signaling pathway.754 In a study of 141 affected individuals, 11 percent decided against bearing children on the basis of the risk.755 These authors noted that only 4 percent of at-risk individuals between 18 and 40 years of age would seek elective abortion for an affected fetus. The importance of accurate presymptomatic tests for potential at-risk kidney donors has been emphasized.756 Organ donation by a sibling of an individual with ADPKD, later found to be affected, has occurred more than once. Since the PKD1 gene abuts the tuberous sclerosis (TSC2) gene, heterozygous deletions may lead to a contiguous gene-deletion syndrome.757 Individuals at 50 percent risk for familial polyposis coli (with inevitable malignancy for those with this mutated gene) who undergo at least annual colonoscopy could benefit from a massive reduction in risk (from 50 percent to 93,000 individuals undergoing expanded carrier screening reveals limitations of ethnicity-based medical guidelines. Genet Med 2020;22(10):1694. 566. Singer A, Sagi-Dain L. Impact of a national genetic carrier-screening program for reproductive purposes. Acta Obstet Gynecol Scand 2020;99:802. 567. Shi L, Webb BD, Birch AH, et al. Comprehensive population screening in the Ashkenazi Jewish population for recurrent disease-causing variants. Clin Genet 2017;91:599. 568. Baskovich B, Hiraki S, Upadhyay K, et al. Expanded genetic screening panel for the Ashkenazi Jewish population. Genet Med 2016;18:522. 569. Bristow SL, Morris JM, Peyser A, et al. Choosing an expanded carrier screening panel: comparing two panels at a single fertility centre. Reprod Biomed Online 2019;38:225. 570. Rosenblum LS, Zhu H, Zhou Z, et al. Comparison of pan-ethnic and ethnic-based carrier screening panels for individuals of Ashkenazi Jewish descent. J Genet Couns 2020;29:56. 571. Arjunan A, Litwack K, Collins N, et al. Carrier screening in the era of expanding genetic technology. Genet Med 2016;18:1214.

572. Gregg AR, Edwards JG. Prenatal genetic carrier screening in the genomic age. Semin Perinatol 2018;42:303. 573. Grzymski JJ, Elhanan G, Morales Rosado JA, et al. Population genetic screening efficiently identifies carriers of autosomal dominant diseases. Nat Med 2020;26(8):1235. 574. Neben CL, Zimmer AD, Stedden W, et al. Multi-gene panel testing of 23,179 individuals for hereditary cancer risk identifies pathogenic variant carriers missed by current genetic testing guidelines. J Mol Diagn 2019;21:646. 575. Tandy-Connor S, Guiltinan J, Krempely K, et al. False-positive results released by direct-to-consumer genetic tests highlight the importance of clinical confirmation testing for appropriate patient care. Genet Med 2018;20:1515. 576. Petersen LM, Lefferts JA. Lessons learned from direct-to-consumer genetic testing. Clin Lab Med 2020;40:83. 577. Laestadius LI, Rich JR, Auer PL. All of your data (effectively) belongs to us: data practices among direct-to-consumer genetic testing firms. Genet Med 2017;19:513. 578. de Pauw A, Schwartz M, Colas C, et al. Direct-to-consumer misleading information on cancer risks calls for an urgent clarification of health genetic testing performed by commercial companies. Eur J Cancer 2020;132:100. 579. Horton R, Crawford G, Freeman L, et al. Direct-to-consumer genetic testing. BMJ 2019;367:I5688. 580. Millward M, Tiller J, Bogwitz M, et al. Impact of direct-to-consumer genetic testing on Australian clinical genetic services. Eur J Med Genet 2020;63:103968. 581. Huml AM, Sullivan C, Figueroa M, et al. Consistency of direct-to-consumer genetic testing results among identical twins. Am J Med 2020;133:143.e2. 582. Garmany R, Lee CJ, Sharp RR, et al. Failure to follow up on a medically actionable finding from direct to consumer genetic testing: a case report. Mol Genet Genomic Med 2020;8:e.1252. 583. Niemiec E, Kalokairinou L, Howard HC. Current ethical and legal issues in health-related direct-to-consumer genetic testing. Per Med 2017;14:433. 584. Committee on Genetics. Committee opinion no. 724: consumer testing for disease risk. Obstet Gynecol 2017;130:e270. 585. ACMG Board of Directors. Direct-to-consumer genetic testing: a revised position statement of

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944. Bartnik M, Derwi´nska K, Gos M, et al. Early-onset seizures due to mosaic exonic deletions of CDKL5 in a male and two females. Genet Med 2011;13:447. 945. Bartsch O, Kress W, Kempf O, et al. Inheritance and variable expression in Rubinstein-Taybi syndrome. Am J Med Genet A 2010;152A:2254. 946. Chiang PW, Lee NC, Chien N. Somatic and germ-line mosaicism in Rubinstein-Taybi syndrome. Am J Med Genet A 2009;149A:1463. 947. Carmignac V, Thevenon J, Adès L, et al. In-frame mutations in exon 1 of SKI cause of dominant Shprintzen-Goldberg syndrome. Am J Hum Genet 2012;91:950. 948. Castronovo C, Rusconi D, Crippa M, et al. A novel mosaic NSD1 intragenic deletion in a patient with an atypical phenotype. Am J Med Genet A 2013;161A:611. 949. Désir J, Cassart M, Donner C, et al. Spondyloperipheral dysplasia as the mosaic form of platyspondylic lethal skeletal dysplasia Torrance type in mother and fetus with the same COL2A1 mutation. Am J Med Genet A 2012;158A:1948. 950. Mineyko A, Doja A, Hurteau J, et al. A novel missense mutation in LIS1 in a child with subcortical band heterotopia and pachygyria inherited from his mildly affected mother with somatic mosaicism. J Child Neurol 2010;25:738. 951. Isodor B, Capito C, Paris F, et al. Familial frameshift SRY mutation inherited from a mosaic father with testicular dysgenesis syndrome. J Clin Endocrinol Metab 2009;94:3467. 952. Dufendach KA, Giudicessi JR, Boczek NJ, et al. Maternal mosaicism confounds the neonatal diagnosis of type 1 Timothy syndrome. Pediatrics 2013;131:e1991. 953. Inbar-Feigenberg M, Choufani S, Cytrynbaum C, et al. Mosaicism for genome-wide paternal uniparental disomy with features of multiple imprinting disorders: diagnostic and management issues. Am J Med Genet A 2013;161A:13. 954. Davis BR, Candotti F. Revertant somatic mosaicism in the Wiskott-Aldrich syndrome. Immunol Res 2009;44:127. 955. Kawai T, Nishikomori R, Izawa K, et al. Frequent somatic mosaicism of NEMO in T cells of patients with X-linked anhidrotic ectodermal dysplasia with immunodeficiency. Blood 2012;119:5458. 956. Yamada M, Okura Y, Suzuki, et al. Somatic mosaicism in two unrelated patients with X-linked chronic granulomatous disease characterized by the presence of a small population of normal cells. Gene 2012;497:110.

957. Twigg SR, Matsumoto K, Kidd AM, et al. The origin of EFNB1 mutations in craniofrontonasal syndrome: frequent somatic mosaicism and explanation of the paucity of carrier males. Am J Hum Genet 2006;78:999. 958. Cheillan D, Joncquel-Chevalier Curt M, Briand G, et al. Screening for primary creatine deficiencies in French patients with unexplained neurological symptoms. Orphanet J Rare Dis 2012;7:96. 959. Chen XL, Zhao Y, Ke HP, et al. Detection of somatic and germline mosaicism for the LAMP2 gene mutation c.808dupG in a Chinese family with Danon disease. Gene 2012;507:174. 960. Juan-Mateu J, Paradas C, Olivé M, et al. Isolated cardiomyopathy caused by a DMD nonsense mutation in somatic mosaicism: genetic normalization in skeletal muscle. Clin Genet 2012;82:574. 961. Vreeburg M, van Geel M, van den Heuij LG, et al. Focal dermal hypoplasia in a male patient due to mosaicism for a novel PORCN single nucleotide deletion. J Eur Acad Dermatol Venereol 2011;25:592. 962. Maas SM, Lombardi MP, van Essen AJ, et al. Phenotype and genotype in 17 patients with Goltz-Gorlin syndrome. J Med Genet 2009;46:716. 963. Margari L, Lamanna AL, Buttiglione M, et al. Long-term follow-up of neurological manifestations in a boy with incontinentia pigmenti. Eur J Pediatr 2013;172:1259. 964. Donsante A, Johnson P, Jansen LA, et al. Somatic mosaicism in Menkes disease suggests choroid plexus-mediated copper transport to the developing brain. Am J Med Genet A 2010;152A:2529. 965. Chénier S, Noor A, Dupuis L, et al. Osteopathia striata with cranial sclerosis and developmental delay in a male with mosaic deletion in chromosome region Xq11.2. Am J Med Genet A 2012;158A:2946. 966. Oegema R, Hulst JM, Theuns-Walks SD, et al. Novel no-stop FLNA mutation causes multi-organ involvement in males. Am J Med Genet A 2013;161:2376. 967. Ducamp S, Schneider-yin X, de Rooij F, et al. Molecular and functional analysis of the C-terminal region of human erythroid-specific 5-aminolevulinic synthase associated with X-linked dominant protoporphyria (XLDPP). Hum Mol Genet 2013;22:1280. 968. Quélin C, Saillour Y, Souville I, et al. Mosaic DCX deletion causes subcortical band heterotopia in males. Neurogenetics 2012;13:367. 969. Ptacek JT, Eberhardt TL. Breaking bad news. JAMA 1996;276:496. 970. Luz R, George A, Spitz E, et al. Breaking bad news in prenatal medicine: a literature review. J Reprod Infant Psychol 2017;35:14-31.

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Genetic Counseling: Preconception, Prenatal, and Perinatal 99

971. Bond CF, Anderson EL. The reluctance to transmit bad news: private discomfort or public display? J Eur Soc Psychol 1987;23:176. 972. Monaghan KG, Leach NT, Pekarek D, et al. The use of fetal exome sequencing in the prenatal diagnosis: a points to consider document of the American College of Medical Genetics and Genomics (ACMG). Genet Med 2020;22:675. 973. Horn R, Parker M. Opening Pandora’s box?: ethical issues in prenatal whole genome and exome sequencing. Prenat Diagn 2018;38:20. 974. Parker M, Lucassen A. Using a genetic test result in the care of family members: How does the duty of confidentiality apply? Eur J Hum Genet 2018;26:955. 975. Robyr R, Bernard JP, Roume J, et al. Familial diseases revealed by a fetal anomaly. Prenat Diagn 2006;26:1224. 976. Donnelly JC, Platt LD, Rebarber, A, et al. Association of copy number variants with specific ultrasonographically detected fetal anomalies. Obstet Gynecol 2014;124:83. 977. Wapner RJ, Martin CL, Levy B, et al. Chromosomal microarray versus karyotyping for prenatal diagnosis. N Engl J Med 2012;367:2175. 978. Hawkins A, Stenzel A, Taylor J, et al. Variables influencing pregnancy termination following prenatal diagnosis of fetal chromosome abnormalities. J Genet Couns 2013;22:238. 979. Blakeley C, Smith DM, Johnstone ED, et al. Parental decision-making following a prenatal diagnosis that is lethal, life-limiting, or has long term implications for the future child and family: a meta-synthesis of qualitative literature. BMC Med Ethics 2019;20:56. 980. Supiano K, Vaughn-Cole B. The impact of personal loss on the experience of health professions: graduate students in end-of life and bereavement care. Death Stud 2011;35:73. 981. Silver J, Caleshu C, Casson-Parkin S, et al. Mindfulness among genetic counselors is associated with increased empathy and work engagement and decreased burnout and compassion fatigue. J Genet Couns 2018;27:1175. 982. Verp M, Bombard A, Simpson J, et al. Parental decision following prenatal diagnosis of fetal chromosome abnormality. Am J Med Genet 1988;29:613. 983. Meryash D, Abuelo D. Counselling needs and attitudes toward prenatal diagnosis and abortion in fragile X families. Clin Genet 1988;33:349. 984. Sandelowski M, Barroso J. The travesty of choosing after positive prenatal diagnosis. J Obstet Gynecol Neonatal Nurs 2005;34(3):307.

985. Lou S, Jensen LG, Peterson OB, et al. Parental response to severe or lethal prenatal diagnosis: a systematic review of qualitative studies. Prenat Diagn 2017;37(8):731. 986. Chow EWC, Watson M, Young DA, et al. Neurocognitive profile in 22q11 deletion syndrome and schizophrenia. Schizophr Res 2006;87:270. 987. Evers LJ, De Die-Smulders CE, Smeets EE, et al. The velo-cardio-facial syndrome: the spectrum of psychiatric problems and cognitive deterioration at adult age. Genet Couns 2009;20:307. 988. Baker K, Vorstman JAS. Is there a core neuropsychiatric phenotype in 22q11.2 deletion syndrome? Curr Opin Neurol 2012;25:131. 989. Murphy KC. Annotation: velo-cardio-facial syndrome. J Child Psychol Psychiat 2005;46:563. 990. Murphy KC. Schizophrenia and velo-cardio-facial syndrome. The Lancet 2002;359:426. 991. McDonald-McGinn DM, Hain HS, et al. 22q11.2 Deletion syndrome. In: Adam MP, Ardinger HH, Pagon RA, et al., eds. GeneReviews [internet]. Seattle (WA): University of Washington, Seattle, 1993–2020, September 23, 1999. 992. 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. 993. Blumberg BD, Golbus MC, Hanson K. The psychological sequelae of abortion performed for a genetic indication. Am J Obstet Gynecol 1975;122:799. 994. Blumberg BD. The emotional implications of prenatal diagnosis. In: Emery, AEH, Pullen IM, eds. Psychological aspects of genetic counselling. London: Academic Press, 1984:202. 995. October T, Dryden-Palmer K, Copnell B, et al. Caring for parents after the death of a child. Pediatr Crit Care Med 2018;19(8S Suppl 2):S61. 996. Udipi S, Veach PM, Kao J, et al. The psychic costs of empathic engagement: personal and demographic predictors of genetic counselor compassion fatigue. J Genet Couns 2008:17;459. 997. Parkes CM. Bereavement. Studies of grief in adult life. London: Tavistock Publications, 1972. 998. Worden JW. Grief counseling and grief therapy, 2nd edn. New York: Springer, 1991. 999. Cacciatore J. Psychological effects of stillbirth. Semin Fetal Neonatal Med 2013;18:76. 1000. 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. 1001. Seller M, Barnes C, Ross S, et al. Grief and midtrimester fetal loss. Prenat Diagn 1993;13:341.

®

100 Genetic Disorders and the Fetus

1002. Fanos JH. Developmental tasks of childhood and adolescence: implications for genetic testing. Am J Med Genet 1997;71:22. 1003. Wertz DC, Fanos JH, Reilly PR. Genetic testing for children and adolescents: who decides? JAMA 1994;272:875. 1004. Clinical Genetics Society (UK). Report of a working party: the genetic testing of children. J Med Genet 1994;31:785. 1005. 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. 1006. Green M, Solnit AJ. Reactions to the threatened loss of a child: a vulnerable child syndrome. Pediatrics 1964;034:58. 1007. McIntosh N, Eldrige C. Neonatal death: the neglected side of neonatal care? Arch Dis Child 1984;59:585. 1008. Bourne S. The psychological effects of a stillbirth on women and their doctors. J R Coll Gen Pract 1968;16:103. 1009. Crowther ME. Communication following a stillbirth or neonatal death: room for improvement. Br J Obstet Gynaecol 1995;102:952. 1010. American College of Obstetricians and Gynecologists, Society for Maternal-Fetal Medicine. Management of Stillbirth: Obstetric Care Consensus No, 10. Obstet Gynecol 2020;135:e110. 1011. Causes of death among stillbirths. Stillbirth Collaborative Research Network Writing Group. JAMA 2011;306:2459. 1012. Laury A, Sanchez-Lara PA, Pepkowitz S, et al. A study of 534 fetal pathology cases from prenatal diagnosis referrals analyzed from 1989 through 2000. Am J Med Genet A 2007;143A:3107. 1013. Korteweg FJ, Bouman K, Erwich JJ, et al. Cytogenetic analysis after evaluation of 750 fetal deaths: proposal for diagnostic workup. Obstet Gynecol 2008;111:865. 1014. Saleem S, Tikmani SS, McClure EM, et al. Trends and determinants from stillbirth in developing countries: results from the Global Network’s Population-Based Birth Registry. Reprod Health 2018;15(Suppl 1):100. 1015. Stanley KE, Giordano J, Thorsten V, et al. Casual genetic variants in stillbirth. N Engl J Med 2020;383:1107. 1016. Barrett PM, McCarthy FP, Evans M, et al. Stillbirth is associated with increased risk of long-term maternal renal disease: a nationwide cohort study. Am J Obstet Gynecol 2020;223:427.e1.

1017. Brookes JAS, Hall-Craggs MA, Sams VR, et al. Noninvasive perinatal necropsy by magnetic resonance imaging. Lancet 1996;348:1139. 1018. Gagnon A, Wilson RD, Allen VM. Evaluation of prenatally diagnosed structural congenital anomalies. J Obstet Gynaecol Can 2009;31:875. 1019. Desilets V, Oligny LL. Fetal and perinatal autopsy in prenatally diagnosed fetal abnormalities with normal karyotype. J Obstet Gynaecol Can 2011;267:1047. 1020. Sethi N, Funamoto K, Ingbar C, et al. Noninvasive fetal electrocardiography in the diagnosis of Long QT syndrome: a case series. Fetal Daign Ther 2020;47(9):711. 1021. Shim SH, Ito M, Maher T, et al. Gene sequencing in neonates and infants with the long QT syndrome. Genet Test 2005;9:281. 1022. Cuneo BF, Kaizer AM, Clur SA, et al. Mothers with long QT syndrome are at increased risk for fetal death: findings from a multicenter international study. Am J Obstet Gynecol 2020;222:263.e1. 1023. Lewkowitz AK, Rosenbloom JI, López JD, et al. Association between stillbirth at 23 weeks of gestation or greater and severe maternal morbidity. Obstet Gynecol 2019;134:964. 1024. Nicholas AM, Lewin TJ. Grief reactions of parental couples: congenital handicap and cot death. Med J Aust 1986;144:292. 1025. Lafarge C, Mitchell K, Fox P. Perinatal grief following a termination of pregnancy for foetal abnormality: the impact of coping strategies. Prenat Diagn 2013;33:1173. 1026. Mashiach R, Anter D, Melamed N, et al. Psychological response to multifetal reduction and pregnancy termination due to fetal abnormality. J Matern Fetal Neonatal Med 2013,26:32. 1027. Lewis E, Bryan E. Management of perinatal loss of a twin. BMJ 1988;297:1321. 1028. Lewis E. Stillbirth: psychological consequences and strategies of management. In: Milunsky A, ed. Advances in perinatal medicine, vol. 3. New York: Plenum, 1983:205. 1029. 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. 1030. 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. 1031. Klaus MH, Kennell JH. Caring for parents of an infant who dies: maternal–infant bonding. St Louis, MO: CV Mosby, 1976.

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1032. Blood C, Cacciatore J. Parental grief and memento mori photography: narrative, meaning, culture and context. Death Stud 2014;38:224. 1033. Flenady V, Boyle F, Koopmans L, et al. Meeting the needs of parents after a stillbirth or neonatal death. BJOG 2014;121(Suppl 4):137. 1034. Furlong RM, Hobbins JC. Grief in the perinatal period. Obstet Gynecol 1983;61:497. 1035. Shulman LP, Grevengood C, Phillips OP, et al. Family planning decisions after prenatal detection of fetal abnormalities. Am J Obstet Gynecol 1994;171:1373. 1036. Rowe J, Clyman R, Green C, et al. Follow-up of families who experience a perinatal death. Pediatrics 1978;62:166. 1037. Forrest GC, Standish E, Baum JD. Support after perinatal death: a study of support and counseling after bereavement. BMJ 1982;285:1475. 1038. Moldovan R, Pintea S, Austin J. The efficacy of genetic counseling for psychiatric disorders: a meta-analysis. J Genet Couns 2017;26:1341. 1039. Caldwell S, Wusik K, He H, et al. Development and Validation of the Genetic Counseling Self-Efficacy Scale (GCSES). J Genet Couns 2018;27:1248. 1040. Keller H, Wusik K, He H, et al. Further validation of the Genetic Counseling Self-Efficacy Scale (GCSES): its relationship with personality characteristics. J Genet Couns 2020;29(5):748. 1041. Borle K, Morris E, Inglis A, et al. Risk communication in genetic counseling: Exploring uptake and perception of recurrence numbers, and their impact on patient outcomes. Clin Genet 2018;94:239. 1042. Clarke A, Parsons E, Williams A. Outcomes and process in genetic counseling. Clin Genet 1996;50:462. 1043. Voorwinden JS, Plantinga M, Ausems M, et al. Cognitive and affective outcomes of genetic counseling in the Netherlands at group and individual level: a personalized approach seems necessary. Eur J Hum Genet 2020;28:1187. 1044. Montgomery SV, Barsevick AM, Egleston BL, et al. Preparing individuals to communicate genetic test

1045. 1046.

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1055.

results to their relatives: report of a randomized control trial. Fam Cancer 2013;12:537. Emery AEH, Raeburn JA, Skinner R. Prospective study of genetic counseling. BMJ 1979;1:253. Sibinga MS, Friedman CG. Complexities of parental understanding for phenylketonuria. Pediatrics 1971;48:216. Reynolds BD, Puck MH, Robinson A. Genetic counseling: an appraisal. Clin Genet 1974;5:177. American College of Medical Genetics and Genomics. ACMG policy statement: updated recommendations regarding analysis and reporting of secondary findings in clinical genome-scale sequencing. Genet Med 2015;17:68. 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. Swerts A. Impact of genetic counseling and prenatal diagnosis for Down syndrome and neural tube defects. Birth Defects Orig Artic Ser 1987;23:61. 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. 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. Milunsky A. Your genetic destiny: know your genes, secure your health, save your life. Cambridge, UK: Perseus Books, 2001. Baars MJH, Scherpbier AJJA, Schuwirth LW, et al. Deficient knowledge of genetics relevant for daily practice among medical students nearing graduation. Genet Med 2005;7:295. Marchant G, Barnes M, Evans JP, et al. From genetics to genomics: facing the liability implications in clinical care. J Law Med Ethics 2020;48:11.

2

Preimplantation Genetic Testing Anver Kuliev and Svetlana Rechitsky Herbert Wertheim College of Medicine, Florida International University, Miami, FL and Reproductive Genetic Innovations, Northbrook, IL, USA

Preimplantation genetic testing (PGT1 ) is a practical option for couples at risk of having offspring with serious/fatal chromosomal or monogenic diseases. It has been used for up to 600 monogenic disorders (PGT-M1 ). Moreover, it has been used for human leukocyte antigen (HLA) typing (PGT-HLA1 ), enabling the births of many children whose matched bone marrows have proved life-saving for siblings with congenital and acquired disorders requiring stem cell transplantation treatment. Analysis of single cells or a few cells with a limited amount of available DNA has always presented a technical challenge, especially when PGT is faced with the need for accurate and rapid results from whole-genome amplification (WGA), followed by polymerase chain reaction (PCR) assays that are robust and sensitive. Next-generation sequencing (NGS) has allowed for accurate identification and transfer of euploid embryos (PGT for aneuploidies (PGT-A)1 ). PGT-M was initially applied for the same indications as prenatal diagnosis,2–4 but was then expanded to conditions that had never been considered, such as late-onset diseases with genetic predisposition and preimplantation HLA typing with or without testing for genetic disorders.5–7 PGT represents a natural evolution of the genetic disease prevention technology, from a period with limited genetic counseling and no prenatal diagnosis or treatment to a time when many options, including PGT, have become available.8 Furthermore, PGT has been applied in order to improve

access to the new treatment methods for some severe conditions by stem cell transplantation, for which no traditional treatment approaches are available. The impact of PGT and stem cell treatment on existing policies for the prevention of genetic disease (see Chapter 36) is clear from the increasing use of PGT to avoid unnecessary termination of many wanted pregnancies and for preimplantation HLA typing.

Approaches to preimplantation genetic testing When prenatal genetic diagnosis was first considered in perspective, in 1984, the World Health Organization (WHO) emphasized the relevance of developing earlier approaches for genetic analysis with the possibility of diagnosis before implantation.9, 10 The following possibilities for PGT were mentioned: genetic analysis of the first or second polar bodies and embryo biopsy at the cleavage or blastocyst stage.10, 11 However, these approaches became possible only after introduction of the PCR assay12 and success in micromanipulation and embryo biopsy. First attempts at PGT were undertaken in mammalian embryos over 30 years ago,13–18 when it was demonstrated that cells could be removed from mammalian preimplantation embryos and analyzed successfully without destroying the viability of the embryo in in vitro fertilization (IVF). PGT for human genetic disease was first demonstrated by Handyside et al.19 for X-linked diseases

Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, Eighth Edition. Edited by Aubrey Milunsky and Jeff M. Milunsky. © 2021 Aubrey Milunsky and Jeff M. Milunsky. Published 2021 by John Wiley & Sons Ltd.

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CHAPTER 2

and by Verlinsky et al.20 for autosomal recessive disorders. Tens of thousands of children without detectable birth defects have been born following these procedures,21–25 demonstrating that PGT can be performed safely in humans. Initially, PGT was based on polar body sampling and embryo biopsy at the cleavage stage, but the present standard shifted to blastocyst biopsy. The polar body approach is still, however, the only possibility for the ethnic groups where no embryos micromanipulation is allowed. The Preimplantation Genetic Diagnosis International Society (PGDIS) and the European Society of Human Reproduction and Embryology (ESHRE) Consortium have published an extensive set of best practice guidelines for PGT.26, 27 These recommendations cover PGT organization, genetic and treatment-related counseling, psychologic evaluation, patient selection, all applicable technical issues, and quality control. The developments of preconception and PGT and the existing problems in the application of these early approaches to clinical practice are presented in this chapter, based on our 30 years’ experience of over 22,000 PGT cycles, including 15,700 PGT-A, 491 PGT-HLA, and 6,778 PGT-M, involving a spectrum of, approximately, 600 different monogenic conditions (Table 2.1). Polar body-based preimplantation genetic testing

The biopsy of gametes opened an intriguing possibility of preconception diagnosis of inherited diseases, because genetic analysis of biopsied gamete material made it realistic to select gametes containing an unaffected allele for fertilization and subsequent transfer.28 In this way, not only was the selective abortion of an affected fetus avoided but also fertilization involving affected gametes, as an option for couples at risk of conceiving a genetically abnormal fetus. Although preconception genetic testing could be achieved by genotyping either oocytes or sperm, the latter approach is still not realistic. Development of methods for culture of primary spermatocytes and spermatogonia followed by genetic analysis of matured spermatides is theoretically possible, but this still remains a subject for future research, such as in the framework of the current attempts at haploidization.29, 30 The technique of sperm duplication has been introduced,

Preimplantation Genetic Testing 103

which may allow testing of the sperm duplicate. However, errors may arise in the reduplication procedure, making the technique of sperm duplication inapplicable for clinical practice.31, 32 The only approach for preconception diagnosis at present, therefore, is genotyping oocytes by biopsy and subsequent genetic analysis of polar bodies. The first attempt to obtain oocyte karyotypes was undertaken in the mouse model by testing the second polar body in the early 1980s, but the technique required much improvement to be considered for clinical application.33 Polar bodies were then used to test the possibility of amplification of β-globin sequences, again in the mouse model.34 The first clinical application of the polar body approach was introduced in 1990.20 It was demonstrated that, in the absence of crossover, the first polar body will be homozygous for the allele not contained in the oocyte and second polar body. However, the first polar body approach will not predict the eventual genotype of the oocytes if crossover occurs, because the primary oocyte in this case will be heterozygous for the abnormal gene. The frequency of crossover varies with the distance between the locus and the centromere, approaching as much as 50 percent for telomeric genes, for which the first polar body approach would be of only limited value, unless the oocytes can be tested further (Figure 2.1). Therefore, the second polar body analysis is required to detect hemizygous normal oocytes resulting after the second meiotic division. In fact, the accumulated experience shows that the most accurate diagnosis can be achieved in cases where the first polar body is heterozygous, so the detection of the normal or mutant gene in the second polar body predicts the opposite mutant or normal genotype of the resulting maternal contribution to the embryo after fertilization.4 To study a possible detrimental effect of the procedure, micromanipulated oocytes were followed and evaluated at different stages of development.3, 4, 35 The absence of any deleterious effect of polar body removal on fertilization, preimplantation, and, possibly, postimplantation development made it possible to consider the polar body approach as a nondestructive test for genotyping the oocytes before fertilization and implantation. In another study, to assess the effect of the second polar body sampling on the viability and

Table 2.1 List of conditions for which preimplantation genetic testing (PGT) was performed and PGT-M outcome: 30 years of original experience. No.

No.

Type of

No.

No.

embryo

embryos

Pregnancy

No.

Conditions

Gene

inheritance

patients

cycles

transfers

transferred

%

deliveries

3-Hydroxyisobutyryl-CoA hydrolase deficiency (HIBCHD)

HIBCH

AR

1

1

1

2

0

0

3-Methylglutaconic aciduria with deafness, encephalopathy, and

SERAC1

AR

1

1

1

1

0

0

Achondroplasia (ACH)

FGFR3

AD

7

6

Achromatopsia 2 (ACHM2)

CNGA3

AR

1

1

1

1

1

1

Achromatopsia 3 (ACHM3)

CNGB3

AR

3

4

4

5

2

2

Leigh-like syndrome (MEGDEL)

Acromesomelic dysplasia, Maroteaux type (AMDM)

NPR2

AR

8

1

17

1

11

2

14

2

1

1

Acyl-CoA dehydrogenase, medium-chain, deficiency

ACADM

AR

3

8

7

14

4

4

Acyl-CoA dehydrogenase, very long-chain; (ACADVL)

ACADVL

AR

5

6

6

11

2

2

Adrenal hyperplasia, congenital, due to 21-hydroxylase deficiency

CYP21A2

AR

23

34

26

42

17

17

Adrenoleukodystrophy (ALD)

ABCD1

XL

17

33

20

29

11

11

Agammaglobulinemia, X-linked (XLA)

BTK

XL

4

7

7

13

3

3

AR

1

2

2

2

1

Alagille syndrome 1 (ALGS1)

JAG1

AD

1

1

1

1

1

1

Albinism, ocular, type i (OA1)

Aicardi–Goutieres syndrome 5 (AGS5 + CF)

GPR143

XL

1

12

5

9

4

3

Albinism, oculocutaneous, type ia (OCA1a)

TYR

AR

4

7

6

9

3

3

Albinism, oculocutaneous, type ii (OCA2)

OCA2

AR

3

6

5

9

3

3

Albinism, oculocutaneous, type iii (OCA3)

TYRP1

AR

1

1

0

0

0

0

Allan–Herndon–Dudley syndrome (AHDS)

SLC16A2

XL

1

2

2

2

1

1

Alopecia universalis congenita (ALUNC)

HR

AR

1

1

1

2

1

1

Alpha-1-antitrypsin deficiency (A1ATD)

SERPINA1

AR

9

16

14

18

9

8

Alport syndrome, autosomal dominant

COL4A3

AR

1

4

0

0

0

0

Alport syndrome, X-linked (ATS)

SAMHD1

8

16

1

COL4A5

XL

15

22

10

9

Alzheimer disease 3

PSEN1

AD

2

3

3

6

3

3

Alzheimer disease 4

PSEN2

AD

1

1

1

2

0

0

Alzheimer disease (AD)

APP

AD

2

3

2

4

2

1

Amegakaryocytic thrombocytopenia, congenital (CAMT)

MPL

AR

1

1

0

0

0

Amyloidosis, hereditary, transthyretin-related

TTR

AD

3

7

5

6

3

2

Amyotrophic lateral sclerosis 1 (ALS1)

SOD1

XL

2

2

2

3

2

1

Amyotrophic lateral sclerosis 4, juvenile (ALS4)

SETX

AD

1

1

1

1

1

0

1

Anemia, nonspherocytic hemolytic, due to g6pd deficiency

G6PD

XL

9

12

12

15

6

6

Angelman syndrome (AS)

UBE3A

AD

2

2

2

3

1

1

Angioedema, hereditary, type i (HAE1)

C1NH

AD

3

4

3

4

1

1

Aniridia (AN)

PAX6

AD

4

7

5

6

4

4

NOTCH1

AD

1

1

2

2

1

1

Argininosuccinic aciduria

ASL

AR

2

3

3

4

1

1

Arterial tortuosity syndrome (ATS)

Aortic valve disease 1 (AOVD1)

SLC2A10

AR

1

2

2

2

1

1

Arthrogryposis, distal, type 2a (DA2a)

MYH3

AD

1

2

2

2

1

1

Arthrogryposis, distal, type 2b (DA2b)

TNNI2

AD

1

2

1

1

0

0

Arthrogryposis, distal, type 2b (DA2b)

TNNT3

AD

1

3

2

3

2

1

Arthrogryposis, distal, type 9 (DA9)

FBN2

AD

1

2

2

2

2

2

Ataxia-telangiectasia (AT)

ATM

AD

5

12

7

8

6

5

Auriculocondylar syndrome 2 (ARCND2)

PLCB4

AR

1

1

0

0

0

0

Axenfeld–rieger syndrome, type 1 (RIEG1)

PITX2

AD

3

13

13

15

5

4

Bardet–Biedl syndrome 10 (BBS10)

BBS10

AR

1

2

3

4

1

1

Bardet–Biedl syndrome 2 (BBS2)

BBS2

AR

1

1

2

2

2

1

BBS4

AR

1

1

2

2

1

1

Bartter syndrome, type 3 (BARTS3)

CLCNKB

AR

1

1

2

2

1

1

Basal cell nevus syndrome (BCNS) (Gorlin)

Bardet–Biedl syndrome 4 (BBS4)

PTCH1

AD

6

7

6

10

4

4

Benign chronic pemphigus (BCPM)

ATP2C1

AD

1

1

1

1

1

0

Beta-ureidopropionase deficiency (UPB1D)

UPB1

AR

1

1

2

2

2

1

Biotinidase deficiency

BTD

AR

3

5

2

3

2

2

Birt–Hogg–Dube syndrome (BHD)

FLCN

AD

1

2

1

1

1

1

Bleeding disorder, platelet-type, 16 (BDPLT16)

ITGB3

AD

1

1

0

0

0

0

Blepharophimosis, ptosis, and epicanthus inversus (BPES)

FOXL2

AD

3

7

5

7

3

3

Blood group – Kell–Cellano system

KEL

AR

14

32

19

32

5

5

Brachydactyly, type B1 (BDB1)

ROR2

AD

1

3

2

4

2

2

Branchiooculofacial syndrome (BOFS)

TFAP2A

AD

1

1

1

2

0

0

Breast cancer

PALB2

AD

2

4

2

2

1

1

Breast–ovarian cancer, familial, susceptibility to, 1 (BROVCA1)

BRCA1

AD

93

175

128

183

89

83

87

122

55

51

Campomelic dysplasia with autosomal sex reversal

SOX9

AD

1

1

0

0

0

0

Camurati–Engelmann disease (CAEND)

Breast–ovarian cancer, familial, susceptibility to, 2 (BROVCA2)

TGFB1

BRCA2

AD

AD

1

64

1

123

1

1

0

0

Canavan disease

ASPA

AR

4

6

5

7

5

5

Carbamoyl phosphate synthetase i deficiency

CPS1

AR

1

1

1

2

0

0

Cardioencephalomyopathy, fatal infantile, due to cytochrome c oxidase

SCO2

AR

2

5

5

10

3

3

Cardiomyopathy, dilated, 1A (CMD1A)

LMNA

AR

7

17

16

25

10

8

Cardiomyopathy, dilated, 1DD (CMD1DD)

RBM20

AD

1

2

2

2

2

2

deficiency 1

(Continued)

Table 2.1 (Continued) No.

No.

Type of

No.

No.

embryo

embryos

Pregnancy

No.

Conditions

Gene

inheritance

patients

cycles

transfers

transferred

%

deliveries

Cardiomyopathy, dilated, 1E (CMD1E)

SCN5A

AD

1

2

2

2

1

1

Cardiomyopathy, dilated, 1G (CMD1G)

TTN

AD

2

2

3

3

1

1

Cardiomyopathy, dilated, 1S (CMD1S)

MYH7

AD

3

6

4

4

2

2

Cardiomyopathy, dilated, with woolly hair, keratoderma, and tooth

DSP

AD

2

3

2

3

2

1

TNNT2

AD

1

2

1

1

1

agenesis (DCWHKTA) Cardiomyopathy, familial hypertrophic, 2 (CMH2)

0

Cardiomyopathy, familial hypertrophic, 4 (CMH4)

MYBPC3

AD

14

22

16

23

11

9

Cardiomyopathy, familial hypertrophic, 7 (CMH7)

TNNI3

AD

1

1

1

1

0

0

Cardiomyopathy, familial hypertrophic, 8 (CMH8)

MYL3

AD

1

2

0

0

0

0

Carnitine deficiency, systemic primary (CDSP)

SLC22A5

AR

1

2

1

2

1

1

Carnitine palmitoyltransferase II deficiency, infantile

CPT2

AR

4

7

4

4

2

2

Cerebral arteriopathy, autosomal dominant

NOTCH3

AD

3

7

6

6

6

4

Cerebral creatine deficiency syndrome 1 (CCDS1)

SLC6A8

XL

1

1

1

2

1

1

Ceroid lipofuscinosis, neuronal 2, late infantile (CLN2)

TPP1

AR

2

3

2

2

2

1

Ceroid lipofuscinosis, neuronal, 10 (CLN10)

CTSD

AR

1

1

2

3

1

1

Ceroid lipofuscinosis, neuronal, 5 (CLN5)

CLN5

AR

1

1

2

3

0

0

Ceroid lipofuscinosis, neuronal, 6 (CLN6)

CLN6

AR

2

2

1

2

0

0

Charcot–Marie–Tooth disease, axonal, type 2A2 (CMT2A2)

MFN2

AD

2

9

6

7

2

Charcot–Marie–Tooth disease, axonal, type 2B (CMT2B)

RAB7A

AD

1

1

2

4

2

1

Charcot–Marie–Tooth disease, axonal, type 2E (CMT2E)

NEFL

AD

1

4

4

7

1

1 0

Charcot–Marie–Tooth disease, axonal, type 2F (CMT2F)

HSPB1

AD

1

1

1

1

0

Charcot–Marie–Tooth disease, demyelinating, type 1A (CMT1A)

PMP22

AD

28

56

38

51

25

2

21

Charcot–Marie–Tooth disease, demyelinating, type 1B (CMT1B)

MPZ

AD

2

5

2

5

0

0

Charcot–Marie–Tooth disease, X-linked, 1 (CMTX1)

GJB1

XL

6

9

9

14

5

5

Cholestasis, benign recurrent intrahepatic, 2 (BRIC2)

ABCB11

AR

1

2

2

4

1

1

Cholestasis, progressive familial intrahepatic, 3 (PFIC3)

ABCB4

AR

1

1

1

2

1

1

Chondrodysplasia punctata 1, X-linked recessive (CDPX1)

ARSE

XL

1

2

2

3

0

0

XL

3

5

5

9

Ciliary dyskinesia, primary, 15 (CILD15)

CCDC40

AR

1

1

1

1

1

1

Ciliary dyskinesia, primary, 3 (CILD3)

Choroideremia (CHM)

DNAH5

CHM

AR

2

2

1

2

1

3

1

3

Citrullinemia, classic

ASS1

AR

4

7

6

8

3

3

Cleidocranial dysplasia (CCD)

RUNX2

AD

1

3

5

5

Cockayne syndrome A (CSA)

ERCC8

AR

1

1

2

2

1

1

Coenzyme Q10 deficiency, primary, 7 (COQ10D7)

COQ4

AR

1

1

1

1

1

2

1

2

Cohen syndrome (COH1)

VPS13B

AR

2

2

2

4

2

2

Colorectal cancer, hereditary nonpolyposis, type 1 (HNPCC1)

MSH2

AD

11

21

14

17

7

6

Colorectal cancer, hereditary nonpolyposis, type 2 (HNPCC2)

MLH1

AD

10

18

15

25

9

Colorectal cancer, hereditary nonpolyposis, type 4 (HNPCC4)

PMS2

AD

1

2

1

1

0

0

Colorectal cancer, hereditary nonpolyposis, type 5 (HNPCC5)

MSH6

AD

5

10

8

11

5

5

Combined oxidative phosphorylation deficiency 13 (COXPD13)

PNPT1

AR

1

1

3

5

0

0

Cone–rod dystrophy 6 (CORD6)

GUCY2D

AD

1

1

1

0

0

9

0

Congenital disorder of deglycosylation (CDDG)

NGLY1

AR

1

1

2

2

1

1

Congenital disorder of glycosylation, type Ia (CDG1A)

PMM2

AR

5

5

4

4

3

3

Congenital disorder of glycosylation, type IIc (CDG2C)

SLC35C1

AR

1

1

2

3

0

0

Congenital disorder of glycosylation, type IIL (CDG2L)

COG6

AR

1

2

2

2

0

0

Congenital disorder of glycosylation, type In (CDG1N)

RFT1

AR

2

2

2

4

1

1 1

Cranioectodermal dysplasia 2 (CED2)

WDR35

AR

1

1

1

1

1

Craniofrontonasal syndrome (CFNS)

EFNB1

XL

1

1

1

1

0

Creutzfeldt–Jakob disease (CJD); Gerstmann–Straussler disease (GSD)

PRNP

AD

6

9

9

12

8

7

Crouzon syndrome

FGFR2

AD

8

16

14

23

9

8

Currarino syndrome

MNX1

AD

1

1

1

2

1

1

Cutis laxa, autosomal dominant 1 (ADCL1)

ELN

AD

2

2

Cutis laxa, autosomal recessive, type IIB (ARCL2B)

PYCR1

AR

1

1

2

2

1

1

Cutis laxa, autosomal recessive, type IIIA (ARCL3A)

ALDH18A1

AR

1

1

1

1

1

1

Cystic fibrosis (CF)

CFTR

AR

1

496

4

748

3

627

4

0

1072

354

314

Cystinosis, nephropathic (CTNS)

CTNS

AR

1

1

1

1

0

0

Danon disease

LAMP2

XL

1

2

2

2

2

2

Darier–White disease (DAR)

ATP2A2

AD

1

1

1

1

1

1

D-bifunctional protein deficiency

HSD17B4

AR

1

1

1

1

0

0

Deafness, autosomal dominant 3b (DFNA3b)

GJB6

AD

1

2

2

3

1

1

Deafness, neurosensory, autosomal recessive 1 (DFNB1)

GJB2

AR

51

68

56

80

33

30

Dentinogenesis imperfecta, shields type III

DSPP

AD

1

2

2

2

2

1

Developmental delay

DHX35

AR

1

1

2

2

1

1

Diabetes insipidus, nephrogenic, X-linked

AVPR2

XL

1

3

3

3

1

1

Diabetes mellitus, permanent neonatal (PNDM)

INS

AD

1

1

1

1

1

1 (Continued)

Table 2.1 (Continued) No.

No.

Type of

No.

No.

embryo

embryos

Pregnancy

No.

Conditions

Gene

inheritance

patients

cycles

transfers

transferred

%

deliveries

Diamond–Blackfan anemia 1 (DBA1)

RPS19

AD

1

1

1

2

1

1

Digeorge syndrome (DGS)

TBX1

AD

1

1

1

1

1

Dihydrolipoamide dehydrogenase deficiency (DLDD)

DLD

AR

1

1

1

1

1

1

Donnai–Barrow syndrome

LRP2

AR

1

1

0

0

0

0

Dyskeratosis congenita, autosomal dominant 3 (DKCA3)

TINF2

AD

1

2

2

3

1

1

1

Dyskeratosis congenita, autosomal dominant 2 (DKCA2)

TERT

AD

1

3

1

1

0

Dyskeratosis congenita, autosomal recessive 5 (DKCB5)

RTEL1

AR

1

1

2

2

1

1

Dyskeratosis congenita, X-linked (DKCX)

DKC1

XL

1

1

1

2

1

1

Dyskinesia, seizures, and intellectual developmental disorder (DYSEIDD)

DEAF1

AR

1

1

1

1

0

0

0

Dystonia 1, torsion, autosomal dominant (DYT1)

TOR1A

AD

16

36

35

63

18

18

Dystonia 28, childhood-onset (DYT28)

KMT2B

AD

1

1

1

1

0

0

Dystonia 3, torsion, X-linked (DYT3)

TAF1

XL

1

1

1

2

1

1

Ectodermal dysplasia 10b, hypohidrotic/hair/tooth type, autosomal

EDAR

AR

1

1

1

2

1

1

recessive (ECTD10B) Ectodermal dysplasia, hypohidrotic, X-linked (XHED)

EDA

XL

6

8

8

10

4

4

Ehlers–Danlos syndrome, classic type

COL5A1

AD

2

4

3

4

3

2

Ehlers–Danlos syndrome, type IV, autosomal dominant

COL3A1

AD

4

6

4

7

4

3

Ehlers–Danlos syndrome, type VI (EDS6)

PLOD1

AR

1

1

2

3

0

0

Emery–Dreifuss muscular dystrophy 1, X-linked (EDMD1)

EMD

XL

3

4

4

7

3

Epidermolysis bullosa dystrophica, autosomal dominant (DDEB)

COL7A1

AR

8

9

8

13

4

4

Epidermolysis bullosa simplex with pyloric atresia (EBSPA)

PLEC1

AR

1

2

1

3

1

1

Epidermolysis bullosa simplex, dowling-meara type (EBSDM)

KRT5

AD

1

2

1

2

1

3

1

Epidermolysis bullosa, junctional, Herlitz type

LAMA3

AR

4

9

7

13

7

7

Epidermolysis bullosa, junctional, non-herlitz type

LAMB3

AR

5

6

5

9

2

2

Epidermolytic hyperkeratosis (EHK)

KRT10

AD

2

3

2

2

2

2

Epileptic encephalopathy, early infantile, 2 (EIEE2)

CDKL5

XL

1

1

1

2

1

Epileptic encephalopathy, early infantile, 3 (EIEE3)

SLC25A22

AR

1

1

0

0

0

0

Epileptic encephalopathy, early infantile, 5 (EIEE5)

SPTAN1

AR

1

1

0

0

0

0

1

Epiphyseal dysplasia, multiple, 1 (EDM1)

COMP

AD

3

4

2

2

1

1

Exostoses, multiple, type I

EXT1

AD

11

21

17

29

12

Exostoses, multiple, type II

EXT2

AD

3

8

6

10

3

3

Fabry disease

GLA

XL

12

19

14

22

9

7

10

Facioscapulohumeral muscular dystrophy 1 (FSHD1)

FRG1

AD

25

51

42

71

23

20

Factor VII deficiency

F7

AR

1

1

1

1

0

0

Familial adenomatous polyposis 1 (FAP1)

APC

AD

23

44

36

57

17

15

Familial cold autoinflammatory syndrome 1 (FCAS1)

NLPR3

AD

1

1

1

Familial Mediterranean fever (FMF)

MEFV

AR

10

18

16

22

11

8

Fanconi anemia, complementation group A (FANCA)

FANCA

AR

2

5

2

3

2

2

Fanconi anemia, complementation group C (FANCC)

FANCC

AR

2

5

4

8

1

1

Fetal akinesia deformation sequence (FADS)

NUP88

1

1

1

AR

1

1

1

2

1

1

Fetal akinesia deformation sequence (FADS)

RAPSN

AR

1

1

1

2

1

0

Fragile-X mental retardation syndrome

FMR1

XL

312

608

450

662

243

214

Fraser syndrome 1 (FRASRS1)

FRAS1

AR

2

2

2

2

1

1

Friedreich ataxia 1 (FRDA)

FXN

AR

2

6

4

7

2

2

Frontotemporal dementia and/or amyotrophic lateral sclerosis 1

c9orf72

AD

1

1

1

1

1

1

ALDOB

AR

2

7

6

7

3

(FTDALS1) Fructose intolerance, hereditary

3

Fumarase deficiency (FMRD)

FH

AR

1

1

0

0

0

0

Galactosemia

GALT

AR

3

7

5

6

2

2

Gastric cancer, hereditary diffuse (HDGC)

CDH1

AD

1

1

1

2

Gaucher disease, type I

GBA

XL

39

52

34

57

24

19

Geroderma osteodysplasticum (GO)

GORAB

AR

1

2

2

4

1

1

1

1

Gitelman syndrome (GTLMNS)

SLC12A3

AR

1

1

1

1

0

0

Glaucoma 3, primary congenital, A (GLC3A)

CYP1B1

AR

1

1

2

2

1

1

Glut1 deficiency syndrome 1 (GLUT1DS1)

SLC2A1

AD

1

2

1

2

0

0

Glutaric acidemia I

GCDH

AR

1

1

1

2

0

0

Glycine encephalopathy (GCE)

GLDC

AR

6

7

6

11

6

6

Glycogen storage disease Ia (GSD1A)

G6PC

AR

1

1

2

2

0

Glycogen storage disease II (GSD2)

GAA

AR

5

7

4

9

1

1

Glycogen storage disease IXa1 (GSD9A1)

PHKA2

XL

1

1

0

0

0

0

Glycogen storage disease VII (GSD7)

PFKM

AR

1

1

1

1

1

1

Gm1-gangliosidosis, type I

GLB1

AR

5

5

5

10

4

0

4

Granulomatous disease, chronic, X-linked (CDGX)

CYBB

XL

4

5

4

6

3

2

Greig cephalopolysyndactyly syndrome (GCPS)

GLI3

AD

1

1

2

2

0

0

Harel–Yoon syndrome (HAYOS)

ATAD3A

AR

1

3

3

3

1

1

Hemoglobin-alpha locus 1 (HBA1)

HBA

AR

14

23

21

38

10

10

470

402

762

192

161

1

0

0

0

0

Hemoglobin-beta locus (HBB)

HBB

AR

301

Hemophagocytic lymphohistiocytosis, familial, 2 (FHL2)

PRF1

AR

1

(Continued)

Table 2.1 (Continued) No.

No.

Type of

No.

No.

embryo

embryos

Pregnancy

No.

