Human Ring Chromosomes: A Practical Guide for Clinicians and Families 3031475291, 9783031475290

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Peining Li Thomas Liehr  Editors

Human Ring Chromosomes A Practical Guide for Clinicians and Families

Human Ring Chromosomes

Peining Li · Thomas Liehr Editors

Human Ring Chromosomes A Practical Guide for Clinicians and Families

Editors Peining Li Laboratory of Clinical Cytogenetics Department of Genetics Yale School of Medicine New Haven, CT, USA

Thomas Liehr Jena University Hospital Institute of Human Genetics Friedrich Schiller University Jena, Germany

ISBN 978-3-031-47529-0 ISBN 978-3-031-47530-6  (eBook) https://doi.org/10.1007/978-3-031-47530-6 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

To the hundreds of genetic researchers and clinicians whose efforts have contributed to the understanding of human ring chromosomes and the improvement of laboratory diagnosis and clinical management for individuals with a ring chromosome. To the thousands of individuals carrying a ring chromosome who charted the genetic cause and disease course for better clinical care and society support of this rare condition.

Preface

Cytogenetics has been a powerful tool to understand the chromosomal and genomic organization of genetic material on a cellular basis for many species. The earliest study of ring-shaped chromosomes in corn provided insights into their cellular behavior and impact on chromosome constitution. As discussed by Barbara McClintock in her paper entitled “A Correlation of Ring-shaped Chromosomes with Variegation in Zea Mays” published in the Proceedings of the National Academy of Science USA, 1932, 18(12):677– 681: “Investigations which should lead to an understanding of the mechanism responsible for the frequent decrease and occasional increase in size of the rings or for their loss have not yet been conducted. Lack of uniformity in the splitting plane could give rise to a double sized ring with two insertion regions or cause split halves of the ring to become interlocked. Subsequent movement of the two insertion regions toward opposite poles at anaphase would cause breaks in the ring chromosomes and thus produce changes in their size and constitution.” Ring chromosomes as a unique chromosomal abnormality causing human diseases were first reported in 1962. Since then, the technologies for chromosome analysis have evolved from various banding techniques, molecular characterization by locus-specific fluorescence in situ hybridization and region-specific assays, to genome-wide chromosomal microarray analysis and next generation sequencing. Clinical cytogenetics has expanded from numerical and structural chromosomal abnormalities to a spectrum of genomic disorders and pathogenic copy number variants. This progress has enabled a better understanding of genomic structures and imbalances and expanding knowledge on clinico-cytogenomic correlations for many cases of ring chromosomes. An international consortium for human ring chromosomes (ICHRC) has been organized to work on laboratory standards and guidelines in analyzing ring chromosomes, to develop an interactive ring chromosome registry, and to promote translational and basic research related to ring chromosomes. The initial effort for ICHRC was proposed by Thomas Liehr, Igor Lebedev, and Peining Li in the 2021 annual meeting of European Cytogenomic Association followed by a concurrent session entitled “Human ring chromosome disorders: An international collaboration toward better diagnosis, interpretations, and clinical management” in the 2022 annual meeting of American College of Medical Genetics and Genomics (ACMG). Barbara R. DuPont vii

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Preface

organized the session with presenters Peining Li on ring chromosome registry, Marco Crimi on ring chromosome 14 international, Thomas Liehr on small supernumerary marker/ring chromosomes, Igor Lebedev on ring chromosome structure and mitotic instability, and Anthony Wynshaw-Boris on ring chromosome correction via reprogramming into induced pluripotent stem cells. The organizing committee of ICHRC include clinical cytogeneticists, clinicians, and basic researchers with regional representatives of Barbara R. DuPont, Peining Li, Jaclyn Murry, and Mary Ann Thomas from North America, Marco Crimi, Igor Lebedev, and Thomas Liehr from Europe, Qiping Hu and Frenny Sheth from Asia, and Maria Isabel Melaragno from South America. The proposal for writing this book was initiated and discussed among members of the ICHRC organization committee. Special thanks for their effort to reach out experts as corresponding authors and co-authors on each chapter. Part I of this book deals with the history of human ring chromosome disorders, the diagnostic methods in current practice, the bioinformatic resources, and patient advocacy activities. Part II includes chapters for constitutional ring chromosomes, supernumerary small ring chromosomes, and importantly real-life experience from individuals and families of ring chromosomes. Part III covers acquired ring chromosomes in tumors of hematopoietic and lymphoid tissues and solid tumors in various tissues. Part IV is about the molecular mechanisms of ring chromosome formation and instability and research approaches using induced pluripotent stem cells and other model organisms. This book represents a systematic effort of ICHRC to summarize laboratory and clinical findings of constitutional ring chromosomes for specific autosomes and sex chromosomes and acquired ring chromosomes in various types of tumors. It serves as a comprehensive desktop reference for diagnostic and clinical geneticists, genetic counselors, clinicians, patients, and their families. To conclude, we would like to thank all authors for their expertise and effort on contributed chapters. We would also like to express our gratitude to Springer Nature publisher, specially to Yogesh Padmanaban, Birke Balia, Viju Falgon, and Tanja Weyandt for their assistance in communicating with all authors and coordinating the process. New Haven, USA Jena, Germany August 2023