Conditions

Gene

inheritance

patients

cycles

transfers

transferred

%

deliveries

Hemophagocytic lymphohistiocytosis, familial, 3 (FHL3)

UNC13D

AR

3

4

4

5

4

Hemophilia A (HEMA)

F8

XL

62

103

88

145

50

42

Hemophilia B (HEMB)

F9

XL

5

6

6

9

6

6

Hereditary leiomyomatosis and renal cell cancer (HLRCC)

FH

AD

1

1

1

2

0

3

0

Hereditary motor and sensory neuropathy, type IIC (HMSN2C)

TRPV4

AD

1

1

2

2

1

1

Hermansky–Pudlak syndrome 1 (HPS1)

HPS1

AR

1

4

3

6

2

2

HLA + myelodysplastic syndrome (MDS)

GATA2

AD

1

2

1

1

1

1 2

HLA + Shwachman–Diamond syndrome (SDS)

SBDS

AR

4

10

3

4

2

HLA + adenosine deaminase deficiency (ADA)

ADA

AR

1

1

1

1

1

1

HLA + adrenoleukodystrophy

ABCD1

XL

3

7

2

2

1

1

HLA + Diamond–Blackfan anemia 1 (DBA1)

RPS19,

AD

6

13

10

15

5

5

HLA + Diamond–Blackfan anemia 2 (DBA2)

RPS20

AD

1

1

1

1

1

1

HLA + Diamond–Blackfan anemia 3 (DBA3)

RPS 24

AD

1

1

1

1

0

0

HLA + Diamond–Blackfan anemia 5 (DBA5)

RPL35A

AD

1

1

1

1

1

1

HLA + Diamond–Blackfan anemia 9 (DBA9)

RPS10

AD

1

1

2

2

1

1

HLA + ectodermal dysplasia, hypohidrotic, with immune deficiency

IKBKG

XL

2

9

6

8

2

2

HLA + Fanconi anemia, complementation group A (FANCA)

FANCA

AR

18

52

29

43

14

10

HLA + Fanconi anemia, complementation group C (FANCC)

FANCC

AR

2

5

5

8

1

1

HLA + Fanconi anemia, complementation group D2 (FANCD2)

FANCD2

AR

1

3

2

3

1

1

HLA + Fanconi anemia, complementation group F (FANCF)

FANCF

AR

2

5

2

3

0

0

HLA + Fanconi anemia, complementation group G (FANCG)

FANCG

AR

2

2

2

3

2

2

HLA + Fanconi anemia, complementation group I (FANCI)

FANCI

AR

1

2

2

3

0

0

HLA + Fanconi anemia, complementation group J (FANCJ)

BRIP1

AR

1

1

1

1

1

1

HLA + Fanconi anemia, complementation group JI (FANCJ)

BRIP1

AR

1

3

1

3

0

0

1

2

2

4

1

0

AR

1

3

2

2

1

1 6

HLA + Glanzmann thrombasthenia (GT, +DMD)

ITGA2B DMD

HLA + granulomatous disease, chronic, autosomal recessive, cytochrome

NCF1

b-positive, type I (CDG1) HLA + granulomatous disease, chronic, X-linked (CDGX)

CYBB

XL

6

16

13

17

7

HLA + hemoglobin-beta locus (HBB)

HBB

AR

92

188

119

177

41

HLA + hyper-IgE recurrent infection syndrome, autosomal recessive

DOCK8

AR

1

1

0

0

0

0

HLA + Krabbe disease

GALC

AR

1

1

1

2

1

1

HLA + myotonic dystrophy 1 (DM1)

DMPK

AD

1

2

1

2

1

1

HLA + neutropenia, severe congenital, 1, autosomal dominant (SCN1)

ELANE

AD

2

3

2

5

2

1

31

HLA + polycystic kidney disease 1 (PKD1)

PKD1

AD

1

1

1

2

1

1

HLA + sickle cell anemia

HBB

AR

18

29

18

27

12

8

HLA + thrombocythemia 1 (THCYT1 )

SH2B3

AR

1

2

2

2

2

1

HLA + thrombotic thrombocytopenic purpura, congenital (TTP)

ADAMTS13

AR

1

2

2

4

1

1

HLA + Wiskott–Aldrich syndrome (WAS)

WAS

XL

1

1

0

0

0

0

HLA immunodeficiency with hyper-IgM, type 1 (HIGM1)

CD40LG

XL

8

15

9

13

5

4

HLA + pyruvate kinase deficiency of red cells

PKLR

AD

1

2

1

1

0

0

Holoprosencephaly 2 (HPE2)

SIX3

AD

1

1

1

2

0

0

Holt–Oram syndrome (HOS)

TBX5

AD

5

8

8

9

4

4

Homocystinuria due to cystathionine beta-synthase deficiency

CBS

AR

4

6

4

9

3

3

Homocystinuria due to deficiency of n(5,10)-methylenetetra-

MTHFR

AR

1

1

1

2

0

0 0

hydrofolate reductase activity MTR

AR

1

2

1

1

0

Human leukocyte antigens

Homocystinuria-megaloblastic anemia, cblG complementation type

HLA

AR

60

119

73

108

25

Huntington disease (HD)

HTT

AD

141

209

171

267

107

97

Hurler syndrome

IDUA

AR

7

10

8

13

3

3

Hyaline fibromatosis syndrome (HFS)

ANTXR2

AR

1

1

1

2

1

1

(HMAG) 20

Hydrocephalus due to congenital stenosis of aqueduct of Sylvius (HSAS)

L1CAM

XL

11

16

16

34

8

6

Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA

HADHA

AR

4

4

4

13

3

3

hydratase, alpha subunit (HADHA) Hyperinsulinemic hypoglycemia, familial, 1 (HHF1)

ABCC8

AR

2

11

8

19

4

2

Hyperuricemic nephropathy, familial juvenile, 1 (HNFJ1)

UMOD

AD

1

1

1

1

0

0

ANOS1

XL

1

1

2

2

0

0

Hypogonadotropic hypogonadism 1 with or without anosmia (HH1)

KAL1

XL

1

2

1

1

1

1

Hypoparathyroidism–retardation–dysmorphism syndrome (HRDS)

Hypogonadotropic hypogonadism 1 with or without anosmia (HH1)

TBCE

1R

1

1

1

2

0

0

Hypophosphatasia, infantile

ALPL

AR

6

7

6

9

4

4

Ichthyosis, congenital, autosomal recessive 1 (ARCI1)

TGM1

AD

2

9

7

10

1

1

Ichthyosis, lamellar, 2 (LI2)

ABCA12

AR

2

2

1

2

0

0

ELOVL4

AR

1

1

1

1

0

0

Ichthyosis, X-linked (XLI)

STS

XL

2

3

3

4

1

1

Ifap syndrome with or without Bresheck syndrome

Ichthyosis, spastic quadriplegia, and mental retardation (ISQMR)

MBTPS2

XL

2

3

2

5

2

1

Immunodeficiency with hyper-IgM, type 1 (HIGM1)

CD40LG

XL

4

14

14

22

6

6

Immunodysregulation, polyendocrinopathy, and enteropathy, X-linked

FOXP3

XL

2

3

3

3

1

1

IKBKG

XL

15

35

28

43

11

(IPEX) Incontinentia pigmenti (IP)

11 (Continued)

Table 2.1 (Continued) No.

No.

Type of

No.

No.

embryo

embryos

Pregnancy

No.

Conditions

Gene

inheritance

patients

cycles

transfers

transferred

%

deliveries

Infantile cerebellar-retinal degeneration (ICRD)

ACO2

AR

1

1

1

2

2

2

Infantile liver failure syndrome 1 (ILFS1)

LARS

AR

1

1

2

2

1

1

Isovaleric acidemia (IVA)

IVD

AR

1

1

1

2

0

0

Joubert syndrome 1 (JBTS1)

INPP5E

AR

1

1

2

2

1

1

Joubert syndrome 17 (JBTS17)

CPLANE1

AD

1

1

1

2

1

1

Joubert syndrome 2 (JBTS2)

TMEM216

AR

1

1

2

2

1

1

Joubert syndrome 21 (JBTS21)

CSPP1

AR

2

5

4

7

1

1

Joubert syndrome 23 (JBTS23)

KIAA0586

AR

1

1

1

2

1

1 0

AR

1

0

0

Joubert syndrome 6 (JBTS6)

TMEM67

AR

2

3

2

2

2

Krabbe disease

Joubert syndrome 3 (JBTS3)

GALC

AR

11

12

11

19

7

5

Larsen syndrome (LRS)

FLNB

AD

2

2

1

1

1

1

Leber congenital amaurosis 2 (LCA2)

RPE65

AR

1

1

0

0

0

0

Leigh syndrome (LS)

AHI1

NDUFS8

1

0

2

AR

1

1

0

0

0

Leigh syndrome (LS)

SURF1

AR

1

1

1

3

0

0

Lesch–Nyhan syndrome (LNS)

HPRT1

XL

1

4

3

3

2

2

0

Leukoencephalopathy with vanishing white matter (VWM)

EIF2B2

AR

1

1

1

2

1

0

Li–Fraumeni syndrome 1 (LFS1)

TP53

AD

16

22

17

24

13

11

Lipoid congenital adrenal hyperplasia (LCAH)

STAR

AR

1

2

2

3

1

1

ARX

XL

0

0

Loeys–Dietz syndrome 1 (LDS1)

TGFBR2

AD

2

5

4

6

2

1

Long QT syndrome 1 (LQT1)

Lissencephaly, X-linked, 2 (LISX2)

KCNQ1

AD

4

5

2

2

2

2

Long QT syndrome 2 (LQT2)

KCNH2

1

1

1

2

AD

3

3

2

2

1

Long QT syndrome 8 (LQT8)

CACNA1C

AD

1

1

1

1

1

1

Lymphedema, hereditary, III (LMPH3)

PIEZO1

AR

1

1

0

0

0

0

1

Lymphoproliferative syndrome, X-linked, 1 (XLP1)

SH2D1A

XL

1

1

2

3

2

2

Lysosomal acid lipase deficiency

LIPA

AR

2

2

2

4

2

2

Machado–Joseph disease (MJD)

ATXN3

AD

4

7

6

8

6

6

Macular dystrophy, vitelliform, 2 (VMD2)

BEST1

AD

1

1

Maple syrup urine disease (MSUD)

BCKDHB

AR

1

2

2

2

1

1

Marfan syndrome (MFS)

FBN1

AD

30

1

58

1

46

1

78

1

27

21

Marinesco–Sjogren syndrome (MSS)

SIL1

AR

1

3

3

5

1

1

Meckel syndrome, type 1 (MKS1)

MKS1

AR

2

Meckel syndrome, type 4 (MKS4)

CEP290

4

6

6

10

4

Meckel syndrome, type 6 (MKS6)

CC2D2A

AR

2

5

5

9

2

2

Meckel syndrome, type 6 (MKS6)

CCD2DA2

AR

1

1

1

2

1

1

Meckel syndrome, type 8 (MKS8)

TCTN2

AR

1

5

1

5

2

9

2

2

1

2 4

1

Mental retardation, autosomal dominant 35 (MRD35)

PPP2R5

AD

1

1

2

2

1

1

Mental retardation, autosomal recessive 38 (MRT38)

HERC2

AR

1

2

2

3

1

1

Metachromatic leukodystrophy due to saposin B deficiency

PSAP

AR

1

1

0

0

0

0

Metachromatic leukodystrophy (MLD)

ARSA

AR

3

4

3

4

4

2

Metaphyseal chondrodysplasia, Schmid type (MCDS)

COL10A1

AD

2

7

3

4

2

2

Methylmalonic aciduria and homocystinuria, cblC type

MMACHC

AR

3

6

6

11

5

5

Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency

MUT

AR

2

4

4

4

2

2

Methylmalonic aciduria, cblB type

MMAB

AR

3

3

2

3

1

1

Microcephalic osteodysplastic primordial dwarfism, type I (MOPD1)

RNU4ATAC

AR

1

1

1

1

1

1

Microcephaly 2, primary, autosomal recessive (MCPH2)

WDR62

AR

1

1

1

1

0

0

Microcephaly 5, primary, autosomal recessive (MCPH5)

ASPM

AR

2

3

2

3

2

2

Microcephaly 6, primary, MCPH6)

CENPJ

AR

1

2

2

2

1

1

Microphthalmia, isolated, with coloboma 3 (MCOPCB3)

VSX2

AR

2

2

1

1

1

1

Midface hypoplasia, hearing impairment, elliptocytosis, and

AMMECR1

XL

2

8

6

9

2

2

nephrocalcinosis (MFHIEN) Migraine, familial hemiplegic, 1 (FHM1)

CACNA1A

AD

1

1

1

2

1

1

Mitochondrial complex i deficiency due to acad 9 deficiency

ACAD9

AR

1

1

1

2

1

1

Mitochondrial DNA depletion syndrome 13

FBXL4

AD

1

1

3

4

1

1

Mitochondrial DNA depletion syndrome 4a (Alpers type) (MTDPS4A)

POLG

AR

3

5

5

5

4

4

Molybdenum cofactor deficiency, complementation group B (MOCODB)

MOCS2

AR

1

1

3

4

0

0

Mosaic variegated aneuploidy syndrome 1 (MVA1)

BUB1B

AR

1

1

1

2

1

0

Mucolipidosis II alpha/beta

GNPTAB

AR

2

3

Mucopolysaccharidosis, type II (MPS2)

IDS

XL

9

20

15

29

10

6

Mucopolysaccharidosis, type IIIA (MPS3A)

SGSH

AR

2

2

2

2

3

2

0

2

0

2

Mucopolysaccharidosis, type IVA (MPS4A)

GALNS

AR

1

4

4

12

2

2

Multinucleated neurons, anhydramnios, renal dysplasia, cerebellar

1

CEP55

AR

1

1

1

2

1

Multiple congenital anomalies–hypotonia–seizures syndrome 2 (MCAHS2)

PIGA

XL

1

1

0

0

0

0

Multiple endocrine neoplasia, type I (MEN1)

MEN1

AD

8

21

16

23

7

4

hypoplasia

Multiple endocrine neoplasia, type IIA (MEN2A)

RET

AD

6

11

11

17

8

Multiple endocrine neoplasia, type IV (MEN4)

CDKN1B

AD

1

3

1

1

1

8 1 (Continued)

Table 2.1 (Continued) No.

No.

Type of

No.

No.

embryo

embryos

Pregnancy

No.

Conditions

Gene

inheritance

patients

cycles

transfers

transferred

%

deliveries

Muscular dystrophy, congenital merosin-deficient, 1A (MDC1A)

LAMA2

AR

6

7

7

14

7

Muscular dystrophy, Duchenne type (DMD)

DMD

XL

69

115

103

169

57

48

Muscular dystrophy, limb-girdle, type 2A (LGMD2A)

CAPN3

AR

1

1

0

0

0

0

6

Muscular dystrophy, limb-girdle, type 2S (LGMD2S)

TRAPPC11

AR

1

1

2

2

2

2

Muscular dystrophy–dystroglycanopathy (congenital with brain and eye

FKRP

AR

1

3

3

3

1

1 2

anomalies), type A, 5 (MDDGA5) Muscular dystrophy–dystroglycanopathy (congenital with brain and eye

FKTN

AR

2

2

2

3

2

Myoglobinuria, acute recurrent, autosomal recessive

LPIN1

AR

1

1

1

1

1

1

Myopathy, areflexia, respiratory distress, and dysphagia, early-onset

MEGF10

AR

1

1

1

1

1

1

anomalies), type A, 4 (MDDGA4)

(EMARDD) Myopathy, centronuclear, X-linked (CNMX)

MTM1

XL

5

6

4

6

4

4

Myopathy, myofibrillar, 1 (MFM1)

DES

AD

1

1

1

1

1

1

Myotonia congenita, autosomal dominant

CLCN1

AD

1

1

1

2

1

1

Myotonic dystrophy 1 (DM1)

DMPK

AD

94

147

107

188

55

46

Myotonic dystrophy 2 (DM2)

CNBP

AD

1

2

2

4

2

2

Nail–patella syndrome (NPS)

LMX1B

AD

3

4

3

4

1

1

Nemaline myopathy 2 (NEM2)

NEB

AR

6

6

6

10

3

3

Nephrotic syndrome, type 1 (NPHS1)

NPHS1

AR

1

3

3

7

1

0

Nephrotic syndrome, type 2 (NPHS2)

NPHS2

AR

1

1

1

1

1

1

Nephrotic syndrome, type 5

LAMB2

AR

1

2

2

4

2

1

Neurofibromatosis, type I (NF1)

NF1

AD

51

90

80

123

46

41

Neurofibromatosis, type II (NF2)

NF2

AD

7

10

9

17

7

7

Neuropathy, hereditary sensory and autonomic, type III (HSAN3)

IKBKAP

AR

13

19

17

28

9

9

Neuropathy, hereditary sensory and autonomic, type VI (HSAN6)

DST

AD

1

2

2

2

2

2

Neutropenia, severe congenital, 1, autosomal dominant (SCN1)

ELANE

AD

1

1

1

1

1

1

Niemann–Pick disease, type A

SMPD1

AR

3

5

3

6

2

2

Nijmegen breakage syndrome (NBS)

NBN

AR

1

1

2

2

1

1

Noonan syndrome 1 (NS1)

PTPN11

AD

5

7

7

9

4

3

Norrie disease (ND)

NDP

XL

5

8

6

12

2

Omenn syndrome

RAG1

AD

2

6

5

12

1

1

Optic atrophy 1 (OPA1)

OPA1

AD

3

5

5

9

1

1

Ornithine transcarbamylase deficiency

OTC

XL

11

24

19

32

11

10

2

Osteogenesis imperfecta, type I (OI1)

COL1A1

AD

24

61

44

72

Osteogenesis imperfecta, type II (OI2)

COL1A2

AD

5

5

5

5

3

2

Osteogenesis imperfecta, type IX (OI9)

PPIB

AR

1

2

2

4

2

2

Osteopathia striata with cranial sclerosis (OSCS)

AMER1

XL

1

1

1

1

17

1

17

1

Osteopetrosis, autosomal recessive 1 (OPTB1)

TCIRG1

AR

5

7

7

13

3

3

Pachyonychia congenita 3 (PC3)

KRT6A

AD

1

2

2

2

2

1

PRSS1

AD

1

2

1

1

Paraganglioma and gastric stromal sarcoma

SDHB

AD

1

1

0

0

0

0

Paramyotonia congenita of von Eulenburg (PMC)

Pancreatitis, hereditary (PCTT)

SCN4A

AD

3

3

3

4

3

2

Pelizaeus–Merzbacher disease (PMD)

PLP1

XL

1

7

1

12

10

15

7

7

Periventricular nodular heterotopia 1 (PVNH1)

FLNA

XL

1

3

3

5

2

1

Peroxisome biogenesis disorder 1A (Zellweger) (PBD1A)

PEX1

AR

3

3

3

6

3

3

Peroxisome biogenesis disorder 2A (Zellweger) (PBD2A)

PEX5

AR

1

2

2

4

0

0

Peroxisome biogenesis disorder 3A (Zellweger) (PBD3A)

PEX12

AR

1

3

3

4

2

1

Peroxisome biogenesis disorder 5A (Zellweger) (PBD5A)

PEX2

AR

1

4

3

5

2

2

STK11

AD

4

9

7

9

6

4

Pfeiffer syndrome

FGFR1

AD

2

2

2

4

2

2

Phenylketonuria (PKU)

Peutz–Jeghers syndrome (PJS)

PAH

AR

15

20

14

16

8

7

Platelet disorder, familial, with associated myeloid malignancy (FPDMM)

RUNX1

AD

1

1

1

1

1

1

Pleuropulmonary blastoma (PPB)

DICER1

AD

1

1

1

1

1

1

Polycystic kidney disease 1 (PKD1)

PKD1

AD

48

84

64

98

37

34

Polycystic kidney disease 2 (PKD2)

PKD2

AD

7

10

9

15

3

3

Polycystic kidney disease, autosomal recessive (ARPKD)

PKHD1

AR

16

29

26

42

17

16

Polymicrogyria, bilateral frontoparietal (BFPP)

ADGRG1

AR

2

2

1

2

1

1

Polymicrogyria, bilateral frontoparietal (BFPP)

GPR56

AR

1

1

1

2

0

0

EXOSC3

AR

2

2

1

1

Popliteal pterygium syndrome (PPS)

IRF6

AD

2

2

1

2

1

1

Porphyria, congenital erythropoietic

Pontocerebellar hypoplasia, type 1B (PCH1B)

UROS

AR

1

1

1

1

1

1

PCCA,

Propionic acidemia

1

1

AR

3

3

3

5

2

2

F2 F5

AR

2

3

3

3

2

2

Pseudovaginal perineoscrotal hypospadias (PPSH)

SRD5A2

AR

1

2

2

4

1

1

Rap guanine nucleotide exchange factor 6 (RAPGEF6)

RAPGEF6

AD

1

2

3

4

3

1

1

1

2

2

1

1

PCCB Prothrombin deficiency, congenital; Factor V deficiency

Renal cell carcinoma, papillary, 1 (RCCP1)

MET

AD

Renal tubular acidosis, distal, autosomal recessive (RTADR)

ATP6V0A4

AR

1

1

2

3

2

1

Renal tubular dysgenesis (RTD)

ACE

AR

1

4

3

4

2

2 (Continued)

Table 2.1 (Continued) No.

No.

Type of

No.

No.

embryo

embryos

Pregnancy

No.

Conditions

Gene

inheritance

patients

cycles

transfers

transferred

%

deliveries

Restrictive dermopathy, lethal

ZMPSTE24

AR

2

2

2

3

1

1

Retinal dystrophy, early-onset, with or without pituitary dysfunction,

OTX2

AD

1

1

0

0

0

0

included XL

1

1

1

2

1

Retinitis pigmentosa 3 (RP3)

RPGR

XL

5

6

6

8

4

3

Retinitis pigmentosa 4 (RP4)

Retinitis pigmentosa 2 (RP2)

RHO

RP2

AD

3

5

2

4

1

0

1

Retinoblastoma (RB1)

RB1

AD

17

31

26

43

14

13

Retinoschisis 1, X-linked, juvenile (RS1)

RS1

XL

1

2

1

2

1

0

Rett syndrome (RTT)

MECP2

XL

3

5

4

4

3

1

Rhabdoid tumor predisposition syndrome 1 (RTPS1)

SMARCB1

AD

1

1

1

1

0

0

Rhesus blood group, D antigen (RHD)

RHD

AD

7

9

9

16

6

6

Sandhoff disease

HEXB

AR

4

6

5

8

4

4

Seckel syndrome 1 (SCKL1)

ATR

AR

1

1

2

2

0

0

Severe combined immunodeficiency, autosomal recessive

IL7R

AR

1

1

2

4

1

Severe combined immunodeficiency, autosomal recessive

RAG2

AR

2

5

4

5

3

3

Severe combined immunodeficiency, X-linked (SCIDX1)

IL2RG

XL

3

4

3

3

2

2

Short stature, idiopathic, X-linked (ISS)

SHOX

XL

2

2

2

3

2

1

2

Short-rib thoracic dysplasia 3 with or without polydactyly (SRTD3)

DYNC2H1

AR

1

1

1

1

1

1

Smith–Lemli–Opitz syndrome (SLOS)

DHCR7

AR

18

30

23

32

15

15

Sonic hedgehog (SHH)

SHH

AD

1

2

2

3

1

1

Sotos syndrome 1 (SOTOS1)

NSD1

AD

2

3

2

2

2

2

Spastic paraplegia 3, autosomal dominant (SPG3A)

ATL1

AD

1

1

1

1

1

1

Spastic paraplegia 4, autosomal dominant (SPG4)

SPAST

AD

6

10

8

12

7

5

Spherocytosis, type 2 (SPH2)

SPTB

AD

1

1

2

2

2

1

Spinal and bulbar muscular atrophy, X-linked 1 (SMAX1)

AR

XL

3

5

5

6

2

1

Spinal muscular atrophy, distal, autosomal recessive, 1 (DSMA1)

IGHMBP2

AR

2

3

2

4

1

1

Spinal muscular atrophy, type I (SMA1)

SMN1

AR

102

151

125

199

78

69

Spinocerebellar ataxia 1 (SCA1)

ATXN1

AD

4

7

8

4

Spinocerebellar ataxia 2 (SCA2)

ATXN2

AD

7

14

14

27

6

8

Spinocerebellar ataxia 6 (SCA6)

CACNA1A

AD

2

5

2

6

3

1

1

4

Spinocerebellar ataxia 7 (SCA7)

ATXN7

AD

2

3

3

7

2

1

Spinocerebellar ataxia 8 (SCA8)

ATXN80S

AD

1

1

1

1

1

1

Spondyloepiphyseal dysplasia tarda, X-linked (SEDT)

TRAPPC2

AD

1

1

2

2

1

1

Stargardt disease 1 (STGD1)

ABCA4

AR

4

10

5

6

2

2

Stickler syndrome, type I (STL1)

Col2A1

AD

4

4

3

5

2

2

Stickler syndrome, type II (STL2)

COL11A1

AD

2

7

6

15

1

1

Stickler syndrome, type II (STL2)

COL18A1

AR

1

1

1

1

1

1

Succinic semialdehyde dehydrogenase deficiency (SSADHD)

ALDH5A1

AR

3

4

4

9

2

2

Sulfocysteinuria

SUOX

2

1

1

AR

1

1

2

Supranuclear palsy, progressive, 1 (PSNP1)

MAPT

AD

2

3

3

5

1

Surfactant metabolism dysfunction, pulmonary, 3 (SMDP3)

ABCA3

AR

1

2

2

4

1

1

Symphalangism, proximal (SYM1)

NOG

AD

1

3

3

7

2

2

Tay–Sachs disease (TSD)

HEXA

AR

25

46

29

52

19

1

17

Telangiectasia, hereditary hemorrhagic, of Rendu, Osler, and Weber (HHT)

ENG

AD

4

11

6

7

3

3

Telangiectasia, hereditary hemorrhagic, type 2 (HHT2)

ACVRL1

AD

4

8

7

8

4

4

Temtamy syndrome (TEMTYS)

C12orf57

AR

1

1

1

2

0

0

Thrombocytopenia-absent radius syndrome (TAR)

RBM8A

AR

4

6

5

7

4

Treacher Collins syndrome 1 (TCS1)

TCOF1

AD

6

8

8

14

7

7

Treacher Collins syndrome 2 (TCS2)

POLR1D

AD

1

1

1

1

0

0

Tuberous sclerosis 1 (TSC1)

TSC1

AD

20

30

27

Tuberous sclerosis 2 (TSC2)

TSC2

AD

8

14

10

14

5

4

Tyrosinemia, type I (TYRSN1)

FAH

AR

1

7

7

13

5

3

Ulnar–Mammary syndrome (UMS)

TBX3

AD

1

3

3

4

1

1

52

16

4

14

Usher syndrome, type I (USH1)

MYO7A

AD

1

3

2

2

1

1

Usher syndrome, type IF (USH1F)

PCDH15

AR

2

4

4

6

4

2

USH2A

AR

2

2

Usher syndrome, type IIC (USH2C)

ADGRV1

AR

1

1

1

2

1

1

Usher syndrome, type IIC (USH2C)

Usher syndrome, type IIA (USH2A)

GPR98

AR

1

3

1

4

0

5

0

6

0

0

3

3

3

Van der Woude syndrome 1 (VWS1)

IRF6

AD

3

3

3

Von Hippel–Lindau syndrome (VHL)

VHL

AD

19

25

21

30

15

Waardenburg syndrome, type 2A (WS2A)

MITF

AD

2

6

6

6

4

4

Wilson disease

ATP7B

AR

3

3

3

5

3

2

Wiskott–Aldrich syndrome (WAS)

WAS

XL

6

15

13

20

9

8

Wolfram syndrome 1 (WFS1)

WFS1

AR

1

2

1

1

1

1

Xeroderma pigmentosum, complementation group g (XPG)

ERCC5

AR

1

1

0

0

0

0

3463

5869

4683

7443

2644

2332

1.59

56.4%

TOTAL

14

118 Genetic Disorders and the Fetus

MI

MII

N

N N

NN CF

CF CF

N CF CF

CF Affected Crossover

MII

CF CF

MII CF

N

N

CFN

CFN N

N

CF

C-1

Affected C-2 CFN CF CF N

CF N

N

Unaffected

Unaffected

developmental potential of the resulting embryo, 343 biopsied and 445 nonbiopsied mouse embryos were compared for the percentage of embryos reaching the blastocyst stage.36 The results of PGT-M performed by polar body biopsy, representing the world’s largest series, is shown in Table 2.2. A total of 1,016 PGT-M cycles were performed, for 538 autosomal recessive, 191 autosomal dominant, and 287 X-linked disorders. Of 1,016 cycles initiated, 838 (82.5 percent)

Figure 2.1 Scheme demonstrating the principle of preimplantation genetic analysis by sequential DNA analysis of the first and second polar body, using the cystic fibrosis (CF) locus as an example. Source: Verlinsky Y, Kuliev AMA. Preimplantation genetic diagnosis. In: Milunsky A, Milunsky JM, eds. Genetic disorders and the fetus: diagnosis, prevention and treatment, 6th edn. Oxford, UK: John Wiley & Sons, 2010.

resulted in transfer of 1,656 embryos (1.98 embryos per transfer on the average), 349 (41.6 percent) clinical pregnancies, and 385 babies born. Only two misdiagnoses were observed in the case of PGT for fragile-X syndrome and muscular dystrophy, which were due to consented transfer of additional embryo with insufficient marker analysis to exclude the probability of allele dropout (ADO) (see later). The example of PGT-M by polar body sampling is shown in Figure 2.2.

Table 2.2 Clinical outcome of PGT-M performed by polar body approach. Conditions/mode of inheritance/ sampling type

Patient

Cycles

Embryo

No.

transfer

embryos

Spontaneous Pregnancy

abortions

Baby

Autosomal recessive Polar bodies

76

131

99

204

36

10

36

Polar bodies + blastomere/blastocyst

254

407

344

701

143

21

168

Subtotal

330

538

443

905

179

31

204

21

Autosomal dominant Polar bodies

29

52

40

84

22

4

Polar bodies + blastomere/blastocyst

79

139

122

233

49

7

61

108

191

162

317

71

11

82

20

Subtotal X-linked Polar bodies

39

86

63

110

22

4

Polar bodies + blastomere/blastocyst

116

201

170

324

77

12

79

Subtotal

155

287

233

434

99

16

99

Total

593

1016

838

1656

349 (41.6%)

58 (17%)

385

Preimplantation Genetic Testing 119

CHAPTER 2

I. 160 158 166 176 111 114 DEL N DOB 11/1984 DEL N DEL N DEL N 108 110 PGT 144 158

II.

III.

PGT

D16S521 D16S525 D16S3395 TSC2 - DELETION D16S664 D16S3082

1 2 TSC2 FET 1 FET 2 Sequential polar body analysis 1

3

5

6

7

8

9 10 11 12 13 14 15 16 17 18 10 AFFECTED 5 NORMAL

Oocyte genotype Trophectoderm analysis Trophectoderm karyotype

160 166 111 DEL DEL DEL DEL 108 144 M

160 166 111 DEL DEL DEL DEL 108 144 M

158 176 114 N N N N 110 158 N

5

160 166 111 DEL DEL DEL DEL 10S 144 M

158 176 114 N N N N 110 158 N

160 166 111 DEL DEL DEL DEL 10S 144 M

160 166 111 DEL DEL DEL DEL 10S 144 M

160 166 111 DEL DEL DEL DEL 10S 144 M

160 166 111 DEL DEL DEL DEL 10S 144 M

7

158 158 176 176 114 114 N N N N N N N N 110 110 158 158 N N

12

FA

160 166 111 DEL DEL DEL DEL 10S 144 M

158 176 114 N N N N 110 158 N

160 166 111 DEL DEL DEL DEL 110 144 MR

160 166 111 DEL DEL DEL DEL 10S 144 M

Embryo #7 46, XY FET-1

17 Embryo #12 46, XX FET-2

45, –14, XX 46,XY FET 1

46,XX FET 2

Figure 2.2 Preimplantation genetic testing for de novo tuberous sclerosis* type II deletion (TSC2 gene, exon 7–10 deletion 16p13.3) and preimplantation genetic testing for aneuploidies by next-generation sequencing (NGS). Of 15 oocytes tested by polar body analysis, ten were affected and five free of deletion. The embryos deriving from deletion-free oocytes were tested for aneuploidy by NGS; three of these were euploid (embryos 7, 12, and 17) and one (embryo 5) with monosomy 14. Two of the mutation-free euploid embryos (embryos 7 and 12, the NGS

Preimplantation genetic testing based on embryo biopsy

Despite clear advantages, the polar body approach does not provide diagnosis of the paternal alleles and the gender of the embryo and therefore cannot be used to avoid the transfer of male embryos in cases of the X-linked disorders, unless specific diagnosis can be achieved on oocytes using the polar body approach (see later). The fact that the genotype of the oocyte is inferred from the genotype of the polar body, rather than determined directly is another weakness. In these situations, embryo biopsy becomes the much more comprehensive approach, with blastocyst biopsy currently being a standard procedure. The first clinical application of embryo biopsy for PGT by Handyside et al.19 was performed at the cleavage stage for X-linked disorders by gender determination.19 The study of the viability

46,XX

results of which are shown bottom right) were transferred in a frozen cycles, resulting in a twin pregnancy and birth of two unaffected children free from deletion. *Tuberous sclerosis complex is an autosomal dominant multisystem disorder characterized by hamartomas in multiple organ systems, including the brain, skin, heart, kidneys, and lung. Central nervous system manifestations include epilepsy, learning difficulties, behavioral problems, and autism. The affected mother had had epilepsy since 3 months old and lympho-angio-leiomyomatosis.

of the biopsied pre-embryos did not reveal any detrimental effect of these procedures: it was shown that more than 70 percent of the manipulated embryos reached blastocyst stage, with no significant reductions in cell number and energy substance (glucose and pyruvate) uptake.37 Embryo biopsy, initially applied at the cleavage stage, has become a method of choice in most centers, resulting in the birth of thousands of children free of genetic disorders.21–25 Nevertheless, some programs have demonstrated significant detrimental effect of the procedure, especially when instead of one blastomere two were removed, or even a single blastomere removed in inexperienced hands (see later). Also, there were problems due to the high rate of allele dropout and the high frequency of mosaicism at this stage (see later). The switch to blastocyst biopsy largely solved these problems and became standard. In

120 Genetic Disorders and the Fetus

fact, blastocyst biopsy was first introduced in the 1960s in rabbits by Gardner and Edwards.38 As the number of cells in human blastocyst increases up to more than 100, a few cells (approximately five cells) are removed from IVF embryos without affecting viability. Another advantage of this approach is that trophectoderm cells are biopsied without affecting the inner cell mass, from which the embryo is derived. Initially the viability of biopsied blastocysts in vitro was studied using morphologic criteria and the patterns of human chorionic gonadotropin (hCG) secretion. Hatching was observed in 38.5 percent of blastocysts, with hCG detected first on day 8, peaking at day 10, and still detectable in some blastocysts at day 14.39 For the individual blastocysts, the pattern of hCG secretion correlated with the assessment of morphology.40 Blastocyst biopsy for clinical purposes was first attempted over 20 years ago41–43 and is currently a PGT standard.44, 45 This is also the method used in uterine lavage, which may soon appear as a realistic approach for PGT without IVF.46 Both mechanical44–46 and laser techniques were used for blastocyst biopsy, which has become a method of choice in most centers, also resulting in improved pregnancy rates, particularly in frozen PGT cycles. The advantage of blastocyst biopsy over cleavage-stage sampling was demonstrated by a well-designed randomized controlled study.47 The procedure is performed as follows: On day 5 of embryo development, when the blastocyst begins to herniate through the zona pellucida, several herniated trophectoderm cells are removed by smooth aspiration into a biopsy pipette with an internal diameter of 30 μm through the zona pellucida, which is opposite the inner cell mass. To break down the tight junctions between trophectoderm cells, three laser shots are applied (with a duration of 0.7 ms pulse for each shot). In the selection of embryos for blastocyst biopsy, poor-quality blastocysts and those with early stage herniation are avoided. It is also possible to perform blastocyst biopsy without the application of laser in order to avoid potential damage to the cells. The technique depends on the use of the force of surface tension on the boundary between the biopsy drop and the mineral oil culture medium, instead of laser pulses. The method can be applied only on grades

2 to 2–3 of the day 5 blastocysts, because the tight junction between trophectoderm cells of the early blastocyst (grades 2 and 2–3) is not as strong as the cell connection in the more advanced blastocyst (grades 3–4, 4, and higher). Grade 2 and 2–3 blastocysts are placed into 5 μL equilibrated culture media drops covered by 2 mL of equilibrated mineral oil, held by holding the pipette on the left side, while the biopsy pipette (interior diameter 25 μm) on the right side is used to suck 5–10 extruded trophectoderm cells into the pipette. The embryo is then moved onto the right edge of the biopsy drop with the biopsy pipette, moving slowly toward the inside of the oil environment, making a cytoplasm bridge between blastocyst and sample thinner, until it is finally removed and placed into a separate 5 μL culture media drop in the same dish. This procedure results in nonsticky trophectoderm samples with no evidence of damaged nuclei, with improved amplification efficiency.48 As the time for analysis is limited by the implantation window, which is less than 24 hours, the technique of vitrification of biopsied blastocysts is now routine, allowing sufficient time before transfer of the tested embryos in a subsequent freeze–thaw cycle. It appears that this approach has also improved implantation and pregnancy rates, which could also be explained by the better receptivity of the uterus in unstimulated cycles. The other advantage of the method is that the embryo has already passed the natural self-correction mechanisms, overcoming the natural errors of the cleavage stage, thus enabling the diagnosis of only persisting abnormalities. Prospects for noninvasive preimplantation genetic testing

Although there is no evidence of the detrimental effect of biopsy procedures on embryo viability, the potential for damage cannot be excluded, so the development of noninvasive preimplantation genetic testing (NIPGT) is of increasing importance. The fact that DNA fragments of approximately 150 bp are present in cell-free DNA of blastocoele fluid and spent culture medium, is the basis of the concept that NIPGT can be developed analogous to noninvasive prenatal testing (NIPT), because these DNA may originate

CHAPTER 2

from breakdown of nuclear DNA derived from cells damaged at biopsy or undergoing apoptosis during cell division. Available reports suggest that the technique may in future be applicable for preselection of euploid embryos for transfer.49–58 Although the usefulness of blastocoele fluid for PGT by different groups lacks consensus, its use has been proposed to identify at-risk embryos from younger patients who otherwise have no accessible indication for PGT-A.49, 50 The other approach, based on the use of cell-free DNA in culture media, represents the genuine noninvasive approach analogous to monitoring cell-free DNA in maternal plasma during pregnancy,51–58 with the concordance studies showing progress. In one of these studies, the test was offered to infertility patients presenting for PGT-A in the format of a clinical trial. Enough DNA for testing was detected in 88% of cases, with 80% concordance to biopsy results.55 In another prospective study, blastocoele samples and spent culture medium samples were compared to diagnostic biopsy samples that were processed for PGT-M and PGT-A. Overall results demonstrated that neither blastocoele samples nor spent medium were sufficiently robust approaches for aneuploidy or single-gene disorders and cannot be applied clinically until the risk of maternal contamination can be excluded. This risk is particularly high in spent culture medium samples due to maternal cumulus DNA contamination.56 In the other study DNA in spent medium was shown to be detectable on day 3, but more reproducibly on day 5, with concordances of 65 and 70 percent with biopsy samples, which are not high enough for practical application.57 Although the origin of the DNA in spent culture medium is not clear, it was postulated that results of the test may have a prognostic utility and better prediction of reproductive outcome if euploid embryos with euploid spent media results are transferred. This is in contrast to the euploid embryo transfer outcome with spent media showing imbalanced results.58 This is also in agreement with the blastocoele data, which suggested a significantly improved embryo transfer outcome for those euploid embryos whose blastocoele fluid has a higher quantity of DNA based on WGA.50 Thus, clearly more research is needed to validate the usefulness of spent culture medium and blastocoele fluid for PGT.

Preimplantation Genetic Testing 121

Preimplantation genetic analysis Initially, PGT was justified only for high-risk pregnancies. Maternal age was not expected to be an indication for such early testing, initially being considered even a contraindication to PGT. However, the development and improvement of the methods for sampling and genetic analysis have made use of PGT for chromosomal disorders a reality. Despite continuing discussion of the impact of PGT-A, it is used routinely worldwide as a tool in assisted reproduction technologies, especially for improving the effectiveness of IVF in poor-prognosis patients, particularly in carriers of chromosomal rearrangements. As a result, the majority of PGT cycles are still done for PGT-A.21–25 Single-gene disorders

DNA analysis for preconception and preimplantation diagnosis is well established, which enables genetic analysis of minute quantities of DNA obtained from a single or few cells.12, 19, 20 Because this also increases the chance of DNA contamination in PGT, decontamination procedures were applied in the initial stages, based on elimination of double-stranded DNA sequences,59 excluding also possible contamination with the embryology and PCR reagents, such as water, salt solutions, oligonucleotides, and Taq polymerase. The major source of contamination in PGT is still cellular contamination, such as cumulus cells, spermatozoa, or cell fragments, which might be amplified simultaneously with polar bodies or embryo biopsies, creating the possibility for erroneous testing. Because potential misdiagnosis of PGT may be caused by sperm DNA contamination, it is currently a routine IVF practice to perform PGT-M for single-gene defects following microsurgical fertilization by intracytoplasmic sperm injection (ICSI). Nevertheless, the major source of misdiagnosis is preferential amplification or allele-specific amplification, referred to as allele dropout (ADO), which may happen in single-cell genetic analysis. As much as 8 percent of ADO was observed in PCR analysis of the first polar bodies, reaching over 20 percent in blastomeres.60 False-negative diagnoses have been observed in PGT for X-linked disorders, myotonic dystrophy, and cystic fibrosis

122 Genetic Disorders and the Fetus

(CF), at the initial stage of PGT clinical application.3, 22, 23, 25, 35, 59 Clearly, the failure to detect one of the mutant alleles in compound heterozygous samples due to ADO will lead to misdiagnosis. However, this is no longer a problem with the application of protocols for simultaneous detection of the causative gene together with highly polymorphic markers closely linked to the gene tested.59 With simultaneous testing of a sufficient number of linked markers amplified together with the gene in question, the risk of misdiagnosis may be substantially reduced or even practically eliminated. The protocol involves a multiplex nested PCR analysis, with the initial first-round PCR reaction containing all the pairs of outside primers, followed by amplification of separate aliquots of the resulting PCR product with the inside primers specific for each site. Following the nested amplification, PCR products are analyzed by restriction digestion, real-time PCR, direct fragment size analysis, or mini-sequencing. Depending on the mutation being studied, different primer systems are designed with special emphasis on eliminating false priming to possible pseudogenes, for which purpose the first-round primers are designed to anneal to the regions of nonidentity with a pseudogene.25, 59 With the introduction of next-generation technologies and the use of WGA prior to DNA analysis, the risk of ADO is further increasing, presenting even more problems in achieving accurate diagnosis.61 To improve the reliability of the test, the use of multiple linked markers became even more important, with importance of not only excluding the presence of the mutant gene, but also confirming the presence of the normal allele(s). Although a sufficient number of informative closely linked markers are usually available for multiplex PCR, this might not be the case in performing PGT by conventional PCR analysis in some ethnic groups.59 Currently available protocols allow an accurate PGT for complex cases, requiring testing for two, three, and even more different mutations. PGT generally requires knowledge of sequence information for Mendelian diseases, but may also be performed when the exact mutation is unknown. With the expanded use of single nucleotide polymorphisms (SNPs), linkage analysis allows PGT for any monogenic disease, irrespective of the

availability of specific sequence information.59–64 This is a more universal approach to track the inheritance of the mutation without actual testing for the gene itself, such as in karyomapping.65 On the other hand, a specific diagnosis is required for X-linked disorders, which may be performed by polar body analysis to preselect the embryos deriving from mutation-free oocytes which may be transferred irrespective of gender or the paternal genetic contribution.66 Polar body analysis (see Table 2.2 and Figure 2.2) also provides the prospect of pre-embryonic diagnosis, which is required in many population groups where objection to the embryo biopsy procedures makes PGT nonapplicable. We performed the first pre-embryonic genetic diagnosis for Sandhoff disease in a couple with a religious objection to embryo destruction.67 Although pre-embryonic genetic diagnosis was previously attempted by first polar body testing,68–71 it is not actually sufficient for accurate genotype prediction without second polar body analysis, as shown in Figure 2.1. It is understood that for pre-embryonic testing the second polar body analysis should be done prior to pronuclei fusion (syngamy), to ensure that only zygotes originating from mutation-free oocytes are allowed to progress to embryo development and to be transferred, avoiding the formation and possible discard of any unaffected embryo. A particular challenge is also presented by PGT for mitochondrial diseases, which still cannot be done reliably. A novel approach has been made to transfer a nuclear genome from the pronuclear stage zygote of an affected woman to an enucleated donor zygote, or to transfer the metaphase II spindle from an unfertilized oocyte of an affected woman to an enucleated donor oocyte.72 As seen from Table 2.1, PGT is no longer restricted to conditions presented at birth; it is gradually expanding to include common diseases with genetic predisposition, such as cancers, performed in 10.5 percent of PGT-M cycles, or nongenetic indications (7.2 percent of cases), such as PGT-HLA with the purpose of stem cell therapy of affected siblings in the family.62 Here we discuss the application of PGT-M to a wider range of disorders, including conditions determined by de novo mutations (DNMs), genetic predisposition for late-onset disorders, and preimplantation HLA matching (Table 2.1).

CHAPTER 2

De novo mutations

PGT is presently applicable to couples who, although they may themselves be noncarriers of a mutation, have been found to have a DNM in their gonads although there is no family history of the genetic disease, or the disease is first diagnosed in one of the parents or their affected children (see Figure 2.2). As neither the origin nor relevant haplotypes may be available for tracing the inheritance of such mutations in single cells biopsied from embryos or in oocytes, the main emphasis is on the identification of the mutation and/or relevant haplotypes enabling mutation detection. Accordingly, PGT strategies for DNM depend on their origin. DNA analysis of the parents and affected children prior to PGT is required for verification of the mutation and polymorphic markers through single sperm testing and polar body analysis, thereby providing the normal and mutant haplotypes to trace the mutation. If the origin of the mutation is paternal, confirmation is first sought on the paternal DNA from blood and total sperm, and then by single sperm typing to determine the proportion of sperm with DNM and relevant normal and mutant haplotypes. It is also useful to test the relevant linked markers for the partner, to exclude misdiagnosis due to possible shared maternal and paternal markers. Where the origin of the mutation is maternal, polar body testing is the method of choice, providing the normal and mutant maternal haplotypes. Again to exclude misdiagnosis caused by possible shared paternal and maternal markers, the relevant paternal haplotypes are established through a single sperm typing. If the mutation was first detected in children, both the maternal and paternal haplotypes are established as described. The other important phenomenon detected in PGT for DNM is gonadal mosaicism, which can be detected in either parent. Although the strategies may differ depending on the type of DNM inheritance, the general approach involves the identification of DNM origin and search for a possible gonadal mosaicism and relevant parental haplotypes. Despite the complexity of PGT for DNM, these strategies may be applied in clinical practice with extremely high accuracy without the traditional requirement for family data, which is not always available. Since the report of our first systematic experience of PGT for 152 families with different

Preimplantation Genetic Testing 123

genetic disorders,73 we have performed 526 cycles from 283 couples for 81 different de novo conditions, resulting in 270 clinical pregnancies and the birth of 234 unaffected children, with no misdiagnosis.48 Late-onset disorders

PGT for late-onset disorders with genetic predisposition was first applied for a couple with inherited cancer predisposition, determined by p53 tumor suppressor gene mutations,74 which are known to determine a strong predisposition to many cancers. Traditionally, these conditions have not been considered as an indication for prenatal diagnosis that would lead to pregnancy termination, which is not justified on the basis of genetic predisposition. Rather, the possibility of choosing embryos free of genetic predisposition for transfer would obviate the need for considering pregnancy termination, as only potentially normal pregnancies are established. Although the application of PGT for these conditions is still controversial, it has been performed for an increasing number of disorders with genetic predisposition that present beyond early childhood and may not even occur in all cases, including inherited cancers and heart disease.6, 7, 25, 74–77 We have performed a total of 874 cycles for 56 different forms of cancers, the most frequent being breast cancer (284 cycles) caused by BRCA1 (159 cycles) and BRCA2 (125 cycles) mutations. A total of 199 PGT cycles for BRCA1/2 resulted in transfer of one or two embryos, yielding 131 pregnancies and birth of 134 children free from genes predisposing to breast cancer.48 The other largest group of cancers for which PGT was performed was neurofibromatosis type 1 and type 2 (NF1/2), for which 103 cycles resulted in transfer of 138 genetic predisposition-free embryos in 88 cycles, 53 clinical pregnancies, and 55 children born free from genes predisposing to neurofibromatosis. Other cancers representing frequent indications for PGT were different types of Fanconi anemia (83 cycles), colorectal cancer (52 cycles), tuberous sclerosis types 1 and 2 (44 cycles) (see Figure 2.2), familial adenomatous polyposis (42 cycles), multiple endocrine neoplasia (34 cycles), and retinoblastoma (31 cycles); under 30 PGT cycles were performed for various other cancers.

124 Genetic Disorders and the Fetus

Overall, 966 genetic predisposition-free embryos were transferred in 634 cycles, resulting in 387 (61.0%) clinical pregnancies and birth of 407 children free from the risk of developing these cancers.25, 48 The other emerging PGT-M indication has been inherited cardiac disease, for which 109 cycles were performed relating to 23 different diseases. The most frequent indications were familial hypertrophic cardiomyopathy, CMH4 (22 cycles), dilated cardiomyopathy, CMD1A (17 cycles), Holt–Oram syndrome, HOS (8 cycles), acyl-CoA dehydrogenase very-long-chain deficiency, ACADVLD (6 cycles), familial hypertrophic cardiomyopathy 1, CMH1 (6 cycles), long QT syndrome 1, LQT1 (6 cycles) and Noonan syndrome 1, NS1 (6 cycles); PGT for another 16 cardiac conditions were performed in five or less number of cycles. Overall, 123 embryos free of genes predisposing to cardiac disease were transferred in 89 cycles (1.38 embryos per transfer on the average), resulting in 55 clinical pregnancies (61.7 percent) and birth of 54 children free from inherited predisposition to these cardiac diseases. If not prevented, many of these conditions may manifest despite presymptomatic diagnosis and follow-up, with their first and only clinical occurrence being a premature or sudden death.78 The couples at risk for producing progeny with inherited cardiac disease usually request PGT prospectively, with no previous pregnancies attempted, given one of the partners being a carrier of the specific mutation. Many couples already going through IVF for fertility treatment may have questions about the implications of genetic susceptibility factors for offspring, and the appropriateness of using PGT in testing for susceptibility to inherited cardiac disease.25, 48 Of special interest are PGT indications for late-onset disorders with inherited predisposition to neurological disorders, including neurodegenerative conditions. A total of 960 PGT cycles were performed for these conditions, including 610 for intellectual disability, 210 for Huntington disease, 42 for different movement disorders, such as torsion or myoclonic dystonia, nine for Alzheimer disease, and nine for Prion disease. As many as 1,110 unaffected or genetic predisposition-free embryos were transferred in 738 cycles (1.5 embryos per transfer on average), yielding 412 clinical pregnancies and birth of 406 infants

unaffected or free of the genes predisposing to the above conditions.48 Thus, PGT provides a nontraditional option for patients who may wish to avoid the transmission of a mutant gene predisposing to late-onset disorders in their future children. Because such diseases that present beyond early childhood and even later may not be expressed in 100 percent of cases, the application of PGT for this group of disorders is still controversial. However, for diseases with no current prospect for treatment, PGT may still be offered as the only relief for at-risk couples. HLA typing

Preimplantation HLA typing is an attractive PGT indication. The first case of preimplantation HLA typing was performed in combination with PGT for Fanconi anemia complementation group C (FA-C), which resulted in a successful hematopoietic reconstitution in the affected sibling by transplantation of stem cells obtained from the HLA-matched offspring resulting from PGT.5 To improve access to the HLA-identical bone marrow transplantation in sporadic bone marrow failures, this approach was then applied with the sole purpose of ensuring the birth of an HLA-identical offspring, not involving PGT, which also resulted in radical treatment of a sibling with a sporadic Diamond–Blackfan anemia (DBA) by stem cell transplantation from an HLA-identical child born following preimplantation HLA typing.79 Preimplantation HLA typing has become one of the most useful indications for PGT, performed currently with or without testing for the causative gene.79–88 Despite the ethical issues involved,80 preimplantation HLA typing procedures have so far been performed in hundreds of cases with affected children requiring HLA-compatible stem cell transplantation, including thalassemia, Fanconi anemia, Wiskott–Aldrich syndrome, X-linked adrenoleukodystrophy, X-linked hyper-IgM syndrome, X-linked hypohidrotic ectodermal dysplasia with immune deficiency, X-linked chronic granulomatous disease, cancer syndromes, incontinentia pigmenti, leukemias, and inherited and sporadic forms of DBA.81–91 We applied PGT-HLA in 485 cycles, including HLA typing alone, or combined with PGT-M for 35 different conditions. Overall, 424 HLA-matched unaffected embryos were detected and transferred

CHAPTER 2

in 291 cycles, resulting in 125 clinical pregnancies and birth of 117 HLA-matched children, as potential donors for their siblings.25, 48, 92 Although the majority of cases were performed for thalassemia, this approach has a great life-saving potential for affected siblings with congenital immunodeficiency. We performed 135 PGT cycles for 18 different immunodeficiency conditions, resulting in the birth of 54 children free of immunodeficiency, stem cell donors for transplantation treatment of affected siblings, with a total cure, such as Fanconi anemia and hyper-IgM syndrome.48 Similar experience has been reported from other large series, such as from Istanbul: 626 PGT-HLA cycles for 312 couples were performed (122 HLA only and 504 with PGT-M), resulting in the birth of 128 thalassemia-free children. Stem cells of 66 of these children were used for cord blood or bone marrow transplantation, which resulted in successful bone marrow reconstitution in all but two of them (transplantation treatment of the remaining 57 sibling pending).93, 94 Chromosomal disorders

The theoretical rate of chromosomally abnormal embryos at fertilization is approximately 40 percent, taking into account both the rate of aneuploidies in oocytes and sperm and fertilization-related abnormalities.95, 96 Mouse data show that most aneuploidies, although compatible with cleavage, are lost during implantation.97, 98 Additional losses of chromosomally abnormal embryos occur after implantation, clinically recognized as spontaneous abortions, more than half of which are caused by chromosomal abnormalities. As a result of this selection against chromosomal abnormalities before and after implantation, only 0.65 percent of newborns have chromosomal disorders, many of which lead to serious disability and early death (see also Chapter 1).

Prevalence and origin of chromosomal errors A wide range in the frequency of chromosomal aneuploidy in human oocytes has been reported (17–70 percent), but most of these studies have been performed on poor-quality oocytes left over after the failure of IVF attempts. A high rate of hypohaploidy observed in oocytes was considered to be artificially induced by spreading techniques,

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so aneuploidy rate was calculated by doubling the number of hyperhaploid oocytes. This ignores chromatid malsegregation and/or chromosome lagging events, contradicting the results of the observation that the rate of hypohaploidy is higher than the rate of hyperhaploidy.99 Cytogenetic analysis of unfertilized oocytes was also improved by parthenogenetic activation of human oocytes.100 As mentioned, an attempt at noninvasive cytogenetic analysis of oocytes was undertaken in the early 1980s through visualization of the second polar body chromosomes by transplanting the polar body into a fertilized egg.33 The success rate of visualization of polar body chromosomes was then improved by different methods, demonstrating the practical implication of polar body analysis for chromosomal errors originating from maternal meiosis,101–103 in contrast to the report on the uselessness of the first polar body for this purpose.104 Various approaches were explored in the attempt to visualize the chromosomes of the first and second polar bodies, as well as of the biopsied blastomeres, including nuclear transfer, electrofusion, and chemical methods.102, 105, 106 However, the major progress in chromosome analysis of oocytes and embryos was achieved with introduction of the fluorescence in situ hybridization (FISH) technique,107–113 microarray technology, and NGS. Our meiosis data based on the analysis of 22,986 oocytes detected 9,812 aneuploid oocytes (46.8 percent), originating comparably from the first and second meiotic divisions. Overall, meiotic division errors were observed in 33.1 percent of oocytes in meiosis I, 38.1 percent in meiosis II, and 28.8 percent in both. Although the aneuploidy rate in embryos is comparable to that in oocytes, the types of anomalies in the oocytes and embryos were significantly different,114–116 also showing inconsistency between the expected and observed frequency of some types of aneuploidies. In our current practice of PGT-A, the analysis of 2,922 blastocysts, 56.0 percent of embryos were aneuploid, comprising 13.0 percent monosomy, 13.0 percent trisomy, 8.0 percent numerical mosaic, 14.0 percent segmental mosaic, and 8.0 percent complex errors.48 It is of interest that no age dependence was revealed for monosomies in embryos, suggesting that the rate of monosomies detected in embryo by PGT-A may be of artefactual nature.

126 Genetic Disorders and the Fetus

A possible explanation for this discordance is that the majority of monosomies detected in embryos are derived from mitotic errors, assuming technical causes are excluded. In fact, a significant proportion of the cleavage-stage monosomies appeared to be euploid after their reanalysis.117, 118 No monosomies, except monosomy 21, are detected after implantation, so either they are eliminated before implantation or have no biological significance, reflecting the poor viability of the monosomic embryos and their degenerative changes. In one relevant study the progression and survival of different types of chromosome abnormalities were followed up in 2,204 fertilized oocytes.119 A variety of chromosome abnormalities was detected, including many types of errors not recorded later in development. However, these appeared to be tolerated until activation of the embryonic genome, after which there were declines in frequency. Nevertheless, many aneuploid embryos still successfully reach the blastocyst stage, even if some chromosome errors present during preimplantation development are not seen in later pregnancy.

Aneuploidies As seen from the previous discussion, without the detection and avoidance of the transfer of chromosomally abnormal embryos, there is at least 50 percent chance of loss during implantation or postimplantation development. In addition to the clear benefit of avoiding the transfer of aneuploid embryos, which contributes to improvement of pregnancy outcome of poor-prognosis IVF patients, this should improve the overall standard of medical practice, upgrading the current selection of embryos by morphologic criteria into aneuploidy testing. This explains a widespread application of PGT-A aimed at the preselection of embryos with the highest developmental potential demonstrating a clinical benefit, in terms of the improved IVF outcome through improved implantation and pregnancy rates, reduction of spontaneous abortions and improved take-home baby rates in IVF patients of advanced reproductive age, repeated IVF failures, and recurrent spontaneous abortions.24, 25, 114–116, 120, 121 The failure to detect a positive effect of aneuploidy testing on reproductive outcome in a few studies may be due to possible methodologic deficiencies.122–124 Despite these methodological

shortcomings, which have been heavily criticized in the literature,125–127 the American Society for Reproductive Medicine Practice Committee did not favor transferring embryos without aneuploidy testing.128 This may mean the alternative of incidental transfer of chromosomally abnormal embryos, as every second oocyte or embryo obtained from poor-prognosis IVF patients is chromosomally abnormal and destined to be lost before or after implantation. In fact, only one in ten of chromosomally abnormal embryos may survive to recognized clinical pregnancy, 5 percent may survive to the second trimester, and only 0.5 percent reach birth. Thus, the majority of chromosomal abnormalities are eliminated before or during implantation, reflecting a poor implantation rate in poor-prognosis IVF patients, and explaining a high fetal loss rate in those patients without PGT-A. This has actually been demonstrated by testing products of conception from poor-prognosis non-PGT IVF patients, confirming the high prevalence of chromosomal aneuploidy in the absence of PGT-A. Of 273 cases tested, 64.8 percent had chromosomal abnormalities, up to 79 percent of which could have been detected and not transferred using PGT.129 Given these data, the current IVF practice of selecting embryos for transfer based on morphologic criteria may hardly be an acceptable procedure for poor-prognosis IVF patients. In addition to giving an extremely high risk of establishing an affected pregnancy from the onset, this will significantly compromise the very poor chances of these patients to become pregnant,130, 131 especially with the current tendency of limiting the number of transferred embryos to only a few or even one. Although culturing embryos to day 5 (blastocyst) before transfer may allow, to some extent, the preselection of developmentally more suitable embryos compared with day 3, some aneuploid embryos will still be capable of developing to the blastocyst stage.132, 133 These abnormal embryos will not be eliminated in the current shift to blastocyst transfer, and may implant and lead to fetal loss, compromising the outcome of pregnancies resulting from the implanted normal embryos in multiple pregnancies. In fact, multiple pregnancies represent a severe complication of IVF, which is currently avoided by preselection and transfer of a single aneuploidy-free blastocyst with

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the greatest developmental potential to result in a healthy pregnancy. However, contrary views also exist about safety, outcome, and efficacy.122–124, 134–137 Randomized controlled studies performed with introduction of next-generation technologies were able to quantify the clinical impact of preselection of aneuploidy-free zygotes, demonstrating the obvious benefit approximating a 15–20 percent increase in pregnancy rates, compared to embryos transferred solely based on morphological criteria, although this was not universal in all age groups. The first randomized controlled trial (RCT) using 24-chromosome analysis was performed in a series of 112 women randomized into two groups:138 transfer of a PGT-A embryo versus transfer of a morphologically normal embryo not biopsied or tested. Of 425 blastocysts tested, 45 percent (191/425) were with aneuploidy, resulting in a 71 percent pregnancy rate compared with 46 percent in the nonbiopsied control group of 389 blastocysts with normal morphology. In the other RCT, involving 72 cases, the transfer of euploid blastocysts resulted in 66 percent implantation and 85 percent delivery rates, compared to 48 percent and 68 percent, respectively, in the control group of 83 morphologically normal embryos but not tested for PGT-A.139 Another RCT did not find differences in pregnancy rates between single euploid embryo transfer and the transfer of two morphologically normal but untested embryos, but a 48 percent twin rate was observed in the latter compared to 0 percent in the single embryo transfer tested group.140 Significant differences between a PGT-A group and control groups were also demonstrated in an RCT performed using a cleavage-stage embryo biopsy.141 Thus, results of RCTs involving 24-chromosome platforms suggest that it is reasonable to inform assisted reproductive technology (ART) patients of advanced maternal age about the utility of PGT-A. The precise age range at which women would benefit is still under study, although the optimal outcome seems to be for the 35–39 age group, as suggested by trials conducted by the Society for Assisted Reproductive Technology (SART)142 and the STAR Study Group.143 The switch of aneuploidy testing from FISH to the next-generation technologies for 24-chromosome testing,144–163 allowing improved detection

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of chromosomally abnormal oocytes and embryos, has, therefore, further confirmed the positive impact of avoiding aneuploid embryos from transfer. In addition to testing all 24 chromosomes, the switch from blastomere sampling to blastocyst biopsy has also contributed to the positive reproductive PGT-A outcome, as only established anomalies are tested. With progress in vitrification procedures, blastocyst biopsy coupled with transfer after freezing in a subsequent cycle has become the major approach for PGT, as it also involves a much higher uterine receptivity than in stimulated cycles. Blastocyst biopsy has also improved PGT accuracy, because instead of using single cells, a number of cells are used for analysis. Blastocyst biopsy and vitrification, coupled with 24-chromosome testing, also simplified the organizational aspects of PGT, because the samples can be processed without the time limits for genetic analysis, also allowing samples to be shipped to specialized centers for more sophisticated testing, if required. Present standards of PGT-A are presented in Figure 2.3. As seen from Figure 2.3, the gold standard for PGT-A is presently NGS, which is also the initial step in PGT-M, also using WGA.160–163 Compared with other PGT-A methods, NGS provides more accurate copy number variations for each chromosome and is, therefore, better able to identify the presence of mosaic aneuploidy within the blastocyst. The detection of mosaicism requires much higher resolution than that provided by array comparative genomic hybridization (array-CGH). This is achieved by NGS, which has improved accuracy of testing, especially in detecting copy number variations that contribute to the mosaicism detection rate. Different labs use different cut-off rates, but PGDIS recommendations currently recommend a 20 percent cut-off: embryos are considered nonmosaic euploid if nonmodel DNA proportions are below 20 percent. Nonmodel DNA over 80 percent is considered nonmosaic aneuploid. Between 20 and 80 percent are considered mosaic.164, 165 Figure 2.4 presents an example of mosaicism detected by NGS. The applicable commercially available kit for NGS is VeriSeqTM PGT Kit (Illumina). Karyomapping supplied by Illumina may also be used for PGT-A, but requires different equipment and reagents than NGS. The primary application of karyomapping is PGT-M.65 The other alternative NGS platform

128 Genetic Disorders and the Fetus

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and cells still undergoing DNA replication, so the results per Figure 2.3 Present standards of preimplantation genetic analysis for aneuploidies (PGT-A). Twenty-four-chromosome embryo derive from proportion of normal (euploid) and aneuploidy testing by measurements of DNA content – not abnormal (aneuploid) DNA. number of cells. DNA content may include damaged cells

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Figure 2.4 Mosaicism for monosomy 3 detected by next-generation sequencing (NGS). NGS results show a 50 percent mosaicism for monosomy 3, with all other chromosomes showing a normal pattern.

is Personal Genome Machine (PGM), developed by Thermo-Fisher Scientific. The commercially available kit for this platform is Ion ReproSeqTM PGS Kit (Thermo-Fisher Scientific). The major concern with NGS is that it is prone to ADO, because WGA must be performed as a first step to generate an adequate amount of DNA for analysis, which, as mentioned earlier, is still extremely inefficient in recovering all genomic sequences. So although NGS allows concomitant PGT-A and PGT-M, without simultaneous testing of a sufficient number of linked markers false-negative results cannot be excluded, which may then lead to misdiagnosis, especially in PGT

for dominant diseases. It can therefore be predicted that the technique should be performed with the use of SNP analysis for this purpose, or to work out the level of deep sequencing that can overcome the problem of ADO or develop more efficient WGA.61, 159

Structural rearrangements The impact of PGT is even higher in translocation patients, with considerable reduction in the spontaneous abortion rate after preimplantation testing, resulting in a corresponding increase in the take-home baby rate.130, 131 Although previous experience with PGT for chromosome structural

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Figure 2.5 Next-generation sequence-based testing for translocation 46,XX, t(6;18)(p21.3;p11.2) (derivative chromosome indicated by red arrows).

rearrangements (PGT-SR) was based on the use of the FISH technique, which is still applicable in some specific cases, the current standard is the utilization of array-CGH or NGS technologies, which improves accuracy of testing and also allows a combined PGT-A. An example of PGT-SR by NGS is shown in Figure 2.5. In our experience of 940 PGT-SR cycles, the comparison of reproductive outcomes of 609 cycles performed by FISH and 331 performed by array-CGH and NGS showed significant improvement of the application of next-generation technologies, resulting in almost doubling pregnancy rate, from 38.8 percent in FISH cycles to 66.5 percent with application of next-generation technologies, and twofold reduction of spontaneous abortion rate, from 18.1 percent to 8.9 percent.48 A few sophisticated approaches based on nextgeneration technologies have been developed for distinguishing noncarrier balanced embryos from normal ones. One such technology involved the use of an SNP microarray.166, 167 However, this method requires the availability of a lot of the unbalanced embryo, as well as parental DNA necessary to serve as a reference for distinguishing balanced translocation from normal blastocysts. The more universal approach is a specially designed NGS technology called mate-pair sequencing (MPS). This involves high-depth MPS to identify breakpoint regions and Sanger sequencing to define the exact breakpoint

needed for designing specific primers required to identify normal and carrier embryos.168 A similar approach, termed nanopore long-read sequencing, also discriminates carrier from noncarrier embryos through high-resolution breakpoint mapping followed by breakpoint PCR.169 Thus, following application of breakpoint PCR, carrier embryos can be discriminated from noncarrier embryos. Both these approaches enable accurate high-resolution breakpoint mapping directly on balanced reciprocal translocation carriers, providing the option of transferring euploid noncarrier embryos. Thus, the current technologies not only insure an acceptable pregnancy outcome for carriers of structural rearrangement, but also enable avoidance of balanced offspring and continuation of the problem in the next generation. The presented data provide strong evidence that PGT is currently an important alternative to prenatal diagnosis, as it widens the options available for couples wishing to avoid the birth of an affected child, and provides the possibility of having children for those who would remain childless because of their objection to termination of pregnancy following prenatal diagnosis. At the same time, PGT is also becoming an integral part of assisted reproduction, by avoiding transfer of chromosomally abnormal and potentially nonviable embryos, thereby contributing to a significant increase in implantation and pregnancy rates in

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IVF, and to a general improvement in the standards of assisted reproduction practices.

Ethical and legal issues Considerations on ethical and legal issues are evolving, along with the evolution of the technology for the control of genetic diseases, and have become one of the key subjects in discussing the acceptability of preconception and preimplantation testing for genetic disorders. Ethical and legal issues determine, to a considerable extent, whether these new approaches are promoted to become an integral part of preventive genetics services or are waived on ethical grounds.8 PGT could be regarded as an ethically acceptable procedure in the context of a general objective of genetic service, which, according to the WHO, is to help genetically disadvantaged people live and reproduce as normally and as responsibly as possible.9 Because PGT is heavily based on IVF, it also is relevant that IVF is considered to be ethically acceptable in many countries.170–174 However, complex ethical and legal issues are confronted differently in various countries (see Chapter 36).175 For example, in Germany, the future of PGT depends on an Embryo Protection Law, which has been in effect since 1991.172, 176 This law is very strict and prohibitive of embryo research. However, it prohibits only destructive research that impairs the chances of the embryo becoming a human being up to the eight-cell stage. In fact, blastocyst biopsy may be possible without any conflict with the law because, together with chorionic villus sampling (CVS), such embryo biopsies are considered beneficial, allowing decisions to be made before replacement. Therefore, there is no conflict about the provision of PGT in Germany before the pronucleate stage, which is currently under way. However, this must be done for diagnostic purposes only, not for research. Even in the case of tripronucleate embryos, only observation is permitted, not experimentation. This approach may also resolve the ethical issues impeding PGT in Austria, Switzerland, Malta, and other predominantly Catholic countries.177 The same holds for other countries where no preventive measures have been allowed on religious grounds. For example, the law has evolved recently in Switzerland, citizens voting to introduce PGT

into clinical practice. In contrast, in France, there seems to be no law at all concerning either PGT or embryo research. However, the attitude of the National Ethical Committee toward PGT is influenced by the fact that the diagnosis is based on genetic analysis of only one or a few cells and that all male embryos are discarded after gender determination, while half of them are completely normal. However, the testing is presently based on specific diagnosis, rather than gender determination. Another concern is that PGT increases the need for IVF, which is provided free of charge in France. Finally, prenatal diagnosis, also provided free of charge, was enough to avoid genetic disorder, so the provision of PGT was considered to be an additional prenatal test, without taking into account the suffering caused by selective abortions after prenatal diagnosis. Nevertheless, there are presently a few well-established centers in France providing a full range of PGT services. In Italy, PGT and many aspects of IVF were forbidden by Act of Parliament for almost 7 years. Only three oocytes were allowed to be aspirated for fertilization in vitro, clearly reflecting the opinions of the hierarchy of the Roman Catholic Church.178 Prior to the law, Italy had been among the most active centers involved in the development and application of PGT for genetic and chromosomal disorders. After the laws were enacted, it was difficult to perform PGT, which nevertheless is presently performed without restriction. In some countries, such as Belgium, the decision on embryo research and PGT rests completely with institutional review boards, so there is no problem with the development of the technique and its implementation into clinical practice. In other countries, such as the Netherlands, PGT is governed by the law on medical experiments, which contains a section on embryo research. It prohibits “cloning,” but probably will not ban PGT research because it provides an alternative to prenatal diagnosis and abortion of genetically affected fetuses. In the United Kingdom, PGT, as well as the practice of IVF and research involving human embryos, is regulated through a statutory body, the Human Fertilisation and Embryology Authority, and the Fertilisation and Embryology Act (1990), allowing research on human embryos up to 14 days of development under an appropriate license. In Spain, although a 1988 law regulating

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human embryo research forbade the fertilization of human oocytes for any purpose other than human procreation, it permitted research on embryos within 14 days of preimplantation development under the supervision of the national health and scientific authorities.179 Therefore, this law did not conflict with the development of research in preimplantation genetics and its application to assisted reproduction practices. In fact, according to a survey on PGT availability in Europe, the number of PGT centers presently available in the country is more than in most of the other European countries. In the United States and Australia, the legal status of PGT and community attitudes differ in different states. For example, in the six states of Australia, only three have laws governing IVF and embryo research. In Victoria, embryo research is prohibited, except for approved experiments, although this law does not actually affect PGT because IVF is allowed for infertile couples, and PGT also can be justified as the procedure for avoiding the risk of transmitting genetic disease to affected children. In Western Australia, PGT cannot be done because of the Experimentation Law, whereas in South Australia it is possible unless destructive to an implantable human embryo. In the United States, the issue of embryo research is closely associated with the debates on abortion and cloning, and there has been no government system for regulating reproductive research projects. Because there is no ethical advisory board (EAB) that is legally given responsibility for reviewing such research proposals, federal funding for human embryo research has not been available. In addition, a wide variation of policy positions exists among different states, mainly being compromised over consideration of the question of when human life begins. However, despite existing differences in current legal restrictions in this field, selection of pre-embryos on genetic grounds may be ethically acceptable based on the premise that the goal of avoiding the birth of offspring with severe genetic handicaps is part of the constitutional rights of procreative liberty.180, 181 Although the National Institutes of Health Revitalization Act of 1993 lifted the requirement (45 CFR 46.204.d) for a federal-level EAB review for IVF research, leaving consideration for clinical research relating to IVF to individual institutional review boards, none of

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the federal funds may be used for research in which a human embryo or embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than allowed for research on fetuses in utero. In Canada, recent legislation to regulate assisted human reproduction technologies has been introduced, entitled an “Act Respecting Assisted Human Reproduction,” which allows PGT for medical reasons but excludes identifying the sex of an embryo for social purposes.182, 183 The Society of Obstetricians and Gynaecologists of Canada have provided valuable guidelines that optimize obstetrical management and counseling for prospective parents undergoing IVF, integral to PGT. Emphasis is given to the increasing evidence that both infertility and subfertility remain as independent risk factors for subsequent complications and adverse perinatal outcomes, even without IVF. Their report also draws attention to the very low but actual risk of imprinting disorders, such as Beckwith–Wiedemann syndrome or Angelman syndrome, estimated to occur in fewer than one in 5,000 patients. Important ethical issues have recently been raised with increasing use of PGT for gender determination for social reasons,184, 185 late-onset disorders with genetic predisposition,6, 7, 186, 187 and PGT-HLA to produce an HLA-compatible donor to treat a family member with fatal bone marrow disease or cancer requiring a stem cell transplantation.5, 188, 189 Although there is no actual difference in the application of PGT for the latter conditions, the controversy can be explained by the fact that in traditional prenatal diagnosis, if the fetus was found to carry the gene predisposing to a late-onset disorder or to be HLA unmatched, a couple would have to make an extremely difficult decision about pregnancy termination, which could hardly be justified by such a finding. Alternatively, PGT technology allows genetic testing of human eggs and embryos before pregnancy is established, making it totally realistic to establish only HLA-matched or potentially normal pregnancies without genetic predisposition to late-onset disorders. Notwithstanding the foregoing considerations, PGT has become an established clinical option in reproductive medicine and is applied using separate consent forms and research protocols approved

132 Genetic Disorders and the Fetus

by institutional ethics committees. Despite recent controversy regarding the quantitation of the impact of PGT-A on clinical outcome, thousands of cases have been performed, resulting in the birth of thousands of healthy children born after PGT-A. However, these protocols still require confirmatory CVS or amniocentesis and follow-up monitoring of their safety and accuracy. Although PGT will help solve some of the longstanding ethical problems, such as the abortion issue (which could largely be avoided as a result of this approach), other issues could become a serious obstacle, particularly those related to “designer babies.” These considerations are highly relevant to the subject of PGT, as well as to any other new methods as we proceed with further development of appropriate technology for avoiding genetic disability.

Conclusion Although the introduction of first-trimester prenatal diagnosis by CVS has considerably improved the possibility of avoiding genetic diseases, selective abortion is an issue in the case of an affected fetus. PGT has been initiated to provide the option of avoiding the birth of an affected child without the need for abortion as an obligatory component in the prevention program. This chapter describes these important developments with the emphasis on addressing the problems of implementation of PGT into clinical practice. Currently, PGT has been applied clinically for up to 600 different conditions, with thousands of

unaffected children born after PGT performed for single-gene and chromosomal disorders. Among the approaches to PGT introduced, blastocyst biopsy is now a standard. This became possible due to the progress in micromanipulation and biopsy and genetic analysis of single cells or small number of cells by PCR and currently by next-generation technologies. The available experience has already demonstrated the practical utility of PGT, and the reliability and safety of this relatively new technology in assisted reproduction. The indications for PGT have been expanded beyond those used in prenatal diagnosis to include couples at high risk of having a child with a genetic disorder (in the face of antipathy toward elective abortion), poor-prognosis IVF patients, couples at risk for producing offspring with late-onset genetic disorders, and preimplantation HLA matching. Because of the high prevalence of chromosomal abnormalities in early pregnancy, the introduction of PGT-A will not only make it possible to avoid the risk of age-related aneuploidies, but will also considerably improve the embryo recovery and pregnancy outcome following PGT and should improve the effectiveness of IVF programs in general. Introduction of NGS-based PGT-A, which uses WGA as the first step of the technique, also makes it possible to perform PGT-M with or without PGT-HLA in the same biopsy material. Concomitant PGT-A with PGT-M, PGT-SR, and PGT-HLA are thus becoming standard procedures toward comprehensive PGT for genetic and chromosomal disorders.

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176. Schreiber HL. The legal situation regarding assisted reproduction in Germany. Reprod Biomed Online 2003;6:8. 177. Feichtinger W. Preimplantation diagnosis – a European clinician’s point of view. J Assist Reprod Genet 2004;21:15. 178. Benagiano P, Gianaroli L. The new Italian IVF legislation. Reprod Biomed Online 2004;9:117. 179. Peinado JA, Russell SE. The Spanish law governing assisted reproduction techniques: a summary. Hum Reprod 1990;5:634. 180. Robertson J. Extending preimplantation genetic diagnosis: the ethical debate. Ethical issues in new uses of preimplantation genetic diagnosis. Hum Reprod 2003;18:465. 181. Marshal E. Embryologists dismayed by sanctions against geneticist. Science 1997;275:472. 182. Gali RP, Woodside JL. Proposed Canadian legislation to regulate reproductive technologies and related research. Reprod Biomed Online 2003;6:114. 183. Society of Obstetricians and Gynaecologists of Canada, Okun N, Sierra S. Pregnancy outcomes after

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assisted human reproduction. J Obstet Gynaecol Can 2014;36:64. Kilani Z, Haj Hassan L. Sex selection and preimplantation genetic diagnosis at the Farah Hospital. Reprod Biomed Online 2002;4:8. Malpani A, Malpani A, Modi D. The use of preimplantation genetic diagnosis in sex selection for family balancing in India. Reprod Biomed Online 2001;4:16. Ethics Committee of the American Society for Reproductive Medicine. Use of preimplantation genetic diagnosis for serious adult onset conditions: a committee opinion. Fertil Steril 2013;100:54. Edwards RG, Angastiniotis M, Antinoty S, et al. Ethics of preimplantation diagnosis: record from the Fourth International Symposiumon Preimplantation Genetics. Reprod Biomed 2003;6:170. Towner D, Loewy RS. Ethics of preimplantation diagnosis for a woman destined to develop early-onset Alzheimer disease. JAMA 2002;287:1038. Damewood MD. Ethical implications of a new application of preimplantation diagnosis. JAMA 2001;285:3143.

3

Amniotic Fluid Constituents, Cell Culture, and Neural Tube Defects Daniel L. Van Dyke 1 and Aubrey Milunsky 2,3 1

Mayo Medical School and Mayo Clinic Cytogenetics Laboratory, Rochester, MN, USA Center for Human Genetics, Cambridge, MA, USA 3 Tufts University School of Medicine, Boston, MA, USA 2

Introduction Amniotic fluid (AF) represents a constantly changing environment that simultaneously reflects and contributes to fetal development. Constituents include growth-promoting and growth-protective factors, and sufficient AF volume (AFV) provides mechanical cushioning and space for fetal movement. Biochemical and molecular components may also reflect fetal disease and maturity and, on occasion, maternal disease or environmental exposures. Analysis of the chemical constituents of AF has yielded valuable information for prenatal diagnosis, allowing assessment of fetal physiology and metabolism. Because the AF can be viewed as an extension of the fetal extracellular space,1, 2 an understanding of its origin, formation, and chemical constitution is crucial to prenatal diagnosis and fetal therapy. Sampling of extracoelomic fluid and AF during the 8th–16th weeks of pregnancy for the purpose of prenatal diagnosis has added valuable knowledge about the origin, formation, and content of AF.

Amniotic fluid Formation and circulation

Fluid exchange between the fetus and the mother occurs via several routes and through different mechanisms, and varies throughout pregnancy. Large volumes of fluid are transferred across the

fetal membranes, which are made up of five layers of amnion and four layers of chorion.3 Electron microscopy of the amnion has revealed a complex system of tiny intracellular canals that are connected to the intercellular canalicular system and the base of the cell.4 Studies in primates suggest that the AF is a transudate of the maternal plasma and becomes like other fetal fluids in the presence of the fetus, which contributes urine and other body secretions to the AF.5 Osmotic or diffusion permeability, hydrostatic pressure, chemical gradients, and other mechanisms are responsible for the fluid exchange between fetus and mother.6 In normal pregnancies, intra-amniotic pressure at 16 weeks ranges between 1 and 14 mmHg.7 Fisk et al.8 studied AF pressure from 7 to 38 weeks and found that it increased with gestational age and may be determined by anatomic and hormonal influences or gravid uterine musculature, but was not influenced by the deepest vertical pool, AF index, maternal age, parity, gravity, fetal sex, twinning, or time of delivery. These authors suggested that AF pressure did not change significantly after removing fluid samples in early or late amniocentesis.9 During the second trimester, total AF turnover is complete within about 3 hours.10 About 20 mL of AF/hour is swallowed by the fetus; that is, approximately 500 mL/day.11 At term, the exchange rate between fetus and mother may approach 500 mL/h.10, 12

Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, Eighth Edition. Edited by Aubrey Milunsky and Jeff M. Milunsky. © 2021 Aubrey Milunsky and Jeff M. Milunsky. Published 2021 by John Wiley & Sons Ltd.

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Although the fetus depends largely on the placenta for nutrient transport, it is also protected from marked fluctuations in maternal metabolism. The increase of creatinine, 𝛼-glutamyl transferase, and 𝛽 2 -microglobulin concentrations in AF after 10 weeks confirms the maturation of fetal glomerular function and reflects the fetal kidney development from the mesonephros to the metanephros.13 Active renal function is evident from the ability of the fetal kidney to concentrate radiopaque substances given intravenously to the mother, thereby allowing visualization of a fetal pyelogram.14 AF is mainly produced by the fetal kidney as pregnancy progresses and oligohydramnios may reflect renal structural anomalies, impaired swallowing, placental pathology, or general growth restriction.15 Volume

Brace16 described three determinants of AFV: (i) movement of water and solutes across the membranes; (ii) physiologic regulation of flow rates, such as fetal urine production and swallowing; and (iii) maternal effects on transplacental fluid movement. Total water accumulation in utero during pregnancy reaches about 4 L (fetus 2,800 mL; placenta 400 mL; AF 800 mL).8 Urine production per kg of body weight increases from 110 mL/kg/24 hour at 25 weeks to 190 mL/kg/24 hour at 35 weeks.17 Interference with disposal in the routes of fluid production by a factor affecting only 1 percent of the volume may increase or decrease total AFV by as much as 1 L in 10 days. AF turnover continues even after fetal death, but it is reduced by about 50 percent,18 implying that membranes may be responsible for about half of the water exchange. This suggests that the membranes play a larger role in water disposal than in production. Indeed, electron microscopic studies19 correlate with an absorptive function of the membranes. It is unlikely that excess AF production results solely from excess urine production or a failure of the fetus to swallow AF.20 The amnion must play a role in the maintenance of AFV and composition. Earlier studies concluded that 25–50 percent of the fluid turnover takes place through the fetus in late pregnancy.21 Abramovich22 challenged the concept that swallowing and voiding are important in controlling the AFV. He showed that some anencephalics may swallow considerable amounts of AF

and that normal volumes were found in esophageal atresia and in the absence of fetal kidneys. Thus, other factors are involved in controlling the AFV. Chamberlain23 has reviewed the studies done on abnormalities of AFV and altered perinatal outcome. Ultrasonic assessment of fetal kidney function in normal and complicated pregnancies revealed that the fetal urinary production rate was 2.2 mL/h at 22 weeks, increasing to 26.3 mL/h at 40 weeks.24 The authors concluded that regulation by the central nervous system does not play a large role in fetal urination control, and that fetal polyuria does not explain polyhydramnios. Polyhydramnios was accompanied by elevated AF pressures.25 Various techniques have been used for the direct estimation of AFV. Comparable results have been reported using dilution techniques, radioactive materials, or various dyes or chemicals.26–33 Abnormal AFV is associated with increased maternal risk and perinatal morbidity and mortality, but the invasive nature of AFV assessment limited its clinical utility.34 The vertical pocket measurement (VPM) is simple but remains semiquantitative with limited accuracy. The AF Index (AFI) is the result of the sum of the four maximum vertical pockets (MVP) from each quadrant of the uterus. A meta-analysis concluded that both AFI and VPM identified abnormal AFVs poorly. The AFI led to more false-positive oligohydramnios findings, and more interventions without improvement in perinatal outcome.35 Population differences for the AFI may also exist.36 Sandlin et al.37 established reference ranges for AFV from 16 to 41 weeks, using dye-dilution techniques and a quantile regression statistical approach (Table 3.1). Refinements in quantifying the noninvasive sonographic assessment of AFV have not significantly improved the predictive ability to identify at-risk pregnancies.38, 39 Polyhydramnios occurs in 1–2 percent of all pregnancies35 and is associated with fetal malformations in about 40 percent of cases.40 Moise defined polyhydramnios in singleton or twin pregnancies as MVP >8 cm in the late second and the third trimesters, and oligohydramnios as MVP 200,000 cells/mL at 16 weeks.5 The number of colony-forming cells is much lower.

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Culture methods The principal difference between the flask and in situ methods resides in the trypsinization step required for a suspension-type harvest. The resulting metaphase spreads can no longer be traced back to an individual parental colony. In contrast, the in situ method leaves colonies intact. When large numbers of cells are required for biochemical or molecular diagnostic studies, culture in T25 flasks remains the method of choice.

10 > 20 CPD 1.5 ± 1.9 n = 20

Frequency

5

3.5 ± 1.8 n = 20

5

0

–1

–2 –3 –4 –5 –6 Clones per mL fluid

–7

–8

Figure 3.9 Cloning efficiency of 20 consecutive amniotic fluid specimens (18 weeks gestational age). Fewer than half of the colonies grew to more than 106 cells per clone (more than 20 cumulative population doublings (CPD)).

Figure 3.9 shows that in platings of 16- to 18-week fluids, an average of 3.5 clones/mL are typically scored at day 12. Only 1.5 colonies per mL reach a clone size of at least 106 cells. Other laboratories report similar values.603 In a series of 14- to 16-week amniocentesis specimens, Hoehn et al.604 observed 3.1 colonies per mL but most were large colonies at day 12. Kennerknecht et al.605 reported high clone counts in 7- to 9-week AF, ranging from 7.9 to 12.2 colonies per mL. Late pregnancy fluids show cloning efficiency of less than 1.5 colonies per mL. Since human AF contains traces of growth and attachment factors such as epidermal growth factor, interleukin-1, tumor necrosis factor 𝛼, fibronectin, and endothelin-1,606 a 1 : 1 mixture of native fluid and growth medium has been recommended.607 Cell growth inhibitors (e.g. IGFBP-1, an insulin-like growth factor binding protein) have also been found in human AF.608 Although the proportion of erythrocytes may vary from 103 to 108 cells/mL, only the most severe blood contamination significantly retards or prevents cell growth. Such specimens can be treated before culture with 0.7 percent sodium citrate hypotonic solution or ammonium chloride lysing reagent (BD Biosciences).

In situ procedure Since the early 1980s, in situ culture and harvest has become the preferred method for cytogenetic studies.609–614 The main advantages are: (i) earlier culture harvest leading to a faster diagnosis, (ii) clonal (or, more correctly, colony) analysis leading to an easier distinction between genuine mosaicism and pseudomosaicism, and (iii) recognition of maternal cell contamination on the basis of clonal morphology. Maternal cell contamination (MCC) occurs in up to 0.5 percent of AFC cultures.615–617 To minimize MCC, some laboratories prefer to discard the first 2 mL of AF. PCR-detectable MCC of AF samples has been described as common (4–17 percent of samples) and probably represents contamination by maternal blood. This contamination can be an important consideration for biochemical or molecular genetic studies.618, 619 However, our local experience is that none of 66 direct AF samples have exhibited variable number of tandem repeat (VNTR)-detectable MCC. This is consistent with the 0.5 percent rate of MCC in AFC cultures identified by PCR analysis by Smith et al.620 This is also consistent with our local experience of finding MCC in 21 of 5,108 (0.41 percent) consecutive AFC karyotype studies (i.e. one or two 46,XX colonies among 15 or more 46,XY colonies in the in situ harvests). MCC rarely confounds the interpretation of cytogenetic results. Guidelines and tables are available detailing the number of metaphases to analyze by either suspension or in situ harvests, to exclude mosaicism at a desired confidence level.603, 621–624 A deficit of these calculations is that they ignore artifactual loss of chromosomes, which is more frequent with suspension than with in situ preparations. Environmental conditions (e.g. relative humidity and temperature) during drying of chromosome

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spreads can influence chromosome spreading, and artifactual aneuploidy is well documented.625, 626 Spurbeck has video-documented the effects of temperature and humidity on metaphase cell spreading.627 To search for mosaicism, the number of colonies sampled is more informative than the number of metaphases analyzed.628, 629 Whether the gold standard should be a 15-colony analysis has been the subject of some debate.630, 631 Guidelines issued by the American College of Medical Genetics632 recommend for the flask technique counting 20 cells from at least two independently established cultures, analyzing five and karyotyping two. For the in situ method, counting a minimum of 15 cells from at least 15 colonies in at least two independently established cultures was recommended. Laboratories using the in situ technique in conjunction with optimal culture media (e.g. Chang or AmnioMAX from Irvine Scientific or GIBCO, respectively) are able to karyotype most specimens in less than two weeks. Longer time intervals result from suboptimal cell growth conditions, adherence to a 5-day work week, or from other administrative rather than biologic limitations. Many laboratories also employ a robotic harvesting system and an environmental control chamber to improve the number and quality of metaphase cells.625, 633, 634 A typical AFC culture protocol was published by Miron in 2012.633 Automated harvesting for in situ chromosome analysis usually employs a Tecan or Scinomix Sci-Prep robotic sample processor,635 which saves personnel time and improves consistency because the timing, rate, and quantity of aspiration and dispensing of media, hypotonic solution, and fixative are automated. An environmental room or chamber that controls temperature, humidity, and airflow is helpful to both optimize the quality of the metaphase spreading and reduce seasonal variations in harvest quality. The system must be optimized in each laboratory but generally provides high-quality preparations in the range of 35–45 percent relative humidity at 25∘ C.625

examined whether maternal activity or uterine agitation before amniocentesis affect the concentration of viable amniocytes in the fluid.636, 637 After correcting for gestational age, both studies were negative. Enrichment of the cell culture innoculum has been attempted via centrifugation of fluids through isopycnic gradients.638 With bloody specimens, these methods might be of some help but they are impractical for routine use. This limitation also holds for an enrichment technique by which the AF is returned to the fetus after aspiration, filtration, and reinjection of AF as early as 12.5 gestational weeks.639–642

Enhancement of amniotic fluid cell growth

Reduction of oxygen supply Other efforts directed at improving the cellular microenvironment take into account that atmospheric oxygen conditions are not optimal for most mammalian cells in culture. Brackertz et al.646 and Held and Sönnichsen647 demonstrated improved

Enrichment techniques Since cloning efficiency is low, it would be advantageous to increase the number of viable cells introduced into the cell cultures. Two studies have

Growth on extracellular matrix surface The culture surface has a definite influence on the rate of attachment and proliferation. To attach to the culture surface, AFCs must create their own microenvironment, consisting of glycoproteins, collagen, laminin, and fibronectin, among others (ECM proteins). Fetal bovine serum contains fibronectin and so does human AF at 15–18 weeks of gestation.643 If serum concentrations of less than 10 percent are used (such as in Chang-type media supplements), the presence of AF in the culture setup might facilitate the coating of fibronectin on plastic surfaces. Chang and Jones607 reported optimal cloning and growth when cultures were initiated with equal parts of AF and growth factor-supplemented medium (including 4 percent fetal bovine serum). Two other studies have shown that precoating the plastic surfaces with ECM improves both cloning and rate of growth of AFCs.644, 645 In both laboratories, ECM-coated dishes were custom made from bovine corneal endothelial cells. The use of such precoated surfaces may be advantageous for cell attachment and cloning if suboptimal media have to be used. It appears impractical for routine use unless the laboratory is prepared to accept the extra expense involved in purchasing precoated dishes. A number of manufacturers offer such “biologic” plastic ware.

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AFC growth under hypoxic conditions. If multigas incubators are opened frequently, however, their humidity tends to decrease, which could disadvantage cells growing in open dishes. Testing and handling fetal bovine serum

The single most important factor in determining the speed and success of prenatal cytogenetic diagnosis is the quality of the growth medium and its supplements.557, 607 Since the traditional medium supplement, fetal bovine serum, represents a complex mixture of growth-promoting substances, considerable effort has been directed toward formulating serum-free media in mammalian cell culture.648 Human AFC culture has benefited greatly from the success of these efforts.649, 650 Fetal bovine serum or Chang-type medium, which includes serum, requires proper storage and handling to preserve its effectiveness. Freeze–thaw cycles and exposure to light are particular problems.651 Defined growth factor supplements

The commercial versions of the growth factorsupplemented media are based on the formulation provided by Chang et al.652, 653 The classic “Chang medium” included transferrin, selenium, insulin, tri-iodothyronine, glucagon, fibroblast growth factor, hydrocortisone, testosterone, estradiol, and progesterone. These factors are added to a 1 : 1 mixture of Dulbecco’s modified Eagles’ medium (DMEM) and Ham’s F12 medium, plus sodium bicarbonate, and small amounts of HEPES buffer and antibiotics. Chang et al. pointed out that their preferred basic medium mixture can be replaced by a number of other formulae (e.g. Ham’s F10 or F12, Coon’s modified Ham’s F12, McCoy’s 5A, RPMI 1640, DMEM, minimal essential medium and TC 199) without detriment. Chang and AmnioMAX media that differ in their buffering systems are available for use in closed or open cell culture systems. As with other aspects of the cell culture art and science, local preferences vary with respect to choice of specialized media, fetal or newborn bovine serum, and whether to mix these media with less costly media.609, 633, 634, 653, 654 It is assumed that the various peptides, hormones, and trace elements act synergistically on the recruitment of cells into the cycle and keep

them from reverting to the G0 stage after completed division. A greater number of cells within a colony will therefore stay in the proliferative pool. The cycle time of individual cells, with the possible exception of the duration of the G1 phase, is not likely to change. Unless Claussen’s micropipette method655 is used, a culture period of 5–7 days will thus remain the minimum time requirement for prenatal cytogenetic diagnosis employing AFC cultures. In our laboratory, we experimented with 12-hour Colcemid exposure and very early harvests. We obtained a small number of metaphase cells after 3 and even 2 days in cell culture but the number of metaphases was insufficient for a complete analysis. A drawback noted by some users of Chang and AmnioMAX media – other than the expense – is their limited shelf-life. Lyophilized or other, more stable media supplements are offered by some manufacturers (e.g. Condimed, UltroSer) but cloning efficiency testing so far has failed to identify a commercial product that consistently yields higher cloning efficiencies than fresh lots of Chang media.604 Use of Chang-type media may augment the incidence of chromosome breakage and chromosomal mosaicism in AFC cultures but rarely to the extent that the cytogenetic interpretation is compromised.656–659 This may in part result from the fact that Chang media can facilitate the growth of E-type colonies and these colonies yield higher rates of random chromosome changes.588, 611 However, the advantages gained by the reduction of turnaround time and the substantial decrease of culture failures using Chang-type media appear to outweigh the potential drawbacks of increased chromosomal breakage and pseudomosaicism. Culture failure

For most laboratories today, reports are completed in 6–14 days with a mean of 7–11 days to the final report, and the rate of culture failure is below 1 percent, depending somewhat on the timing of the amniocentesis,660 and in many laboratories averages closer to 0.1–0.2 percent (van Dyke, unpublished data). There are multiple reasons for cell culture problems and outright failure.661 With the increased experience of the obstetrician and the universal use of high-resolution ultrasound, maternal urine is now rarely received as an AF sample. Anecdotal evidence of some labs suggests

170 Genetic Disorders and the Fetus

that the risk of culture failure is higher in cases of fetal aneuploidy. In one published retrospective study, 56 (0.7 percent) of 7,872 AF samples did not grow.662 Twenty-four of these were judged technically inadequate and 10 were from women whose fetuses had died. Of the remaining 32 cases, 4 had proven (determined by repeat amniocentesis) and 4 had possible (extrapolated from fetal phenotype) aneuploidy. This 25 percent rate of growth failure associated with proven or likely chromosomal aberrations was not confirmed in a similar study comprising 6,369 cases and a growth failure rate of 1.2 percent.663 A study of 14,615 cases identified a higher incidence of culture failure in advanced pregnancies with abnormal ultrasound findings but no association with aneuploidy.664 In addition to a baseline level of less than 1 percent unexplained culture failure (the standard set by the American College of Medical Genetics is 2 percent),623, 624, 665 a number of known hazards can interfere with cell growth.

Syringe toxicity and delayed transportation One serious hazard is transmittal of AF in toxic syringes or tubes.666, 667 AF samples should not be transported in syringes; rather, the fluid should always be promptly transferred and transported in conical centrifuge tubes with plastic caps, spinal tap tubes or similar specimen transport containers. Rubber-capped tubes and stoppered syringes should not be used as storage or transport containers for AF. Problems reported in the United States prompted one manufacturer to recommend minimizing both the time of AF in the syringe and contact with the stopper attached to the plunger rod. Although it is advisable to deliver AF samples to the laboratory without delay, in our experience with AF specimens transported by courier and various delivery services, cell viability is maintained for at least 5 days, assuming the sample is not exposed to extreme temperatures. There is one report of successful cell cultures after unfortunate delays of more than 2 weeks.668 Microbial contamination Microbial contamination is a rare cause of culture failure in experienced laboratories and is largely preventable. As noted earlier, AF itself has bacteriocidal properties. If overwhelming microbial

contamination is apparent within 24 hours after setup, it is probably due to improper handling of the specimen between amniocentesis and delivery to the laboratory (e.g. leakage from loose screw caps or poorly packaged syringes). Approximately 10–20 percent of all samples are cell rich and their turbidity should not be a source of anxiety with regard to possible contamination. This also holds for brownish fluids containing cellular debris and granules in addition to erythrocytes. Seguin and Palmer669 measured cell growth from clear, cloudy (cell-rich), bloody, and dark brown fluids. They showed that cloudy fluids yield better growth than clear ones. They confirmed earlier observations670 that very bloody fluids adversely affect the cloning efficiency. If bacterial or yeast contamination arises during the course of cell culture, it is by no means hopeless to attempt to salvage such cultures. Penicillin-, streptomycin-, or fungicide-supplemented media are used to feed cultures daily after initial frequent washings. Increased chromosomal breakage rates and elevated rates of pseudomosaicism may be observed in such salvaged cultures but if the metaphase cells are analyzable, this rarely interferes with interpretation of the results.

Mycoplasma Mycoplasma is not a significant problem in AFC culture, due mainly to better quality control by the serum manufacturers but also to the awareness of cell culturists that AFC cultures should not share incubator space with established cell lines. A shared water bath used for heating media and trypsin can be a source of mycoplasma contamination because permanent cell lines, frequently shipped from laboratory to laboratory, remain the prevailing source of mycoplasma contamination. As additional protection, many laboratories heat-inactivate their sera before use. Commercial test kits are available for the detection of mycoplasma infections in cell cultures.671 Plastic ware and media storage There have been occasional batches of cell culture-grade plastic that do not, or barely, support cell attachment and growth. As with any component of the cell culture system, it is advisable to test new and old plastic ware in parallel for toxicity and ability to support growth in vitro.