Peining Li Thomas Liehr

About This Book

Chromosomes are the structures, which carry in a normal human cell the 6 billion base pairs constituting the human genome. Accordingly, there are 46 differently long, densely packed DNA-strands per cell, normally organized linear with one centromeric constriction, each. Variations from this norm can be incredibly diverse and be or not be connected with a clinical phenotype for the corresponding carrier. The state that one (or more) of the human chromosomes may form a ring shape is extremely rare. This must be the reason that only 60 years after its first description an international consortium for human ring chromosomes (ICHRC) was initiated, mainly by the initiative of Peining Li and the support from colleagues with diagnostic and research experience in this abnormality. This book and all in preface described activities are owed to his enthusiasm in first place. As the co-editor of this book, I sincerely thank him, together with all authors of this book for all his work. July 2023

Thomas Liehr

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Contents

Part I  Introduction 1

Historical Perspective of Human Ring Chromosomes. . . . . . . . . 3 Peining Li and Thomas Liehr

2

Diagnostic Methods for Ring Chromosomes . . . . . . . . . . . . . . . . 17 Benjamin Hilton and Barbara R. DuPont

3

Genetic Databases and Online Ring Chromosome Registry. . . . 31 Qiping Hu, Deqiong Ma, Peining Li and Thomas Liehr

4

Advocate Activities and Patient-Centred Approaches. . . . . . . . . 43 Marco Crimi and Allison Watson

Part II  Constitutional Ring Chromosomes 5

Ring Chromosome 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Sainan Wei and Sheila Saliganan

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Ring Chromosome 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Jaclyn B. Murry and Ying S. Zou

7

Ring Chromosome 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Maria Isabel Melaragno and Bruna Burssed

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Ring Chromosome 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Kathleen M. Bone, Judy Chernos and Mary Ann Thomas

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Ring Chromosome 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Jingwei Yu

10 Ring Chromosome 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Frenny Sheth, Jhanvi Shah and Harsh Sheth 11 Ring Chromosome 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Thomas Liehr 12 Ring Chromosome 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Anna A. Kashevarova and Igor N. Lebedev

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13 Ring Chromosome 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Anna M. Szekely and Peining Li 14 Ring Chromosome 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Bruna Burssed and Maria Isabel Melaragno 15 Ring Chromosome 11. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Liming Bao 16 Ring Chromosome 12. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Hugh S. Taylor and Jiadi Wen 17 Ring Chromosome 13. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Peining Li and Mei Ling Chong 18 Ring Chromosome 14. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Alessandro Vaisfeld, Marco Crimi and Berardo Rinaldi 19 Ring Chromosome 15. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Qin Wang, Hui Guo, Yong-Hui Jiang and Weiqing Wu 20 Ring Chromosome 16. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 Young Mi Kim, Holly Johnson, Xianfu Wang, Neelam Lama, Xianglan Lu, Ying Liu and Shibo Li 21 Ring Chromosome 17. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Zhongxia Qi 22 Ring Chromosome 18. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 Jannine D. Cody 23 Ring Chromosome 19. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Jiadi Wen and Mei Ling Chong 24 Ring Chromosome 20. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Kenneth A. Myers 25 Ring Chromosome 21. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Hui Zhang and Hongyan Chai 26 Ring Chromosome 22. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 Katy Phelan 27 Ring Chromosome X. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Nikhil Sahajpal and Barbara R. DuPont 28 Ring Chromosome Y. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 Barbara R. DuPont 29 Small Supernumerary Ring Chromosomes . . . . . . . . . . . . . . . . . 353 Thomas Liehr 30 Ring Chromosomes from Patients’ Perspective. . . . . . . . . . . . . . 363 Thomas Liehr, Claire Andersen, Sarah Wynn and Anna Pelling

Contents

Contents

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Part III  Somatic Ring Chromosomes 31 Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 Ying S. Zou, Hanadi El Achi, Guilin Tang, Brian H. Phan, Michael H. Phan, Taylor N. Anderson and Jaclyn B. Murry 32 Acquired Ring Chromosomes in Solid Tumors . . . . . . . . . . . . . . 475 Jiadi Wen and Mei Ling Chong Part IV  Ring Chromosome Research 33 Molecular Mechanisms of Ring Chromosome Formation and Instability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493 Stanislav A. Vasilyev and Igor N. Lebedev 34 iPSC Models of Ring Chromosomes, Genome Editing, and Chromosome Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505 Tatiana V. Nikitina and Igor N. Lebedev 35 Genetic Mosaic Analysis in Model Organisms. . . . . . . . . . . . . . . 517 Hui Zong