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Hoehn’s laboratory switched several times between Corning and Falcon and tested additional brands because of considerable fluctuations in quality.604

Incubator failure Incubator failure is not a trivial cause of culture loss. The main threats are breakdown of the gas supply or equipment. AF cells cannot tolerate a pH close to or higher than 8 for more than 6–8 hours. On the other hand, a pH of less than 7.0 (for example, due to excess CO2 in the incubator) causes cells to stop dividing. A second danger is overheating of incubators due to mechanical failure or human error. Connection of incubators to emergency power sources is important. Temperature- and gas-sensitive alarm systems are advisable. Record keeping and quality control With the advent of highly standardized cell culture methods, culture hazards have become a much rarer cause for concern in the prenatal diagnosis laboratory. Due to the greater number of specimens processed by the average laboratory, a variety of quality-control measures need to be followed to avoid mistakes ranging from culture mixups to diagnostic errors. The most common and potentially serious laboratory errors are human errors in labeling and cross-contamination of samples. Labeling errors can occur at any stage where cells are transferred between vessels: in the amniocentesis procedure room, at culture initiation, feeding and subculture, harvest, slide making, and even microscope analysis. Cross-contamination of cells between patient samples is most common at the time of cell culture harvest, especially for suspension harvests. For these reasons, quality control and quality assurance programs must include a nonpunitive recording system for all laboratory events. A regular review of those events should seek patterns of error that can be eliminated by continuing education of laboratory staff or (often more effective) process improvement directed at reducing the opportunity for human error. Laboratory directors and supervisors should be familiar with the College of American Pathologists Laboratory General and Cytogenetics checklists and the American College of Medical Genetics Standards and Guidelines.665 Laboratories should also participate in a peer review system such as the CAP proficiency surveys.

Safety in the laboratory

It is the responsibility of the laboratory director and all the laboratory staff to protect the rights, privacy and health of employees, ancillary staff and patients alike. AF specimens and all cultures up to the stage of fixation should be treated as potentially hazardous. Universal precautions are essential. Available resources include the CAP Safety Checklist and excellent reviews of laboratory safety and management.672–674

Mesenchymal stem cells in amniotic fluid

Multipotent mesenchymal stem cells (MSCs) can be obtained from several tissue sources and are of great interest for their potential uses in gene therapy and tissue repair. Those derived from adult bone marrow or other sources apart from AFCs have some drawbacks including their relative rarity and slow rate of proliferation in vitro. In contrast, MSCs derived from AFCs have distinct advantages.675–679 MSCs comprise about 1 percent of the cells in midtrimester AF and likely derive from fibroblastic F-type cells.675 Recent advances in the isolation and culture of MSCs from AF are welcomed because these cells apparently do not form teratomas and are not tumorigenic even after many passages. Amniotic MSCs proliferate well and have stable normal telomeres, cytogenetics, and cell surface markers of pleuripotency, similar to embryonic stem cells. Amniotic MSCs also circumvent ethical objections associated with the use of embryonic stem cells. Although more research is needed, amniotic MSCs appear to have immunogenic characteristics that are favorable for allogenic transplantation.680 They can be coaxed into differentiation along many lineages such as adipogenic, osteogenic, myogenic, endothelial, neurogenic, pancreatic, and hepatic, and including mesodermal, ectodermal, and endodermal lineages.480, 676, 678, 681–683 These cells are likely to find utility in a wide variety of cellular therapies,481, 675 including anticancer combination therapy.684 In a demonstration of whole-tissue engineering for early repair of congenital malformations, heart valves have been fabricated using amniotic MSCs. The engineered heart valves exhibited normal endothelial surfaces and adequate opening and closing behavior.479

172 Genetic Disorders and the Fetus

Under appropriate conditions, amniotic MSCs can form bone.676 The potential exists for amniotic MSCs to be employed for engineering tissues in time to be implanted shortly after birth to repair

malformations of the heart, skin, bladder, or diaphragm.479, 685–687 This approach is unlikely, at least in the short term, to be helpful in the fetal therapy of spina bifida (see Chapters 29 and 30).

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596. Burton BK, Gerbie AB, Nadler HL. Biochemical and biological problems and pitfalls of cell culture for prenatal diagnosis. In: Milunsky A, ed. Genetic disorders and the fetus: diagnosis prevention, and treatment. New York: Plenum Press, 1979:369. 597. Hoehn H, Bryant EM, Fantel AG, et al. Cultivated cells from diagnostic amniocentesis in second trimester pregnancies. III. The fetal urine as a potential source of clonable cells. Humangenetik 1975;29:285. 598. Chang HC, Jones OW. Amniocentesis: cell culture of human amniotic fluid in a hormone supplement. In: Sirbasku DA, Pardee AB, Sato GH, eds. Growth of cells in hormonally defined media (Cold Spring Harbor Conferences on Cell Proliferation). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1982:1187. 599. Medina-Gomez P, Bard JB. Analysis of normal and abnormal amniotic fluid cells in vitro by cinemicrography. Prenat Diagn 1983;3:311. 600. Harris A. Glycoproteins that distinguish different cell types found in amniotic fluid. Hum Genet 1982;62:188. 601. Felix JS, Sun TT, Littlefield JW. Human epithelial cells cultured from urine: growth properties and keratin staining. In Vitro 1980;16:866. 602. von Koskull H, Aula P, Trejdosiewicz LK, et al. Identification of cells from fetal bladder epithelium in human amniotic fluid. Hum Genet 1984;65:262. 603. Richkind KE, Risch NJ. Sensitivity of chromosomal mosaicism detection by different tissue culture methods. Prenat Diagn 1990;10:519. 604. Hoehn HW. Fluid cell culture. In: Milunsky A, ed. Genetic disorders and the fetus, 4th edn. Baltimore, MD: The Johns Hopkins University Press, 1998:128. 605. Kennerknecht I, Baur-Aubele S, Grab D, et al. First trimester amniocentesis between the seventh and 13th weeks: evaluation of the earliest possible genetic diagnosis. Prenat Diagn 1992;12:595. 606. Casey ML, Word RA, MacDonald PC. Endothelin-1 gene expression and regulation of endothelin mRNA and protein biosynthesis in avascular human amnion. Potential source of amniotic fluid endothelin. J Biol Chem 1991;266:5762. 607. Chang HC, Jones OW. Reduction of sera requirements in amniotic fluid cell culture. Prenat Diagn 1985;5:305. 608. Liu L, Brinkman A, Blat C, et al. IGFBP-1, an insulin like growth factor binding protein, is a cell growth inhibitor. Biochem Biophys Res Commun 1991;174:673. 609. Priest JH, Rao KW. Prenatal chromosome diagnosis. In: Barch MJ, ed. The AGT cytogenetics laboratory manual, 3rd edn. Philadelphia: Lippincott Williams & Wilkins, 1997:199.

610. Schmid W. A technique for in situ karyotyping of primary amniotic fluid cell cultures. Humangenetik 1975;30:325. 611. Hoehn H, Rodriguez ML, Norwood TH, et al. Mosaicism in amniotic fluid cell cultures: classification and significance. Am J Med Genet 1978;2:253. 612. Boué J, Nicolas H, Barichard F, et al. Le clonage des cellules du liquide amniotique, aide dans l’interpretation des mosaiques chromosomiques en diagnostic prenatal. Ann Genet 1979;22:3. 613. Hecht F, Peakman DC, Kaiser-McCaw B, et al. Amniocyte clones for prenatal cytogenetics. Am J Med Genet 1981;10:51. 614. Tabor A, Lind AM, Andersen AM, et al. A culture vessel for amniotic fluid cells allowing faster preparation of chromosome slides. Prenat Diagn 1984;4:451. 615. Benn PA, Hsu LY. Maternal cell contamination of amniotic fluid cell cultures: results of a U.S. nationwide survey. Am J Med Genet 1983;15:297. 616. Bui TH, Iselius L, Lindsten J. European collaborative study on prenatal diagnosis: mosaicism, pseudomosaicism and single abnormal cells in amniotic fluid cell cultures. Prenat Diagn 1984;4 Spec No:145. 617. Worton RG, Stern R. A Canadian collaborative study of mosaicism in amniotic fluid cell cultures. Prenat Diagn 1984;4 Spec No:131. 618. Batanian JR, Ledbetter DH, Fenwick RG. A simple VNTR-PCR method for detecting maternal cell contamination in prenatal diagnosis. Genet Test 1998;2:347. 619. Frederickson RM, Wang HS, Surh LC. Some caveats in PCR-based prenatal diagnosis on direct amniotic fluid versus cultured amniocytes. Prenat Diagn 1999;19:113. 620. Smith GW, Graham CA, Nevin J, et al. Detection of maternal cell contamination in amniotic fluid cell cultures using fluorescent labelled microsatellites. J Med Genet 1995;32:61. 621. Claussen U, Schafer H, Trampisch HJ. Exclusion of chromosomal mosaicism in prenatal diagnosis. Hum Genet 1984;67:23. 622. Sikkema-Raddatz B, Castedo S, TeMeerman GJ. Probability tables for exclusion of mosaicism in prenatal diagnosis. Prenat Diagn 1997;17:115. 623. American College of Medical Genetics. Standards and guidelines for clinical genetics laboratories, 2nd edn. Bethesda, MD: American College of Medical Genetics, 1999. 624. American College of Medical Genetics. Standards and guidelines for clinical genetics laboratories, 2008. https://www.acmg.net/ACMG/Medical-GeneticsPractice-Resources/Technical_Standards_and_ Guidelines.aspx (accessed July 8, 2020).

194 Genetic Disorders and the Fetus

625. Spurbeck JL, Zinsmeister AR, Meyer KJ, et al. Dynamics of chromosome spreading. Am J Med Genet 1996;61:387. 626. Henegariu O, Heerema NA, Lowe Wright L, et al. Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing. Cytometry 2001;43:101. 627. Spurbeck JL. The dynamics of chromosome spreading. Compact disk copyright 2013. Distributed by Association of Genetic Technologists (AGT), 2014. 628. Cheung SW, Spitznagel E, Featherstone T, et al. Exclusion of chromosomal mosaicism in amniotic fluid cultures: efficacy of in situ versus flask techniques. Prenat Diagn 1990;10:41. 629. Featherstone T, Cheung SW, Spitznagel E, et al. Exclusion of chromosomal mosaicism in amniotic fluid cultures: determination of number of colonies needed for accurate analysis. Prenat Diagn 1994;14:1009. 630. Cheng EY, Luthy DA, Dunne DF, et al. Is the 15-in situ clone protocol necessary to detect amniotic fluid mosaicism? Am J Obstet Gynecol 1995;173:1025. 631. Ing PS, van Dyke DL, Caudill SP, et al. Detection of mosaicism in amniotic fluid cultures: a CYTO2000 collaborative study. Genet Med 1999;1:94. 632. American College of Medical Genetics. Standards and guidelines for clinical genetics laboratories. E4.1.3 amniotic fluid. Bethesda, MD: American College of Medical Genetics, 2006. 633. Miron PM. Preparation, culture, and analysis of amniotic fluid samples. Curr Protoc Hum Genet 2012:8.4. 634. van Dyke DL, Roberson JR,Wiktor A. Prenatal cytogenetic diagnosis. In: McClatchey KD, ed. Clinical laboratory medicine, 2nd edn. Philadelphia: Lippincott Williams & Wilkins, 2002:636. 635. Spurbeck JL, Carlson RO, Allen JE, et al. Culturing and robotic harvesting of bone marrow, lymph nodes, peripheral blood, fibroblasts, and solid tumors with in situ techniques. Cancer Genet Cytogenet 1988;32:59. 636. Carlan SJ, Papenhausen P, O’Brien WF, et al. Effect of maternal–fetal movement on concentration of cells obtained at genetic amniocentesis. Am J Obstet Gynecol 1990;163:490. 637. Fischer RL, LaMotta J, McMorrow LE, et al. Effect of pre-amniocentesis uterine manipulation on amniocyte concentration and culture duration: a randomized, clinical trial. Prenat Diagn 1996;16:673. 638. Melnyk JH, Persinger G, Teplitz RLA. A micromethod for processing amniotic fluid cells. In Vitro 1979;15:200. 639. Byrne DL, Marks K, Braude PR, et al. Amniofiltration in the first trimester: feasibility, technical aspects

640.

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652. 653.

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and cytological outcome. Ultrasound Obstet Gynecol 1991;1:320. Sundberg K, Smidt-Jensen S, Lundsteen C, et al. Filtration and recirculation of early amniotic fluid. Evaluation of cell cultures from 100 diagnostic cases. Prenat Diagn 1993;13:1101. Sundberg K, Bang J, Brocks V, et al. Early sonographically guided amniocenteses with filtration technique: follow-up on 249 procedures. J Ultrasound Med 1995;14:585. Sundberg K, Lundsteen C, Philip J. Early filtration amniocentesis for further investigation of mosaicism diagnosed by chorionic villus sampling. Prenat Diagn 1996;16:1121. Kunsela P, Seppala MB, Brock DJH, et al. Amniotic fluid fibronectin in normal pregnancy and in pregnancies with anencephalic fetus. Biomedicine 1978;29:296. Vlodavsky I, Voss R, Yarkoni S, et al. Stimulation of human amniotic fluid cell proliferation and colony formation by cell plating on a naturally produced extracellular matrix. Prenat Diagn 1982;2:13. Crickard K, Golbus MS. Influence of extracellular matrix on the proliferation of human amniotic fluid cells in vitro. Prenat Diagn 1982;2:89. Brackertz M, Kubbies M, Feige A, et al. Decreased oxygen supply enhances growth in culture of human midtrimester amniotic fluid cells. Hum Genet 1983;64:334. Held KR, Sönnichsen S. The effect of oxygen tension on colony formation and cell proliferation of amniotic fluid cells in vitro. Prenat Diagn 1984;4:171. Barnes D, Sato G. Serum-free cell culture: a unifying approach. Cell 1980;22:649. Gospodarowicz D, Moran JS, Owashi ND. Effects of fibroblast growth factor and epidermal growth factor on the rate of growth of amniotic fluid-derived cells. J Clin Endocrinol Metab 1977;44:651. Porreco RP, Bradshaw C, Sarkar S, et al. Enhanced growth of amniotic fluid cells in presence of fibroblast growth factor. Obstet Gynecol 1980;55:55. Art to Science. Freezing and thawing serum and other biological materials: optimal procedures minimize damage and maximize shelf-life. HyClone Laboratories, Inc, 1992;11:1. Chang HC, Jones OW. A new growth medium for human amniotic fluid cells. Karyogram 1981;7:54. Chang HC, Jones OW, Masui H. Human amniotic fluid cells grown in a hormone-supplemented medium: suitability for prenatal diagnosis. Proc Natl Acad Sci U S A 1982;79:4795. Biddle WC, Kuligowski S, Lockwood DH, et al. AmnioMAXTM-C100: a new specialized cell culture

CHAPTER 3

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medium for the propagation of human amniocytes. Focus 1992;14:80. Claussen U. The pipette method: a new rapid technique for chromosome analysis in prenatal diagnosis. Hum Genet 1980;54:277. Salk D, Disteche C, Stenchever MR, et al. Routine use of Chang medium for prenatal diagnosis: improved growth and increased chromosomal breakage. Am J Hum Genet 1983;35:151A. Masia A, Jenkins EC, Duncan C. Chromosomal abnormalities in cultures with Chang medium. Am J Hum Genet 1986;39:260. Krawczun MS, Jenkins EC, Masia A, et al. Chromosomal abnormalities in amniotic fluid cell cultures: a comparison of apparent pseudomosaicism in Chang and RPMI-1640 media. Clin Genet 1989;35:139. Eiben B, Goebel R, Hansen S, et al. Early amniocentesis – a cytogenetic evaluation of over 1500 cases. Prenat Diagn 1994;14:497. Assel BG, Lewis SM, Dickerman LH, et al. Single operator comparison of early and mid-second-trimester amniocentesis. Obstet Gynecol 1992;79:940. Corning guide for identifying and correcting common cell growth problems. http://www.bioprocessonline. com/doc/identifying-correcting-cell-growthproblems0001 (accessed May 19, 2015). Persutte WH, Lenke RR. Failure of amniotic-fluid cell growth: is it related to fetal aneuploidy? Lancet 1995;345:96. Elejalde BR, de Elejalde MM, Soto A. Amniotic-fluid cell growth and fetal aneuploidy. Lancet 1995;345:924. Lam YH, Tang MH, Sin SY, et al. Clinical significance of amniotic-fluid-cell culture failure. Prenat Diagn 1998;18:343. American College of Medical Genetics Standards and Guidelines for Clinical Genetics Laboratories. https:// www.acmg.net/ACMG/Medical-Genetics-PracticeResources/Technical_Standards_and_Guidelines.aspx (accessed July 8, 2020). Milunsky A, Bender CS. Failure of amniotic-fluid cell growth with toxic tubes. N Engl J Med 1979;301:47. Kohn G. Failure of amniotic fluid cell cultures due to syringe toxicity. Prenat Diagn 1981;1:233. Chiesa J, Bureau JP. Nothing ventured, nothing gained! Prenat Diagn 1999;19:894. Seguin LR, Palmer CG. Variables influencing growth and morphology of colonies of cells from human amniotic fluid. Prenat Diagn 1983;3:107. Felix JS, Doherty RA. Amniotic fluid cell culture II. Evaluation of a red blood cell lysis procedure for culture of cells from blood-contaminated amniotic fluid. Clin Genet 1979;15:215.

671. Johansson KE, Bolske G. Evaluation and practical aspects of the use of a commercial DNA probe for detection of mycoplasma infections in cell cultures. J Biochem Biophys Methods 1989;19:185. 672. Knutsen T. Laboratory safety, quality control and regulations. In: Barch MJ, ed. The AGT cytogenetics laboratory manual, 3rd edn. Philadelphia: Lippincott Williams & Wilkins, 1997:597. 673. Holtge GA. Laboratory safety. In: McClatchey KD, ed. Clinical laboratory medicine, 2nd edn. Philadelphia: Lippincott Williams & Wilkins, 2002:78. 674. Travers EM. Basic laboratory management. In: McClatchey KD, ed. Clinical laboratory medicine, 2nd edn. Philadelphia: Lippincott Williams & Wilkins, 2002:3. 675. Tsai MS, Lee JL, Chang YJ, et al. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod 2004;19:1450. 676. De Coppi P, Bartsch G, Jr., Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100. 677. Guillot PV, Gotherstrom C, Chan J, et al. Human first trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC. Stem Cells 2007;25:646. 678. Roubelakis MG, Pappa KI, Bitsika V, et al. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells. Stem Cells Dev 2007;16:931. 679. Zhou J,Wang D, Liang T, et al. Amniotic fluid-derived mesenchymal stem cells: characteristics and therapeutic applications. Arch Gynecol Obstet 2014;290:223. 680. In’t Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003;102:1548. 681. Trounson A. A fluid means of stem cell generation. Nat Biotechnol 2007;25:62. 682. Liu YW, Roan JN, Wang SP, et al. Xenografted human amniotic fluid-derived stem cell as a cell source in therapeutic angiogenesis. Int J Cardiol 2013; 168:66. 683. Petsche Connell J, Camci-Unal G, Khademhosseini A, et al. Amniotic fluid-derived stem cells for cardiovascular tissue engineering applications. Tissue Eng Part B Rev 2013;19:368. 684. Kang NH, Hwang KA, Kim SU, et al. Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells. Cancer Gene Ther 2012;19:517.

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685. Kaviani A, Perry TE, Dzakovic A, et al. The amniotic fluid as a source of cells for fetal tissue engineering. J Pediatr Surg 2001;36:1662. 686. Gucciardo L, Lories R, Ochsenbein-Kolble N, et al. Fetal mesenchymal stem cells: isolation, properties

and potential use in perinatology and regenerative medicine. BJOG 2009;116:166. 687. Murphy SV, Atala A. Amniotic fluid and placental membranes: unexpected sources of highly multipotent cells. Semin Reprod Med 2013;31:62.

4

Molecular Aspects of Placental Development Wendy P. Robinson 1,2 and Deborah E. McFadden 1,2 1 2

BC Children’s Hospital Research Institute, Vancouver, BC, Canada University of British Columbia, Vancouver, BC, Canada

Overview The placenta is a fetal organ that is discarded after birth, but is essential to ensuring normal development in utero. It regulates fetal growth, protects the fetus from infection and other adverse exposures, as well as generally programming the fetus for good health after birth. Screening for placental disease is an important component to the assessment of the fetus in pregnancy. Reduced placental efficiency can lead to fetal growth restriction (FGR) and/or maternal preeclampsia (PE). This can be caused by genetic changes within the placenta or by environmental influences, such as maternal stress or drug exposure. In this chapter, causes of placental disease and the role of the placenta in diagnosis of fetal health will be reviewed with a focus on genetic associations.

Placental structure Evaluating the placenta requires an understanding of its unique structure and development. The chorionic villi that compose the placenta are organized into 50–70 distinct tree-like structures that grow in a clonal manner outwards from the chorionic plate into the basal plate (which interface with the maternal decidua).1 These villi are bathed in maternal blood, from which they sponge up nutrients important for fetal growth.

The maternal blood is in direct contact with the outer trophoblast bilayer of the chorionic villi. This bilayer is made up of a multinucleated syncytium derived by fusion of the cytotrophoblast cells that form a single-cell layer below the syncytium. In addition, some cytotrophoblasts form columns that migrate into and anchor the placenta to the uterine wall. Invasive cells that detach from these columns are termed extravillous trophoblasts (EVTs) and include the interstitial cytotrophoblasts (iCTBs) found in the decidual stroma and those that remodel maternal blood vessels, termed endovascular cytotrophoblasts (eCTBs).1 The inner core of the villi is the chorionic mesenchyme, which includes structural components, and a mix of cells including fetal blood vessels, fibroblasts, pericytes, and Hofbauer cells (placental macrophages). These extraembryonic cells derive from the epiblast of the blastocyst, from which the fetus is also derived. Placental size is strongly correlated with fetal size; however, there is considerable variation in placental size for any given birthweight.2 The efficiency of the placenta depends on the surface area for exchange, thickness, and density of transporter proteins,3 and birthweight is highly associated with placental weight.4 Interestingly, mean placental size can vary between populations and even within a population over time because of changes in maternal nutrition or other environmental conditions.5

Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, Eighth Edition. Edited by Aubrey Milunsky and Jeff M. Milunsky. © 2021 Aubrey Milunsky and Jeff M. Milunsky. Published 2021 by John Wiley & Sons Ltd.

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198 Genetic Disorders and the Fetus

Placental development and function The placenta is responsible for many functions, which change as pregnancy proceeds.1 In early gestation, the primary roles of the placenta include invasion into the maternal endometrium, remodeling of maternal vasculature, and secretion of hormones important to maintain pregnancy. The placenta subsequently regulates blood flow and nutrient delivery to the fetus, buffers the fetus from adverse environmental effects, and generally performs the functions of multiple organs (lung, brain, kidney, immune system, etc.). Implantation

During the invasion process, the early trophoblasts produce molecules to help them attach to and invade the uterine wall (e.g. integrins), prevent menstruation (e.g. human chorionic gonadotropin (hCG)), destroy the uterine matrix (e.g. matrix metalloproteinases), and suppress the maternal immune system (e.g. corticotropin-releasing hormone (CRH)).6, 7 hCG (encoded by CGA and CGB) is one of the earliest hormones expressed from syncytiotrophoblast and stimulates many other processes. Multiple growth factors are important in regulating trophoblast proliferation, including placental growth factor (PlGF), epidermal growth factor (EGF), and transforming growth factor β (TGF-β). In addition, microparticles, such as microvesicles (0.1–2 μm) and exosomes (30–100 nm), are released by the placental syncytium into maternal decidua and blood and may play a role in early maternal immune suppression and vascular remodeling.8–11 These microparticles provide a rich source of placental derived material detectable in maternal blood early in pregnancy that has the potential to be used in monitoring the pregnancy. Failure of implantation and invasion can lead to early miscarriage. The majority of miscarriages occurring in the first trimester of pregnancy are associated with chromosome abnormalities, with trisomy, triploidy, and 45,X accounting for the vast majority of these.12, 13 The reasons for implantation failure in chromosomally abnormal cases are likely complex, involving dysregulation of multiple important genes that then impede trophoblast growth and invasion. Trisomy for chromosomes

1, 11, and 19 are rarely observed even in early miscarriages, and presumably do not survive to clinical detection of pregnancy. Chromosome 19 not only has the highest gene density of any chromosome, but is sometimes referred to as the “placenta chromosome” because of the numerous placenta-specific genes located on it, including the highly expressed pregnancy-specific glycoprotein cluster (PSG),14 and the maternally imprinted chromosome 19 miRNA cluster (C19MC), the largest microRNA cluster in humans.15 Failure of implantation among genetically normal conceptuses can be due to a nonreceptive maternal environment resulting from disturbances in maternal hormone levels, immune health, anatomical interference, or a variety of maternal health conditions.16 Angiogenesis

Placental angiogenesis and vasculogenesis serves to increase both uterine (maternal) and umbilical (fetal) blood flow. This process is dependent on a balance between pro- and antiangiogenic factors.17 Early in pregnancy, EVTs invade and plug the maternal uterine arteries, helping to maintain a low-oxygen environment needed for trophoblast proliferation.18 Trophoblast cells (eCTBs) also migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. This expands the diameter of the maternal vessels and, in combination with gradual disintegration of the spiral artery plugs, results in a dramatic increase in blood flow after 12 weeks gestational age, which is needed to support fetal growth later in pregnancy. In turn, placental vasculature develops, and increases throughout gestation as the needs of the fetus grow.19 FGR can result from poor spiral artery remodeling or reduced vascular development within the placenta. Furthermore, insufficient remodeling of the maternal spiral arteries can result in a prolonged state of hypoxia and increased reoxygenation stress. This leads to increased syncytiotrophoblast apoptosis and necrosis, causing increased debris circulating in the maternal blood that has been associated with maternal PE.20 In addition to FGR and PE, abnormal spiral artery remodeling has been associated with placental abruption, preterm premature rupture of membranes, and intrauterine fetal death.21

CHAPTER 4

Nutrient delivery

Fetal growth is dependent on efficient nutrient delivery to the fetus. This is determined by maternal availability, maternal blood flow to the placenta, the amount of placental surface in contact with maternal blood, and the efficiency of placental transport.22, 23 Transport of substances across the placenta can occur by (i) passive transport (simple or facilitated diffusion); (ii) active transport; and (iii) vesicular transport, by which large molecules are captured by microvesicles. A well-functioning placenta can be extremely efficient at extracting nutrients for the fetus even when maternal supplies are low. For example, there is a threefold increase in folate concentration in the placenta compared with maternal blood;24 this is accomplished via several folate receptors highly expressed in the human placenta, including folate receptor 1 (FOLR1), proton-coupled high-affinity folate transporter (PCFT), and reduced folate carrier (RFC).25 As the fetus grows and requires more nutrients, the placenta alters gene expression to increase nutrient supply to the fetus;26 for example, upregulation of System A transporters can increase delivery of amino acids.3, 27 There is also an increase in iron transport proteins, which absorb iron from maternal blood,28 and of placental CRH, which increases the production of maternal glucose needed to support the growing fetal brain.29 One pathway by which increased cortisol can lead to growth restriction is by interfering with CRH-driven glucose production.29 Immune function

The placenta employs a number of mechanisms that protect the embryo/fetus from infection. Genes involved in immune regulation are among the most differentially expressed30, 31 and differentially methylated32 in the placenta across different gestational ages. The human placenta is not only the source of hematopoiesis early in pregnancy, but remains a hematopoietic organ throughout gestation.33, 34 The placenta also contains a large number of Hofbauer cells (placental macrophages), which may play roles in placental angiogenesis and prevent pathogens crossing from mother to fetus.35 Exosomes and microvesicles also appear to provide protection against viruses, which may be partially attributable to transmission of members of the chromosome 19

Molecular Aspects of Placental Development 199

placenta-specific paternally expressed microRNA cluster (C19MC).36 Understanding how the placenta protects from infection is an important question in the study of preterm birth (PTB). Chorioamnionitis (CA), or intra-amniotic infection, an inflammation of the chorion and amnion usually caused by bacterial infection, is associated with the majority of extremely (T (p.Glu6Val) variant in the HBB gene, or achondroplasia caused predominantly by two pathogenic variants in FGFR3, c.1138G>A or c.1138G>C (both resulting in p.Gly380Arg substitution). For such conditions, robust assays with accurate estimates of test sensitivity and specificity as well as clinical utility can be achieved. However, single-gene disorders are more commonly caused by one of a number of different types of variants throughout a gene, or sometimes in a

CHAPTER 8

Noninvasive Prenatal Diagnosis and Screening for Monogenic Disorders Using Cell-Free DNA 323

set of related genes. For other conditions, only a subset of patients have pathogenic variants in the known disease gene(s), while for others the cause is unknown. Finally, not all have mutations detectable by sequencing of cfDNA fragments. These include structural chromosomal variants, smaller CNVs such as single exon deletions, complex insertion–deletion variants, or repeat expansions. Such issues are not unique to cfDNA testing but must be considered when these tests are introduced into the clinic.

Current status of noninvasive single-gene testing by cell-free DNA analysis Overview

The demonstration that Y chromosome-specific sequences could be amplified from maternal plasma in women who were pregnant with a male fetus provided the first evidence for the presence of fetal DNA in maternal plasma during pregnancy.10 This was soon followed by the first report in 2000 of single-gene disorder diagnosis for myotonic dystrophy,50 and several tests are now approved for clinical use.18 Table 8.1 summarizes reported experience with single-gene disorder cfDNA-based NIPD for diagnosis of autosomal dominant, autosomal recessive, and X-linked disorders in the fetus (not including sex determination or fetal RHD status). As methods become more refined newer reports are showing promising results with multiplexed assays for several diseases at once using NGS-based approaches.95 The next paragraphs highlight the reported experience with some more common conditions for which such testing has been developed and is now clinically available in some countries: fetal sex determination, Rhesus hemolytic disease of the fetus and newborn (HDFN), sickle cell disease, α- and β-thalassemia, Duchenne and Becker muscular dystrophy, CYP21A2-related congenital adrenal hyperplasia (CAH), spinal muscular atrophy (SMA), cystic fibrosis and common de novo dominant skeletal dysplasias, and other rare monogenic conditions. A wide variety of technologies have been described for delivering cfDNA-based NIPD; although the most common analysis in clinical use is now NGS, some indications are best delivered by digital PCR. Ultimately, platforms used may

vary across the globe and will also depend on an individual laboratory’s experience. However, clinical laboratories offering an accredited NIPD service must have equipment dedicated to NIPD to avoid cross-contamination. Noninvasive fetal sex determination

Cell-free DNA-based prenatal testing for fetal sex determination can be performed reliably from 7 weeks of gestation16, 18 and has value for pregnancies at increased risk of X-linked conditions, such as fragile X syndrome, hemophilia A,96 Duchenne muscular dystrophy (DMD), or X-linked adrenoleukodystrophy to help guide which pregnancies will most benefit from invasive diagnostic testing.15 Another indication is for conditions that differentially affect male and female fetuses, such as CAH.57, 97 Fetal CAH can cause virilization of the female external genitalia preventable with maternal prenatal steroid treatment.98 However, more recently, a specific noninvasive test for CAH has been developed (see later).68 Sex determination by cfDNA-based targeted testing is also helpful for the clarification of fetal ultrasound findings such as abnormalities of the fetal external genitalia or to provide additional information for diagnosing genetic conditions where genital ambiguity or sex reversal is a feature of the condition (see Chapter 12). Cell-free DNA assays for fetal sex determination usually employ real-time quantitative PCR (RT-qPCR) to identify Y chromosome-specific sequences.99 The most common determine the presence of the SRY gene or the multicopy DYS14 sequence with no significant difference in performance between these two.15 The presence of the Y chromosome sequence indicates that the fetus is male. To avoid falsely reporting a female result if Y chromosome sequences are not amplified due to technical amplification failure or low fetal fraction, the presence of fetal cfDNA must be confirmed by parallel or co-amplification of a control universal fetal cfDNA or cfRNA marker or polymorphism not present in the mother.100–102 A systematic review and meta-analysis that included 57 studies with a combined 3,524 pregnancies with male fetuses and 3,017 pregnancies with female fetuses concluded that cfDNA for fetal sex determination is reliable after 7 weeks in pregnancy.15 Nevertheless, individual tests had a failure rate of up to 5 percent

324 Genetic Disorders and the Fetus

Table 8.1 Selected studies reporting noninvasive prenatal diagnosis (NIPD) for single-gene disorders. Accredited for Condition

Method

Reference

clinical use

Autosomal dominant conditions Achondroplasia

Restriction digest

48, 130

MALDI-TOF MS

49

PCR–RED

81

QF-PCR

131

PCR–RED, NGS

90

ddPCR

86

NGS

19, 133

Multiplex PCR

134

High-resolution melting point analysis with

93

Yes – superseded by NGS Yes Yes

confirmatory melting point analysis Apert syndrome

Crouzon

AS RT PCR

211

PCR–RED

212

NGS panel

19

PCR–RED

212

Yes

NGS panel and bespoke testing

19

Huntington disease

QF-PCR

51, 213, 214

Myotonic dystrophy

Nested PCR

50

Thanatophoric dysplasia type

PCR–RED

82

Yes – superseded

PCR–RED, NGS

90

Yes

Targeted capture sequencing

72

NGS

19

Yes – panel

NGS

19

Yes – bespoke

1 (TD1) and type 2 (TD2)

Torsion dystonia

Yes

by NGS

Autosomal recessive – parents carrying different mutations Congenital adrenal

Fluorescent SNPs

57

hyperplasia Cystic fibrosis

PCR-RFLP

52

NGS for paternal exclusion

6

SnaPshot

56

MEMO fluorescent PCR

142

Digital PCR and sequencing

59

Nested PCR and restriction digestion

150

Semi-nested and nested real-time PCR for

151

Yes – superseded by RHDO

Fraser syndrome Hemoglobin E

three different mutations HB Lepore

AS-PCR

163

Leber congenital amuarosis

Denaturing HPLC

52

Polycystic kidney disease

Digital PCR and sequencing

59

Propionic acidaemia

SnaPshot; melt curve analysis.

215

Spinal muscular atrophy

NGS and RHDO

76, 139

α-Thalassemia

RT nested PCR

173

QF-PCR

176

AS RT-PCR for paternal exclusion

178

Yes

(Continued)

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Noninvasive Prenatal Diagnosis and Screening for Monogenic Disorders Using Cell-Free DNA 325

Table 8.1 (Continued) Accredited for Condition

Method

Reference

β-Thalassemia

COLD-PCR

167

AS-PCR for SNPs

164

APEX

165

Genome-wide MPS and SPRT analysis

21

AS RT fluorescent PCR for paternal exclusion

53

SABER and MS assay for paternal exclusion

160

Size selection and peptide nucleic clamp

54

Size fractionation and MALDI-TOF

161

COLD-PCR and microarray for paternal

168

clinical use

exclusion Modified fast temperature gradient

169

COLD-PCR Restriction analysis PCR

170

Quantitative genotyping and direct

171

mutation analysis Targeted NGS for paternal exclusion

89, 172

Autosomal recessive – parents carrying the same mutations Congenital adrenal

Targeted MPS and haplotype analysis

68

Targeted MPS and haplotype

69

hyperplasia analysis – hidden Markov model NGS and RHDO

19

Undergoing

Cystic fibrosis

NGS with RHDO

77, 139

Yes

Methylmalonic academia

Relative mutation dosage with ddPCR and

64

validation

parental SNP analysis Sickle cell disorder

Relative mutation dosage using digital

62

RT-PCR Pyrophosphorolysis-activated

152

polymerization (PAP) PCR-amplification with NGS

149

High-resolution melting analysis

153

Targeted MPS and RMD

156

Spinal muscular atrophy

NGS and RHDO

139

α-Thalassemia

AS RT-PCR

178

Targeted capture sequencing

180

β-Thalassemia

Semi-conductor sequencing

181

Relative mutation dosage using digital PCR

61

Targeted MPS and relative haplotype

65

Yes

dosage MPS and RMD

21

Amplicon sequencing and RMD

89

Semi-conductor sequencing

91

Targeted locus amplification and NGS

75

ddPCR

84

Relative mutation dosage using digital PCR

63

X-linked Hemophilia A and B

(Continued)

326 Genetic Disorders and the Fetus

Table 8.1 (Continued) Accredited for Condition

Method

Reference

Retinitis pigmentosa

Sequencing

58

NGS and RHDO

70, 71, 73, 139

Direct haplotyping and RHDO

140

clinical use

(X-linked) Duchenne and Becker

Yes

muscular dystrophies

This table details selected reports of NIPD for monogenic disorders. It is impossible to detail all tests done, not least since in the United Kingdom there is an accredited service for bespoke by-family testing using next-generation sequencing for a wide range of rare diseases where the mutation is known, either inherited paternally or having arisen de novo in a previous pregnancy.19 Here we also show which tests are being, to our knowledge, delivered clinically from an accredited laboratory. APEX, arrayed primer extension; AS, allele-specific; COLD, co-amplification at lower denaturation temperature; dd, droplet digital; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy; MEMO, modified oligonucleotide; MPS, massively parallel sequencing; MS, mass spectroscopy; NGS, next-generation sequencing; PCR, polymerase chain reaction; QF, quantitative fluorescent; RED, restriction enzyme digest; RFLP, restriction fragment length polymorphism; RHDO, relative haplotype dosage; RMD, relative mutation dosage; RT, real time; SABER, single allele-based extension reaction; SnaPshot, single base primer extension; SNP, single-nucleotide polymorphism; SPRT, sequential probability ratio test. Source: Based on Drury S, Mason S, McKay F, et al. Implementing non-invasive prenatal diagnosis (NIPD) in a National Health Service laboratory; from dominant to recessive disorders. Adv Exp Med Biol 2016;924:71.19

in some studies, likely due to low fetal fraction,15, 16 highlighting the importance of including strategies for optimal sample collection and processing to minimize the number of inconclusive cases.103 Fetal sex determination using cfDNA analysis is available as a clinical service in several countries97, 100–102, 104 and its clinical utility has been demonstrated. An audit of UK public sector accredited laboratories found that the most common indications for testing were X-linked disorders (81.2 percent, of which 20.8 percent were for hemophilia), and CAH (11.3 percent). Of these, only 43 percent of women at risk of X-linked conditions (excluding hemophilia) and 38 percent at risk of CAH subsequently underwent invasive testing.16 A multicenter audit of fetal sex determination for 258 pregnancies at risk of CAH (134 male and 124 female fetuses) conducted in France demonstrated that prenatal steroid treatment had been avoided in 68 percent of pregnancies with a male fetus.97 The introduction of NIPD for fetal sex determination in the UK was shown to be cost-effective because of the reduction in invasive procedures and was well-received by patients because of practical and psychological benefits.105–107

Currently, determination of fetal sex is included in most of the commercial cfDNA tests for aneuploidy screening of chromosomes 13, 18, and 21 although this is not routinely reported in several countries (the Netherlands, China, United Kingdom, etc.). It is important to recognize that these approaches are not based on a targeted assay but rather on counting of cfDNA fragments, or dosage analysis of polymorphic SNPs across the X or Y chromosomes, which are affected by mosaicism. Although mosaicism can make interpretation complex, this information may have clinical utility and cannot be obtained with targeted testing for fetal sex but may also complicate analysis when fetal sex is required to target invasive testing for X-linked disorders. Fetal RHD and other blood group genotyping

The most common cause of HDFN is Rhesus D (RhD) blood group incompatibility between the mother and fetus. When a RhD-negative (RhD−) mother is pregnant with a RhD-positive (RhD+) fetus, she can develop anti-RhD antibodies which can cross the placenta and cause fetal red cell lysis, resulting in HDFN in subsequent pregnancies with a RhD+ fetus. Most cases can be prevented

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by administration of anti-RhD immunoglobulins during pregnancy and/or after delivery to RhD− women at risk for alloimmunization along with monitoring antibody titers during pregnancy. The Rhesus blood group system is encoded by two adjacent genes on chromosome 1, the RHD gene and the RHCE gene. Most individuals with an RhD− blood type have a deletion of both copies of the RHD gene. RhD+ individuals have at least one functional copy of the RHD gene. Of these, 45 percent have two intact RHD genes and men with this genotype will always have RhD+ offspring while the other 55 percent have only one intact RHD gene, in most cases due to a heterozygous RHD gene deletion. If the father of the pregnancy of an RhD− women is heterozygous RhD+, there is a 50 percent chance the fetus will be RhD+ and at risk for HDFN. Until the development of noninvasive tests for fetal RHD genotyping, the only way to unequivocally determine if the fetus is RhD− was through amniocentesis, which is not done for routine monitoring of pregnancies at risk for sensitization, and many women would unnecessarily receive anti-RhD immunoprophylaxis to prevent alloimmunization. This motivated early development of noninvasive cfDNA-based fetal RHD genotyping.11 This can be done with a “paternal exclusion” type assay designed to detect RHD gene sequences in cfDNA since most women lack the RHD gene. Because the RHD gene structure is complex, with more than 200 rare variants and an RHD pseudogene, most assays are actually designed to analyze at least two17, 108 or more RHD exons109–111 to improve accuracy. A 2006 meta-analysis of 37 studies found an overall diagnostic accuracy of 94.8 percent for NIPD for fetal RHD genotyping, with 16 of the included studies reporting 100 percent accuracy.112 More recent large-scale studies using robust high-throughput methods suitable for routine screening reported diagnostic accuracy of greater than 99 percent from 10–11 weeks of gestation onwards,113–121 but rare false-positive, false-negative, and inconclusive results remain an issue. A false-positive result, often due to a pseudogene, would only result in unnecessary anti-RhD immunoprophylaxis, but a false-negative result could place women at risk of sensitization and potential HDFN in future pregnancies. This could be mitigated by postnatal prophylaxis if the neonate is found to be RhD+ by serology,

and since the test has >99 percent accuracy the overall risk of alloimmunization would increase minimally.117 Although the best timing for offering cfDNA-based fetal RHD genotyping by cfDNA testing is still debated,117, 122 it has now been implemented as standard practice at a national level for management of pregnancies at high risk of HDFN in a growing number of countries, largely those that did not have a robust immunoprophylaxis program already in place.108, 121, 123 It can avoid exposing women who are not at risk to unnecessary immunoprophylaxis.124 Research with women and health professionals in the United Kingdom suggests that introduction of this service would be welcomed.125 Although the test is now commercially available, it is not universally used in the United States,126 where its cost-effectiveness over routine prophylaxis has been questioned by some.127, 128 HDFN can also be due to maternal–fetal incompatibility for other Rhesus blood antigens, Rhesus C/c and Rhesus E/e, as well as incompatibility for the Kell (K/k) antigens.109 The C/c and E/e Rhesus blood types are determined by SNVs in the RHCE gene and the Kell blood type results from variants in the KEL gene, which can be tested for by sensitive quantitative PCR assays. Other technologies for predicting blood group phenotypes have also been explored. For example, massively parallel sequencing (MPS) has been used to test noninvasively for the fetal KEL genotype,129 as well as for development of a multiplexed assay for a panel of blood group genes.122

Noninvasive prenatal diagnosis of monogenic disorders Here we describe the use of cfDNA testing for the diagnosis of monogenic disorders in families at known increased risk of specific conditions, either because of a relevant family history or because of sonographic findings suggestive of a genetic condition. In this situation analysis is targeted to a known mutation or range of mutations. This testing, once validated and introduced into clinical practice, is considered diagnostic and does not require confirmation by analysis of a fetal sample obtained by invasive testing. Some of the analyses we describe are already in clinical practice

328 Genetic Disorders and the Fetus

(Table 8.1), but others, as noted, are still under development. Skeletal dysplasias

Although there are over 400 different skeletal dysplasias, the ones more commonly detected prenatally (see Chapter 20) are severe lethal conditions, such as thanatophoric dysplasia caused by autosomal dominant pathogenic variants in FGFR3, or lethal osteogenesis imperfecta, caused by pathogenic variants in COL1A1 or COL1A2. These genes are also implicated in other skeletal dysplasias that are not lethal. Achondroplasia is caused by one of two variants in FGFR3 and mutations in COL1A1 and COL1A2 can cause milder forms of osteogenesis imperfecta. Making an early diagnosis is important to differentiate these different forms from each other and from other conditions that may be recessive and have different recurrence risks or that can be associated with other health problems in addition to the skeletal dysplasia. Furthermore, many of these conditions cannot be diagnosed by early ultrasound examinations and are detected only later in gestation. These considerations have sparked strong interest in developing cfDNA-based NIPD assays for the most common skeletal dysplasias. The first studies used PCR or quantitative fluorescent PCR and restriction enzyme digestion (PCR–RED), directly or combined with a fragment size selection protocol to detect the common achondroplasia c.1138G>A mutation in FGFR3 in maternal plasma cfDNA from a patient whose fetus was known to have achondroplasia.48, 130, 131 Subsequently, size fractionation was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with good results on two patients.49 Chitty et al. reported success with PCR–RED for cfDNA-based NIPD of fetal achondroplasia in four affected and two unaffected pregnancies that had growth restriction.81 They successfully used a similar PCR–RED approach for diagnosing thanatophoric dysplasia from maternal plasma cfDNA in three of 42 women referred for ultrasound findings consistent with severe skeletal dysplasia by detecting the c.742C>CT (p.Arg248Cys) or c.1948A>AG (p.Lys650Glu) variant in FGFR3.82 ddPCR with minisequencing of amplicons containing c.1138G nucleotide provided 100 percent sensitivity and specificity

in analysis of 26 maternal plasma samples, five of which were from women carrying a fetus with achondroplasia.86 These approaches are useful when there is predominantly one mutation that causes the condition, such as for achondroplasia. When there are multiple causative mutations in de novo conditions, such as in thanatophoric dysplasia, it becomes too complex and time consuming to test for a range of mutations using PCR-based methods that can only test for one mutation at a time. To address this, MPS was introduced to enable testing for a panel of mutations in a single assay. Comparison of PCR–RED to a newly developed NGS-based method for NIPD of achondroplasia and thanatophoric dysplasia showed in 47 maternal plasma cfDNA samples that NGS was more accurate (96.2 percent) with fewer inconclusive results.90, 132 Tests that combined multiplex PCR with NGS for 19 FGFR3 pathogenic variants that cause achondroplasia, thanatophoric dysplasias I and II and one for the five common mutation hotspots in FGFR3 yielded 100 percent sensitivity and specificity on maternal plasma samples from women with affected and unaffected pregnancies.133, 134 A prospectively conducted multicenter study that included 86 samples from women with pregnancies with achondroplasia and 65 from controls on the performance of high-resolution melting analysis (HRM) with confirmatory SNaPshot minisequencing confirmed high accuracy and suitability for introducing this test into clinical care.93 These studies remained focused on conditions caused by FGFR3 mutations, but did not address other skeletal dysplasias. Such tests are available in small panels or as bespoke analysis for individual at-risk families, for example for FGFR2 variants causing Apert syndrome or Crouzon syndrome and for COL1A1 or COL1A2 mutations causing osteogenesis imperfecta. In the United Kingdom, bespoke analysis for rarer skeletal dysplasias caused by dominant mutations that may have arisen de novo in a previous pregnancy, or which are carried by the father or for autosomal recessive variants where parents are heterozygous for different mutations is available for high-risk families.135 One group reported development of a targeted capture sequencing panel cfDNA-based NIPD that includes 16 genes associated with the most common autosomal dominant

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and autosomal recessive lethal skeletal dysplasias: achondrogenesis IA (TRIP11), achondrogenesis IB (SLC26A2), achondrogenesis II or hypochondrogenesis (COL2A1), thanatophoric dysplasia I and II (FGFR3), short rib-polydactyly syndrome I, IIB, and III (DYNC2H1), short rib-polydactyly syndrome IIA (NEK1), fibrochondrogenesis I (COL11A1), atelosteogenesis I and III (FLNB), atelosteogenesis II (SLC26A2), perinatal osteogenesis imperfecta (COL1A1, COL1A2, CRTAP, LEPRE1, PPIB, BMP1), and hypophosphatasia (ALPL). This assay was tested on parental genomic DNA and maternal plasma cfDNA for three families with fetuses affected with a skeletal dysplasia caused by a de novo pathogenic variant, which were all detected and for two control families, which were both negative. They also were able to identify 97 percent of nonpathogenic variants that were present in the fetus but not in the mother.72 This was combined with pseudotetraploid genotyping which directly deduces the combined maternal and fetal genotype in one patient and resulted in identification of a de novo COL1A1 mutation.136 Duchenne and Becker muscular dystrophy

DMD and the milder Becker muscular dystrophy (BMD) are caused by deletions and pathogenic variants of differing severity in the X-linked DMD gene. DMD affects 1:3,500–1:6,000 male births. Boys with DMD have early normal development, followed by progressive skeletal muscle weakness of lower limbs and later also develop dilated cardiomyopathy and most become wheelchair-dependent in their early teenage years. Two-thirds of affected boys inherit the mutation from asymptomatic carrier mothers who have a 50 percent chance of having an affected son. Among the observed mutations, 60-65 percent are deletions and 5–10 percent duplications of one or more DMD exons, while 20–35 percent are SNVs or small insertions/deletions.137, 138 ECS panels currently exist that can detect the carrier mothers, but one-third of affected boys have a de novo mutation in the DMD gene, and for those carrier screening is not useful. In both scenarios, a cfDNA-based DMD NIPD test could identify affected pregnancies before the end of the first trimester.

The early noninvasive approaches were based on cfDNA-based fetal sex determination to triage women with male pregnancies for invasive testing but did not directly assay the disease-causing mutation. Beginning in 2015, several groups reported fetal DMD genotyping from cfDNA. In most of these the maternal haplotype that carries the pathogenic variant is determined first by capture NGS of the DMD gene and flanking regions containing polymorphic SNPs in parents and the affected proband, after which cfDNA-targeted sequence results are compared to the maternal haplotypes and pathogenic mutations. Prediction of the fetal genotype from maternal SNPs was 99.98 percent accurate in eight families at risk for transmitting DMD.70 Yoo et al. showed similar success in detecting deletions, duplications, and a pathogenic SNV for four mother–proband pairs.71 Parks et al. validated cfDNA analysis using an RHDO approach in seven healthy and two carrier pregnant women which was accurate in all samples with a fetal fraction >4 percent.73 NIPD using RHDO for DMD and BMD has since been introduced into clinical practice in the United Kingdom and this group have reported their experience with 35 cases at risk of these conditions.139 In five women with pregnancies at risk of BMD, four fetuses inherited the low-risk maternal haplotype and in the fifth there was a recombination event close to the familial variant, giving an inclusive result. Of the 35 pregnancies at risk of DMD, 11 were unaffected because they inherited the low-risk haplotype and 13 inherited the high-risk haplotype. Eight of these were pregnancies from known DMD carriers, but for the other five there was increased risk of parental germline mosaicism (see Chapter 14) and invasive testing was required to determine the fetal status.139 These methods still require availability of the proband DNA to determine the haplotype that carries the mutation. In 2018, Jang et al. and in 2020, Chen et al. reported success with direct targeted haplotype phasing without needing to analyze DNA from the proband or other family members in high-risk families where the mother is a carrier for DMD.79, 140 They resolved maternal haplotypes that carried the pathogenic variants by targeted linked-read sequencing of a captured region including the DMD gene and polymorphic SNPs and predicted the fetal genotype by integrating the maternal

330 Genetic Disorders and the Fetus

haplotype data with the targeted sequence data from the cfDNA to infer the fetal sex, DMD gene variants, and fetal haplotype.140 Cystic fibrosis

With an incidence of 1:2,500 livebirths in people of Northern European descent, cystic fibrosis (see Chapter 15) is one of the most common autosomal recessive disorders for which parental carrier or newborn screening is routinely offered in several countries. It is also one of the first conditions for which noninvasive testing on maternal plasma cfDNA was explored. With developments in multidisciplinary care the life expectancy of patients with severe cystic fibrosis is increasing, with a median survival now at 44 years.141 The earliest approaches focused on exclusion of the paternally inherited pathogenic variant when parents carry different mutations.6, 55, 56, 142 This approach has significant limitations since invasive testing would still be required for 50 percent of cases where the paternal mutation was detected. In addition, there is a common mutation in CFTR (p.Phe508del) that is present in approximately 4 percent of people of Northern European descent such that an estimated 47 percent of carrier couples would be ineligible for paternal mutation testing as they both carry the same mutation. However, studies in the United Kingdom indicated that NIPD would be welcomed by parents at risk of transmitting cystic fibrosis. Although 56 percent of parents said they would decline or had declined invasive testing, 94.9 percent (including those who had previously declined invasive testing) said they would choose NIPD if it were available to prepare for the birth of an affected child.6 Subsequently, Chandler et al.77 developed and validated an NGS assay of the region flanking CFTR and applied RHDO from sequencing proband and parental samples to correctly determine which alleles were inherited by the fetus for 13 cases. Following the validation and accreditation of this method, the assay was brought into clinical service in the United Kingdom with samples accepted from 9 weeks of gestation. In the first 2 years of clinical service, 36 of 38 referrals had conclusive results with known outcomes. There were two inconclusive cases, one due to a recombination event on the paternal allele and the other due to no heterozygosity around the

CFTR gene likely due to consanguinity. A similar assay has also been successfully implemented into clinical service in another UK laboratory.139 Although successful, this approach is limited by the requirement for a proband and is not suitable for use with consanguineous couples. In 2017, Vermeulen et al. reported success in implementing a proband-free RHDO approach using targeted-locus amplification of the region surrounding the CFTR gene and MPS with correct outcomes for nine pregnancies at risk of cystic fibrosis.75 Spinal muscular atrophy

SMA is an autosomal recessive group of neuromuscular disorders caused by mutations in the SMN1 gene that result in the loss of motor neurons and progressive muscle wasting.143 The severity of symptoms and age of onset varies by the type. Although rare, it is the second most common autosomal recessive condition in Northern European populations with most types apparent at birth or in the early months and years and is a significant cause of early mortality and morbidity.143 Parks and colleagues reported using RHDO in six pregnancies where parents were carriers of SMA to correctly define the fetal genotype.76 This test has since been introduced into clinical practice in the United Kingdom and this group has reported on 81 cases undergoing NIPD for SMA. They correctly identified 23 unaffected, 34 carrier, and 15 affected fetuses. Complete diagnosis was not possible in the remaining nine cases due to low fetal fraction, recombination, or lack of informativity regarding the maternal allele.139 Congenital adrenal hyperplasia

CAH is an autosomal recessive inborn error of cortisol biosynthesis. More than 90–95 percent of CAH cases are caused by steroid 21-hydroxylase deficiency caused by mutations in the CYP21A2 gene, a condition that affects 1:14,000–1:18,000 liveborns.98, 144, 145 CAH is characterized by impaired glucocorticoid and mineralocorticoid synthesis, which causes excess androgen secretion that can result in virilization of female fetuses and premature adrenarche in males.98, 144, 145 The three main clinical presentations of CAH due to 21-hydroxylase deficiency – classic salt-wasting CAH, classic simple virilizing CAH, and nonclassic

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Noninvasive Prenatal Diagnosis and Screening for Monogenic Disorders Using Cell-Free DNA 331

CAH – can be differentiated by the degree by which mutations affect protein function. Although close to 300 different CYP21A2 mutations are known, about half of all classic CAH are caused by large deletions and a common splice site mutation in intron 2 of the CYP21A2 gene.98 Prenatal treatment of CAH with maternal dexamethasone can reduce the prenatal virilization of the external genitalia of female fetuses, but to be effective such treatment has to be initiated before the onset of genital development which begins at 9 weeks of gestation.98, 144, 145 This management is controversial in view of the potential adverse effects on neuronal development and in some countries is no longer used.146 CAH and fetal gender can be diagnosed prenatally by CVS between 11 and 13 completed weeks of gestation or by amniocentesis after 15 weeks. This means that seven out of eight women with male fetuses or unaffected female fetuses were unnecessarily exposed to the side effects of dexamethasone treatment.68 Initially, cfDNA-based noninvasive screening for fetal sex was done to avoid treating male fetuses,97 but even with this, 75 percent of treated female at-risk pregnancies are actually unaffected. The initial attempts for disease-specific NIPD for CAH relied on sex determination and subsequent paternal exclusion testing to identify the fetuses that are predicted to be either unaffected or carriers for the disease.57 New and colleagues first reported their development of NIPD methods for fetal CAH in 2012,147 and in 2014 described a collaborative study wherein they successfully determined the fetal genotype of seven affected, five carriers, and two unaffected fetuses in 14 at-risk pregnancies by RHDO as early as 5–6 weeks of gestation for some.68 To achieve this, they developed and validated an MPS assay of a captured 6 Mb polymorphic region flanking CYP21A2 and deduced the parental haplotypes from sequencing the affected proband and parental samples, which allowed them to subsequently determine which alleles were inherited by the fetus.68 An additional case of successful NIPD for CAH using a haplotype-based approach on the sequenced exome was reported shortly after this.148 In 2016, Drury et al. reported on a similar assay targeting 6,700 heterozygous SNPs in a 7 Mb CYP21A2 region that was successfully validated in two sets of three families;19 validation is ongoing

to implement this test in the UK National Health Service. Ma et al. reported 14 samples from 12 independent families using an RHDO approach as early as 8 weeks of gestation.69 However, because adequate fetal fractions are required for the RHDO approach, a later gestation of at least 10–11 weeks is recommended. Hemoglobinopathies

Hemoglobinopathies, including sickle cell anemia, α-thalassemia, β-thalassemia, and anemias caused by rarer globin variants, are the most common autosomal recessive disorders worldwide (see Chapter 27), with many cases occurring in low-resource countries149 and many groups have reported development of cfDNA-based NIPD assays for these hemoglobinopathies. As with other recessive conditions, initial attempts focused on exclusion of the paternally inherited alleles,150–153 but this was soon followed by approaches to directly diagnose the fetal genotype noninvasively. However, to our knowledge this is yet to be offered routinely in an accredited molecular genetic diagnostic service. This is largely due to the fact that they are recessive conditions with multiple causative mutations, but whereby parents frequently carry the same mutation, making analysis complex.

Sickle cell anemia More than 90 percent of sickle cell anemia (see Chapter 27) is caused by biallelic inheritance of the hemoglobin β gene (HBB) gene rs334 variant (p.Glu6Val, HbS or βS) causing HbSS disease, or by co-inheritance in trans of this variant with the rs33930165 (p.Glu6Lys, HbC or βC) variant, causing milder HbSC disease. Other forms of sickle cell disease include compound heterozygous inheritance of HbS with a β-thalassemia variant or HbS with other rarer β-globin variants.95, 154 Due to the protective effect of the HbS variant against malaria, carrier frequencies for sickle cell trait (HbAS) are 1:12 for African Americans and more than 20 percent in sub-Saharan Africa with more than 200,000 births worldwide and 1,000 in the United States annually with sickle cell anemia.149, 155 In the first proof-of-principle study of cfDNA-based NIPD for sickle cell anemia, digital PCR was used for RMD for the pathogenic variant along with an assay for

332 Genetic Disorders and the Fetus

DYS14 for male pregnancies and a panel of biallelic polymorphic indel markers for female pregnancies to quantify the fetal fraction. This yielded an overall accuracy of 82 percent (37/45) in male and 75 percent (15/20) in female pregnancies and 100 percent accuracy with a fetal fraction above 7 percent.62 More recently, optimized assays have been developed.95, 149, 156 PCR amplification of HbS variant-containing amplicons combined with NGS and fetal fraction determination by RASSF1A promoter methylation analysis on 57 cfDNA samples from women between 8 and 17 weeks of gestation yielded overall sensitivity of 94 percent, specificity of 88 percent, positive predictive value (PPV) of 75 percent, and negative predictive value (NPV) of 98 percent. Specificity and PPV were 100 percent when fetal fraction was above 4 percent.149 A sensitive sequencing-based molecular counting assay with barcoded spiked-in Quantitative Counting Templates (QCTs) for cfDNA-based NIPT for various autosomal recessive single-gene disorders, including sickle cell anemia, α-thalassemia, β-thalassemia, cystic fibrosis, and SMA had >98 percent analytical sensitivity and >99 percent analytical specificity during assay development, but no actual samples from carriers of sickle cell trait were tested during validation.95 A targeted MPS-based RMD assay with incorporated unique molecular identifiers (UMIs) for NIPD of fetal sickle cell anemia, without need for paternal or proband samples, was tested on cfDNA samples from 42 pregnant HbS carriers, 15 with HbSS disease, and seven with HbSC disease at or above 8 weeks of gestational age. It yielded 100 percent clinical sensitivity and specificity with fetal fractions >4 percent.156 One sample with a fetal fraction 90–95 percent had at least one cell scorable for aneuploidy only.203 Although promising, evaluation of performance for single-gene disorder diagnosis from circulating trophoblast has been limited. One group demonstrated success with diagnosis of cystic fibrosis from circulating trophoblast cells isolated from maternal blood, but we are not aware of additional follow-up studies with this method.192 A recent study attempted diagnosis of Huntington’s disease and other single-gene disorders from circulating

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trophoblasts with moderate success.205 Although the access to pure fetal genome is attractive, the biggest challenge with intact fetal (trophoblast) cell analysis is the small number of cells that can be recovered. In addition, significant validation is needed to determine how many cells are required to avoid the possibility of inaccurate results due

to allele dropout that has already been reported in individual cells. This will require approaches to improve cell recovery and assays that are optimized for small samples, similar to strategies developed for preimplantation genetic testing for monogenic disorders. How this will compare to cfDNA-based testing remains to be determined.

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169. Byrou S, Makrigiorgos GM, Christofides A, et al. Fast temperature-gradient COLD PCR for the enrichment of the paternally inherited SNPs in cell free fetal DNA; an application to non-invasive prenatal diagnosis of beta-thalassaemia. PLoS One 2018;13(7):e0200348. 170. Liu S, Chen L, Zhang X, et al. Primer-introduced restriction analysis polymerase chain reaction method for non-invasive prenatal testing of beta-thalassemia. Hemoglobin 2015;39(1):18. 171. Breveglieri G, Travan A, D’Aversa E, et al. Postnatal and non-invasive prenatal detection of beta-thalassemia mutations based on Taqman genotyping assays. PLoS One 2017;12(2):e0172756. 172. Papasavva T, van Ijcken WF, Kockx CE, et al. Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to beta-thalassaemia. Eur J Hum Genet 2013;21(12):1403. 173. 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 N Y Acad Sci 2006;1075:103. 174. Sirichotiyakul S, Charoenkwan P, Sanguansermsri T. Prenatal diagnosis of homozygous alpha-thalassemia-1 by cell-free fetal DNA in maternal plasma. Prenat Diagn 2012;32(1):45. 175. Pornprasert S, Sukunthamala K, Kunyanone N, et al. Analysis of real-time PCR cycle threshold of alpha-thalassemia-1 Southeast Asian type deletion using fetal cell-free DNA in maternal plasma for noninvasive prenatal diagnosis of Bart’s hydrops fetalis. J Med Assoc Thai. 2010;93(11):1243. 176. Ho SS, Chong SS, Koay ES, et al. Noninvasive prenatal exclusion of haemoglobin Bart’s using foetal DNA from maternal plasma. Prenat Diagn 2010;30(1):65. 177. Li X, Yang T, Li CS, et al. Prenatal detection of thalassemia by cell-free fetal DNA (cffDNA) in maternal plasma using surface enhanced Raman spectroscopy combined with PCR. Biomed Opt Express 2018;9(7):3167. 178. Yan TZ, Mo QH, Cai R, et al. Reliable detection of paternal SNPs within deletion breakpoints for non-invasive prenatal exclusion of homozygous alpha-thalassemia in maternal plasma. PLoS One 2011;6(9):e24779. 179. Ge H, Huang X, Li X, et al. Noninvasive prenatal detection for pathogenic CNVs: the application in alpha-thalassemia. PLoS One 2013;8(6):e67464. 180. Wang W, Yuan Y, Zheng H, et al. A pilot study of noninvasive prenatal diagnosis of alpha- and beta-thalassemia with target capture sequencing of

344 Genetic Disorders and the Fetus

cell-free fetal DNA in maternal blood. Genet Test Mol Biomarkers 2017;21(7):433. 181. Yang J, Peng CF, Qi Y, et al. Noninvasive prenatal

193. Beaudet AL. Using fetal cells for prenatal diagnosis: History and recent progress. Am J Med Genet C Semin Med Genet 2016;172(2):123.

detection of hemoglobin Bart hydrops fetalis via mater-

194. Breman AM, Chow JC, U’Ren L, et al. Evidence for

nal plasma dispensed with parental haplotyping using

feasibility of fetal trophoblastic cell-based noninvasive

the semiconductor sequencing platform. Am J Obstet Gynecol 2020;222(2):185 e1.

prenatal testing. Prenat Diagn 2016;36(11):1009. 195. Normand E, Qdaisat S, Bi W, et al. Comparison of

182. ACOG. Practice advisory: cell-free DNA to screen

three whole genome amplification methods for detec-

for single-gene disorders, 2019, updated Feb 21,

tion of genomic aberrations in single cells. Prenat Diagn

2019. https://www.acog.org/Clinical-Guidance-andPublications/Practice-Advisories/Cell-free-DNA-toScreen-for-Single-Gene-Disorders 183. Lewis C, Hill M, Chitty L. Non-invasive prenatal diagnosis for single gene disorders: experience of patients. Clin Genet 2014;85(4):336. 184. Hill M, Oteng-Ntim E, Forya F, et al. Preferences for prenatal diagnosis of sickle-cell disorder: a discrete choice experiment comparing potential service users and health-care providers. Health Expect 2017;20(6):1289. 185. National Health Service. UK national genomic test directory, 2018, updated 20 March 2019. https://www .england.nhs.uk/publication/national-genomic-testdirectories/ 186. Wilkie AOM, Goriely A. Gonadal mosaicism and non-invasive prenatal diagnosis for ‘reassurance’ in sporadic paternal age effect (PAE) disorders. Prenat Diagn 2017;37(9):946. 187. Deans Z, Hill M, Chitty LS, et al. Non-invasive prenatal testing for single gene disorders: exploring the ethics. Eur J Hum Genet 2013;21(7):713. 188. Skirton H, Goldsmith L, Chitty LS. An easy test but a hard decision: ethical issues concerning non-invasive prenatal testing for autosomal recessive disorders. Eur J Hum Genet 2015;23(8):1004.

2016;36(9):823. 196. Kolvraa S, Singh R, Normand EA, et al. Genome-wide copy number analysis on DNA from fetal cells isolated from the blood of pregnant women. Prenat Diagn 2016;36(12):1127. 197. Vestergaard EM, Singh R, Schelde P, et al. On the road to replacing invasive testing with cell-based NIPT: five clinical cases with aneuploidies, microduplication, unbalanced structural rearrangement or mosaicism. Prenat Diagn 2017;37(11):1120. 198. Huang CE, Ma GC, Jou HJ, et al. Noninvasive prenatal diagnosis of fetal aneuploidy by circulating fetal nucleated red blood cells and extravillous trophoblasts using silicon-based nanostructured microfluidics. Mol Cytogenet 2017;10:44. 199. Chen F, Liu P, Gu Y, et al. Isolation and whole genome sequencing of fetal cells from maternal blood towards the ultimate non-invasive prenatal testing. Prenat Diagn 2017;37(13):1311. 200. Wei X, Ao Z, Cheng L, et al. Highly sensitive and rapid isolation of fetal nucleated red blood cells with microbead-based selective sedimentation for non-invasive prenatal diagnostics. Nanotechnology 2018;29(43):434001. 201. Vossaert L, Wang Q, Salman R, et al. Reliable detection

189. Westgren M, Gotherstrom C. Stem cell transplantation

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190. Verhoef TI, Hill M, Drury S, et al. Non-invasive pre-

202. Huang ZW, Fong CY, Gauthaman K, et al. Biol-

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2018;38(9):673.

191. Jenkins LA, Deans ZC, Lewis C, et al. Delivering an

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192. Mouawia H, Saker A, Jais JP, et al. Circulating tro-

204. Huang Y, Situ B, Huang L, et al. Nondestructive identi-

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205. Cayrefourcq L, Vincent MC, Pierredon S, et al. Single circulating fetal trophoblastic cells eligible for non invasive prenatal diagnosis: the exception rather than the rule. Sci Rep 2020;10(1):9861. 206. Imudia AN, Suzuki Y, Kilburn BA, et al. Retrieval of trophoblast cells from the cervical canal for prediction of abnormal pregnancy: a pilot study. Hum Reprod 2009;24(9):2086. 207. Bolnick AD, Fritz R, Jain C, et al. Trophoblast retrieval and isolation from the cervix for noninvasive, first trimester, fetal gender determination in a carrier of congenital adrenal hyperplasia. Reprod Sci 2016;23(6):717. 208. Jain CV, Kadam L, van Dijk M, et al. Fetal genome profiling at 5 weeks of gestation after noninvasive isolation of trophoblast cells from the endocervical canal. Sci Transl Med 2016;8(363):363re4. 209. Kadam L, Jain C, Kohan-Ghadr HR, et al. Endocervical trophoblast for interrogating the fetal genome and assessing pregnancy health at five weeks. Eur J Med Genet 2019:103690. 210. Pfeifer I, Benachi A, Saker A, et al. Cervical trophoblasts for non-invasive single-cell genotyping and prenatal diagnosis. Placenta 2016;37:56.

211. Au PK, Kwok YK, Leung KY, et al. Detection of the S252W mutation in fibroblast growth factor receptor 2 (FGFR2) in fetal DNA from maternal plasma in a pregnancy affected by Apert syndrome. Prenat Diagn 2011;31(2):218. 212. Raymond FL, Whittaker J, Jenkins L, et al. Molecular prenatal diagnosis: the impact of modern technologies. Prenat Diagn 2010;30(7):674. 213. Bustamante-Aragones A, Trujillo-Tiebas MJ, Gallego-Merlo J, et al. Prenatal diagnosis of Huntington disease in maternal plasma: direct and indirect study. Eur J Neurol 2008;15(12):1338. 214. Gonzalez-Gonzalez MC, Garcia-Hoyos M, Trujillo-Tiebas MJ, et al. Improvement in strategies for the non-invasive prenatal diagnosis of Huntington disease. J Assist Reprod Genet 2008;25(9–10):477. 215. Bustamante-Aragones A, Perez-Cerda C, Perez B, et al. Prenatal diagnosis in maternal plasma of a fetal mutation causing propionic acidemia. Mol Genet Metab 2008;95(1–2):101.

9

Amniocentesis, Chorionic Villus Sampling, and Fetal Blood Sampling Anthony O. Odibo University of South Florida, Tampa, FL, USA

Introduction

Amniocentesis

Amniocentesis, chorionic villus sampling (CVS), and, to a lesser extent, fetal blood sampling are the most common prenatal diagnostic techniques. Amniocentesis was first used in Germany in the early 1880s to treat polyhydramnios.1, 2 Earlier uses of amniocentesis included aiding in evaluating the fetus to localizing the placenta, and even as a method of pregnancy termination by injecting hypertonic saline into the amniotic cavity.3, 4 In 1950, Alvarez performed amniocentesis to assess fetal wellbeing.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 In the mid-1950s amniocentesis for exclusively genetic indications evolved when several investigators demonstrated that fetal sex could be determined by X-chromatin analysis of amniotic fluid cells (AFCs).7–9 Over the next few years, several reports of successful diagnosis of a wide variety of chromosomal and metabolic disorders were published, helping to establish amniocentesis as an integral part of modern obstetric care.10–16 This chapter addresses current techniques and the safety of genetic amniocentesis, CVS, and fetal blood sampling. Indications and methods of prenatal diagnosis are considered in detail throughout this text.17–22

Prerequisites

Ideally, couples should have the opportunity to discuss their genetic risks and available antenatal testing options before pregnancy.17, 21, 23 This may necessitate genetic counseling and 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 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 is experienced in this procedure, has high-quality ultrasonography available, and has access to a laboratory with experience in performing prenatal diagnostic studies.20, 24, 25 Only obstetricians should perform the procedure 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

Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, Eighth Edition. Edited by Aubrey Milunsky and Jeff M. Milunsky. © 2021 Aubrey Milunsky and Jeff M. Milunsky. Published 2021 by John Wiley & Sons Ltd.

346

CHAPTER 9

Amniocentesis, Chorionic Villus Sampling, and Fetal Blood Sampling 347

either perform the abortion or refer the family to a provider who will act on their request.26 Timing

Amniocentesis is typically performed between 15th and 16th weeks of gestation, when 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. Since most couples wait for the findings of detailed anatomic ultrasound survey prior to amniocentesis, the majority of procedures are now performed between the 18th and 20th weeks of gestation. Early amniocentesis performed before 14 weeks of gestation is discussed below. Transvaginal amniocentesis is only of historical interest, because of its technical difficulty and because of associated infection and spontaneous abortion.28 Technique

Amniocentesis for genetic diagnosis is typically performed in an outpatient facility. A careful ultrasonographic examination is performed and a needle insertion site is selected. The needle is inserted, employing concurrent ultrasound guidance to correspond to the location of the optimal pocket of AF while avoiding the fetus. Although Tabor et al.29 reported that transplacental needle insertion increased the risk of the procedure, this has not been confirmed by others.30, 31 The umbilical cord and its insertion site should be especially identified and avoided. The maternal bowel and bladder should also be located and avoided. A local anesthetic (e.g. 2–3 mL of 1 percent xylocaine) may or may not be used; however, local anesthesia, including the use of creams or subfreezing the needle, does not appear to affect the level of pain of the procedure.32–35 It has been suggested that needle insertion through the upper third of the uterus is less painful than insertion through the lower two-thirds; however, data to substantiate this claim are limited.36 Counseling before amniocentesis should emphasize that the actual pain and anxiety experienced during the procedure are significantly lower than expected.37, 38 After the maternal skin has been cleansed with an iodine-based and/or alcohol-based solution, sterile

drapes are placed around the needle insertion site to help maintain an aseptic field. A disposable 22-gauge spinal needle with stylet is most frequently used and recommended. During the entire procedure, two-dimensional, real-time ultra-sonographic monitoring with continuous visualization of the needle should be performed. Use of four-dimensional ultrasound guidance has been suggested, but there are no objective data to indicate improved outcomes.39 Ultrasound gel is applied adjacent to the insertion site, and a real-time transducer is held in position by an assistant such that the ultrasound beam is directed at a 15–20∘ angle from the parallel of the planned needle track (Figure 9.1). After assurance that the needle is in its proper location, the stylet is moved and a 10 or 20 mL syringe attached. The tip is typically more easily identified on removal of the stylet. If freely flowing AF is not obtained on aspiration, the needle must be repositioned with stylet in place. The first several milliliters are theoretically most likely to contain maternal cells from blood vessels, the abdominal wall, or the myometrium; therefore, this initial sample is usually discarded or set aside for AF 𝛼-fetoprotein (AFAFP) assay. Twenty to thirty milliliters of AF are aspirated into sterile, disposable plastic syringes, although as little as 3–5 mL of AF has been shown to suffice for reliable prenatal cytogenetic results.40,41 Maternal cell contamination appears to occur more frequently in genetic amniocentesis samples that are obtained by physicians who perform 40 years (5.1 percent). Women with a history of vaginal bleeding during the current pregnancy also had a higher fetal loss rate (6.5 percent) compared with controls (2.8 percent). Women with a history of previous spontaneous abortions/terminations had a fetal loss rate of 8 percent, compared with a 2.8 percent loss rate among controls.

First- and Second-Trimester Evaluation of Risk (FaSTER) Trial Research Consortium In 2006, Eddleman et al. reported the procedurerelated fetal loss rate after midtrimester amniocentesis using the database from the NICHD-sponsored multicenter First- and Second-Trimester Evaluation of Risk (FaSTER) trial designed to compare first-trimester Down syndrome screening with nuchal translucency, pregnancy-associated plasma protein A, and free β-human chorionic gonadotropin (β-hCG) to second-trimester screening with AFP, hCG, unconjugated estriol, and inhibin A.148 Among a total of 35,003 patients who were enrolled in the FaSTER trial, 3,096 underwent midtrimester amniocentesis (study group) and 31,907 did not (control group). The rate of fetal loss 1,300

Huntington disease

AD

CAG

6–36

–

35–121

Kennedy disease (spinal bulbar muscular atrophy)

X

CAG

12–34

–

40–62

Machado–Joseph disease

AD

CAG

13–36

–

68–79

Myotonic dystrophy type 1

AD

CTG

5–37

–

50 to >2,000

Myotonic dystrophy type 2d

AD

CCTG

500

Spinocerebellar ataxia type 1

AD

CAG

6–39

–

41–81

Spinocerebellar ataxia type 2

AD

CAG

15–29

–

35–59

Spinocerebellar ataxia type 6

AD

CAG

4–16

–

21–27

Spinocerebellar ataxia type 7

AD

CAG

4–18

–

37–130

Spinocerebellar ataxia type 8

AD

CTG

16–37

–

>90 >19,000

Spinocerebellar ataxia type 10e

AD

ATTCT

10–22

–

Spinocerebellar ataxia type 12

AD

CAG

7–28

–

66–78

Spinocerebellar ataxia type 17

AD

CAG

27–44

–

>45 >2,500

Spinocerebellar ataxia type 31e

AD

TGGAA

Not detected

–

Spinocerebellar ataxia type 36f

AD

GGCCTG

3–14

–

650 to 2,500

Spinocerebellar ataxia type 37e

AD

ATTTC

Not detected

–

31–75

a AD,

autosomal dominant; AR, autosomal recessive; X, X-linked.

b Variable

ranges reported and overlapping sizes may occur.

c Mutation

may not involve an expansion.

d Expansion

involves four nucleotides.

e Expansion

involves five nucleotides.

f Expansion

involves six nucleotides.

Sources: La Spada AR, Taylor JP. Repeat expansion disease: progress and puzzles in disease pathogenesis. Nat Rev Genet 2010;1:247;119 López Castel A, Cleary JD, Pearson CE. Repeat instability as the basis for human diseases and as a potential target for therapy. Nat Rev Mol Cell Biol 2010;11:165;120 Mootha VV, Gong X, Ku HC, et al. Association and familial segregation of CTG18.1 trinucleotide repeat expansion of TCF4 gene in Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci 2014;55:33;121 Rodriguez CM, Todd PK. New pathologic mechanisms in nucleotide repeat expansion disorders. Neurobiol Dis 2019;130:104515;122 Tian Y, Wang JL, Huang W, et al. Expansion of human-specific GGC repeat in neuronal intranuclear inclusion disease-related disorders. Am J Hum Genet 2019;105:166;123 Swinnen B, Robberecht W, Van Den Bosch L. RNA toxicity in non-coding repeat expansion disorders. EMBO J 2020;39:e101112;124 Ueyama M, Nagai Y. Repeat expansion disease models. Adv Exp Med Biol 2018;1076:63;125 Babi´c Leko M, Župunski V, Kirincich J, et al. Molecular mechanisms of neurodegeneration related to C9orf72 hexanucleotide repeat expansion. Behav Neurol 2019;2019:2909168;126 Goodman LD, Bonini NM. Repeat-associated non-AUG (RAN) translation mechanisms are running into focus for GGGGCC-repeat associated ALS/FTD. Prog Neurobiol 2019;183:101697;127 LaCroix AJ, Stabley D, Sahraoui R, et al. GGC repeat expansion and exon 1 methylation of XYLT1 Is a common pathogenic variant in Baratela-Scott syndrome. Am J Hum Genet 2019;104:35;128 Stoyas CA, La Spada AR. The CAG-polyglutamine repeat diseases: a clinical, molecular, genetic, and pathophysiologic nosology. Handb Clin Neurol 2018;147:143;129 Saade JS, Xing C, Gong X, et al. Instability of TCF4 triplet repeat expansion with parent-child transmission in Fuchs’ endothelial corneal dystrophy. Invest Ophthalmol Vis Sci 2018;59:4065;130 and Nguyen L, Cleary JD, Ranum LPW. Repeat-associated non-ATG translation: molecular mechanisms and contribution to neurological disease. Annu Rev Neurosci 2019;42:227.131

582 Genetic Disorders and the Fetus

anticipation has also been reported.134 Clinical presentations include psychosis, hallucinations, delusions, bulbar involvement, dementia, and motor disability. More widespread knowledge of the manifestations of C9orf72 expansion mutations and the 50 percent risk of transmission is likely to invoke a greater use of prenatal diagnosis. Although technical problems in assays for the C9orf72 hexanucleotide expansions have been noted,136 Rollinson et al.137 have reported a repeat expansion mutation initially undetectable by Southern blotting. In the two brothers studied they identified a 10-base pair deletion adjacent to the expansion that interfered with the genotyping. In spinocerebellar ataxia (SCA) type 10, an autosomal dominant neurodegenerative disorder, interruptions in the pentanucleotide (ATTCT) repeat expansions are associated with a paradoxical contraction in intergenerational repeat size.138 An inverse correlation has been noted between the expansion size and the age of onset.138 For many of the SCAs (Table 14.2), clinical onset is in the reproductive years, posing personal challenges for affected individuals (self-extinction) considering prenatal diagnosis. It is important to recall that unstable repeat expansions occur in the following SCA types: 1, 2, 3, 6, 7, 8, 10, 12, 17, 31, and 36, while conventional mutations characterize other SCAs, including types 5, 11, 13, 14, 15, 20, 23, 27, 28, 35, and 38.139, 140 SCA type 37 is caused by an intronic insertion of the pentanucleotide ATTTC in between two polymorphic ATTTT tracks in the DAB1 gene.141 Long-range PCR with Sanger sequencing should reliably detect the pathogenic ATTTC repeat insertion that is typically 31–75 repeats.142 However, repeat primed PCR may also detect the ATTTT repeats and should not be used due to the potential for false-negative/positive results. Autosomal dominant myotonic muscular dystrophy (DM1) (discussed in Chapter 1), due to a CTG expansion, occurs in 1 in 8,000, while the more common DM2 (1 in 1,830 in Finland) is often undiagnosed. DM2, due to a CTTG expansion, is milder, rarely has anticipation, and does not cause congenital myotonic dystrophy.143 Distinction between these two types is important because prenatal diagnosis is unlikely to be requested in DM2. Children with congenital myotonic muscular

dystrophy (DM1) are almost exclusively born to affected mothers. Fuchs endothelial corneal dystrophy is a common genetically heterogeneous disorder with the late-onset type 3 affecting approximately 4 percent of the population older than 40 years, resulting in impaired vision. This type is caused by a heterozygous CTG expansion in the TCF4 gene.144 An expanded allele >40 repeats increases the risk of the disorder (>30-fold increase).121 More severe disease is correlated with higher expansions145 similar to other triplet repeat disorders. However, given the documented incomplete penetrance146 it is not well suited for prenatal diagnosis. There are at least nine heritable disorders due to the expansion of polyalanine tracts.147 For those that are autosomal recessive or X-linked in origin, prenatal diagnosis is feasible. Only rarely would there be similar options for those transmitted as autosomal dominant. Similar to the unstable repeat expansions, clinical severity increases with the length of the expanded polyalanine tract in some of these genes (e.g. PHOX2B).148 Mosaicism

Somatic mosaicism is the consequence of postzygotic de novo mutations occurring in a portion of all cells that constitute our organs and is almost certainly underestimated in its frequency. Moreover, due to a broad range of factors, because of subtle phenotypic changes, technical limitations, and tissue differences, mosaicism is often undetected or undiagnosed. Indeed, many mutations that may be present in all cells may affect some tissues more than others. De novo mutations likely arise in the sperm or ovum of a parent in whom the fateful change is not determinable. Once transmitted, the mutations are found in all tissues of the offspring. Disease-causing mutations may occur during mitotic cell divisions, leading ultimately to offspring who are mosaic. Somatic mutations occur frequently in the preimplantation stage or during early embryogenesis.149, 150 It is surprising that somatic mosaicism is not more commonly encountered, as 48–90 percent of embryos studied were mosaic for at least chromosomal abnormalities.151 Further to the discussion in Chapter 1, the distinction between somatic and gonadal mosaicism may often depend upon the sensitivity of the assay used. Standard clinical assays missed detection of

CHAPTER 14

mutant alleles subsequently identified by a specialized assay with greater sensitivity.152–156 Campbell et al.157 studied 100 families with children who had genomic disorders due to rare deletion CNVs thought to be de novo. In that study, four cases of low-level somatic mosaicism were found in a parent’s blood, clearly (but unwittingly) increasing future risk. Of course, limited mosaicism in another tissue may not be known (or discoverable), yet be in association with concomitant undeterminable gonadal mosaicism. De novo somatic mutations are now recognized as a cause of neuronal migration and brain overgrowth disorders as well as intellectual disability, autism spectrum disorders, and epileptic encephalopathies.158 The occurrence and description of transmissible somatic mosaicism is well known for a wide range of genetic disorders (see Chapter 1). The extant issues of limited assay reliability in the face of low percentage mosaicism in blood introduces a significant reservation to conclusions that infer gonadal mosaicism for many described genetic disorders (Table 14.3). Clinical caution is therefore advised when considering a genetic disorder as sporadic or due to gonadal mosaicism. The risks of recurrence are likely to be significantly higher if undetected somatic mosaicism is present. Most new mutations are paternal in origin and increase in frequency with advancing age.271 Moreover, recurrence risk, according to in silico analysis, depends on the parent of origin, with paternal age being an important variable that modifies risk.272, 273 Young fathers who transmit a mutant gamete appear to have higher recurrence risks. In addition, it is has been demonstrated that de novo deletions and insertions that are associated with autism spectrum disorder are predominantly paternally derived.274 Somatic mutations are more common for deletions (more often related to a severe phenotype) than for other types of mutations in neurofibromatosis type 1.275 Microdeletions in the NF1 gene are reportedly associated with somatic mosaicism in 40 percent of cases.276 In neurofibromatosis type 2, estimates indicate a high frequency of somatic mosaicism (25–30 percent).277, 278 A study of 10,362 consecutive patients by exon-targeted high-resolution whole-genome oligonucleotide array concluded that somatic chromosomal mosaicism occurred in 0.55 percent of cases.279

Molecular Genetics and Prenatal Diagnosis 583

In disorders with significant frequencies of somatic mosaicism, pursuit of the diagnosis may require examination of other tissue besides blood. A good example is the Cornelia de Lange syndrome, which is characterized by distinctive craniofacial dysmorphism, growth restriction, intellectual disability, and limb malformations.280 This autosomal dominant or X-linked congenital malformation syndrome is known to arise as a consequence of mutations in seven different genes.281 Limited sensitivity of Sanger sequencing to detect somatic mosaicism occurring at a 10–20 percent level prompted the recommendation to examine skin fibroblasts or buccal epithelial cells, enabling confirmation of mosaicism.282–284 A similar example has occurred in Costello syndrome (facial dysmorphism and cardiovascular, skin, and musculoskeletal abnormalities along with tumor predisposition) in which no detectable mutations in blood DNA were observed. Subsequently the typical HRAS mutation was found in repeated buccal epithelial cells.285 More routine utilization of NGS technologies has yielded increased detection of somatic mosaicism in many disorders.155, 156 As each base pair is interrogated from dozens to hundreds of times, detection of lower levels of mosaicism (even as low as 0.4 percent) is now possible.286 Some monogenic disorders exist only in the mosaic state (e.g. Proteus syndrome), suggesting that a full complement of mutations would be lethal. Prenatal diagnosis of Proteus syndrome by ultrasound has been made following findings that included a cystic abdominal mass and malpositioned fingers287 and abdominal and pelvic cystic lymphangioma.288 Of these disorders (such as the Maffuci syndrome – overgrowth of vascular, lymphatic, soft and bone tissues) most probably exist primarily in the mosaic state with germline mutations being lethal.275 Somatic PIK3CA pathogenic variants have been described in a variety of overgrowth disorders of the brain and body segments.289–293 The phenomenon of revertant mosaicism is not likely to complicate efforts of parental or fetal studies aimed at prenatal diagnosis. Fetal skin biopsy to diagnose epidermolysis bullosa has now been effectively replaced by gene sequencing, thereby circumventing the possibility of sampling normal skin patches, which occur in about a third of these patients.294

584 Genetic Disorders and the Fetus

Table 14.3 Selected monogenic disorders with reported gonadal mosaicism Disorder

Inheritance

Reference

Achondrogenesis type II

AD

159

Achondroplasia

AD

160

Acro-cardio-facial syndrome

AR

161

Adrenoleukodystrophy

X-L rec

162, 163

Albright hereditary osteodystrophy

AD

164

𝛼-Thalassemia mental retardation syndrome

X-L

165

Alport syndrome

X-L, AR, AD

166, 167

Amyloid polyneuropathy

AD

168

Aniridia

AD

169

Ankyloblepharon–ectodermal defects–cleft

AD

170

lip/palate (AEC syndrome) Apert syndrome

AD

171

Arboleda–Tham Syndrome

AD

172

Bainbridge–Ropers syndrome

AD

173

Becker muscular dystrophy

X-L rec

174

Cantu syndrome

AD

175

Cardiofaciocutaneous syndrome

AD

176

Cerebellar ataxia with progressive macular

AD

177

dystrophy (SCA7) CHARGE syndrome

AD

178

Chondrodysplasia punctata

X-L rec

179

Coffin–Lowry syndrome

X-L dom

180

Congenital contractural arachnodactyly

AD

181

Congenital fibrosis of extraocular muscles

AD

182

Congenital central hypoventilation

AD

183, 184

syndrome Conradi–Hunnermann–Happle syndrome

X-L dom

185

Cornelia de Lange syndrome

AD

186

Costello syndrome

AD

187

Crouzon syndrome

AD

188

Danon disease

X-L

189

Dejerine–Sotas syndrome (HNSN III) with

AD

190

stomatocytosis Duchenne muscular dystrophy

X-L rec

191, 192

Dyskeratosis congenita

X-L

193

EEC syndrome (ectrodactyly, ectodermal

AD

194, 195

dysplasia, orofacial clefts) Epidermolysis bullosa, dystrophic

AR

196

Fabry disease

X-L rec

197

Facioscapulohumeral muscular dystrophy

AD

198

Factor X deficiency

AR

199

Familial adenomatous polyposis

AD

200, 201

Familial focal segmental glomerulosclerosis

AD

202

Familial hypertrophic cardiomyopathy

AD

203

Fibrodysplasia ossificans progressiva

AD

204

Fragile X syndrome (deletion type)

X-L

205

Fraser syndrome

AD

206

Glass syndrome

AD

207

Gonadal dysgenesis

AD

208 (Continued)

CHAPTER 14

Molecular Genetics and Prenatal Diagnosis 585

Table 14.3 (Continued) Disorder

Inheritance

Reference

Hartsfield syndrome

AD

209

Hemophilia B

X-L rec

210, 211

Hereditary angioedema (C1 inhibitor

AD, AR

212

Hereditary hemorrhagic telangiectasia

AD

213

Herlitz junctional epidermolysis bullosa

AR

214

Holt–Oram syndrome

AD

215

Hunter syndrome

X-L rec

216

Hypophosphatemic rickets

X-L dom

217

Incontinentia pigmenti

X-L dom

218

deficiency)

Karsch–Neugebauer syndrome

AD

219

Keratitis–ichthyosis deafness syndrome

AD

220

L1 syndrome

X-L rec

221

Lissencephaly (males); “subcortical band

X-L rec

222

heterotopia” (almost all females) LMNA-associated muscular dystrophy

AD

223

Malan syndrome

AD

224

Megalencephaly syndrome

AD

225

Microdeletion 1p36

AD

226

Microdeletion 15q11–q13 (Angelman

AD

227

Microdeletion 19p13.13

AD

228

Myotubular myopathy

X-L rec

229, 230

Nemaline myopathy

AD, AR

231

Neurodegeneration with brain iron

X-L

232

AD

233

AD

234

syndrome)

accumulation Neurodevelopmental disorder with involuntary movements (NEDIM) Neurodevelopmental disorder with severe motor impairment and absent language (NEDMIAL) Neurofibromatosis type 1

AD

235, 236, 237

Neurofibromatosis type 2

AD

238

Noonan syndrome

AD

239, 240

Oculocerebrorenal syndrome of Lowe

X-L

241

Oculofaciocardiodental

X-L dom

242

Ornithine transcarbamylase deficiency

X-L rec

243

Osteogenesis imperfecta

AD

244

Osteopathia striata

X-L dom

245

Otopalatodigital syndrome

X-L dom

246

Pallister–Hall syndrome

AD

247

Polycystic kidney disease

AD

248

Polycythemia–paraganglioma syndrome

AD

249

Pseudoachondroplasia

AD

250

Renal coloboma syndrome

AD

251

Retinoblastoma

AD

252, 253

Rett syndrome

X-L dom, AD

254, 255, 256

Rhabdoid tumor predisposition

AD

257, 258

Schizophrenia susceptibility

AD

259

syndrome/congenital cataracts

(Continued)

586 Genetic Disorders and the Fetus

Table 14.3 (Continued) Disorder

Inheritance

Reference

Severe combined immunodeficiency disease

X-L rec

260

Shprintzen–Goldberg syndrome

AD

261

Smith–Kingsmore syndrome

AD

262

Spondyloepimetaphyseal dysplasia

AD

263

Sotos syndrome

AD

264

Tuberous sclerosis

AD

265, 266

von Willebrand disease (type 2b)

X-L rec

267

Waardenburg syndrome

AD

268, 269

Wiskott–Aldrich syndrome

X-L rec

270

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

Imprinting and uniparental disomy

Epigenetic modifications that enable parent-oforigin preferential gene expression result in genomic imprinting.295 DNA methylation at CpG dinucleotides is the predominant modification that enables detection by methylation-sensitive PCR approaches to characterize this group of imprinting disorders. Imprinted domains containing more than 100 genes are clustered on several chromosomes, including 6, 7, 11, 14, 15, and 20.296–299 In the early embryo the previous methylation at these locations is reset to establish new parent-specific imprints.300, 301 Deletions and duplications of these imprinted regions can result in imprinting disorders. Uniparental disomy (see Chapter 1) in which two copies of an imprinted region are derived from one parent also results in these disorders (Table 14.4). Indications for the prenatal diagnosis of these disorders may include nonhomologous Robertsonian translocations involving chromosome 14 and/or 15 (risk significantly less than 1 percent),329, 330 the detection of chromosomal mosaicism,331 or the use of assisted reproductive technologies (ART).332–336 Genotype–phenotype correlations

Well-defined genotype–phenotype correlations exist for multiple disorders (see Chapter 1). For example, specific FBN1 mutations result in isolated ectopia lentis in patients that do not meet Ghent criteria for Marfan syndrome.337 More severe disease and earlier age of onset of neonatal Marfan syndrome has been documented for mutations in exons 24–32 of FBN1.338 Different types of

mutations within the same gene may lead to the same disorder with striking differences in severity. For the COL3A1 gene, significant difference in morbidity and mortality in vascular Ehlers–Danlos syndrome is associated with missense mutations, in contrast to truncating/frameshift/deletion mutations.339 Furthermore, compound heterozygosity for different types of mutations (i.e. leading to severe or mild effects on the protein) typify the resultant CBAVD (CFTR gene).340–342 For monogenic disorders many studies have identified modifier genes including microRNAs that influence the expression and severity of disease.343–345 Complicating matters further is the well-known variable expressivity and penetrance issues seen even within families affected by common genetic disorders.346 Complex genetic mechanisms exist, including digenic inheritance,347 which often confound accurate genetic counseling in these cases. Whole-exome studies have revealed multiple causative alleles for many conditions.348 Some of these conditions have differential tissue-specific results of specific classes of mutations. In addition, there are deleterious mutations that individually are necessary but not sufficient to cause disease without additional mutations.349 Hence, except for specific well-characterized known mutations, it is very difficult to predict a precise phenotype from genotype in the prenatal setting. Additional cautions and considerations

Extreme caution attends the use of all prenatal diagnostic modalities, given the potential for irreversible decisions concerning continuation (or

CHAPTER 14

Molecular Genetics and Prenatal Diagnosis 587

Table 14.4 Examples of imprinting and human disease Syndrome

Chromosomal location

Parental origin

Selected references

Albright hereditary osteodystrophy

20q13.32

Maternal

302

Angelman syndrome

15q11-q13

Maternal

303, 304

Autism

15q11-q13

Maternal

305

Beckwith–Wiedemann syndrome

11p15.5

Paternal

306, 307

Birk–Barel syndrome

8q24

Maternal

308

Congenital hyperinsulinism

11p15

Maternal

309

Congenital myotonic muscular dystrophy

19q13.3

Maternal

310

Early embryonic failure

21

Maternal

311

Familial paraganglioma

11q23

Paternal

312

Hereditary myoclonus–dystonia

7q21

Paternal

313

Intellectual disability and dysmorphism

14

Paternal

314

Intrauterine and postnatal growth restriction

7

Maternal

315

Intrauterine growth restriction or miscarriage

16

Maternal

316

Intrauterine growth restriction

11p15.5

Paternal

317

MatUPD14-like (Temple syndrome)

14

Maternal

318

PatUPD14-like syndrome

14

Paternal

319

Prader–Willi syndrome

15q11-q13

Paternal

320 321

Progressive osseous heteroplasia

20q13.3

Paternal

Pseudohypoparathyroidism 1a

20q13.3

Maternal

302

Pseudohypoparathyroidism 1b

20q13.3

Paternal

322

Pseudohypoparathyroidism

20q13.32

Paternal

323

Russell–Silver syndrome

7p11.2

Maternal

324

11p15

Maternal

325

11p15

Paternal

317

Schaaf–Yang syndrome

15q11.2-q12

Paternal

326

Short stature

14

Maternal

327

Transient neonatal diabetes

6q24.2

Paternal

328

not) of pregnancy. Complex considerations are necessary, with examples of a broad range (but commonly shared) of key caveats and cautions emphasized in the following discussion. There is a wide range of genetic disorders that result in cardiomyopathy. Causal genes and their mutations have been identified for many of these disorders, with more genes to be discovered. In families even with adult onset of hypertrophic cardiomyopathy, prenatal diagnosis could be a consideration, given that at least 16 genes have been identified, each encoding different components of the sarcomere. However, thus far, mutations have been recognized in only up to 60 percent of those affected and who have a family history.350 Complicating matters further, about 6 percent of affected individuals may possess more than one gene variant or, worse still, variants in more than one gene.351 Any consideration of prenatal

diagnosis for dilated cardiomyopathy requires precise molecular diagnosis focusing on the more than two dozen genes and their causal mutations. Again a wide range of primarily myopathic disorders can be considered, including the typical myopathies, limb-girdle muscular dystrophy type 2J, Barth syndrome, and the dystrophinopathies. Prenatal diagnosis can proceed only with precise determination of the culprit mutation. In addition to Noonan syndrome, discussed earlier, another RASopathy is the cardiofaciocutaneous syndrome (see Table 14.1). In this condition, hypertrophic cardiomyopathy, cellular dysplasias, septal defects, and dysrhythmias also occur. Thus far, four genes are known to be causally related to this syndrome with BRAF gene mutations accounting for about 75 percent of cases and the MAP2K2 for about 25 percent of cases. Given that neurological and variable intellectual disability is

588 Genetic Disorders and the Fetus

a constant in those with this disorder, prenatal diagnosis becomes a consideration. As the vast majority of cases occur from a de novo mutation, consideration of prenatal diagnosis would arise only if midtrimester ultrasound signs raised the question of a RASopathy. Craniofacial features may be similar to those described in Noonan syndrome, which, if suspected by ultrasound study, should lead to the offer of RASopathy molecular analysis (discussed earlier). An arrhythmogenic right ventricular cardiomyopathy may present in early childhood or at the mean age of diagnosis at 31 years. At least 14 genes are known to be casually related to this serious/lethal disorder.352 Where autosomal dominant inheritance applies (the frequency of de novo mutations is not known) and given the serious nature of this disorder, prenatal diagnosis or PGT would be a consideration once a precise mutation has been identified. Complicating this potential issue further is the fact that digenic inheritance might occur in a different but related arrhythmogenic cardiomyopathy gene, or the offspring could inherit one or two mutations.353 Digenic inheritance (heterozygous mutations in two separate autosomal recessive genes that only together result in the disorder) is well documented in families with hearing loss (e.g. connexin-26 and connexin-30)354, 355 and retinitis pigmentosa.356 These not-so-rare circumstances need careful attention when providing genetic counseling and prenatal diagnosis. Furthermore, triallelic inheritance has been described in Bardet–Biedl syndrome.357 The prenatal diagnosis and PGT of neurologic disorders were discussed in Chapters 1 and 2, respectively. Experience in the Netherlands has indicated that most individuals at 50 percent risk of inheriting Huntington disease (the paradigm for presymptomatic or predictive testing) prefer not knowing their possible presymptomatic status.358 It is likely that patients in the rest of the Western world have generally similar views. The Dutch investigators showed that, in their experience, exclusion prenatal diagnosis or exclusion PGT were acceptable for all the couples they studied. They emphasized yet again the importance of comprehensive and timely nondirective counseling, as well as professional and psychological support during and after the entire process of testing.

The study of cohorts of patients with neurodevelopmental and psychiatric disorders, using a wide range of technologies, has yielded inherited and de novo CNVs and rearrangements as well as single-nucleotide variants (SNVs).79, 359–364 Specifically, whole-exome/genome sequencing has revealed a plethora of de novo pathogenic variants, especially in autism.365–369 The study of many of these disorders by microarray previously revealed what was thought to be singularly pathogenic CNVs responsible for complex phenotypes such as autism. More recent studies have supported a multiple-hit model of autism spectrum disorders in some cases requiring both a pathogenic SNV as well as a susceptibility CNV to yield the specific clinical phenotype.370–372 Hence, care is needed in the prenatal diagnosis and counseling of these families that may only have one potential pathogenic variant in a multiple-hit model. In some of these cases, a maternally inherited CNV when combined with a paternally derived de novo single-nucleotide pathogenic variant may result in a specific neuropsychiatric phenotype, whereas inheritance of the maternal CNV alone may not. Thrombocytopenia-absent radius (TAR) syndrome is a disorder in which neonatal thrombocytopenia is present in combination with various radial ray defects (typically bilateral absence of the radii) with preservation of the thumbs. Various additional anomalies may be present in this autosomal recessive disorder. Most patients have a chromosome 1q21.1 deletion373 on one allele encompassing the RBM8A gene in trans with a typically hypomorphic (partial loss of gene activity) heterozygous RBM8A allele.374 About 50–75 percent of affected individuals have inherited the deletion from an unaffected carrier parent. The carrier frequency of one of the hypomorphic alleles is about 3 percent.375 Interestingly, TAR syndrome does not occur when two hypomorphic alleles in trans are detected. The rare occurrence of a homozygous chromosome 1q21.1 deletion is thought to be lethal. Hence, prenatal ultrasound detection of bilateral radial ray anomalies with present thumbs376 necessitates not only CNV interrogation for the chromosome 1q21.1 deletion, but also RBM8A gene sequencing to establish the molecular diagnosis of TAR syndrome.377, 378

CHAPTER 14

Determination of the pathogenic variants in a family will allow additional carrier screening and the opportunity for future PGT and prenatal diagnosis. There is a paucity of reports on the prenatal diagnosis of SCA.379–381 Currently there are at least 40 types of SCA, and a number associated with other neurological findings such as deafness. In a Cuban study of patients with SCA type 2, 28 out of 51 known presymptomatic carriers had affected fetuses, and 20 (71.43 percent) elected to terminate their pregnancies.380 These authors subsequently have drawn attention to couples where one has an expanded and a large normal allele, which may result in occurrences greater than 50 percent.381 Earlier, authors in Taiwan drew attention to the prenatal diagnosis of SCA3, in a case in which there was a contraction of repeat expansions from 78 in the father to 74 in the fetus.379 In another study of SCA patients examining the perceptions regarding reproductive options, the majority were most interested in PGT to avoid having affected children.382 A special caveat exists when performing prenatal diagnosis for repeat expansion disorders. Not infrequently, a fetus will appear to be homozygous for a normal allele (e.g. SCA2). It is important to know the allele sizes of the affected parent (and often the unaffected parent) in order not to miss a very large fetal expansion. Use of repeat primed PCR assay provides added reassurance of a normal-size homozygous allele.383, 384 Major advances have occurred in the understanding of the neurogenetic basis of epilepsy. Scores of newly recognized genes and their causal mutations have been documented, representing primarily de novo autosomal dominant genes, but also autosomal recessive and X-linked ones.385–389 Molecular diagnosis of those individuals with syndromic and nonsyndromic epilepsy with or without intellectual disability has improved anticipatory guidance for those affected, allowed more appropriate targeted therapies, and the ability to recognize family members who may be mildly affected.390, 391 A recent study of patients with epilepsy and intellectual disability has demonstrated an additional 10 percent positive molecular diagnosis if the previously reported negative WES data was reanalyzed 6–12 months later.392 Hence, as the databases and literature are updated, so too should our previously negative data be

Molecular Genetics and Prenatal Diagnosis 589

reanalyzed, especially in those in whom a VUS has been reported.393, 394 Additional molecular approaches including high-throughput automated patch clamping to assess the functional properties of a detected VUS have proven to be beneficial in the classification of these variants in the SCN5A gene.395 A parent with a history of seizures needs careful clinical assessment as gene analysis would be available and prenatal diagnosis and PGT would be an option for such conditions as tuberous sclerosis, neurofibromatosis, incontinentia pigmenti, and any gene in which the pathogenic variant(s) are established. The genetic basis of many myopathies has been resolved providing opportunities for prenatal diagnosis and PGT after mutation detection. Myotonic dystrophies (DM) types 1 and 2 as well as DMD/Becker muscular dystrophy were discussed in Chapter 1. A major concern is the affected pregnant woman with DM1 giving birth to a child with the severe congenital DM1, given the high risk of anticipation. A contrasting concern for DMD/Becker muscular dystrophy “noncarrier” females who have had one affected son is the 4–8 percent risk of recurrence due to gonadal mosaicism.98 Mutation detection in the proband is therefore crucial prior to further childbearing. Rarely two deletions or two duplications may be found in the DMD gene even if one such change did not disrupt the reading frame.396 A large study of 1,053 Chinese patients with DMD/Becker muscular dystrophy noted large rearrangements in the dystrophin gene in 70.56 percent of probands, 59.35 percent and 11.21 percent having deletions or duplications, respectively.397 A salutary lesson has emerged from a Dutch study of DMD during the periods 1961–1974 and 1993–2002.98 Remarkably the incidence of DMD did not decline, with the percentage of first affected boys increasing from 62 percent to 88 percent. The authors concluded that it took up to 5 years for a first diagnosis of DMD. Consequently recurrence was common. Moreover, female fetuses were not tested for their carrier status and years later went on to have affected children. They pressed for a change in policy with special reference to determining female carrier status during prenatal diagnosis and introducing neonatal screening of males. Requests for prenatal diagnosis of severe skin disorders are uncommon. Originally fetal skin

590 Genetic Disorders and the Fetus

biopsy was the only means to achieve prenatal detection. Subsequent recognition of causative genes has enabled molecular prenatal diagnosis. Consequently, Harlequin ichthyosis has been detected prenatally via sequencing of the ABCA12 gene.398 Prenatal sonographic features have been described.399 Despite familial phenotypic variability, prenatal diagnosis has also been successfully achieved or the diagnosis excluded by sequencing of the TGM1 gene in families with lamellar ichthyosis.400, 401 Prenatal diagnosis of epidermolytic ichthyosis or hyperkeratosis has so far been achieved by molecular analysis of ABCA12. In fact, for all three clinical phenotypes mentioned, mutations in this gene represent a major cause of severe autosomal recessive congenital ichthyoses.402, 403 X-linked steroid sulfatase deficiency (STS), most often suspected by the finding of low maternal serum estriol in the second trimester of pregnancy, is usually determined prenatally by demonstration of a deletion in the STS gene present in about 90 percent of families.404, 405 In about 10 percent of families, however, a contiguous gene deletion syndrome may be present and include the association of Kallman syndrome and chondrodysplasia punctata.406 Larger deletions in the STS gene are likely to be associated with intellectual disability and hypogonadism. Prenatal diagnosis of steroid sulfatase deficiency by FISH or microarray has been made repeatedly.407, 408 Cases of epidermolysis bullosa come to attention for prenatal diagnosis either because an unexplained elevation of amniotic fluid 𝛼-fetoprotein is observed or following the birth of a previously affected child. PGT has also been achieved with exclusion of the disorder by haplotyping in a fetus with Herlitz junctional epidermolysis bullosa.409 There are four categories and multiple subcategories of the autosomal recessive epidermolysis bullosa. For many years, fetal skin biopsy was used for prenatal diagnosis of lethal epidermolysis bullosa subtypes. This has now been replaced by NGS approaches.410 The prenatal detection of adult-onset potentially lethal disorders has steadily gained traction, including the neurodegenerative disorders, cardiomyopathies, and malignancies. The prenatal detection of a retinoblastoma, initially by linkage analysis and subsequently by routine molecular techniques, has been available for decades.

DNA analysis detects about 92 percent of the RB1 gene mutations in either bilateral and/or familial cases.411 An important potential pitfall is the presence of mosaicism.412, 413 Incidental detection of a fetal chromosomal abnormality involving the retinoblastoma gene locus on 13p following detection, for example, of increased nuchal translucency, could infer a likely future retinoblastoma.414 Although first- or second-trimester prenatal diagnosis for breast/ovarian cancer gene mutations are available, a likely more common future choice is PGT.415, 416 Similarly, patients at risk of having offspring with familial adenomatous polyposis, hereditary nonpolyposis colon cancer, or multiple endocrine neoplasia may select PGT or prenatal diagnosis.417–420 Noninvasive prenatal screening for these disorders may be available with known mutations (see Chapter 8).

Prenatal diagnosis of mitochondrial disorders Mitochondrial disorders are more common than originally thought and have an estimated prevalence of at least one case in 7,500 individuals.421, 422 Mitochondrial disorders may be due to mutations in either mtDNA (∼ 1/3) or nuclear DNA (∼ 2/3).423, 424 Primary mitochondrial diseases are due to defects in mtDNA, which are transmitted via maternal inheritance. mtDNA is a double-stranded circular molecule with 37 genes. This means that all mitochondria in the zygote derive from the ovum, thus far with rare exceptions.425–427 Hence, an affected mother with a mitochondrial disorder would transmit to all of her children, but subsequently her sons, while affected, would not be transmitters. A characteristic feature of this form of inheritance is that the pathogenic mutations in mtDNA are present in some but not necessarily all mitochondria. This situation, known as heteroplasmy, will be highly variable with greater clinical manifestations, reflecting high mutant loads. Moreover, the highly variable distribution of mutations within mtDNA will vary from tissue to tissue. Indeed, heteroplasmy may also occur in single mitochondria in which both normal and mutated mtDNA can be found. Tissues that are highly dependent on oxidative metabolism, such as the brain, heart, skeletal muscle, retina, renal

CHAPTER 14

tubules, and endocrine glands, are particularly vulnerable to the effects of mutations in mtDNA. Prenatal diagnosis of mitochondrial disorders is fraught with challenges. Extremely careful genetic counseling will inform an affected mother of the likely 100 percent transmission of her mutation to all her offspring. Key to the clinical manifestations is the size of the mutant load transmitted and the tissue distribution of the abnormal mitochondria. Hence, at-risk parents will have to understand that assessment of the mutant load from chorionic villi or amniocyte cells may not necessarily reflect ultimate fetal health and welfare.428 A low mutant load may not necessarily result in a child with few or no clinical features and, moreover, may be uninformative about future health. Vachin et al. have shown more reliable mutant load determinations from amniocytes than from chorionic villi.429 Nesbitt et al. describe their experience with prenatal diagnosis of mitochondrial disorders in the United Kingdom.430 Their approach includes ascertaining the degree of maternal heteroplasmy (blood and urine) and that of affected and unaffected maternal relatives. Utilization of NGS as well as quantitative real-time PCR assays have improved the accurate detection of heteroplasmy in the tissue being studied.431–433 Notwithstanding the obvious lack of guarantees in these circumstances, a number of cases have been reported for the prenatal diagnosis of Leigh syndrome, more specifically of the m.T8993G mutation434–437 and the m.T8993C mutation.438 “Successful” prenatal diagnosis of MELAS (myopathy, encephalopathy, lactic acidosis and stroke-like syndrome) has been reported.439 As some of the mitochondrial disorders, especially with significant heteroplasmy, have symptomatic presentation in adulthood, caution is needed with any prenatal prognostications. Prenatal diagnosis of nuclear mitochondrial genes transmitted as monogenic disorders is feasible if the familial mutations are known. Similarly, PGT is available for nuclear mitochondrial genes with known mutations (see Chapter 2). Amato et al. described novel three-parent in vitro fertilization (IVF) techniques designed to avoid the maternal transmission of mitochondrial disorders.440 Maternal spindle transfer is an approach in the setting of IVF to prevent the transmission of mtDNA mutations.441–443 Further larger studies are needed to determine efficacy and success rates.

Molecular Genetics and Prenatal Diagnosis 591

Reporting incidental (secondary) results SNP microarray analysis, WES, and WGS have inevitably led to the recognition of genomic abnormalities that were not the purpose of the intended study. When such unexpected observations reveal mutations that enable prediction with significant life-threatening risk, a responsibility inures to communicate with the patient or family. The American College of Medical Genetics and Genomics (ACMG) has published recommendations for reporting incidental findings444 and provided a “minimum” list of 56 (now 59) serious monogenic disorders requiring communication445, 446 (Table 14.5). This list, to which other disorders will undoubtedly be added, focuses on conditions where treatment is available or surveillance is necessary. Not included in the list are structural variants (e.g. translocations and inversions), repeat expansions, or CNVs. The ACMG report emphasized that a negative incidental findings report should not be misconstrued to imply the absence of a pathogenic variant. Moreover, the ACMG weighed the practical issues and limitations of genetic counseling in clinical practice as well as patients’ autonomy. A fiduciary duty to prevent harm by warning patients was recognized for both clinicians and reporting laboratories. Informed consent for patients undergoing sequencing about possible incidental findings provides them with the choice to opt out. A recent Dutch study examining 1,640 anonymized healthy individuals by WES, identified 1 in 38 with a medically actionable dominant (likely) pathogenic variant in the 59 ACMG recommended genes to be reported.447 Additional studies of varying sizes examined medically actionable pathogenic variants found in 56–114 genes and reported less than 3.4 percent.116, 448, 449 These results vary depending on gene selection, population studied, and criteria utilized to determine variant pathogenicity. Studies regarding patients’ perspectives,450 methods for return of the findings,451 as well as management,452 and implications for genetic counseling, are germane to prenatal diagnosis.68 Policies not to perform presymptomatic or predictive genetic testing of children have been in place

592 Genetic Disorders and the Fetus

Table 14.5 Conditions, genes, and variants recommended for return of incidental findings in clinical sequencing Phenotype Hereditary breast and ovarian cancer

Gene

Inheritancea

Variants to reportb

BRCA1

AD

KP and EP KP and EP

BRCA2 Li–Fraumeni syndrome

TP53

AD

Peutz–Jeghers syndrome

STK11

AD

KP and EP

Lynch syndrome

MLH1

AD

KP and EP

MSH2 MSH6 PMS2 Familial adenomatous polyposis

APC

AD

KP and EP

MYH-associated polyposis; adenomas,

MUTYH

ARc

KP and EP

multiple colorectal, FAP type 2; colorectal adenomatous polyposis, autosomal recessive, with pilomatricomas Juvenile polyposis

BMPR1A SMAD4

AD

KP and EP

Von Hippel–Lindau syndrome

VHL

AD

KP and EP

Multiple endocrine neoplasia type 1

MEN1

AD

KP and EP

Multiple endocrine neoplasia type 2

RET

AD

KP

Familial medullary thyroid cancer (FMTC)

RET

AD

KP

PTEN hamartoma tumor syndrome

PTEN

AD

KP and EP

Retinoblastoma

RB1

AD

KP and EP

Hereditary

SDHD

AD

KP and EP

paraganglioma–pheochromocytoma

SDHAF2

KP

syndrome

SDHC

KP and EP

SDHB Tuberous sclerosis complex

TSC1

KP and EP AD

KP and EP

TSC2 WT1-related Wilms tumor

WT1

AD

KP and EP

Neurofibromatosis type 2

NF2

AD

KP and EP

Ehlers–Danlos syndrome – vascular type

COL3A1

AD

KP and EP

Marfan syndrome, Loeys–Dietz syndromes,

FBN1

AD

KP and EP

and familial thoracic aortic aneurysms

TGFBR1

and dissections

TGFBR2 SMAD3 ACTA2 MYH11

Hypertrophic cardiomyopathy, dilated cardiomyopathy

MYBPC3

AD

KP and EP

MYH7

KP

TNNT2

KP and EP

TNNI3

KP

TPM1 MYL3 ACTC1 PRKAG2 GLA

XL

KP and EP (hemi, het, hom)

MYL2

AD

KP and EP

AD

KP

LMNA Catecholaminergic polymorphic ventricular

RYR2

tachycardia (Continued)

CHAPTER 14

Molecular Genetics and Prenatal Diagnosis 593

Table 14.5 (Continued) Phenotype

Gene

Inheritancea

Arrhythmogenic right ventricular

PKP2

AD

cardiomyopathy

Variants to reportb KP and EP

DSP

KP

DSC2

KP and EP

TMEM43 DSG2 Romano–Ward long QT syndromes types 1, 2, and 3, Brugada syndrome

KCNQ1

AD

KP and EP

KCNH2 SCN5A

Familial hypercholesterolemia

LDLR

SD

KP and EP

APOB

SD

KP

PCSK9

AD

Wilson disease

ATP7B

ARc

KP and EP

Ornithine transcarbamylase deficiency

OTC

XL

KP and EP (hemi, het, hom)

Malignant hyperthermia susceptibility

RYR1

AD

KP

CACNA1S a Some

conditions that may demonstrate semidominant inheritance (SD) have been indicated as autosomal dominant

(AD) for the sake of simplicity. Others have been labeled as X-linked (XL). b KP:

known pathogenic, sequence variation is previously reported and is a recognized cause of the disorder; EP: expected

pathogenic, sequence variation is previously unreported and is of the type which is expected to cause the disorder. The recommendation not to report expected pathogenic variants for some genes is due to the recognition that truncating variants, the primary type of expected pathogenic variants, are not an established cause of some diseases on the list. c We

recommend searching only for individuals with biallelic mutations.

Source: Modified from Kalia SS, Adelman K, Bale SJ, et al. Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics [published correction appears in Genet Med. 2017 Apr;19:484]. Genet Med 2017;19:249.446

for at least two decades.453–455 In the face of discovery of an incidental finding that may have serious health implications for a parent or concerning future reproduction, the age bar for testing has been lifted.

Ethical considerations in prenatal testing As our molecular and bioinformatic technologies rapidly progress, a crucial element in providing

prenatal diagnostic genomics is the recognition and understanding of parental perceptions and ethical implications (see Chapter 37 for in-depth discussion). Studies that examine parental, clinician, and researchers’ perspectives on prenatal genomics can promote more optimal care.456–459 The essential discussion of the ethical and counseling challenges of prenatal genomics will hopefully continue to positively impact the provision of standard prenatal care.460–463

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441. Costa-Borges N, Spath K, Miguel-Escalada I, et al. Maternal spindle transfer overcomes embryo developmental arrest caused by ooplasmic defects in mice. Elife. 2020;9:e48591. 442. Mobarak H, Heidarpour M, Tsai PJ, et al. Autologous mitochondrial microinjection; a strategy to improve the oocyte quality and subsequent reproductive outcome during aging. Cell Biosci 2019;9:95. 443. Zhang J, Liu H, Luo S, et al. Live birth derived from oocyte spindle transfer to prevent mitochondrial disease [published correction appears in Reprod Biomed Online. 2017 Jul;35:49. Konstandinidis, Michalis [corrected to Konstantinidis, Michalis]] [published correction appears in Reprod Biomed Online 2017;35:750]. Reprod Biomed Online 2017;34:361. 444. Green RC, Berg JS, Grody WW, et al. ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing. Genet Med 2013;15:565. 445. ACMG policy statement: updated recommendations regarding analysis and reporting of secondary findings in clinical genome-scale sequencing. Genet Med 2015;17:68. 446. Kalia SS, Adelman K, Bale SJ, et al. Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics [published correction appears in Genet Med. 2017 Apr;19:484]. Genet Med 2017;19:249. 447. Haer-Wigman L, van der Schoot V, Feenstra I, et al. 1 in 38 individuals at risk of a dominant medically actionable disease. Eur J Hum Genet 2019;27:325. 448. Olfson E, Cottrell CE, Davidson NO, et al. Identification of medically actionable secondary findings in the 1000 genomes. PLoS One 2015;10:e0135193. 449. Amendola LM, Dorschner MO, Robertson PD, et al. Actionable exomic incidental findings in 6503 participants: challenges of variant classification. Genome Res 2015;25:305. 450. Hart MR, Biesecker BB, Blout CL, et al. Secondary findings from clinical genomic sequencing: prevalence, patient perspectives, family history assessment, and health-care costs from a multisite study [published correction appears in Genet Med 2019 Jan 22]. Genet Med 2019;21:1100. 451. Schwartz MLB, McCormick CZ, Lazzeri AL, et al. A model for genome-first care: returning secondary genomic findings to participants and their healthcare providers in a large research cohort. Am J Hum Genet 2018;103:328.

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452. Katz AE, Nussbaum RL, Solomon BD, et al. Management of secondary genomic findings. Am J Hum Genet 2020;107:3. 453. American Society of Human Genetics Board of Directors and American College of Medical Genetics Board of Directors. Points to consider: ethical, legal, and psychosocial implications of genetic testing in children and adolescents. Am J Hum Genet 1995;57:1233. 454. Committee on Bioethics. Ethical and policy issues in genetic testing and screening of children. Pediatrics 2013;131:620. 455. Ross LF, Saal HM, David KL, et al. Technical report: ethical and policy issues in genetic testing and screening of children. Genet Med 2013;15:234. 456. Kalynchuk EJ, Althouse A, Parker LS, et al. Prenatal whole-exome sequencing: parental attitudes. Prenat Diagn 2015;35:1030. 457. Wou K, Weitz T, McCormack C, et al. Parental perceptions of prenatal whole exome sequencing (PPPWES) study. Prenat Diagn 2018;38:801.

458. Brew CE, Castro BA, Pan V, et al. Genetics professionals’ attitudes toward prenatal exome sequencing. J Genet Couns 2019;28:229. 459. Horn R, Parker M. Health professionals’ and researchers’ perspectives on prenatal whole genome and exome sequencing: ‘We can’t shut the door now, the genie’s out, we need to refine it’. PLoS One 2018;13:e0204158. 460. Dondorp WJ, Page-Christiaens GC, de Wert GM. Genomic futures of prenatal screening: ethical reflection. Clin Genet 2016;89:531. 461. Harris S, Gilmore K, Hardisty E, et al. Ethical and counseling challenges in prenatal exome sequencing. Prenat Diagn 2018;38:897. 462. Richardson A, Ormond KE. Ethical considerations in prenatal testing: Genomic testing and medical uncertainty. Semin Fetal Neonatal Med 2018;23:1. 463. Horn R, Parker M. Opening Pandora’s box? Ethical issues in prenatal whole genome and exome sequencing. Prenat Diagn 2018;38:20.

15

Prenatal Diagnosis of Cystic Fibrosis Wayne W. Grody UCLA School of Medicine and UCLA Medical Center, Los Angeles, CA, USA

For many practitioners in the prenatal setting, cystic fibrosis (CF) represents their first exposure to a “molecular disease” – one that is screened at the DNA level to reveal risks to the fetus that are otherwise not apparent. Indeed, since heterozygous carriers of recessive mutations for the disease are entirely normal, as are the fetuses in most cases, prenatal screening and diagnosis for CF was not even feasible until the discovery of the causative gene in 1989. Unlike the trisomies and neural tube defects that obstetricians could visualize by ultrasound and screen for using biochemical signs of the disorders (maternal α-fetoprotein and other serum markers), CF is, except in the minority of cases with meconium ileus/echogenic bowel, an invisible disorder to the obstetrician and one that moreover had formerly been exclusively within the purview of pediatricians. Thus, the advent of population-based carrier screening for CF mutations in the prenatal setting was a true paradigm shift for obstetricians at the time, although, as we now know, it was merely the harbinger of even more sophisticated DNA-based tests to come.

Genetics and epidemiology CF is often considered the most common lifethreatening autosomal recessive disorder in North America. It is clearly inherited in an autosomal recessive fashion, in which the parents of an affected child are both obligate carriers. Though

physiologically entirely normal themselves, the at-risk couple has a 1-in-4 chance with each pregnancy of having a child with CF. The disorder is most common among Caucasians of European ancestry (including Ashkenazi Jews), in whom the birth incidence is about 1 in 3,500. The prevalence of the disorder is about 30,000 in the United States and 60,000–70,000 worldwide.1, 2 Likewise, the carrier rate is highest among non-Hispanic Caucasians at 1 in 25, but also found at appreciable levels in other ethnic and racial groups (Table 15.1). It is because of this panethnic carrier rate that universal population carrier screening was deemed useful in order to identify couples at risk who otherwise would only learn of their carrier status through the birth of an affected child.

Clinical features Although usually thought of as a pulmonary disease, CF actually affects many different organs and tissues. What most of these features have in common is abnormal ion transport and abnormally viscous or salty secretions. In the classic form of the disease, these viscous secretions in the lung are not readily cleared by the usual mechanisms and become culture media for bacteria that, over the years, become resistant to the available antibiotics. The most notorious species in this regard is Pseudomonas aeruginosa, and many patients ultimately

Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, Eighth Edition. Edited by Aubrey Milunsky and Jeff M. Milunsky. © 2021 Aubrey Milunsky and Jeff M. Milunsky. Published 2021 by John Wiley & Sons Ltd.

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612 Genetic Disorders and the Fetus

Table 15.1 Cystic fibrosis mutation carrier frequencies and detection rates in various ethnic/racial groups Percentage

Approximate

of Group

carrier risk

mutations

Carrier

detected

frequency screening

after negative

African American

64

1/61

1/170

Ashkenazi Jewish

94

1/24

1/380

Asian American

49

1/94

1/180

Caucasian

88

1/25

1/200

72

1/58

1/200

Diagnosis

(non-Hispanic) Hispanic

fallopian tube secretions, but this is nowhere near as dramatic as in the male patients.3, 4 In the sweat glands, abnormal ion transport results in elevated levels of sodium and chloride secreted in the sweat. This is the basis of the well-known sweat test for diagnosis of CF5 and, in earlier days, clinicians licking the skin of suspected patients to assess the salty taste.

Source: Adapted from American College of Medical Genetics and Genomics, Technical Standards and Guidelines for CFTR Mutation Testing, 2006. Available at http://www.acmg.net

die from resistant infections by this organism or from the chronic lung damage it causes. Up until that time, they exhibit features of chronic obstructive pulmonary disease, with coughing, bronchitis and bronchiectasis, hypoxia, and recurrent pneumonias that often require admission to the hospital for treatment. In the gastrointestinal tract, viscous secretions can result in meconium ileus and intestinal obstruction. This affects under 10 percent of CF patients and is an emergency in the newborn period. In about 85 percent, the pancreatic ducts become obstructed, resulting in chronic malabsorption of fats and proteins and leading to failure to thrive in infants and young children. In the reproductive tract, almost all males with CF exhibit a congenital malformation called congenital bilateral absence of the vas deferens (CBAVD), which causes obstructive azoospermia and infertility. Of note, sperm production is normal, and these men can father children through the procedure of sperm aspiration. Conversely, some patients with atypical CF may experience only the infertility and be identified when they present to infertility clinics and are tested for CFTR mutations (which is now part of the standard workup for male infertility). (The CFTR genotypes associated with isolated CBAVD are quite complex and are discussed in detail later in this chapter.) There are some reports of compromised fertility in female CF patients, presumed due to viscous cervical or

Until the identification of the gene in 1989, diagnosis of CF in affected individuals relied primarily on clinical history with attention to the features described above. Family history is also contributory in those cases where there is a previously affected sibling or other relative, but more often the proband is the first case in the family. Two biochemical measurements are also used. For newborn screening, which is now offered in all 50 states and many other countries, measurement of immunoreactive trypsinogen (IRT) in the serum can be used as an early sign of pancreatic obstruction; this is in every sense a screening test, not a diagnostic one, and false-positive and false-negative results are common.6 IRT is elevated in neonates with early pancreatic changes; once full-blown pancreatic insufficiency develops, the IRT level falls below normal. For postnatal diagnosis, the sweat test, or sweat chloride test, has been the mainstay of clinical chemistry diagnosis of CF for many decades. Chloride concentrations above 60 mmol/L are generally considered diagnostic. Sweat chloride levels generally trend with the more classic or severe mutations, but there are exceptions and it is possible to have classic lung disease with normal sweat chloride, or vice versa. The sweat test requires skill and experience to perform properly, so false results are possible; also, it is said to be less reliable in the immediate newborn period.5 Lastly, diagnosis can be made at the molecular level by the observation of two mutations in the CFTR gene. Yet there are patients with undisputed clinical CF in whom only a single mutation is found, even by complete gene sequencing. It is assumed that such cases involve a second mutation in a noncoding region of the gene (not addressed by the sequencing test).

CHAPTER 15

Treatment Although there is as yet no cure for CF, advances in supportive therapies have dramatically increased the life expectancy, with median survival progressing from under 1 year 80 years ago to the mid-40s today. Treatments consist of everything from basic physical therapy to clear lung secretions to specific mutation-targeted molecular therapies. The viscosity of these secretions can be reduced for easier clearance by the administration of deoxyribonuclease-𝛼 (Pulmozyme , Genentech).7 Clearly a major credit for the increased lifespan is owed to the ever-expanding arsenal of antibiotics for treatment of new and resistant infections. The availability of pancreatic enzyme supplements addresses the problems of malabsorption, malnutrition, and failure to thrive. Steroidal and nonsteroidal anti-inflammatory drugs improve outcome by reducing reactive damage from the infections. For patients who eventually reach end-stage lung disease, lung transplant has proved a savior, assuming the patient survives the surgery and immunosuppression. For patients with concomitant cardiac disease secondary to pulmonary hypertension, combined heart–lung transplants have proven effective.8 Of course, the ultimate dream in CF management is correction of the basic defect by gene replacement therapy. Alas, despite a great deal of work in this area, significant hurdles remain in the form of adequate gene delivery to the target tissue (bronchial epithelium) and host response against the commonly used viral vectors. More recently, the advent of gene editing for mutation correction has offered promise, though again there are many technical challenges to overcome.9 In the interim, however, there have been exciting advances in what might be called the next best thing to gene therapy: direct targeting of specific mutations by small-molecule drugs, analogous to what is being done in oncology. These drugs are collectively called “CFTR modulators;” they represent an elegant example of “personalized medicine,” in that the treatment is tailored to the patient’s specific, personal mutation(s).10 The first such drug to emerge from the research phase and come on the market was ivacaftor (Kalydeco , Vertex Pharmaceuticals), which specifically targets the G551D mutation that is found in about 5

®

®

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613

percent of CF patients.11 The drug improves CFTR ion transport and therefore is called a “CFTR potentiator.” Other drugs have since emerged that target more common mutations, including the predominant mutation ΔF508, so that at present a triple-drug cocktail can be effective in about 90 percent of CF patients.12 It is worth considering the advent of these targeted therapies in the context of this chapter, since carrier screening is predicated on the fact that there is no cure for CF, yet these drugs would seem to put that goal within reach. The obvious question is whether couples will be less inclined to terminate affected pregnancies when the fetus carries one of these “targetable” mutations.13

Discovery of the cystic fibrosis gene The causative gene for CF was identified in 1989, which predates by 1 year the launch of the Human Genome Project. Thus, this gene, the target of an intense search for many years, was isolated the old-fashioned way, by linkage analysis within families followed by successive cloning, DNA “walking,” and DNA “jumping” to narrow down the region on chromosome 7, and finally the characterization of candidate genes within the region to see which one seemed to fit the features of CF based on tissue-specific gene expression. The identified gene, expressed in lung, nasal epithelium, sweat glands, colon and liver,14 was found to carry a mutation in the few subjects analyzed (the famous ΔF508 mutation) and thus fit the requirements. Like dystrophin before it, the protein product was named after the disease, which is unfortunate since the protein did not evolve merely to cause cystic fibrosis. Nevertheless, we are left with the ungainly name of cystic fibrosis transmembrane conductance regulator, with the gene name being CFTR. The CFTR gene and its protein product

The CFTR gene is relatively large, spanning about 250 kb and containing 27 exons.15 It is located on the long arm of chromosome 7 at 7q31.2. The CFTR protein contains 1,480 amino acid residues and has a molecular weight of 168 kDa. Its shared homology with known cell membrane transport proteins gave the first clue that it functions as

614 Genetic Disorders and the Fetus

an ion channel. Further study revealed it to be an active chloride transporter, dependent upon ATP for energy. The protein itself consists of both membrane-spanning domains (the pores) and a nucleotide-binding domain where ATP binds and is hydrolyzed.16 This deduced function was satisfying in that it could explain many of the symptoms and features of cystic fibrosis. CFTR mutations and variants

As just noted, the first few patients studied at the time of the gene discovery all had a specific three-nucleotide deletion of codon 508, designated ΔF508 (also referred to, by purists, as c.1521_1523delCTT using the formal Human Genome Variation Society nomenclature).17 This finding prompted early hopes that a CF carrier screening test would be simple and easily implemented. However, it was not long before those hopes were dashed, as a great number of additional mutations began to be catalogued, many of them extremely rare or confined to single families (so-called “private” mutations). While ΔF508 accounts for about 70 percent of Caucasian carriers of northern European ancestry, the next most common mutation in that population, G542X, is found in only about 1.5 percent of carriers, and after the next few mutations the carrier frequency drops to well under 1 percent. Thus, a screening test for CFTR carrier mutations was not to come easily. Indeed, we now know that the mutational spectrum of the gene is extremely heterogeneous, with close to 2,000 mutations catalogued thus far (available at the Cystic Fibrosis Mutation Database: http://www.genet.sickkids.on.ca). They are spread across all the exons of the gene, with no particular “hotspots.”18 At time of this writing, 39 percent of the mutations are missense, 16 percent are frameshift, 11 percent splicing defects, 8 percent nonsense, and about 5 percent are insertions or deletions. It is largely because of this mutational heterogeneity that population carrier screening for

CF mutations took 12 years to be launched from the time the gene was discovered. Though not necessarily cited as such in CFTR testing reports, some laboratorians and practitioners have found it useful to delineate classes of mutations based on their effect on the protein product of the gene. There are five classes (Table 15.2), as follows: class I mutations are the most severe, resulting in truncation of the protein product, as caused by nonsense mutations or splice variants leading to exon skipping (e.g. the nonsense mutation G542X and the splice-junction mutation 1717–1G→A); class II mutations result in proteins that are intrinsically functional but are not processed properly after translation and never reach the cell membrane (e.g. the microdeletion mutation ⊗F508); class III mutations result in proteins that do not properly bind or hydrolyze ATP (e.g. the missense mutation G551D); class IV mutations result in proteins that do bind ATP but are inefficient in chloride transport (e.g. the missense mutation R117H); and class V mutations result in decreased levels of normally functioning proteins, usually due to alternative splicing (e.g. 3849+10kb C→T).19 Again, although most clinical laboratory reports will not specify the mutational result by classes, they are worth keeping in mind for their mechanistic implications as we move further into the era of mutation-specific therapies (discussed earlier).11, 12

Genotype–phenotype correlation The spectrum of severity of clinical symptoms in CF is extraordinarily broad, ranging from infants who die in the newborn period of meconium ileus to middle-aged adults who experience only chronic sinusitis and have no idea that they carry two CFTR mutations. It goes without saying that mutations associated with the former, along with serious lung disease and pancreatic insufficiency, are classified as “severe,” while those associated

Table 15.2 Classes of CFTR mutations Class

I

II

III

IV

V

Defect

Protein truncation

Post-translational processing

ATP-binding

Cl− transport

Decreased product

Examples

G542X; 1717–1G→A

ΔF508

G551D

R117H

3849+10kb C→T

CHAPTER 15

with the latter “atypical” forms are classified as “mild.” Pancreatic insufficiency to a degree requiring pancreatic enzyme supplementation (seen in 85 percent of CF patients) tends to track with the “severe” mutations.20, 21 Unfortunately, however, those designations are not absolutely predictive: some patients with so-called “mild” mutations may have classical disease replete with pancreatic insufficiency, while there are homozygotes for the most classic “severe” mutation, ΔF508, who experience only mild or subclinical lung disease well into adult life.22–25 Variable expressivity even extends to the biochemical abnormalities, in that a patient may (though rarely) have classic lung disease but normal or near-normal sweat chloride levels.26, 27 The reason, clearly, is that the expression of all of these mutations is subject to the action of various known and unknown modifier genes and other host factors (see later). But aside from academic interest, this relatively poor genotype–phenotype correlation makes genetic counseling for the results of prenatal and postnatal diagnostic tests fraught with uncertainty. If a prenatal mutation test result is poorly predictive as to expected mild or severe disease, how are parents expected to make an informed choice regarding continuation or termination of the pregnancy?

Congenital bilateral absence of the vas deferens Perhaps the most striking example of clinical variability in CF involves the feature of male infertility due to CBAVD. This malformation is seen in almost all males with CF but, surprisingly, it can also be seen as an isolated feature in the absence of any other signs of CF, associated with certain CFTR mutations and variants.28 Most patients with isolated CBAVD have a classical CF mutation on one allele and a more “mild” or atypical variant on the other allele. It may be that the male reproductive apparatus requires higher levels of CFTR activity for proper development than do the lungs and pancreas for proper function, so a partial compromise not dramatic enough to cause classical CF symptoms can still cause CBAVD.29 This has led to some debate over the primary diagnosis in such individuals. Do they have an extremely mild/atypical form of CF or do they in fact have an entirely different disease?30, 31 (Examples of other genes causing two

Prenatal Diagnosis of Cystic Fibrosis

615

different diseases are not unknown in medical genetics.) Whatever the nomenclature, it is now widely accepted that otherwise healthy adult males presenting to infertility clinics should be checked for CBAVD and then for CFTR mutations. The two mutations most commonly associated with CBAVD are R117H and 3849+10kb C→T, with the former being by far the more common.28, 32 Both mutations are influenced in their expressivity and penetrance by a tandem repeat polymorphism in intron 8 of the CFTR gene consisting entirely of thymidines. The alleles commonly seen in the population are runs of 5, 7, or 9 thymidines (designated 5T, 7T, and 9T, respectively). The repeat is located toward the 3′ -end of intron 8, near the splice-acceptor site, and the 7T and 5T alleles result in reduced RNA splicing efficiency and hence gene expression (with 5T worse than 7T). The details of the interaction are rather complex (Table 15.3) and depend upon whether the 5T or 7T variants are in cis or trans (i.e. on the same or opposite chromosome) with a variable mutation such as the two noted above.33, 34 In general, having 5T in cis with a mutation like R117H (and in the presence of another CF mutation on the opposite allele) causes enough of a decrease in gene expression to result in CF symptoms, albeit often fairly mild, whereas 5T in trans with R117H is more likely to result in CBAVD (if anything). Likewise, 7T in cis with R117H is more likely to result in CBAVD (or nothing at all). 7T in trans with R117H has no effect as long as the mutation has the “normal” 9T variant in cis. Lastly, homozygosity for 5T, even in the absence of any exonic CFTR mutation, is associated with CBAVD.35 And to make matters even more complicated, the effect of the 5T variant is further influenced by another tandem repeat within intron

Table 15.3 R117H/polyT genotype–phenotype correlations Genotype

Phenotype includes

R117H-5T/CF mutation

Nonclassic CF, PS CF

R117H-7T/CF mutation

Asymptomatic female, CBAVD,

5T/CF mutation

Asymptomatic, nonclassic CF,

nonclassic CF, PS CF (including R117H) 7T or 9T/CF mutation

CBAVD Asymptomatic

CF, cystic fibrosis; PS, pancreatic sufficiency; CBAVD, congenital bilateral absence of the vas deferens.

616 Genetic Disorders and the Fetus

8, a polyTG tract adjacent to the polyT tract. Eleven TG repeats counters the adverse function of the 5T, whereas 12 or 13 TG repeats are associated with the abnormal phenotypes.36 At present, this is largely of academic interest, as measurement of the TG tract is not routinely performed by clinical laboratories, unlike measurement of the polyT tract.

Modifier genes The variable penetrance and expressivity of CFTR mutations in different patients has called into question whether CF is truly a “single-gene disorder,” as it has in many other Mendelian conditions. This variance must be caused by something extraneous to the CFTR gene itself, whether environmental, epigenetic, or genetic (and most likely a combination of all three). It is well known that environmental factors such as cigarette smoke, exposure to infectious agents, general nutrition level, socioeconomic status, and compliance with antibiotic and physical therapies may adversely affect outcomes in patients with identical mutations.29, 37 But there are clearly genetic host factors as well. Just as the intron 8 polyT tract can affect clinical expressivity, so can a variety of other genes, both known and unknown. Much of the work in this area is in its infancy, but variants in a number of genes and putative regulatory regions appear to impact severity or prevalence of lung, pancreatic, or gastrointestinal manifestations.29, 38 However, as most of these findings are the products of genome-wide association studies (GWAS), they are as yet not sufficiently predictive for practical use in CF genetic testing. As genome-level DNA sequencing (whole-exome or whole-genome) becomes more routine, it is expected that many of these factors will be further defined in CF patients, hopefully allowing for improved prognostication, surveillance, and prevention of complications.

Ethnic variation in mutation frequencies As noted, the vast majority of the approximately 2,000 mutations identified in the CFTR gene are extremely rare or even “private.” Of the recurrent mutations, ΔF508 is by far the most common, but its frequency, along with those of many

others, varies markedly among different ethnic and racial populations. Worldwide, it accounts for 68 percent of CF chromosomes (at least of those tested);1 it is at higher frequency in individuals of northern European ancestry. In contrast, it accounts for only about 50 percent of African American carriers39 and Hispanic Americans.40 In Ashkenazi Jews, some of the ΔF508 frequency is supplanted by a mutation unique to this population, W1282X,41 while in African Americans the second most-common mutation is 3120+1 G→A. Beyond ΔF508, other recurring (though much less common) mutations detected in the general population include G542X, R553X, R334W, R117H, 3849+10kb C→T, G551D, 621+1 G→T, and N1303K. As CF is a rare disease among Asians, not much is known about common or unusual mutations in that racial group (and the same for Native Americans).40

Development and implementation of public policy for CF population carrier screening and the core mutation panel As is true for all single-gene disorders, prenatal diagnosis for CF cannot proceed unless and until the mutational genotypes of both parents are known; otherwise, one does not know what to look for in the fetus. But as a pure recessive disease, the majority of carriers do not know they are carriers, and the majority of at-risk couples do not know they are at risk until they give birth to their first affected child. That is the impetus for initiating population carrier screening, in order to identify carriers and couples at risk so that they may be offered prenatal diagnosis and the option to terminate an affected pregnancy and thereby avoid the birth of that first affected child. Of course, that is the same impetus for past successful carrier screening programs such as those for thalassemia in the Mediterranean region and for Tay–Sachs and other recessive diseases in the Ashkenazi Jewish population. The difference is that CF is not restricted or markedly concentrated within a single ethnic or racial population but rather is panethnic. That is the reason why CF was the first universal population carrier screen to be implemented, offered to all pregnant couples or to those planning

CHAPTER 15

a pregnancy. And, as noted at the start of this chapter, it was the first program to be instituted using molecular (DNA-based) technology. But how did we get to this place where universal CF carrier screening is the standard of care? It was a long and tortuous road, interrupted by speed bumps and controversies. It started when the CFTR gene was discovered in 1989. At that time a consortium representing the National Center for Human Genome Research (now the National Human Genome Research Institute [NHGRI]), the American College of Medical Genetics (now the American College of Medical Genetics and Genomics [ACMG]) and the American College of Obstetricians and Gynecologists (ACOG) decided that CF population carrier screening was worth considering but should not be initiated until a number of feasibility and ethical issues could be sorted out via pilot studies and expert committees (in fact, an actual moratorium was laid down until these things could be accomplished).42, 43 Of particular concern was the mutational heterogeneity of the gene and the fact that no screening test of reasonable cost (at that time) would be capable of picking up all carriers. Would such an imperfect test actually do more harm than good? Would it make many couples anxious without providing them with actionable information (for example, those couples in whom one partner tests positive and the other negative)? A number of pilot studies were funded under the sponsorship of the Ethical, Legal and Social Implications (ELSI) program of NHGRI. For several years these studies explored the practical feasibility, acceptance, and understanding of carrier screening in those with and without a family history of CF. The settings varied from large to medium-sized cities, from mostly Caucasian to multi-ethnic populations, from university-based to HMO clinics, and from pregnant couples to those not yet pregnant.44–47 All of the studies demonstrated feasibility of the screening programs, but the highest uptake (interest in being screened) occurred in the author’s own study,47 probably because it was set entirely within prenatal clinics and exclusively targeted couples who were pregnant. This is not surprising, as early carrier screening programs had demonstrated higher interest among couples who were already pregnant than in those not yet thinking about optimizing reproductive outcomes.

Prenatal Diagnosis of Cystic Fibrosis

617

In any case, these findings influenced the expert consensus panel that was convened at NIH in 1997 to evaluate the results of the pilot studies and ultimately recommended that CF carrier screening “be offered to all pregnant couples and those planning a pregnancy.”48 The expert panel did not address a number of practical implementation questions, leaving those to future expert groups. Such questions included: Should CF screening be offered only to those in the “higher risk” groups such as Caucasians and Ashkenazi Jews, or opened up to all individuals regardless of ethnicity? Should screening be offered in a sequential fashion – first to the pregnant woman and then to her reproductive partner only if she tests positive? How many of the 1,500 (at that time) known CFTR mutations should constitute a minimal core screening panel? Can practitioners go beyond the core panel if they or the patients wish? How should test results and residual risks be reported? These questions were indeed taken up by a steering committee and various subcommittees, with representatives from ACMG, ACOG, and NHGRI.49 As is now well known, it was decided that screening would be universal/panethnic, using a minimal core panel of the 25 most common and most phenotypically severe mutations (recognizing that genotype–phenotype correlations are far from perfect) (Table 15.4), based on data from the Cystic Fibrosis Foundation and large clinical reference laboratories; any mutation representing at least 0.1 percent of the mutant alleles in the affected CF patients within these databases was included.50 The resulting number, 25, was substantially greater than the number of mutations tested during the pilot study phase, which was initially just 6: ⊗F508, G542X, W1282X, N1303K, G551D, and R553X.47 Though the patient databases used were predominantly Caucasian, effort was made to include mutations unusually prevalent in other groups as long as they contributed at least 0.1 percent to the overall mixed population; thus, for example, the most common African mutation, 3120+1G→A, was included, as was the common Ashkenazi Jewish mutation, W1282X. Three years later, the core panel was modified with the removal of two variants: 1078delT and I148T:51 the former was discovered to be so rare that it was virtually never seen in screening and should not have reached the 0.1 percent threshold

618 Genetic Disorders and the Fetus

Table 15.4 The original American College of Medical Genetics and Genomics core mutation panel for population cystic fibrosis carrier screening50 ΔF508

ΔI507

G542X

G551D

W1282X

N1303K

R553X

621+1G>T

R117H

1717-1G>A

A455E

R560T

R1162X

G85E

R334W

R347P

711+1G>T

1898+1G>A

2184delA

1078delTa

3849+10kbC>T

2789+5G>A

3659delC

I148Ta

3120+1G>A a These

two mutations were removed in 2004.51

Source: Grody WW, Cutting GR, Klinger KW, et al. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 2001;3:149.50

set for inclusion, while the latter was discovered not to be a mutation at all but merely a benign variant (polymorphism); those CF patients who had I148T in the original affected cohort were later found to carry another, truly pathogenic mutation elsewhere in the gene on the same chromosome.52–54 So the revised core mutation panel for population screening was and remains 23, though there is now more flexibility in going well beyond that number (see “Expanded panels”). After careful consideration, it was decided that either the “couple” or “sequential” model of offering carrier screening could be used, as long as test results were given to both members of the couple; on that point it excluded one particular model in which couples testing positive–negative are reported as “negative” in order to spare them the resulting risk uncertainty.55, 56 It was felt to be unethical not to disclose a test result to a patient who had consented to it, and, moreover, in that model one loses the ancillary benefit of alerting other family members of the person who tests positive – the so-called “cascade” effect of genetic screening.57 In an Appendix to the recommendations, model test reports were provided covering various combinations of results, including relation of mutation R117H to the polyT tract, as well as a table showing the a priori carrier rates of various racial/ethnic populations and the residual risk of still being a carrier after testing negative with the core mutation panel (adapted here as Table 15.1).50 These risks are used by genetic counselors in the post-test counseling session. As shown, the test panel has the highest clinical sensitivity, 94 percent, in those of Ashkenazi Jewish descent (thanks largely to the combined prevalence of the ⊗F508 and W1282X

mutations), compared to 88 percent in the general non-Hispanic Caucasian population, and progressively less in the other ethnic/racial groups. Although the core panel was designed strictly for use in carrier screening in individuals with no family history of CF, many laboratories began to use the same assay for other purposes, such as diagnostic testing in symptomatic patients. One can do the math and figure out the sensitivity for detecting two mutations instead of one, which comes out to about 78 percent for the Caucasian population (about 21 percent of affected patients will show only a single mutation – which is supportive but not conclusive evidence for a diagnosis of CF – and about 1 percent will show no mutation). This should not be an issue in prenatal diagnosis, however, because one would not attempt to test the fetus without first knowing the identity of the mutations in both the father and mother. Since the R117H mutation exhibits different phenotypes depending on the length of the associated polyT tract in intron 8, the recommendations call for measuring the number of Ts – but only as a reflex test after R117H is identified in the person being screened. The reason for that approach is that the 5T allele is rather common in the general population and if both parents screened positive for the 5T, their baby, if male, would be at risk of CBAVD (homozygous 5T, even in the absence of any other CFTR mutation). Since the goal of this effort was to screen for risk of CF, not male infertility, there was a fear that too many 5T carriers, with no inherent risk of CF, would be identified in a first pass if everyone were screened for it. That is why it was designated a reflex test. In fact, since R117H even in cis with 5T is likely to produce mild disease, there was much debate

CHAPTER 15

about whether to include it at all in the core screening panel. In the end it was felt to be too common to completely ignore. On the other hand, a multinational European consensus group, considering the same question for their populations, decided against including R117H (and polyT).58 The committees fully recognized that this type of complex molecular screening for a disease previously in the domain of pediatricians and pulmonologists would represent a significant change for obstetricians who would be the primary providers of the test in the prenatal setting. In conjunction with ACMG, ACOG developed educational materials that were sent to all their members, and a “ramp-up” period of at least 6 months was specified in order to give laboratories and providers adequate time to incorporate this new paradigm into their practice. Indeed, laboratories faced their own unique challenge in suddenly having to test for 25 mutations in a multiplex type of assay when no commercial kits or reagents for doing so were available at the time. Fortunately, the law of supply-and-demand prevailed, as equipment and reagent vendors perceived a lively market once millions of couples were going to be screened; previously, diagnostic molecular genetics was a sort of backwater in the commercial world, compared to the much higher volume tests in molecular microbiology and molecular oncology. The result was a plethora of kits and platforms for purchase that were tailored to assaying precisely those 25 mutations (see “Laboratory methods”). In actual practice, it took something like 2–3 years for the majority of obstetricians to get on board, and even several years into the program there were still appreciable numbers of practitioners not routinely offering CF carrier screening to their patients.59, 60 Given that the procedure had been deemed standard of care by ACOG, the risk of liability from an affected child born to a couple who had not been offered screening is only too obvious.

Laboratory methods As stated, developing a multiplex assay for detection of 25 mutations is no simple task. Whereas detection of one or two mutations in a gene is relatively straightforward and is often accomplished using in-house, laboratory-developed methods, most laboratories had to depend on commercial

Prenatal Diagnosis of Cystic Fibrosis

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vendors for the array-based and other assays designed to cover the ACMG core mutation panel. Laboratory directors soon had a diverse array of platforms to choose from, utilizing techniques such as microarrays, reverse-hybridization strips (also called dot blots), amplification-refractory mutation system (ARMS), oligonucleotide ligation assay (OLA), liquid bead arrays, fluorescence resonance energy transfer (FRET), and microbead suspension hybridization assays.61 The starting material for all of them is DNA which can be isolated from amniocytes or chorionic villus samples for prenatal testing, or from blood, saliva or buccal brushes for carrier screening. A summary of the technical advantages and disadvantages of the various methods is presented in Table 15.5. In general, all of them have produced robust accuracy in blinded proficiency tests,62, 63 and clinical practitioners need not concern themselves with the nuances, though they should strive to use only those laboratories that are CLIA- and College of American Pathologists (CAP)-certified.

Expanded panels Commercial device and reagent manufacturers are not the only ones who saw a large and appealing market in universal CF carrier screening. Commercial reference laboratories were also obliged to re-evaluate clinical molecular diagnostics as a potentially lucrative sector of the clinical laboratory market. But to capture larger market share, they needed to do something to distinguish themselves from their competitors, and the way they chose to do it was to boast of ever larger CFTR mutation panels. Despite the fact that the original ACMG recommendation strongly discouraged going beyond the 25 mutations in the core panel,50 the ink was hardly dry on that statement when commercial laboratories began publishing and advertising that they could offer larger panels with 40 or 70 or 100 or more mutations, allegedly promising greater sensitivity at detecting more carriers than the standard panel.64, 65 Considering that the carrier percentages of the additional mutations are all well under 0.1 percent, it seems hard to believe that their addition could noticeably increase clinical test sensitivity, but arguments were made that some of the additional mutations were specific to non-Hispanic/non-Caucasian ethnic

Table 15.5 Methods of mutation detection Method

Principle

Advantages

Disadvantages

Allele-specific oligonucleotide

Individual wild-type or mutant probes hybridize

Can be automated

Platform design and

hybridization (ASO)

to target (patient) DNA bound to membrane

Multiplex possible

interpretation of results can be complex

Reverse dot-blot hybridization

Probe pairs (wild-type and mutant) are bound to membrane and hybridized with target

Capable of high throughput Multiplex possible

Difficult to customize or add new mutations

(patient) DNA Rapid and robust assay Amplification-refractory mutation system (ARMS), also

PCR primers designed to amplify only a specific

Rapid and reliable

(typically mutant) sequence

Absence of product implies negative result if there is no

called allele-specific

paired wild-type reaction

amplification

Assays without paired wild-type reactions cannot differentiate between the heterozygous and homozygous mutant state

Oligonucleotide ligation assay (OLA)

Allele-specific PCR followed by ligation with probes to identify mutant and wild-type sequence

Liquid bead arrays

Multiplex PCR followed by hybridization to beads with covalently bound universal tags or allele-specific capture probes. A fluorochrome coupled to a reporter molecule

Capable of high throughput Software automatically analyzes data and

Detection requires use of automated DNA sequencer

creates summary reports Capable of high throughput Software analyzes data automatically and

Detection requires the use of specialized equipment

enables results for reflex polymorphisms to be revealed only as appropriate or on demand

quantifies hybridization Fluorescence resonance energy transfer (FRET)

Hybridization of patient DNA to a normal or mutant probe forms a structure that is

Capable of high throughput Rapid

recognized and cleaved by a proprietary enzyme. The released fragment hybridizes to

Software analyzes data automatically and

a cassette containing a reporter and

enables results for reflex polymorphisms to be

quencher molecule forming a second

revealed only as appropriate or on demand

structure, which is enzymatically cleaved, generating fluorescence signal

Detection requires the use of specialized equipment

Microarray hybridization

Hybridization of patient DNA to probes on a microarray (or chip)

Capable of high throughput Rapid Relatively easy to customize or add new

Detection requires the use of specialized equipment Expensive

mutations DNA sequencing (Sanger method)

Sequence individual regions, exons or complete CFTR gene

Can theoretically identify all mutations within the amplicons

Relatively expensive Relatively slow Cannot identify large deletions Likelihood of revealing variants of uncertain clinical significance (VUS)

DNA sequencing (next-generation method)

Sequence individual regions, exons or complete CFTR gene

(NGS)

High throughput Allows for integration of CF screening with

Requires very expensive equipment

expanded carrier screening for many other

Limited ability to identify large

disorders

deletions Likelihood of revealing variants of uncertain clinical significance (VUS)

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

Requires expensive, specialized

Robust Automated

equipment

622 Genetic Disorders and the Fetus

groups that had been overlooked by the original Cystic Fibrosis Foundation cohort, which consisted mainly of northern European Caucasians.66, 67 On the other hand, some of these panels included variants of uncertain pathogenicity, such as D1270N, D1252, G662D, L997F, and R117C.68–70 Such mutations were deliberately not included in the ACMG panel so as not to present parents with ambiguous decision making. Some suggested that the competitive quest for ever larger CFTR screening panels at that time was not only unseemly but also unscientific, as summarized in a commentary by this author and colleagues.71 The selection of additional mutations was in some sense arbitrary, and because they were so rare, very little was known about them, so how could one be sure of their true penetrance and pathogenicity? Remember that the original ACMG core panel contained a serious error – the inclusion of the I148T variant which turned out to have no pathogenicity whatsoever – despite almost 3 years of careful vetting; this experience no doubt haunted the original committees and produced a certain hesitancy in dramatically expanding the mutation screening panel. How many other “I148Ts” may be lurking among the expanded panels currently offered? Nevertheless, it must be recognized that much has changed in the last 20 years in both molecular technologies and our understanding of the many variants in the CFTR gene. With the cost of DNA sequencing ever-diminishing since the introduction of next-generation sequencing (NGS), one may ask why not begin screening for CF carriers by that technique, enabling the easy detection of either a larger panel of targeted mutations or indeed any and all variants in the coding regions of the CFTR gene? DNA sequencing, even by the slower Sanger method, has long been accepted for postnatal CF diagnosis, especially for those patients with atypical or ambiguous clinical features and inconclusive targeted mutation tests. The resistance to its implementation in carrier screening and prenatal diagnosis has been because of the risk that it will pick up all sorts of single-nucleotide changes (missense variants) that have not been seen or reported before, with nothing known about their pathogenicity or lack thereof, and found in a parent or fetus with no phenotype to give us any clues. In the

sequencing world, these are the notorious “variants of uncertain significance” (VUS), and they could potentially place a pregnant couple in the untenable position of trying to make an irreversible reproductive decision based on little or no predictive information. The first move in this direction was the clearance by the US Food and Drug Administration (FDA), in 2013, of a targeted 139-variant CFTR panel on the Illumina MiSeq, a popular NGS instrument. In fact, this was the first FDA-approved NGS assay of any type in laboratory medicine.72 Importantly, these variants were not arbitrarily selected, as in so many previous expanded panels, but are based on a sophisticated study of all 2,000 CFTR mutations for prevalence (>0.01 percent of the total), phenotype in patients, and molecular and functional studies in vitro, the results of which are accessible in a database called CFTR2 (https://cftr2.org/). After a process of elimination, the investigators were left with these specific mutation targets that were concluded to be disease-causing.73 In light of these developments, along with widely accepted criteria (also promulgated by ACMG) for objective classification of novel sequence variants,74 ACMG has revisited the initial CF carrier screening recommendations.75 While stopping short of abandoning the original minimum screening panel (now affectionately referred to as “the ACMG-23”), the new guideline leaves open to laboratory discretion the use of larger panels or even complete gene sequencing. In part this was a recognition of the contemporary state of genetic testing technology, but also a recognition that “the cat is out of the bag” and more comprehensive gene and genomic sequencing is already here to stay and being applied to both diagnostic and screening indications. One reflection of this state is the heavy marketing and popular uptake of highly expanded carrier screening panels encompassing as many as 300 or more diseases, again made possible by the high-throughput nature of NGS. Aside from covering a great many genes, most of these offerings utilize comprehensive exome sequencing, essentially delivering an unlimited mutation screening panel for CF and all the other diseases as well. However, all the concerns raised above regarding the handling and reporting of VUSs and prediction of likely disease severity apply here as well.76

CHAPTER 15

Outcomes of the CF carrier screening program At time of this writing, it has now been almost 20 years since the official launch of population-based CF carrier screening in the United States. Even allowing for some delay in the ramping-up period, that should have been enough time to assess its outcomes. Strangely, however, hard outcomes data are difficult to come by, as the US healthcare system is so fragmented and no registry is kept of CF screening results. The best early data came out of northern California, where the incidence of CF births appeared to have decreased by about 50 percent compared to the prescreening days.77 A more recent meta-analysis found a somewhat broader range in various cited studies.78 It appears that much of the residual birth incidence can be ascribed to couples who consented to the carrier screening procedure but then got withdrew, either at the prenatal diagnosis or pregnancy termination step and decided to go on with the pregnancy regardless (D. Witt, personal communication). This is understandable for a disease like CF, which exhibits no mental retardation or major physical malformations. In addition, as would also be expected, the concurrent presence of a fetal abnormality on ultrasound (such as echogenic bowel; see later) influenced the choice for pregnancy termination. Given that the lifetime cost of medical care for a patient with CF living to the median life expectancy is US$1–2 million, one can draw a general conclusion that the carrier screening program has been cost effective.

Special prenatal diagnosis situations Despite the complexity of CFTR mutations, the majority of pregnant couples undergoing this screening will be handled in a straightforward manner, usually not requiring any special genetic counseling. If screening is conducted in the more common sequential manner, the majority of women will test negative; and of the few who test positive, the majority of their partners will test negative, and that will be the end of it. If the woman tests positive and her partner also tests positive, prenatal diagnosis by either amniocentesis or chorionic villus sampling will be offered, and

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the molecular test will involve looking for the two parental mutations in the fetus; there is a 1-in-4 chance that both will be found, in which case referral for genetic counseling should be made and the option for pregnancy termination will be offered. However, there are certain situations that require special consideration, such as positive–negative couples, positive family history, echogenic bowel, assisted reproduction and preimplantation diagnosis, and newborn screening. Positive–negative couples

The situation where one member of the couple (usually the mother, if sequential screening is done) tests positive for a CFTR mutation and the other tests negative presents some additional concern. The problem with this scenario is that the couple’s risk to have an affected child is now higher than it was before they were tested, yet the guidelines state that nothing further need be offered to them. The reason is that if the negative partner does carry a mutation, it is a rare one not covered by the ACMG panel (or whatever targeted panel was used) and therefore not detectable in the fetus either. It is not recommended to proceed with targeted prenatal diagnosis in this situation because if the one mutation is found in the fetus, there is no way to know whether the fetus is affected or (more likely) a healthy carrier of just the one mutation. Given the current availability of NGS, however, it may be reasonable to offer a larger panel or even whole-gene sequencing and deletion analysis to the negative partner, in the hopes of identifying (or, conversely, lowering the possibility of) a very rare CFTR mutation. But again, the possibility of identifying a problematic VUS in the other partner always exists when going down this route. Positive family history

Although much of this chapter has been concerned with population-based carrier screening in those with no family history of CF, there are those who will have an affected relative, and it is essential that such family history be sought because it dramatically changes the a priori risks of being a carrier (and in so doing, one should also inquire about a family history of male infertility). Even before the launch of population carrier screening, such testing was always made available to individuals with a positive family history. Moreover, it is perfectly

624 Genetic Disorders and the Fetus

acceptable to use expanded panels in this situation, because one wants to optimize the chances of identifying the familial mutation. Ideally, testing will be done first on the affected index case in the family so that the familial mutation will already be known. The bottom line is that for couples with a positive family history of CF, one should go the extra mile and not rely solely on guidelines for routine population carrier screening. Echogenic bowel

Although we have generally described CF in this chapter as a disease which shows no visible signs in the fetus, that is not entirely true. A small proportion of CF fetuses will show echogenic bowel on ultrasound, though the finding is also seen in a wide variety of other conditions, both acquired (e.g. infection) and congenital, and may be present when there is nothing wrong with the fetus at all.79 Combining the results of several series, it appears that an isolated finding of echogenic bowel carries a 1–2 percent risk that the fetus has CF.80 This is considered high enough to warrant CFTR mutation testing in the fetus. If two mutations are found, the conclusion is straightforward, but if only one is found, there will be much uncertainty and anxiety. This may be one of the few cases where full-gene sequencing and deletion analysis may be warranted in the prenatal screening situation, in order to provide the best possible chance of identifying the second mutation, if it is there.75

“allele dropout” that can produce false results, with disastrous consequences. Another, less expensive option, of course, is to use an unrelated sperm or egg donor (who has already been screened negative for CFTR mutations). Sometimes it will be an adult male CF patient himself who presents for assisted reproductive technologies (ART). The reason, of course, is the problem of CBAVD. Such men are not truly infertile; they do make sperm, but it cannot get out into the ejaculate. However, many of these men have fathered children by sperm aspiration.82 Remember that genetic counseling and partner screening are essential in such cases, because the father has a 100 percent chance of passing a CFTR mutation or less penetrant variant to his offspring. Newborn screening

CF is one of the diseases chosen for expanded newborn screening, and all US states and many countries are now including it, usually by measurement of IRT followed by limited mutation testing. Its many ramifications are beyond the scope of this chapter, except to note that newborn screening is another way in which carrier parents will be identified (indirectly). This knowledge will in turn influence their attitudes toward prenatal diagnosis in a future pregnancy. In this sense, newborn screening and carrier screening represent a dynamic interaction, with each affecting the frequencies and attitudes of the other.83

Assisted reproduction and preimplantation diagnosis

Future directions

As can be gleaned from the discussion in “Outcomes of the CF carrier screening program”, there are many couples who find the prospect of pregnancy termination for CF problematic. For those that do but would still like to avoid having a child with CF, in vitro fertilization followed by preimplantation genetic testing (PGT) by single-cell biopsy of the embryo is a possible option. It is expensive and not always covered by insurance, so it is largely available only to those couples who can afford it. But it is certainly feasible, as long as both parental CFTR mutations are known from previous testing. In fact, CF was the first disease for which PGT was successfully performed.81 The technique is available in only a handful of centers and is technically challenging, subject to artifacts like

In addition to the other strengths of NGS discussed in this chapter, we cannot conclude without at least mentioning the NGS test most familiar to obstetricians: noninvasive prenatal screening (NIPS). Its advantages over traditional prenatal specimen collection by amniocentesis and CVS are well known, allowing for testing earlier in the pregnancy and at no risk to the fetus (see Chapter 8). Its primary application has been in screening for trisomies and other aneuploidies, for which it boasts far better predictive value than maternal serum screening.84 Since it involves whole-genome sequencing, it can also theoretically go beyond merely counting chromosome “reads” to detect subchromosomal deletions and insertions, and some NIPS laboratories now claim to screen for several

CHAPTER 15

known syndromes of that type, such as 22q11 deletion/DiGeorge syndrome. Extending the resolution even farther, it should be possible to sequence and reconstruct the entire fetal genome, opening the way for noninvasive prenatal diagnosis of single-gene disorders such as CF.85 Although some laboratories have begun offering such tests for a variety of disorders, including CF,86 it is not without significant challenges, most prominently the need to tease out a fetal sequence change from the much more abundant background of maternal DNA.87 Nevertheless, given the rapid pace of advances in technology, it is reasonable to predict that such capabilities will improve enough for routine use in the next few years. At that point, for pregnant women already being offered NIPS for aneuploidy detection, CF and any number of other single-gene disorders could be added in at that time, obviating the need for prior carrier screening of the parents. In this way, NIPS could become the universal reproductive screening test, assimilating much of the purpose of both newborn and carrier screening. Further in the future, we can expect a proteomics revolution analogous to the genomics revolution which we are now living through. Its complexity can only be imagined at this point, but

Prenatal Diagnosis of Cystic Fibrosis

625

one of its fruits will hopefully be a more definitive understanding of the effect of gene mutations at the protein level – this would be a boon to both carrier screening and prenatal diagnosis of CF, where our current ability to predict genotype–phenotype correlations is less than optimal. It would also presumably help to identify and characterize the function of CFTR-modifier genes.88 Will CF as a disease ever be completely eliminated through carrier screening and prenatal diagnosis? Such a goal is virtually impossible for any recessively inherited disease. Hypothetically, it would require truly universal uptake of parental carrier screening and 100 percent of at-risk couples choosing to terminate affected pregnancies, something we are unlikely to see with this particular disease. Some ethicists might argue that it is not even the goal we should be striving for. Our mission in this area, as in all aspects of medical genetics, should always be to provide patients with the most current, unbiased information and the full range of interventional options open to them. Thanks to the power of the modern molecular genetic technologies, we can certainly do that in a way that previous generations could hardly have conceived.

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43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55. 56.

Health workshop on population screening for the cystic fibrosis gene. N Engl J Med 1990;323:70. Caskey CT, Kaback MM, Beaudet AL. The American Society of Human Genetics statement on cystic fibrosis screening. Am J Hum Genet 1990;46:393. Tambor ES, Bernhardt BA, Chase GA, et al. Offering cystic fibrosis carrier screening to an HMO population: factors associated with utilization. Am J Hum Genet 1994;55:626. Loader S, Caldwell P, Kozyra A, et al. Cystic fibrosis carrier population screening in the primary care setting. Am J Hum Genet 1996;59:234. Clayton EW, Hannig VL, Pfotenhauer JP, et al. Lack of interest by nonpregnant couples in population-based cystic fibrosis carrier screening. Am J Hum Genet 1996;58:617. Grody WW, Dunkel-Schetter C, Tatsugawa ZH, et al. PCR-based screening for cystic fibrosis carrier mutations in an ethnically diverse pregnant population. Am J Hum Genet 1997;60:935. NIH Consensus Statement. Genetic Testing for Cystic Fibrosis. NIH Consensus Statement Online, April 14–16, 1997. http://consensus.nih.gov/1997/ 1997GeneticTestCysticFibrosis106html.htm Grody WW, Desnick RJ. Cystic fibrosis population carrier screening: here at last – are we ready? Genet Med 2001;3:87. Grody WW, Cutting GR, Klinger KW, et al. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 2001;3:149. Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Genet Med 2004;6:387. 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. Buller A, Olson S, Redman JB, et al. Frequency of the cystic fibrosis 3199del6 mutation in individuals heterozygous for I148T. Genet Med 2004:6:108. Monaghan KG, Highsmith WE, Amos J, et al. Genotype-phenotype correlation and frequency of the 3199del6 cystic fibrosis mutation among I148T carriers: Results from a collaborative study. Genet Med 2004;6:421. Wald NJ. Couple screening for cystic fibrosis. Lancet 1991;338:1318. Doherty RA, Palomaki GE, Kloza EM, et al. Couple-based prenatal screening for cystic fibrosis in primary care settings. Prenat Diagn 1996;16:397.

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57. Super M, Schwartz MJ, Malone G, et al. Active cascade testing for carriers of cystic fibrosis gene. Lancet 1991;338:1318. 58. Castellani C, Duff B, Dalla Piccola, B, et al. Benchmarks for cystic fibrosis carrier screening: a European consensus document. J Cyst Fibrosis 2010;9:165. 59. 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. 60. 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. 61. Brennan ML, Schrijver I. Cystic fibrosis: a review of associated phenotypes, use of molecular diagnostic approaches, genetic characteristics, progress, and dilemmas. J Mol Diagn 2016;18:3. 62. Palomaki GE, Bradley LA, Richards CS, Haddow JE. Analytic validity of cystic fibrosis testing: a preliminary estimate. Genet Med 2003;5:15. 63. Lyon E, Schrijver I, Weck KE, et al. Molecular genetic testing for cystic fibrosis: laboratory performance on the College of American Pathologists external proficiency surveys. Genet Med 2015;17:219. 64. Heim RA, Sugarman EA, Allitto BA. Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic mutation panel. Genet Med 2001;3:168. 65. Amos JA, Bridge-Cook P, Ponek V, Jarvis MR. A universal array-based multiplexed test for cystic fibrosis carrier screening. Expert Rev Mol Diagn 2006;6:15. 66. Alper OM, Wong LJ, Young S, et al. Identification of novel and rare mutations in California Hispanic and African American cystic fibrosis patients. Hum Mutat 2004;24:353. 67. Schrijver I, Ramalingam S, Sankaran R, et al. Diagnostic testing by CFTR gen mutation analysis in a large group of Hispanics: Novel mutations and assessment of a population-specific mutation spectrum. J Mol Diagn 2005;7:289. 68. Claustres M, Altiéri JP, Guittard C, et al. Are p.I148T, p.R74W and p.D1270N cystic fibrosis causing mutations? BMC Med Genet 2004;5:19. 69. Grody WW. Expanded carrier screening and the law of unintended consequences: from cystic fibrosis to fragile X. Genet Med 2011;13:996. 70. Strom CM, Redman JB, Peng M. The dangers of including nonclassical cystic fibrosis variants in population-based screening panels: p.L997F, further genotype/phenotype correlation data. Genet Med 2011;13:1042.

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71. Grody WW, Cutting GR, Watson MS. The cystic fibrosis mutation “arms race”: When less is more. Genet Med 2007;9:739. 72. Grosu DS, Hague L, Chelliserry M, et al. Clinical investigational studies for validation of a next-generation sequencing in vitro diagnostic device for cystic fibrosis testing. Expert Rev Mol Diagn 2014;14:605. 73. Sosnay PR, Siklosi KR, Van Goor F, et al. Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene. Nat Genet 2013;45:1160. 74. Richards S, Aziz N, Bale S, et al.; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405. 75. Deignan JL, Astbury C, Cutting GR, et al.; ACMG Laboratory Quality Assurance Committee. CFTR variant testing: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med 2020;22:1288. 76. Grody WW. Where to draw the boundaries for prenatal carrier screening. JAMA 2016;316:717. 77. Witt DR, Schaefer C, Hallam P, et al. Cystic fibrosis heterozygote screening in 5,161 pregnant women. Am J Hum Genet 1996;58:823. 78. Kessels SJM, Carter D, Ellery B, et al. Prenatal genetic testing for cystic fibrosis: a systematic review of clinical effectiveness and an ethics review. Genet Med 2020;22:258. 79. Saha E, Mullins EW, Paramasivam G, et al. Perinatal outcomes of fetal echogenic bowel. Prenat Diagn 2012;32:758.

80. Carcopino X, Chaumoitre K, Shojai R, et al. Fetal magnetic resonance imaging and echogenic bowel. Prenat Diagn 2007;27:272. 81. 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. 82. 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. 83. Hale JE, Parad RB, Comeau AM. Newborn screening showing decreasing incidence of cystic fibrosis. N Engl J Med 2008;358:973. 84. Ashwood ER, Palomaki GE. A new era in noninvasive prenatal testing. N Engl J Med 2013;369:2164. 85. Lo YM. Noninvasive fetal whole-genome sequencing from maternal plasma: feasibility studies and future directions. Clin Chem 2013;59:601. 86. Chandler NJ, Ahlfors H, Drury S, et al. Noninvasive prenatal diagnosis for cystic fibrosis: Implementation, uptake, outcome, and implications. Clin Chem 2020;66:207. 87. Tsao DS, Silas S, Landry BP, et al. A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT. Sci Rep 2019;9:14382. 88. Lim SH, Legere EA, Snider J, Stagljar I. Recent progress in CFTR interactome mapping and its importance for cystic fibrosis. Front Pharmacol 2018;8:997.

16

Prenatal Diagnosis and the Spectrum of Involvement from Fragile X Mutations Randi J. Hagerman and Paul J. Hagerman UC Davis Health System, Sacramento, CA, 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, which ranges from intellectual disability and autism to mild learning or emotional difficulties in the context of normal IQ. In addition, developmental and late adult-onset neurological, cognitive, psychiatric, and medical problems arise in some premutation carriers (fragile X-associated tremor ataxia syndrome (FXTAS)).1–3 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 important for synaptic maturation and plasticity.4 Since FMRP specifically downregulates the translation of a number of postsynaptic proteins, production of these proteins is significantly upregulated in the absence of FMRP.5 One important pathway 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.4, 6 The associated neuroanatomical phenotype includes long, thin (“immature”) synaptic connections, which are thought to be the cause of the intellectual disability in FXS. Recent research has led to a number of treatment trials for FXS using various mGluR5 antagonists, GABA agonists, metformin, and cannabidiol (CBD) which have been shown to reverse at least some of the neuroanatomical and clinical phenotype of FXS in animal models.6, 7 For CGG-repeat expansions in the premutation range, both clinical involvement and its pathogenesis (elevated FMR1 mRNA) are distinct from the FMRP-deficit model for full-mutation alleles and FXS. The molecular pathogenesis for premutation-associated clinical involvement involves a toxic gain-of-function of the expanded-repeat FMR1 mRNA,3, 8 which is produced at elevated levels in the premutation range, in contrast to the reduced/absent levels of FMR1 mRNA in the full-mutation range. However, some patients who are in the upper premutation range or in the full-mutation range without methylation can have both lowered FMRP and elevated FMR1 mRNA, also termed a double hit or a dual mechanism of involvement.3 Although most individuals with the premutation possess normal intellectual abilities, some individuals do experience developmental problems

Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment, Eighth Edition. Edited by Aubrey Milunsky and Jeff M. Milunsky. © 2021 Aubrey Milunsky and Jeff M. Milunsky. Published 2021 by John Wiley & Sons Ltd.

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that include attention deficit hyperactivity disorder (ADHD) and/or social deficits, ranging from social anxiety to autism spectrum disorder (ASD).9–12 These problems are more common in males (both children and adults) than in females, although the psychiatric problems, including anxiety and depression, are common in women and the term fragile X-associated neuropsychiatric disorder (FXAND) is an umbrella term to encompass these neuropsychiatric disorders.13 In addition, some adults with the premutation may develop clinical symptoms related to FXTAS,14, 15 including neuropathy;16, 17 autoimmune problems, such as fibromyalgia and hypothyroidism;18 emotional difficulties, including depression and anxiety;19, 20 and dementia.21, 22 The abnormally elevated mRNA triggers a cascade of events in neural cells that ultimately leads to the clinical involvement described later in this chapter.8, 23 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 will review epidemiology, clinical involvement, genetic counseling, prenatal diagnostic procedures, and treatment in the fragile X spectrum disorders.

Epidemiology Newborn screening studies in the United States and internationally have found a high prevalence of the premutation in the general population: 1 in 156–250 females and 1 in 250–810 males, with variability depending on the location of the screening (reviewed in refs).24–26 A 2014 review of prevalence studies places the prevalence of the full mutation at approximately 1 in 5,000 to 7,000;27 however, newborn screening studies have not yet confirmed this prevalence because of the large numbers needing to be screened.24 Prevalence may vary in different parts of the world where pockets of families with FXS, presumably from a founder effect, are found; this situation is more common in developing countries such as Ricaurte, Colombia, and Samarang, Indonesia, where poverty may constrain people to the region where they were born. One important correlate with 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, since about 40 percent of male carriers may be affected by FXTAS. Also, if we define FXS or the fragile X phenotype as the presence of lowered FMRP leading to developmental problems, then a subgroup of premutation carriers on the high end of the CGG spectrum may be labeled as FXS, even though they do not have a full mutation.28 The emergence of data demonstrating that some individuals with neurodevelopmental or neuropsychiatric problems have lowered FMRP levels in blood and/or brain but without a fragile X mutation has stimulated research regarding FMRP levels across neurodevelopmental disorders.6 Examples of such disorders include schizophrenia, where the age of onset and IQ both correlate with a level of FMRP that is lower than that seen in the general population.29, 30 In addition, Fatemi and colleagues have found lowered FMRP levels in the brain of people with neuropsychiatric conditions, including bipolar disorder, depression, autism, and schizophrenia.31–33 The FMRP function may also be dysregulated by hypoxia and seizures.34 Following early life seizures in the rat, FMRP moves away from the dendrites and localizes near the nucleus so that synaptic plasticity is dysregulated without FMRP at the synapse.34 The increase in seizure frequency can worsen the severity of autism from any cause, including tuberous sclerosis, neurofibromatosis, FXS, premutation involvement, and even idiopathic autism.35, 36 As FMRP regulates the translation of approximately 30 percent of all of the genes associated with autism, particularly those involved with synaptic plasticity,37 the dysregulation of FMRP by seizures would possibly dysregulate many other genes associated with autism, and therefore the social deficits would increase.36 Further populations likely will be found with a deficit of FMRP that go beyond those with an FMR1 mutation, so our definition of what constitutes the fragile X phenotype may be broadened substantially in the future.

Clinical involvement in those with the full mutation Most males with FXS have intellectual disability with a mean IQ in the 40s,38 though the floor effect of most IQ assessment measures does not accurately score IQs below 45.39 Only approximately 15

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percent of males with FXS will have an IQ above 70; these individuals have either significant mosaicism (high percentage of cells with the premutation in addition to the full mutation) or a lack of methylation of the full-mutation alleles.40, 41 Differences in methylation status are often found between blood leukocytes and fibroblasts, but the level of FMRP in either tissue correlates positively with the IQ, and the level of methylation correlates inversely with the level of FMRP.42 Prenatal diagnostic techniques cannot determine the methylation status of the fetus because methylation may not set in for the full mutation until later in gestation, after prenatal testing is carried out. Both the European EMQN guidelines for fragile X testing43 and the American College of Medical Genetics Standards and Guidelines for fragile X testing26 recommend avoiding methylation testing in prenatal diagnosis, particularly chorionic villus sampling (CVS). If an expanded allele is detected, then amniocentesis is recommended to confirm either a premutation or a full-mutation allele.26 It is therefore 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 with FXS.26 Approximately 70 percent of females with the full mutation will have an IQ of 85 or lower.44 Although only 25 percent 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.38, 45–49 These individuals typically require significant interventions during their schooling.50 Approximately 25 percent of females with the full mutation will have a normal IQ without learning disabilities, though emotional problems, such as anxiety, are still common in this group.51 Recent quantitative methods to measure FMRP include western blot,52, 53 enzyme-linked immunosorbent assay (ELISA),54 fluorescence resonance energy transfer (FRET),28, 55 and a Luminex assay.56 The last three techniques are more quantitative than western blot analysis or earlier immunocytochemical methods and are best used to detect mild deficits of FMRP in carriers or in other disorders without an FMR1 mutation.29 As these techniques move into clinical use, those at risk for FMRP deficits will be more easily

631

recognized. Further research will be stimulated with regard to the medications or environmental changes that can upregulate FMRP levels. Psychopathology is common in those with the full mutation and includes ADHD in approximately 80 percent of boys and 25 percent of girls;57, 58 autism or ASD in 60 percent of boys and 10–20 percent of girls;59–63 anxiety or mood disorder in 25–70 percent of boys and girls;64 and psychosis in less than 10 percent.57 Often the behavioral problems, particularly impulsivity, anxiety, mood instability, and aggression, cause the main problems for the family caring for children with FXS. Maternal stress is associated with more severe behavioral problems and anxiety in children with FXS, and mothers with the lowest activation ratio are at greatest risk for stress.65 These difficulties in children with the full mutation often lead to medical interventions and diagnosis, which occur typically between 2.5 and 4 years of age.66, 67 There are many medications and behavioral interventions that can help with these behavioral problems,68 and new targeted treatments for FXS have shown some promising results;6, 7, 69 however, a multidisciplinary intervention plan is necessary, including special education supports and therapies, such as speech and language therapy, occupational therapy, and psychotherapy.70, 71 The physical features of FXS classically include a long face, prominent ears, hyperextensible finger joints, flat feet, and macro-orchidism at puberty. Approximately 30 percent 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.72 Many individuals are diagnosed with autism or ASD before the diagnosis of FXS, so all children with ASD should have FMR1 testing.72 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 fewer than 5 percent of individuals. Hernias are more common (15 percent); in males, the weight of the large testicles combined with the loose connective tissue lead to the hernias. Recurrent otitis media is the most common medical problem (85 percent), followed by strabismus (36 percent), and seizures (20

632 Genetic Disorders and the Fetus

percent).57, 73 The lack of FMRP leads to a seizure phenotype in the fragile X knockout (KO) mouse that improves with the use of mGlur5 antagonists or other targeted treatment for FXS, as described below.74

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). Then in 1991, premature ovarian failure (POF; menses stopping before age 40) was described in approximately 20 percent of premutation daughters of these males.75 Subsequent studies have found that this problem increases with increasing CGG-repeat numbers in the premutation carriers, but the prevalence of POF decreases somewhat after 120 repeats.76 This problem has been renamed fragile X-associated primary ovarian insufficiency (FXPOI) because some women with this diagnosis may subsequently become pregnant. The cause of FXPOI seems to be related to the toxicity of elevated FMR1 mRNA on the ovum or the supporting cells of the ovum. For a number of years, psychological problems in male and female carriers with the premutation have been reported with some controversy.77–79 With the identification of FXTAS, there was a clear neurological phenotype, involving an intention tremor, ataxia, neuropathy, autonomic dysfunction, and cognitive decline in some older males and occasional females with the premutation.80 The incidence of FXTAS increases with increasing age, such that only approximately 15 percent of males in their 50s have symptoms, but 75 percent of males in their 80s develop FXTAS.81 In females, approximately 16 percent of carriers older than 50 years develop FXTAS but their symptoms are less severe and cognitive decline is rare.3, 18, 82 Brain atrophy combined with white matter disease is part of the diagnostic criteria in FXTAS,83 with approximately 60 percent 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 percent of females with FXTAS.84 Further radiological studies have demonstrated white matter disease in the splenium of the corpus callosum in approximately 50 percent

of patients with FXTAS, so this finding has been added to the diagnostic criteria of FXTAS.85, 86 Additional reports have expanded the phenotype in women with the premutation to include hypothyroidism (50 percent) and fibromyalgia (40 percent) in those with neurological symptoms, suggesting that an autoimmune component occurs in some female carriers.18, 87 For example, multiple sclerosis (MS) is found in approximately 2–3 percent of carriers.18 MS was reported in addition to FXTAS in one case documented at autopsy.86 The elevated mRNA in the premutation leads to upregulation of a number of proteins including 𝛼B-crystallin, which is a primary antigen for MS.88 There is increasing evidence that neurodevelopmental problems are associated with a subgroup of children, particularly boys, with the premutation. Reports of ADHD, social anxiety, and ASD are common in boys with the premutation who present as the proband of the family,9, 10, 67, 89, 90 and although most have a normal IQ, some have intellectual disability.91 The presence of seizures in premutation boys is associated with both ASD and intellectual disability.15 In carriers with ASD, intellectual disability, and/or neurological problems, approximately 20 percent demonstrate a second genetic hit, which is likely to have an additional and cumulative deleterious effect on the individual.92 Premutation neuronal cell cultures demonstrate decreased branching, fewer synaptic connections, earlier cell death, and slower moving mitochondria compared with control neurons.93, 94 In the retina, FXS mice show a 47 percent deficit of rhodopsin and deficits in retinal stimulation,95 which may relate to the visual–perceptual deficits that have been demonstrated in babies and toddlers with the full mutation and also the premutation.96

Pathogenesis of the premutation-associated disorder FXTAS Of the premutation-associated disorders, we understand the most about the pathogenic mechanisms linked to FXTAS; however, similar mechanisms may be at play for both the neurodevelopmental and neuropsychiatric involvement covered under FXAND and in the early menopausal features (FXPOI).13, 97, 98 Therefore, the following paragraphs will focus on FXTAS.

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Neuropathology

Gross neuropathological features of the brains examined postmortem 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 magnetic resonance images (MRIs) in the same individuals.84, 99–101 The primary neuropathological 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.100, 101 The greatest concentration of inclusions is found in the hippocampal formation (up to 40 percent of nuclei-bearing inclusions in some cases), with lower inclusion densities (2–10 percent) in cortical neurons and the near absence of inclusions in Purkinje cells of the cerebellum, despite substantial Purkinje cell dropout. Inclusion counts are highly correlated with the number of CGG repeats within the premutation range.101 More recently, inclusions have also been observed in tissues outside of the central nervous system, including in both anterior and posterior pituitary, in the Leydig and myotubular cells of the testes,102 and in ganglion cells of adrenal medulla, dorsal root ganglia, paraspinal sympathetic ganglia, mesenteric ganglia, and subepicardial autonomic ganglia.103 Finally, the presence of inclusions in neuronal nuclei within the hypoglossal cranial nerve nucleus may represent a neuropathological correlate to the late-stage swallowing difficulties experienced by many FXTAS patients.101 Molecular pathogenesis

Several lines of evidence indicate that the pathogenesis of FXTAS involves a “toxic” gain-of-function of the FMR1 mRNA. First, FXTAS is almost exclusively confined to carriers of premutation alleles, where the gene is active; although FXTAS has been reported in carriers with rare, unmethylated full mutations, where the gene is still active.104–106 This observation indicates that FXTAS is not the result of the loss of FMRP, because FMRP levels are only moderately lowered within the premutation range, yet profoundly lowered or absent in individuals with full mutation, fully methylated FMR1 alleles (i.e. those with FXS). Second, the absence of FXTAS

633

among individuals with large, full-mutation, fully methylated 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 range,52, 107, 108 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.109–111 Although the mechanism(s) by which the expanded-repeat mRNA triggers the molecular pathogenesis of FXTAS has not been established, at least two models have been put forward (Figure 16.1). In the first model, the expanded CGG-repeat element as RNA binds increased numbers of specific proteins, thereby “sequestering” those proteins from their normal cellular roles. A precedent for this model is the sequestration of muscleblind-like protein 1 (MBNL1) by the expanded CUG-repeat element in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene of myotonic dystrophy.112–114 For FXTAS, a number of candidate proteins have been identified and listed in table 1 of reference 115 .116–120 The most candidates for sequestration are the pair of proteins (DGCR8 and DROSHA) comprising the “microprocessor” that processes micro (mi)RNA precursors in the nucleus.121 Diminished levels of a broad range of miRNAs, and increased levels of their precursors, are observed in the brains of FXTAS cases, and restoration of DGCR8 and DROSHA in cell culture models is capable of reversing some of the cellular defects. However, a specific link between specific miRNAs and FXTAS has not been established. A second model for FXTAS pathogenesis is the aberrant translation of the expanded CGG-repeat region from a non-AUG start codon upstream of the CGG-repeat element. This process, termed repeat-associated non-AUG (RAN) translation, generates a peptide, FMRpolyG, that contains a polyglycine element that is translated from the +1 (GGC) codon frame.122–124 In normal translation of FMRP, a canonical AUG start codon downstream of the CGG repeat is used; thus, the CGG (+0 frame) is not translated. Although FMRpolyG

634 Genetic Disorders and the Fetus FMR1 gene

CGGn

AUG

FMRP coding

5′ UTR

3′ UTR

15 mm) (Figure 17.7). It may be unilateral or bilateral as well as asymmetrical. Hydrocephaly is commonly used when severe ventriculomegaly is present, leading to progressive macrocephaly due

652 Genetic Disorders and the Fetus

(a)

(b)

(c)

(d)

(e) Figure 17.4 (a) Transverse section of the fetal head at the level of the septum cavum pellucidum, demonstrating the “lemon” sign (scalloping of the frontal bones). (b) Suboccipital bregmatic view, demonstrating the “banana” sign (anterior curvature of the cerebellar

hemispheres and obliteration of the cisterna magna). (c–e) Second-trimester fetus at 20 weeks of gestation with open spina bifida visible on coronal plane (c, d) and sagittal plane (e).

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Prenatal Diagnosis of Fetal Malformations by Ultrasound 653

(a)

(b)

(c)

(d)

Figure 17.5 A fetus with spina bifida in the first trimester at 13 weeks of gestation. (a) Transverse section of the fetal head with dried up brain. (b) The “crash” sign. (c) Sagittal plane showing the absence of intracranial translucency. (d) Mid-sagittal plane presenting the spina bifida.

to increased intracranial pressure, usually caused by obstruction. The level of the obstruction is defined by examining the aqueduct of Sylvius and the third and fourth ventricles. When spina bifida is present, macrocephaly may be absent, since in those cases the head can be relatively small. Ventriculomegaly is a relatively frequent finding during the 20 week scan with a prevalence of 1:675.40 The prevalence of congenital hydrocephalus is more difficult to define, mostly because the clinical criteria and definition vary between studies. The prevalence has been reported as between 1 and 32 per 10,000 live births.41 When a prenatal diagnosis of fetal ventriculomegaly is made prenatal counseling is challenging since mild ventriculomegaly may not always be an anomaly but rather a sonographic sign. Males are more frequently affected than females.42 Ventriculomegaly may result from a heterogeneous

spectrum of conditions including spina bifida, chromosome anomalies (most frequently Down syndrome),43–45 genetic syndromes,46 intrauterine hemorrhage, and congenital infection such as cytomegalovirus and toxoplasmosis,42 although the majority of cases remain unresolved. In up to 30–50 percent of patients presenting to a fetal medicine unit with ventriculomegaly, additional findings will be present.42, 47 The overall prevalence of abnormal chromosomes is around 5 percent. However, when considering fetuses with and without associated findings separately, the prevalence of chromosome anomalies was 1 in 12 for associated and 1 in 33 for isolated cases.47 Thus, the fetal and neonatal mortality and morbidity and neurological development of ventriculomegaly are strongly related to the presence of other malformations and chromosomal defects. Isolated mild ventriculomegaly is associated with a good prognosis and

654 Genetic Disorders and the Fetus

Figure 17.6 Meckel–Gruber syndrome with the association of multicystic kidneys in a coronal view (left), posterior encephalocele in coronal, sagittal, and axial lanes (middle). and hexadactyly (right).

Source: Ville YG, Bault J-P. Diagnosis of fetal malformations by ultrasound. In: Milunsky A, Milunsky JM, eds. Genetic disorders and the fetus, 7th edn. Hoboken, NJ: John Wiley & Sons, 2016, chapter 13.

significant hydrocephalus is poor, with high fetal wastage or perinatal death due mainly to the associated anomalies. Severe intellectual disability is common among the survivors. Hydranencephaly

Figure 17.7 A fetus with severe ventriculomegaly – hydrocephaly.

the risk of abnormal neurodevelopmental delay is around 8 percent.47 One of the causes of hydrocephalus may be congenital stenosis of the aqueduct of Sylvius. One of the most frequent heritable causes of isolated hydrocephalus in males is a mutation in L1 cell adhesion molecule gene (L1CAM).48 Associated findings are adducted thumbs, although these may be difficult to demonstrate prenatally. Other genetic syndromes associated with ventriculomegaly are the RASopathies (see Chapter 14) (including Noonan and Costello syndrome), ciliopathies (including Joubert and Meckel–Gruber syndrome), megalencephaly-associated syndromes, dystroglycanopathies (Walker–Warburg syndrome), and syndromes associated with skeletal anomalies such as craniosynostosis (Apert) or dwarfism (achondroplasia).46, 49 The prognosis in cases of

Congenital absence of the cerebral hemispheres with preservation of the midbrain and cerebellum may result from widespread vascular occlusion in the distribution of the internal carotid arteries, prolonged severe hydrocephalus, an overwhelming infection such as toxoplasmosis or cytomegalovirus, or defects in embryogenesis.50 The condition is generally not inherited and is usually incompatible with survival beyond early infancy. Ultrasonographically, the complete absence of echoes–resonance from the anterior and middle fossae distinguishes hydranencephaly from severe hydrocephalus, in which a thin rim of remaining cortex and the midline echo can always be identified. Holoprosencephaly

Holoprosencephaly is classically defined as incomplete cleavage of the forebrain (prosencephalon) that originates from failed midline delineation during early development. There are three classic types, from the most to least severe: alobar, semilobar, and lobar and a milder subtype – the middle interhemispheric (MIH) variant.51 Holoprosencephaly is associated with facial anomalies of varying severity ranging from cyclopia and proboscis to ocular hypotelorism. Facial midline defects are also often

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Prenatal Diagnosis of Fetal Malformations by Ultrasound 655

Figure 17.8 A fetus with holoprosencephaly at 14.6 weeks of gestation.

present. The prevalence of this brain abnormality is around 1.3 in 10,000 births.7, 52 Holoprosencephaly may be associated with chromosome anomalies in 22 percent of cases of which 14 percent occurred de novo and 8 percent were inherited and caused by parental translocations.53 The most common chromosomal abnormality is trisomy 13. Additionally, another frequent cause of holoprosencephaly are monogenic disorders. The Sonic Hedgehog (SHH) gene was the first discovered holoprosencephaly gene and subsequently many more genes have been involved, all of which encode for proteins belonging to brain development pathways and involved in the regulation of SHH activity.54–56 Prenatal diagnosis by ultrasonography is based on the demonstration of a single dilated midline ventricle replacing the two lateral ventricles or partial segmentation of the ventricles in the standard transverse view of the fetal head for measurement of the biparietal diameter (Figure 17.8). Microcephaly

The definition of microcephaly is usually defined as a head circumference of more than 3 standard deviations (SD) below the age- and sex-related mean. It has a birth incidence of approximately 1 per 10,000 and is commonly found in the presence of other brain abnormalities, such as encephalocele or holoprosencephaly. Prenatal diagnosis is based on the demonstration of a decrease in the head-to-abdomen circumference ratio in the presence of associated abnormal intracranial pathology and normal growth in the abdominal circumference. Another common

feature is a sloping forehead. Since the decrease in head circumference may not become apparent before the third trimester, prenatal diagnosis in the second trimester is low and around 10 percent.30 Microcephaly has been associated with chromosomal defects (trisomy 13 and 18), microdeletions, monogenic disorders, or malformation syndromes. Other etiologic factors include fetal hypoxia, intrauterine infection (such as cytomegalovirus and Zika virus),57, 58 and exposure to radiation or other teratogens, including the anticoagulant warfarin. Autosomal recessive primary microcephaly (MCPH) is a static developmental anomaly and to date multiple MCPH genes have been identified. The inactivation of most MCPH genes leads to neurogenesis defects, while the upregulation of some MCPH genes is associated with different kinds of carcinogenesis.59 Prognosis depends on the underlying cause, but in approximately 90 percent of the cases, there is severe intellectual disability. Corpus callosum agenesis

The corpus callosum is a large interhemispheric tract connecting the cerebral hemispheres and appearing as a hypoechoic structure located between the cavum septum pellucidum and the cingulate gyrus. Both 2D and 3D techniques allow imaging of the corpus callosum from 18–20 weeks of gestation. Prior to 18 weeks the pericallosal arteries can be visualized using color Doppler and this visualization is an indirect sign that a normal corpus callosum will develop.60 Corpus callosum agenesis can be either complete or partial. In complete corpus callosum agenesis both the corpus callosum and the cavum septum pellucidum are absent. The normal pericallosal arteries cannot be identified when using color Doppler. In the axial plane the frontal horns appear narrow and laterally displaced, and the atria and occipital horns are slightly dilated (colpocephaly); the shape is similar to a teardrop.61 On coronal section, the falx cerebri can be seen in a broad interhemispheric fissure which meets the third ventricle; the lateral ventricles are widely separated and vertically oriented (“Viking’s helmet” sign) (Figure 17.9). The suspicion of a corpus callosum agenesis should rise when the cavum septum pellucidum cannot be visualized during the 20 week scan. The cavum septum pellucidum should be

656 Genetic Disorders and the Fetus

(a)

(b)

Figure 17.9 (a) Transverse and (b) coronal plane of corpus callosum agenesis at 21.2 weeks of gestation.

seen in all fetuses at 18 and 37 weeks of gestation. This is specifically a second-trimester diagnosis which can reach detection rates of 95 percent.30 When in doubt, fetal magnetic resonance imaging (MRI) (see Chapter 19) may provide additional information, however, the actual diagnostic accuracy of fetal MRI in isolated corpus callosum agenesis is still debated.62 Corpus callosum agenesis can be an isolated finding or associated with other structural anomalies. In the case of isolated or partial corpus callosum the rate of chromosome anomalies is 5.7 percent.63 The risk of associated anomalies detected at fetal MRI is about 8 percent and 12 percent, whereas associated anomalies detected postnatally can occur in about 5 percent and 14 percent of fetuses with complete and partial corpus callosum agenesis, respectively. Neurodevelopmental outcome in complete corpus callosum agenesis is normal in 76 percent and in partial corpus callosum agenesis in 71 percent.63

Mega cisterna magna

In mega cisterna magna the cisterna magna is >10 mm in an oblique transverse plane which visualizes the cavum septum pellucidum, cerebellum, cisterna magna, and nuchal fold. The cerebellum is normally developed. Even at 11–13 weeks an abnormal appearance of the fourth ventricle–cisterna magna complex is present.65 In a recent systematic review, in 7 percent of cases the prenatal diagnosis of mega cisterna magna was not confirmed postnatally.66, 67 When associated with other structural anomalies, the risk of chromosome anomalies and genetic syndromes is high, with trisomy 18 being the most common.68 Mega cisterna magna was not significantly associated with additional anomalies detected at prenatal MRI or detected after birth. The outcome in isolated cases is generally good.67

Blake’s pouch cyst Posterior fossa malformations

During a 20 weeks anomaly scan the transcerebellar plane allows evaluation of the posterior fossa or the cisterna magna.8 The size of the cisterna magna is around 5 mm with an upper limit of 10 mm.64 An increased amount of fluid in the cisterna magna should raise the suspicion of pathology. Posterior fossa malformations embody a heterogeneous spectrum of conditions. It is difficult to delineate the prenatal detection rate in these malformations, since isolated cases may have a completely normal outcome and may remain undiagnosed during life.

The Blake’s pouch is an embryological cystic structure bulging into the fourth ventricle (Figure 17.10). When fenestration fails or is delayed, a cyst may develop. Resolution of the cyst may occur until 26 weeks. The prenatal diagnosis is false positive in up to 10 percent of cases. The rates of associated central nervous system and other structural anomalies are 12 percent and 25 percent, respectively. The risk of chromosome anomalies is around 2 percent. The risk of additional postnatal findings is low and the outcome in isolated cases is generally good.66, 67

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Prenatal Diagnosis of Fetal Malformations by Ultrasound 657

(a)

(b)

Figure 17.10 (a) Transverse and (b) sagittal plane of Blake’s pouch cyst in a fetus at 19.2 weeks of gestation.

Vermian hypoplasia

In vermian hypoplasia of Dandy–Walker variant, the vermis is formed but smaller than normal. The rates of associated central nervous system and other structural anomalies are 56 percent and 49 percent, respectively. The risk of chromosome anomalies is around 3 percent. When isolated, neurodevelopmental outcome is normal.66, 67 Dandy–Walker malformation

The sonographic findings in Dandy–Walker malformation are an enlarged fossa posterior with hypoplasia of the cerebellar vermis, dilatation of the fourth ventricle and a persistent Blake’s pouch cyst (Figure 17.11). Dandy–Walker malformation is a nonspecific marker of chromosomal abnormalities, microdeletions, genetic syndromes, congenital infection, or teratogens. In general, the associated mortality rate is high and intellectual development among survivors is poor. Associated central nervous system and other structural anomalies were present in 61 percent and 43 percent, with ventriculomegaly being the most common finding in 31 percent. The prevalence of chromosomal abnormalities is 16 percent. Abnormal neurodevelopmental occurs in 58 percent.66, 67

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 sonographic examination planes – sagittal, coronal,

Figure 17.11 Transverse plane of a fetus with a Dandy–Walker malformation.

and axial. The coronal plane provides the best view of the upper and lower lips. Additionally, the nose, cheeks, eyelids, lenses, and forehead can be visualized in this plane. In the transverse plane the orbits, nasal bones, mandible, palate, alveolus (tooth buds), and lips should be evaluated and in the sagittal plane the fetal profile, including the chin, forehead, nasal bone, and nasal bridge. Recently, it has been demonstrated that by following specific steps in the mid-sagittal section of the fetus, the secondary palate can be well demonstrated.69 Moreover, the fetal uvula can also be demonstrated and presents as an “equals sign” in both the transverse and mid-sagittal plane. The absence of this sign can be used to diagnose an isolated cleft palate, since the presence of the uvula and consequently visualization of the “equal sign” proves an intact palate.70 With the improvement in ultrasound

658 Genetic Disorders and the Fetus

techniques an integrated approach of 2D and 3D ultrasound has been suggested to evaluate the fetal face to rule out a cleft lip and or palate. The 3D/4D ultrasound can be used for screening for facial clefts but is mandatory and an integral part in cleft analysis. The multiplanar reconstruction mode allows the simultaneous analysis of the three reference planes.71 The surface-rendering mode can be used to identify facial dysmorphologies and clefts. Cleft lip and/or palate

Anomalies of the lip and palate vary greatly, from isolated unilateral cleft lip (CL) or palate (P), to large defects involving the bilateral lip, alveolus, and palate. The prevalence of cleft lip with or without cleft palate (CL ± P) is around 1 per 1,000 compared to a much lower prevalence of isolated cleft palate of 1.3 to 25 per 10,000.72, 73 The prenatal detection rate of CL ± P depends on the population screened and varies greatly from 9 percent to 100 percent in low-risk populations and from 60 percent to 100 percent in high-risk populations, with an average around 40 percent.74 With the introduction of a routine 20 weeks scan, prenatal detection rates increased significantly, however, and now reach 70–88 percent in skilled hands.74–77 This impact could mainly be explained by the significant increase in prenatal detection rates of isolated CL ± P, since the prenatal detection rates of associated CL ± P were already high and remained stable.76, 77 Moreover, with the introduction of screening programs, the gestational age at the time of diagnosis also decreased gradually, allowing more time for additional testing.76, 77 The prenatal detection rate of isolated cleft palate remains low, however, between 0 and 22 percent,30 although some studies claim better results.74 In the case of micrognathia, the presence of a cleft palate should always be considered. When comparing the accuracy of the fetal anomaly scan in determining the oral cleft type, the postnatal findings were in accordance with the prenatal findings in 77 percent.78 Underestimation of the defect was more common than overestimation in 19 percent versus 4 percent, respectively.78 The amount of amniotic fluid may be helpful in recognizing the involvement of the palate in the case of CL ± P, since involvement of the palate was significantly more common in the presence

of polyhydramnios.79 Mistakes in differentiating unilateral as opposed to bilateral defects are rare.78 In the first trimester the prenatal diagnosis of CL ± P is more difficult but feasible. Thirty-five percent of the fetuses with a cleft lip and palate were diagnosed while the remaining 65 percent became apparent at the 20 weeks anomaly scan30 (Figure 17.12). Isolated cleft lip, however, was never diagnosed in the first trimester and 14 percent were only diagnosed after birth.30 Fetal facial anomalies can be isolated or associated with chromosomal anomalies or various multiple malformation conditions. In a systematic review a cleft lip had additional anomalies in only 13 percent versus 54 percent in fetuses with a cleft lip and palate.74 Although chromosome analysis is generally offered when CL ± P is found prenatally, the risk of chromosome anomalies is only 0.9 percent in isolated cleft lip versus 51 percent in associated cases.80 The most common chromosome anomaly found in isolated CL ± P is 22q11 microdeletion syndrome.81 In associated CL ± P, trisomy 18 is present in 56 percent of the fetuses, followed by trisomy 13 in 30 percent.80 In trisomy 13 the defect is frequently positioned in the midline of the face since it is often associated with holoprosencephaly. Besides chromosome anomalies, CL ± P may be present in many genetic syndromes. Micrognathia and retrognathia

Both micrognathia and retrognathia refer to an abnormal mandible and are an ominous sign.82 In micrognathia, the mandible is small, while in retrognathia it is displaced posteriorly. Most fetuses, however, will have a combination of both. Complete absence of the mandible, agnathia, is rare and may result in malposition or even fusion of the fetal ears at the level of the mandible.83 Both micrognathia and retrognathia may be diagnosed from the first trimester onwards in a mid-sagittal view, with the fetus showing an overbite (Figure 17.13). Care must be taken, however, not to misinterpret a long philtrum with micrognathia. The retronasal triangle view is also a useful technique to evaluate the fetal chin. This technique captures the coronal plane of the face in which the primary palate and the frontal processes of the maxilla are visualized simultaneously. Normal first-trimester fetuses display a characteristic gap between the right and left

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 659

(a)

(b)

(c)

(d)

Figure 17.12 Fetus with a unilateral cleft lip and/or palate presenting at 19.2 weeks of gestation in transverse plane (a), coronal plane (b), and sagittal plane (c, d).

body of the mandible in this view (the “mandibular gap”).84 In the case of micrognathia, this gap is either absent or replaced by a bony structure. The majority of cases, however, will be detected in the second trimester.30 When isolated, micrognathia may be familial or part of the Pierre–Robin sequence, a craniofacial anomaly comprising mandibular hypoplasia, cleft secondary palate, and glossoptosis leading to life-threatening obstructive apnea and feeding difficulties during the neonatal period.85 When ear anomalies are present, Treacher Collins syndrome86 or hemifacial microsomia should be considered.87 Most of the fetuses with micrognathia have additional anomalies and will have underlying chromosome anomalies or genetic syndromes. Common chromosome anomalies are 22q11 microdeletion, trisomy 18 and 13, and triploidy.

Figure 17.13 Micrognathia demonstrated in a sagittal plane with the fetus showing an overbite.

660 Genetic Disorders and the Fetus

Pulmonary and thoracic abnormalities Congenital pulmonary airway malformation

Two of the most common long masses that may develop before birth are congenital pulmonary airway malformation (CPAM), previously known as congenital cystic adenomatoid malformation (CCAM) and bronchopulmonary sequestration (BPS). Neither is considered a genetic or hereditary condition, and both are unlikely to recur in a subsequent pregnancy. In a recent study the prenatal incidence of CPAM was 43 in 100,997 pregnancies.30 CPAM is a mass of abnormal fetal lung tissue that forms in pregnancy. Its exact cause is unknown, but it is hypothesized to be related to dysregulation of cell proliferation and cell apoptosis in the terminal bronchioles.88 CPAMs are classified sonographically as either macrocystic, with one or more cysts greater than 5 mm in diameter, or microcystic lesions, which appear as solid, echogenic masses89 (Figure 17.14). The mass may displace lung tissue and heart. BPS is a very similar condition, with a mass within the chest; however, it has a large vessel feeding it from the aorta. In general, CPAM is not visible on ultrasound during the first trimester, as this is a typical second-trimester diagnosis. However, an increased nuchal translucency (NT) was observed in 12 percent of all CPAM cases.30 Fetal hydrops may develop in large CPAMs because of compression of the heart and major

(a)

blood vessels in the thorax. The CCAM volumeto-head circumference ratio (CVR) is a tool that can be used to predict the development of hydrops, survival, need for fetal intervention, and the need for ventilation support or extracorporeal membrane oxygenation (ECMO), and length of hospital stay postnatally. It is calculated by dividing the volume of the mass (length × width × height × 0.52) by the head circumference.90 A CVR 1.6 is associated with adverse outcomes. When the CVR exceeds 1.6, steroid therapy can be considered.91 The effect of steroids should not be overestimated, as only small case series are present and no randomized trials exist. As in 20 percent of cases spontaneous regression of the CPAM occurs, especially in small, microcystic lesions, randomized trials are necessary to evaluate the value of steroid treatment.92 When hydrops develops, fetal intervention may be considered such as cyst aspiration or thoracoamniotic shunts. Postnatally, the management of CPAM or BPS is very similar. When symptomatic, there is little controversy that resection is indicated. When a lesion is asymptomatic there is greater debate regarding the benefit of resection versus continued observation.93 Arguments in favor of operation are decrease of pulmonary infections, poor diagnostic tools to distinguish a CPAM from a malignant pleuro-pulmonary blastoma, and better development of the remaining lung tissue after removal.94

(b)

Figure 17.14 Macrocystic congenital pulmonary airway malformation visible as a solid, echogenic structure in transverse plane (a) and sagittal plane (b).

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 661

Congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a developmental discontinuity of the diaphragm that allows the abdominal organs to herniate into the chest. Although surgery can correct the diaphragmatic defect, in utero herniation of organs can result in pulmonary hypoplasia and pulmonary hypertension due to abnormal pulmonary vascular development and function. In a large study in 100,997 pregnancies 24 fetuses were diagnosed with CDH.30 An analysis of data from 31 population-based European registries revealed that there were 3,373 CDH cases reported among 12,155,491 registered births. Of 3,131 singleton cases, 353 (10 percent) were associated with a chromosomal anomaly, genetic syndrome or microdeletion and 784 (28 percent) were associated with other major structural anomalies. The total prevalence was 2.3 per 10,000 births for all CDH cases and 1.6 for isolated CDH cases.95 When cases resulting in termination of pregnancy and intrauterine fetal demise (IUFD) are added, the total incidence of CDH was 3.5 in 10,000 liveborn. The mortality rate between 1987 and 2013 was 36 percent: 44 percent during the first time period 1987–1999 and 27 percent in the later period 2000–2013.96 Prenatally, the diaphragm is imaged by ultrasonography as an echo-free space between the thorax and abdomen. CDH, which results from failure of closure of the posterolateral pleuro-peritoneal fold at 8–9 weeks of gestation, can be diagnosed by the demonstration of stomach and intestines (90 percent of the cases) or liver (50 percent) in the thorax and the associated mediastinal shift to the opposite side. Herniation is on the left in 80–85 percent of cases, on the right in 10–15 percent of cases, and bilateral in 45

15–25

Severe

20

5 mm110 (Figure 17.16). The prenatal incidence was found to be 8 in 100,997 pregnancies.30 This is a nonspecific finding in a wide variety of fetal and maternal

disorders, including hematologic, chromosomal, cardiovascular, renal, pulmonary, gastrointestinal, hepatic, and metabolic abnormalities, congenital infection, neoplasms, and malformations of the placenta or umbilical cord. Fetal hydrops can be of immune or nonimmune origin. In immune hydrops, the passage of maternal alloantibodies across the placenta cause fetal red cell destruction and in some cases bone marrow suppression, resulting in fetal anemia and subsequent hydrops. This disease process can affect both the fetus and neonate and is better labeled as hemolytic disease of the fetus and newborn (HDFN). Anemia and maternal alloantibodies are key in the diagnosis of immune hydrops fetalis. Hydrops is typically a second-trimester diagnosis and can be suspected from Doppler ultrasonographic assessment of the peak velocity of

(a)

(b)

(c)

(d)

Figure 17.16 Bilateral pleural effusion on a transverse plane of the fetal thorax (a), fetal abdomen (b), and fetal bladder with the umbilical arteries (c). (d) Coronal plane of hydrops in all compartments with clearly visible separation of the compartments by the diaphragm.

664 Genetic Disorders and the Fetus

systolic blood flow in the middle cerebral artery.111 Intravascular intrauterine erythrocyte transfusion can be offered to correct anemia. Livebirth rates as high as 95 percent have been reported in recent years,112 and when intrauterine transfusion is available, the outcome of fetuses with immune hydrops is just as good as nonhydropic fetuses with HDFN.113 With the widespread introduction of early alloantibody screening, immunoprophylaxis and the successful treatment of rhesus disease by fetal blood transfusions, lethal alloimmune hydrops has practically disappeared.113 Nonimmune fetal hydrops (NIHF) is the presence of ≥2 abnormal fetal fluid collections in the absence of red cell alloimmunization. NIHF is the cause in >85 percent of all affected individuals.114 The most common etiologies include cardiovascular, chromosomal, and hematologic abnormalities, followed by structural fetal anomalies, complications of monochorionic twinning, infection, and placental abnormalities (Table 17.2). Although in many instances the underlying cause may be determined by genetic screening, including WES, maternal antibody and infection screening, fetal

ultrasound scanning including echocardiography and Doppler studies, often the abnormality remains unexplained even after expert postmortem examination. In specific cases there are treatment options, like maternal transplacental administration of antiarrhythmic medication(s) for tachyarrhythmia, laser in twin-to-twin transfusion, intrauterine transfusion in anemia due to ParvoB19 infection, or a thoracoamniotic shunt in case of a chylothorax.115 The spontaneous resolution of hydrops has not been reported and the prognosis for this condition, irrespective of the underlying pathology, is extremely poor, with reported mortality rates of 50–95 percent.116, 117

Cardiovascular defects Congenital heart disease (CHD) is the most common congenital malformation. Gross structural abnormalities of the heart, or of major blood vessels that could actually or potentially affect the proper functioning of the heart are found in 8 per 1,000 livebirths118, 119 (see Chapter 1). The majority of the CHD is compatible with life, while

Table 17.2 Etiologies of nonimmune hydrops fetalis (before introduction of whole-exome sequencing). Etiology

Percent

Cause

Cardiovascular

17–35

Increased central venous pressure

Chromosomal

7–16

Cardiac anomalies, lymphatic dysplasia, abnormal myelopoiesis

Hematologic

4–12

Anemia, high output cardiac failure; hypoxia (α-thalassemia

Infectious

5–7

Anemia, anoxia, endothelial cell damage, and increased capillary

Thoracic

6

Vena caval obstruction or increased intrathoracic pressure with

Twin–twin transfusion

3–10

hypervolemia and increased central venous pressure

Urinary tract

2–3

Urinary ascites; nephrotic syndrome with hypoproteinemia

Gastrointestinal

0.5–4

Obstruction of venous return; gastrointestinal obstruction and

permeability) impaired venous return

infarction with protein loss and decreased colloid osmotic pressure Lymphatic dysplasia

5–6

Impaired venous return

Tumors including chorioangioma

2–3

Anemia, high output cardiac failure, hypoproteinemia

Skeletal dysplasia

3–4

Hepatomegaly, hypoproteinemia, impaired venous return

Syndromic

3–4

Various

Inborn errors of metabolism

1–2

Visceromegaly and obstruction of venous return, decreased

Miscellaneous

3–15

Unknown

15–25

erythropoiesis and anemia, and/orhypoproteinemia

Source: Society for Maternal-Fetal Medicine, Norton ME, Chauhan SP, Dashe JS. Society for maternal-fetal medicine (SMFM) clinical guideline #7: nonimmune hydrops fetalis. Am J Obstet Gynecol 2015;212(2):127. © 2015, Elsevier.115

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Prenatal Diagnosis of Fetal Malformations by Ultrasound 665

between 20–30 percent is severe, defined as being potentially life-threatening and requiring surgery within the first year of life.120–122 The cause of heart defects is heterogeneous and probably depends on the interplay of multiple genetic and environmental factors (see Chapter 1). Environmental factors that have been implicated in the causation of cardiac defects include maternal diabetes mellitus or collagen disease, exposure to drugs, and viral infections such as rubella. When a previous sibling has had a congenital heart defect, in the absence of a known genetic syndrome, the risk of recurrence is 2–3 percent. Specialist echocardiography in these cases at around 20 weeks of gestation can identify most critical cardiac defects. Since the majority of CHD, however, will occur in fetuses from women with no prior risk factor, the major challenge in prenatal diagnosis is to identify the high-risk group for referral to specialist centers and fetal echocardiography. When screening for CHD the cardiac screening examination should include both the four-chamber and outflow tract views.123 A normal heart is situated on the left side and occupies around one third of the area in the thorax. The cardiac axis points to the left by about 45 ± 20 degrees (2 SD). Normal heart rhythm rate lies between 110 and 160 beats per minute, but mild transient bradycardia may be observed when scanning the fetus. When assessing the four-chamber view one should evaluate if both atria and ventricles are equal in size, with the right side showing a moderator band. In the left atrium the foramen ovale flap should be seen and the entrance of the pulmonary veins. The cardiac crux should be intact as well as the ventricular septum. The offset of the right atrioventricular valve is slightly lower than the left. Views of the left and right ventricular outflow tracts should be an integral part of the fetal cardiac screening examination. To achieve this, angulation of the transducer from the four-chamber view is necessary for the demonstration of the normal aortic and pulmonary arterial origins from the left and right ventricles, respectively, to assess the normal size of the vessels and for the diagnosis of great arterial malalignment. When a fetal abnormality is suspected and women are referred for fetal echocardiography, pulsed Doppler studies for measurement of blood velocity across the valves should be added to the evaluation of the fetal heart,

since they are helpful in the assessment of fetal cardiac function (see Chapter 14). Moreover, in the setting of a fetal medicine unit, real-time directed M-mode echocardiography improves the accuracy of the cross-sectional scan in the diagnosis of cardiac defects and provides additional information on cardiac geometry and function. This method is particularly useful in the diagnosis and evaluation of dysrhythmias and the monitoring of in utero antiarrhythmic therapy. With the advancement in technology, assessment of the position of the fetal heart and the four-chamber view is now an integrated part of the first-trimester anomaly scan.123 In addition, since there is a greater incidence of cardiac anomalies among fetuses with a large NT,124, 125 tricuspid regurgitation,126 and abnormal ductus venosus,127 these findings in the first trimester warrant fetal echocardiography. These first-trimester markers subsequently lead to an increased number of fetuses with cardiac anomalies being diagnosed. Over the years prenatal detection rates have significantly increased. When national screening programs were first introduced and screening was primarily based on examination of the four-chamber view of the heart at the 20 weeks scan, only about 25 percent of the critical cardiac defects were prenatally diagnosed.128 The last decade detection rates in a low-risk population have slowly increased with the highest reported rate now being 60 percent,129 which is remarkably high in an unselected population, compared to similar programs that never exceeded 45 percent.95, 128, 130–132 For isolated CHD, however, detection rates are relatively low, not exceeding 44 percent.129 The detection rate very much depends on the severity of the anomaly, with CHD affecting the four-chamber view, such as hypoplastic left heart syndrome, being more often diagnosed prenatally compared to smaller defects, such as ventricular septal defects (Figure 17.17). The recognition that the introduction of the three-vessel view would significantly contribute to the prenatal detection of CHD had a dramatic impact.133 In a recent study it was demonstrated that the prenatal diagnosis of transposition of the great arteries (TGA) increased from 44 percent in the period without inclusion of the three-vessel view to 82 percent in the period including the

666 Genetic Disorders and the Fetus

Figure 17.17 A fetus with hypoplastic left heart syndrome.

three-vessel view. For tetralogy of Fallot the detection rate increased from 44 percent to 68 percent, respectively.134 With improved expertise and equipment, basic echocardiography for the examination of the four-chamber view, the connection of TGA, and the detection of dysrhythmias should be incorporated in the routine ultrasound screening programs for all pregnancies. Suspected anomalies can then be referred to specialized centers for further elucidation of the problem. Even in the first trimester, fetal echocardiography can pick up the majority of major CHD.135 When isolated, some of the cardiac defects resolve spontaneously (e.g. ventricular septal defect), some are correctable and carry intermediate operative risks (e.g. TGA), while others carry high operative risks (e.g. truncus arteriosus and left hypoplastic heart syndrome). Cardiac anomalies

Depending on the type of defect, CHD is more or less associated with other anomalies. TGA is generally isolated and carries a good prognosis, while tetralogy of Fallot and double outlet right ventricle are associated with chromosome anomalies and genetic syndromes. The presence or absence of other structural anomalies significantly adds to the risk of a more generalized problem. When other anomalies are present, CHD especially has a high risk of chromosome anomalies with a reported incidence around 20 percent, the most common being trisomy 21, trisomy 18, and 22q11 microdeletions.136, 137 Overall, it is advocated to offer invasive prenatal testing for fetal karyotyping or QF-PCR or FISH

and chromosomal microarray analysis whenever CHD is detected in a fetus, irrespective of how small or large the defect is. In a recent systematic review, array comparative genomic hybridization (array CGH) yielded additional clinically valuable information in 7.0 percent of fetal CHD cases, even after karyotyping and 22q11 FISH analysis were normal.138 More recently, WES has been added to the prenatal workup in some settings.139, 140 Cardiac anomalies can be the stimulus for a detailed search of anomalies; the opposite is also true, since in most genetic syndromes, other anomalies are likely to be detected first and only after a thorough evaluation of the fetal heart is a cardiac anomaly identified. Extracardiac defects, particularly craniospinal, gastrointestinal, and renal, are found in approximately one-third of cases. Based on the combination of anomalies, certain chromosome anomalies or genetic syndrome should be considered and rapid aneuploidy testing and chromosome microarray analysis should be part of standard care.141 However, since many genetic syndromes remain undetected with these tests, addition of WES had a positive effect on making a definite diagnosis.142 Ventricular septal defect

A ventricular septal defect (VSD) is a defect in the ventricular septum resulting in communication between the ventricular cavities (Figure 17.18). A VSD usually occurs as an isolated anomaly, but may also be present in TGA and tetralogy of Fallot. A recent study investigating the incidence and nature of abnormal chromosomal microarray analysis (CMA) results in a large cohort of pregnancies with VSD, showed that the rate of abnormal CMA findings (1.4 percent) in isolated VSD did not differ from that in pregnancies without a VSD. Atrioventricular septal defect

Atrioventricular septal defects (AVSDs) are characterized by a defect of the septal crux immediately above and below the atrioventricular valves (Figure 17.19). Coarctation of the aorta is the most frequent associated cardiac anomaly, while in 75 percent these defects are associated with other cardiac malformations.143 In cases of prenatally diagnosed AVSD up to 44 percent of the fetuses will be affected with Down syndrome.144

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 667

(a)

(b)

Figure 17.18 Demonstrating a ventricular septal defect in transverse plane with (a) and without (b) color Doppler flow.

(a)

(b)

Figure 17.19 Demonstrating an atrioventricular septal defect with the atrioventricular valves during diastole (a) and systole (b). The septum primum is clearly missing.

Transposition of the great arteries

TGA is characterized by atrioventricular concordance and ventriculo-arterial discordance in which the aorta arises from the right ventricle and the pulmonary artery from the left ventricle. The diagnosis should be triggered when in the three-vessel view only two vessels are seen. Moreover, in three-vessel tracheal view a parallel course of great arteries may be observed. This cyanotic heart disease accounts for 5–7 percent of all CHDs and is usually an isolated finding.145 The prenatal diagnosis influences postnatal outcomes and therefore requires planned delivery and perinatal management (Figure 17.20). Tetralogy of Fallot

Tetralogy of Fallot consists of three main components that are the result of malalignment of the

infundibular septum: VSD, overriding aorta, and pulmonary stenosis (Figure 17.21). It occurs in 8–12 percent of fetuses with a cardiac anomaly.146 Sometimes the right ventricle may show hypertrophy. A right aortic arch is the most frequent cardiac anomaly in 22 percent. An abnormal cardiac angle will be noticed in 24 percent of fetuses with tetralogy of Fallot.147 Aneuploidy and 22q11 microdeletion are frequent associations.

Abdominal wall defects From approximately 7 weeks of gestation, the embryonic development of the intra-abdominal viscera can be followed by ultrasound.148, 149 At this gestation, the anterior abdominal wall is already

668 Genetic Disorders and the Fetus

(a)

(b)

Figure 17.20 Fetus with transposition of the great arteries presenting at 20.4 weeks of gestation. (a) Transverse plane of the normal four-chamber view. (b) Outflow tracts – transposition of the great arteries is clearly visible.

(a)

(b)

(c)

(d)

Figure 17.21 Outflow tract of the overriding aorta with ventricular septal defect (a, b), showing transverse plane at the level of the three-vessel view, presenting as the “U sign,” which corresponds to a right aortic arch, without and with color Doppler (c and d)).

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 669

formed, but there is evidence of physiological herniation of the bowel into the cord: the abdominal cavity is too small to accommodate its contents. This physiological herniation is due to 90 degrees of counterclockwise rotation of the midgut and sonographically evident from 9 to 11 weeks of gestation. At approximately 12 weeks of gestation, the midgut will further rotate to 270 degrees and returns to the abdominal cavity. Beyond 12 weeks of gestation, midgut herniation is no longer physiologic.149 The liver is never present in physiological herniation, since it does not undergo physiologic migration outside the abdominal cavity. Omphalocele (exomphalos)

Omphalocele is an anterior abdominal wall defect with an incidence of 2 per 10,000 livebirths.150 The defect is herniated through the base of the umbilical cord, containing abdominal intestines like liver or spleen, and is covered by three layers: amnion, peritoneum, and Wharton’s jelly. Omphaloceles are divided into two groups: nonliver-containing or liver-containing omphalocele. Eighty percent of omphaloceles belong to the liver-containing group. Although not completely established, the pathogenesis of omphalocele is thought to have two mechanisms. The first one describes the failing of the 270 degrees counterclockwise rotation of the midgut back into the abdominal cavity. The second one is based on failure of closing of the lateral folds, resulting in a large abdominal wall defect through which abdominal content can herniate.151 Chromosomal and other associated structural anomalies are common in omphalocele. In the literature, associated structural anomalies are described in 35–70 percent of cases. Chromosomal anomalies are more prevalent in nonliver-containing omphalocele; approximately 60 percent are associated with fetal aneuploidy.152, 153 Trisomy 18, trisomy 13, Turner syndrome, and triploidy are the most reported chromosomal anomalies.154 Survival depends primarily on whether other malformations or chromosomal defects are present. In isolated cases, the malformation is correctable and survival rates of more than 90 percent are reported.155–157

First trimester Prenatal diagnosis of an omphalocele is made in 90 percent of cases and mostly in the first

trimester.30, 158 In a large prospective study with 44,859 pregnancies, fetal anomalies were detected in 488 cases and 213 were successfully identified through a first-trimester scan at 11+0 to 13+6 weeks of gestation, including all 104 cases of exomphalos, gastroschisis, megacystic, and body stalk anomaly.17 Since the liver is never a physiological finding outside the abdominal cavity, large, liver-containing omphalocele can be detected early in the first trimester, at approximately 9–10 weeks of gestation. Suggestive of liver is a homogeneous mass greater than 5–10 mm, present at the physiological herniation area.159, 160 The diagnosis of a nonliver-containing omphalocele is only possible after 12 weeks of gestation, when the physiologic midgut herniation has resolved.149 The diagnosis of omphalocele is made on the dorsal plane of the abdominal wall, demonstrating the herniated sac with its visceral contents and the umbilical cord insertion at the apex of the sac (Figure 17.22). Gastroschisis

Unlike omphalocele, gastroschisis is a paraumbilical abdominal wall defect in which the viscera herniate through the abdominal wall, usually located to the right side of the umbilical cord. Evisceration of the bowel occurs, sometimes in combination with other abdominal organs, floating freely in the amniotic fluid. Because the loops of intestines lie uncovered in the amniotic fluid, they become thickened, edematous, and matted. Several hypotheses have been proposed to explain the pathogenesis of gastroschisis, all

Figure 17.22 Large omphalocele at 13 weeks of gestation containing liver.

670 Genetic Disorders and the Fetus

involving defective formation or disruption of the body wall in the embryonic period with gut herniation. The latest hypothesis proposes that abnormal folding of the body wall results in a ventral body defect through which the gut herniates.161 The incidence of gastroschisis is increasing worldwide, approaching 1 in 4,000 live births in recent years.162 A consistent risk factor that has been reported in epidemiological studies is young maternal age. One European study found that compared with mothers aged 25–29, the relative risk was 7.0 (95 percent confidence interval 5.6–8.7) for mothers under 20 and 2.4 (95 percent CI 2.0–3.0) for mothers aged 20–24 years.163 Smoking, recreational drug use, and low BMI are also strongly associated with gastroschisis.162 It is usually an isolated finding and chromosomal anomalies are rare.158, 164 The prevalence of chromosomal anomalies in fetuses with isolated gastroschisis is not increased as compared to the normal population. Therefore, isolated gastroschisis is not a strong indication to pursue invasive diagnostics. However, other structural malformations are found in up to 16.6 percent of cases.165, 166 Over the past years, the postoperative survival rate increased from 75 percent to 96 percent and the incidence of complications decreased.166, 167

First trimester Most cases will be detected in first trimester. Like omphalocele, the prenatal detection rate is over 90 percent as a result of routine ultrasound screening.30 Diagnosis of gastroschisis is made

(a)

at the transverse plane, when demonstrating the normally situated umbilicus. It typically presents as a para-umbilical abdominal wall defect, to the right side of the midline, with herniation of free-floating bowel into the amniotic cavity168 (Figure 17.23).

Second and third trimester A fetus with gastroschisis will be monitored frequently because of fetal growth restriction that can occur in second or third trimester. Underestimation of the severity of the growth restriction is frequently seen, since measuring the abdominal circumference is complicated. Sonographers should also be alert to intra-abdominal bowel dilatation (Figure 17.24), gastric dilatation, and polyhydramnmios, since these findings are associated with bowel atresia.169 The number of cases of “sudden” intrauterine fetal death occurring in the last trimester of pregnancy is still significant (up to 13.6 percent). Usually the cause of death is not found170, 171 and occurs in both growth-delayed and nongrowth-delayed fetuses.170, 172 The hypothesis is that as a result of fetal hypo-albuminemia due to protein loss through the intestine, hypovolemia occurs with cardiovascular problems as a result.173 Regular fetal monitoring seems to decrease the intrauterine fetal deaths and results in premature birth. Body stalk anomaly

Body stalk anomaly is a severe and fatal condition with a reported incidence of 1 in 14,000 to

(b)

Figure 17.23 Gastroschisis in first trimester at 12.0 weeks of gestation. (a) Crown–rump length view of the fetus in mid-sagittal plane. (b) Gastroschisis in a transverse plane.

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 671

(a)

(b)

Figure 17.24 (a) Transverse plane showing gastroschisis in the second trimester, detected at 19.3 weeks of gestation. (b) Coronal plane showing bowel dilation in the same fetus at 26.5 weeks of gestation.

(a)

(b)

Figure 17.25 Body stalk anomaly at 13 weeks of gestation. (a) Mid-sagittal plane with a large abdominal wall defect containing liver intestines and the bladder. (b) Severe kyphoscoliosis and a short umbilical cord.

1 per 31.000 pregnancies in large epidemiologic studies.155 It results from a developmental failure of the cephalic, caudal, and lateral body folds. In this severe abdominal defect the intrathoracic and abdominal organs lie outside the abdominal cavity, covered with a sac attached directly to the placenta.174 There is an absent or extremely short umbilical cord.175 Multiple additional defects may coexist, including skeletal, limb, and craniofacial abnormalities.176 Severe kyphoscoliosis is often present (Figure 17.25). Prenatal diagnosis is made after visualization of an anterior abdominal wall defect attaching the fetus to the placenta or uterine wall (Figure 17.26).

Bladder exstrophy and cloacal exstrophy

Bladder exstrophy is a defect of the caudal fold of the anterior abdominal wall. Whereas a small defect may cause epispadias alone, a large defect leads to exposure of the posterior bladder wall. Cloacal exstrophy is a severe multisystem congenital malformation involving the urinary, gastrointestinal, musculoskeletal, and neurological tracts. The disorder is also known as OEIS (omphalocele–exstrophy–imperforate anus–spinal dysraphism) complex.177 It most likely results from a very early defect in the closure of the ventral body wall rather than an abnormality related to premature rupture of the cloacal membrane.178

672 Genetic Disorders and the Fetus

(a)

(b)

(c) Figure 17.26 Body stalk anomaly at 10.3 weeks of gestation. (a, b) Mid-sagittal plane (a) and a transverse plane (b) showing the sac attached to the placenta. (c) Color Doppler showing the umbilical cord.

The incidence of bladder exstrophy is 1 per 20,000–40,000 with female predominance. Cloacal exstrophy is extremely rare, with a birth incidence of 1 per 200,000–400,000.178 A genetic basis is possible, but it is more likely to be a sporadic problem because it leads to sterility. Survival rates of 83–100 percent have been reported but involve technically challenging surgical management of the bowel and genitals. Although survival rates have improved due to advances in surgical reconstruction and techniques, the quality-of-life remains a concern.179 Accurate prenatal diagnosis of cloacal exstrophy appears to be less than 25 percent due to the rarity of the disorder.180 Major and minor criteria were defined in fetuses with cloacal exstrophy.180 Major criteria were nonvisualization of the urinary bladder, large midline infraumbilical anterior abdominal wall defect, omphalocele,

and meningomyelocele. Minor criteria were renal anomalies, lower extremity defects, and ascites.180 Prenatal ultrasound findings associated with bladder exstrophy are absence of bladder filling, low-set umbilicus, pubic bone diastasis, diminutive genitalia, and a lower abdominal mass that increases in size when the pregnancy progresses181, 182 (Figure 17.27). Prenatal detection is usually made in second trimester.182

Gastrointestinal anomalies On ultrasound, the fetal stomach is visible as a sonolucent structure in the upper left quadrant of the abdomen. A fluid-filled stomach should be detectable between 11 and 14 weeks of gestation, since the fetus begins to swallow amniotic fluid. The bowel is variably echogenic until the third trimester, when prominent meconium-filled loops

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 673

Figure 17.27 Bladder exstrophy with nonvisualization of the urinary bladder.

Figure 17.28 The “pouch sign” in a fetus with esophageal atresia.

of large bowel are commonly seen. The normal esophagus is typically not visualized during ultrasound, it is collapsed and the amount of fluid is too small to be seen.

percent of cases, with trisomy 21 and trisomy 18 as most reported chromosomal anomalies.186 Prenatal diagnosis of esophageal atresia is poor and remains challenging, with reported detection rates between 23 percent to 32 percent.187 It should be suspected when fetal ultrasound fails to demonstrate the fetal stomach or a small stomach is visible, frequently in combination with a polyhydramnios. Ultrasound findings differ between types of esophageal atresia, depending on the presence of a fistula. In type A, without a fistula, the three key findings are: (i) polyhydramnios, (ii) absent stomach or a collapsed stomach, and (iii) blind-ending dilated upper esophageal pouch (“pouch sign”) (Figure 17.28). Prenatal diagnosis of esophageal atresia with a TEF is more difficult since the fistula allows the stomach to fill with amniotic fluid. In a systematic review of 1,760 affected fetuses, 77.9 percent of cases were correctly identified prenatally and only 21.9 percent of cases with a TEF.184 Ultrasound findings alone are thought to have a high false-positive rate for the diagnosis of esophageal atresia. Therefore, it is proposed that MRI should be used for establishing or ruling out a prenatal diagnosis of esophageal atresia.188

Esophageal atresia

Esophageal atresia refers to a congenitally interrupted esophagus and has an incidence of 2.4 in 10,000 births.183 Normally, during the 4th week of gestation, the primitive foregut separates into the trachea and the esophagus. Esophageal atresia arises from a developmental disruption of this separation. In 90 percent of esophageal atresia cases, a trachea-esophageal fistula (TEF) is present. Five types of esophageal atresia have been described in literature: type A – esophageal atresia without TEF (7 percent); type B – esophageal atresia with a TEF to the proximal esophageal segment (2 percent); type C – esophageal atresia with a TEF to the distal esophageal segment (86 percent); type D – esophageal atresia with TEF to both the proximal and distal esophageal segments (10 mm is always pathological and a reason for further evaluation. In the third trimester the cut-off for the diagnosis of ANH increases to 10 mm. Bilateral cases should have follow-up at higher frequency (Figure 17.31). In a recent study of 279 infants with secondtrimester isolated ANH (cut-off 5 mm), ANH had normalized (APPD 12 mm is seen in the first trimester the prognosis is extremely poor and parents often opt for termination of pregnancy.227 Posterior urethral valves are responsible for over 63 percent of LUTO, followed by urethral atresia or stenosis in 17 percent of cases.225 The male fetus is predominantly affected with posterior urethral valves, while in female fetuses urethral atresia is more common. Rare presentations of LUTO are megacystis–microcolon–intestinal hypoperistalsis syndrome (MMIHS),228 which may present with prune belly syndrome.229 When LUTO is suspected, ultrasound evaluation should include gender, associated anomalies such as heart (vertebral defects, anal atresia, cardiac

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 679

defects, tracheo-esophageal fistula, renal anomalies limb anomalies [VACTERL association]); omphalocele (omphalocele exstrophy imperforate anus [OIES] complex); growth (overgrowth syndromes); and amniotic volume should be assessed. At the first-trimester scan, in over 30 percent of pregnancies with a megacystis, a trisomy was diagnosed,230 and in total 10 percent LUTO is associated with trisomy 21, 18, or 13.218 Moderate megacystis is in general more frequently associated with trisomies than severe megacystis.230 Although vesicoamniotic shunting seemed promising in treating LUTO caused by posterior urethral valves (PUV), thus far it seems to improve survival without improving renal function or decreasing the need for renal replacement therapy231 (see Chapter 29). Structural kidney malformations

Dysplastic kidneys Multicystic dysplastic kidneys

Multicystic dysplastic kidneys (MCDK) is a severe form of renal dysplasia in which the renal tissue is displaced by cysts. As a result the kidney consists of multiple noncommunicating cysts which differ in size and are separated by dysplastic parenchyma. Primitive nephrons filled with urine are the basis of the multiple cysts, which are seen initially at the periphery of the kidney. The kidney is enlarged and the overall shape is abnormal. The affected kidney is nonfunctional and frequently undergoes involution when the cysts shrink from absence of urine production. This leads to a picture similar to renal

(a)

agenesis. Unilateral MCDK is prevalent in 1:4,300 births, with the left kidney and males slightly more often affected232 (Figure 17.32). Bilateral MCDK occurs in 1:10,000 births. Typically the diagnosis of MCDK is made in the second trimester, but recent publications suggest that it may be visible by 2D and 3D ultrasound in the first trimester already233 (Table 17.4). Unilateral MCDK without additional anomalies is often sporadic,234 but bilateral MDCK should raise suspicion of aneuploidy or inherited conditions.218 Associated renal anomalies, often found in the contralateral kidney, include VUR and obstruction of the ureteropelvic junction, duplex kidney, agenesis, or pelvic kidney. The most common extrarenal abnormalities are heart defects, esophageal or intestinal atresia, spinal abnormalities, and the VACTERL association. Bilateral MDCK, formerly known as Potter type 2, can lead to “Potter sequence,” that is pulmonary hypoplasia secondary to impaired renal function and anhydramnios. A recent study in 53 cases of prenatally suspected MCDK, of which 46 cases were liveborn and confirmed post natally (38 survivors, 8 nonsurvivors) revealed that extrarenal anomalies, bilateral MCDK, contralateral renal anomalies, and anhydramnios were significantly associated with death or need for dialysis (all p < 0.0001).235

Polycystic kidney disease Polycystic kidney disease is typically due to terminal epithelial differentiation disruption. This includes autosomal recessive polycystic kidney

(b)

Figure 17.32 Unilateral multicystic dysplastic kidney demonstrated in transverse plane (a) and coronal plane (b).

680 Genetic Disorders and the Fetus

Table 17.4 Conditions associated with large and echogenic kidneys from Yulia and Winyard218 . Amniotic

Renal

APPKD

Renal

fluid

cysts

dilatation

size

Macrosomia

Extrarenal abnormalities

Inheritance

Obstruction

N/oligo

+/−

+

+

−

+/−

Sporadic

Dysplasia

N/oligo

+/−

+/−

+

−

+/−

10 AD

ARPKD

N/oligo

−

−

+

−

−

AR

ADPKD

N/oligo

+

−

+

−

−

AD

Beckwith–Wiedeman

N/poly

−

+/−

+

+

+

AD or disomy

Perlman

N/oligo

−

+/−

+

+

+

AR

+

+

+

X-linked

Meckel–Gruber

Oligo

−

−

+

−

+

AR

Nephrocalcinosis

N

−

−

−

−

−

Sporadic

Simson–Golabi–Behmel

ARPKD, autosomal recessive polycystic kidney disease; ADPKD, autosomal dominant polycystic kidney disease; AD, autosomal dominant; AR, autosomal recessive. Source: Yulia A, Winyard P. Management of antenatally detected kidney malformations. Early Hum Dev 2018;126:38. © 2018, Elsevier.218

disease (ARPKD), which occurs in approximately 1:20,000 births.236 It is characterized by multiple microscopic cysts in the distal collecting ducts. It is caused by mutations in the PKHD1 gene. Characteristic ultrasound findings are two large, hyperechogenic kidneys (Figure 17.33). The differential diagnosis includes Beckwith–Wiedeman syndrome, trisomy 13, or Meckel–Gruber syndrome. The timing and presentation may vary, but when anhydramnios is present, it is almost always a lethal condition. Formerly this was also referred to as infantile polycystic kidney disease or Potter 1 renal dysplasia. Autosomal dominant polycystic kidney disease (ADPKD), or adult polycystic kidney disease, is far more common with an incidence of 1:500–1,000 livebirths.237 Ultrasound findings are similar to ARPKD, with bilateral enlarged (hyperechogenic) kidneys with or without cysts. It can either be isolated or with cardiovascular and biliary tract involvement.238 In adulthood, ADPKD accounts for 5–10 percent of all end-stage renal disease. Symptoms with cysts formation and renal failure usually start in middle age, but prenatal presentation may occur in 2–5 percent of cases, mainly in the third trimester, and is associated with adverse outcomes. ADPKD is associated with mutations in the PKD1 or PKD2 genes.239

Nephronophthisis (former Potter 3 renal dysplasia) Nephronophthisis (NPH) is a renal ciliopathy and is characterized by reduced renal concentrating ability, chronic tubulo-interstitial nephritis, cystic renal disease, and progression to end-stage renal disease (ESRD).240 It includes autosomal recessive renal diseases in which multiple cysts are formed within the cortico-medullary region. Nephronophthisis is responsible for 2.4–15 percent of ESRD in children.241 Mutations can be found in one of the 19 known NPH-related genes involved in ciliary structure or function. Well-known nephronophthisis syndromes are Meckel–Gruber syndrome, characterized prenatally by hyperechogenic kidneys, polydactyly, and encephalocele, and Bardet–Biedl syndrome, characterized by hyperechogenic kidneys and polydactyly.242 Renal agenesis

Unilateral renal agenesis Unilateral renal agenesis, defined as congenital absence of one kidney, accounts for 5 percent of renal malformations243 (Figure 17.34). Unilateral renal agenesis occurs in 1 in 1,000 births. Although it may be secondary to a chromosomal abnormality or part of a genetic syndrome, such as Fraser syndrome, more commonly it is an isolated finding. It is diagnosed either antenatally or in a workup for a urinary tract infection. In nonsyndromic cases, the risk of recurrence is approximately 3 percent. However, 4.5 percent of first-degree relatives of affected

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 681

(a)

(b)

Figure 17.33 Polycystic kidney disease presenting two large hyperechogenic kidneys in transverse plane (a) and sagittal plane (b).

Figure 17.34 Unilateral renal agenesis demonstrated in coronal plane.

Renal agenesis is accompanied by absence of both ureters and renal arteries, and results from failure of ureteric buds to either form or reach the metanephric mesenchyme which results in apoptosis.219 Anhydramnios around 18–20 weeks of gestation is the most striking feature on ultrasound that will lead to the diagnosis of bilateral renal agenesis. Color Doppler flow of the renal arteries will facilitate the detection of bilateral renal agenesis, especially when imaging due to oligo- or anhydramnios is difficult.216 If either unilateral of bilateral renal agenesis is identified, a detailed ultrasound of all other organ systems must be done because the risk of a genetic syndrome may be as high as 30 percent.242, 246

infants have unilateral renal agenesis themselves, against 0.3 percent in a control population.244 Unilateral renal agenesis is easily overlooked as amniotic fluid is normal due to hyperfiltration and compensatory hyperplasia of the contralateral kidney and normal bladder filling. Differential diagnosis includes, among others, a pelvic kidney.

Abnormalities of pelvic migration

Bilateral renal agenesis Bilateral renal agenesis complicates approximately 1 in 10,000 to 3 in 10,000 births.242 In general, it is a lethal condition, leading to pulmonary hypoplasia because of lack of amniotic fluid in the critical stage of lung development between 16 and 20 weeks of gestation.245 First-trimester diagnosis is possible, but more difficult as amniotic fluid may appear normal. However, the bladder is not filled with urine.

Pelvic kidney A pelvic kidney may be considered when the kidney cannot be visualized at its usual anatomical site, but is present in the pelvis. The diagnosis can easily be missed because the adrenal glands can be mistaken for kidneys. Its incidence is approximately 1 in 1,700 pregnancies.247 In most cases a pelvic kidney is isolated, but it has also been associated with genetic disorders, including Turner

Disruption of the normal embryologic migration of the kidneys can result in renal ectopia (pelvic kidney) and/or fusion abnormalities (horseshoe kidney). Most patients are asymptomatic but complications such as urinary tract infection or reflux occur.

682 Genetic Disorders and the Fetus

syndrome (45,X), Williams–Beuren syndrome, and Antley–Bixler syndrome.248–250 In most of these conditions, a pelvic kidney is not isolated and additional anatomic abnormalities are present. A recent study revealed no difference in copy number variants (CNVs) detected with chromosomal microarray analysis in fetuses with a pelvic kidney as compared to a normal population.251 Therefore invasive procedures solely for isolated pelvic kidneys can be questioned.

Horseshoe kidney A horseshoe kidney is seen in 1:400 persons. A horseshoe kidney is a result of fusion of the upper or lower poles of the kidneys before 4–6 weeks of embryological development and before the kidneys have migrated upward and rotated on their long axis.252

Skeletal anomalies There are a wide range of skeletal anomalies with different underlying pathologies such as skeletal dysplasia, idiopathic limb reduction, chromosome anomalies, teratogens, genetic syndromes, and metabolic conditions. Skeletal dysplasias are relatively rare, each with a specific mode of inheritance, genotype, phenotype, recurrence risk, and implications for neonatal survival and quality of life (see Chapter 20). In the past, skeletal anomalies were usually only recognized postnatally, but with the introduction of routine scanning, many may be discovered in the first trimester because of specific sonographic findings.253, 254 In the case of skeletal dysplasia it is important to at least differentiate between the lethal and nonlethal types. Knowledge on the specific prenatal features that are present in skeletal dysplasia, however, will greatly facilitate diagnosis. Gene discovery and WES (see Chapters 14 and 20) adds to an accurate prenatal diagnosis in additional cases. A definitive diagnosis facilitates parental counseling necessary to provide parents with accurate information regarding the prognosis and the option to make autonomous reproductive choices. When a skeletal anomaly is found during a routine ultrasound or a skeletal dysplasia is prenatally suspected, a systematic examination of all the skeletal structures and all the organ systems is recommended to arrive at the correct diagnosis.255

All limbs must be evaluated for length, shape, mineralization, and movement, and associated abnormalities in other systems, particularly the head, thorax, and spine, should be sought (see Chapter 20). A putative diagnosis may then allow definitive confirmation via mutation analysis. The majority of bones of the appendicular system can be imaged from the first trimester onwards, and several nomograms relating the length of long bones to gestational age or biparietal diameter have been published.256 For other skeletal structures, nomograms relating to the head, clavicles, mandible, and thorax are widely available.257–259 Prenatal assessment of the fetus suspected of a skeletal anomaly will usually start with measurement of all the long bones. The absence or degree of hypoplasia should be evaluated. Anomalies can be both symmetrical and asymmetrical and the pattern of shortening may be variable. Most often the trigger leading to fetal evaluation for skeletal dysplasia will be the finding of a short femur. It should be recognized that the most common cause of a short femur is a genetically small but healthy child, either due to being small for gestational age or due to fetal growth restriction (Table 17.5). The most frequent anomaly in fetuses with a short femur is Down syndrome. In the case of a short femur and the prenatal suspicion of achondroplasia, usually in the third trimester because of a sudden decline in femoral growth velocity, the proximal diaphysis–metaphysis angle of the femur should be measured. From 20 weeks onwards, this angle is generally wider in fetuses with achondroplasia as compared to fetuses affected by intrauterine growth restriction (IUGR), small for gestational age (SGA) fetuses, and normal fetuses, and exceeds 130 degrees260, 261 (Figure 17.35). Severe lethal skeletal dysplasia such as osteogenesis imperfecta type II, achondrogenesis, thanatophoric dysplasia, and diastrophic dysplasia, have a first-trimester detection rate of 70 percent.30 In the case of achondroplasia, however, the diagnosis may not become obvious until the third trimester because of a sudden decline in femoral growth velocity.262 When examining the bones it is also important to assess whether bowing, fractures, or ephiphyseal stippling is present. A minor degree of lateral curvature of the femur is commonly seen in normal fetuses. Fetuses with achondroplasia may have mildly bowed femora.262

CHAPTER 17

Prenatal Diagnosis of Fetal Malformations by Ultrasound 683

Table 17.5 Differential diagnosis in short femur. Feature

SGA

FGR

Down syndrome

Achondroplasia

Ultrasound anomalies

None

None

Markers AVSD

Frontal bossing

Femur length