Contributors

Hanadi El Achi  Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Claire Andersen  Unique, The Stables, Surrey, UK Taylor N. Anderson  Department of Biomedical Engineering, The Johns Hopkins University Whiting School of Engineering, Baltimore, MD, USA Liming Bao Department of Pathology and Laboratory Medicine, Weill Cornell Medical College of Cornell University, New York, NY, USA Kathleen M. Bone Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI, USA Bruna Burssed  Genetics Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil Hongyan Chai  Department of Genetics, Yale University School of Medicine, New Haven, CT, USA Judy Chernos Department of Medical Genetics, University of Calgary, Calgary, AB, Canada Mei Ling Chong Laboratory of Clinical Cytogenetics, Department of Genetics, Yale University School of Medicine, New Haven, CT, USA Jannine D. Cody  Department of Pediatrics, University of Texas Health San Antonio, San Antonio, TX, USA; Chromosome 18 Registry and Research Society, San Antonio, TX, USA Marco Crimi Ring14 International, Reggio Emilia, Italy; Kaleidos SCS, Scientific Office, Bergamo, Italy Barbara R. DuPont  Cytogenomics Laboratory, Greenwood Genetic Center, Greenwood, SC, USA Hui Guo Forensic Evidence Laboratory, Genetic and Prenatal Disease Diagnosis Center, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Benjamin Hilton Cytogenomics Laboratory, Greenwood Genetic Center, Greenwood, SC, USA

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Qiping Hu Department of Cell Biology and Genetics, School of Basic Medical Sciences, Guangxi Medical University, Nanning, Guangxi, China Yong-Hui Jiang Departments of Genetics, Neuroscience, and Pediatrics, Yale University School of Medicine, New Haven, CT, USA Holly Johnson  Specialty Laboratory, Department of Pediatrics, College of Medicine, Oklahoma University Health Science Center, Oklahoma City, OK, USA Anna A. Kashevarova  Laboratory of Cytogenetics, Research Institute of Medical Genetics, Tomsk National Research Medical Center of Russian Academy of Sciences, Tomsk, Russia Young Mi Kim  Specialty Laboratory, Department of Pediatrics, College of Medicine, Oklahoma University Health Science Center, Oklahoma City, OK, USA Neelam Lama  Specialty Laboratory, Department of Pediatrics, College of Medicine, Oklahoma University Health Science Center, Oklahoma City, OK, USA Igor N. Lebedev  Laboratory of Ontogenetics, Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation Peining Li  Laboratory of Clinical Cytogenetics, Department of Genetics, Yale University School of Medicine, New Haven, CT, USA Shibo Li Specialty Laboratory, Department of Pediatrics, College of Medicine, Oklahoma University Health Science Center, Oklahoma City, OK, USA Thomas Liehr Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, Jena, Germany Ying Liu Specialty Laboratory, Department of Pediatrics, College of Medicine, Oklahoma University Health Science Center, Oklahoma City, OK, USA Xianglan Lu Specialty Laboratory, Department of Pediatrics, College of Medicine, Oklahoma University Health Science Center, Oklahoma City, OK, USA Deqiong Ma  Department of Genetics, Yale School of Medicine, New Haven, CT, USA Maria Isabel Melaragno  Genetics Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil Jaclyn B. Murry  The Johns Hopkins Cytogenomics Laboratory, Department of Pathology, Division of Molecular Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

Contributors

Contributors

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Kenneth A. Myers Research Institute of the McGill University Health Centre, Division of Neurology, Department of Pediatrics, Department of Neurology and Neurosurgery, Montreal Children’s Hospital, McGill University Health Centre, Montreal, QC, Canada Tatiana V. Nikitina Laboratory of Ontogenetics, Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russia Anna Pelling  Unique, The Stables, Surrey, UK Brian H. Phan  The College of William and Mary, Williamsburg, VA, USA Michael H. Phan  The Johns Hopkins University, Baltimore, MD, USA Katy Phelan  Genetics Department, Florida Cancer Specialists and Research Institute, Fort Myers, FL, USA Zhongxia Qi  Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA Berardo Rinaldi  Medical Genetics Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy Nikhil Sahajpal Cytogenomics Laboratory, Greenwood Genetic Center, Greenwood, SC, USA Sheila Saliganan Ambry Genetics Corporation, Aliso Viejo, California, USA Jhanvi Shah Department of Cytogenetics and Molecular Cytogenetics, FRIGE’s Institute of Human Genetics, Ahmedabad, India Frenny Sheth Department of Cytogenetics and Molecular Cytogenetics, FRIGE’s Institute of Human Genetics, Ahmedabad, India Harsh Sheth  Advanced Genomic Technologies Division, FRIGE’s Institute of Human Genetics, Ahmedabad, India Anna M. Szekely Department of Neurology, Yale University School of Medicine, New Haven, CT, USA Guilin Tang  Department of Hematopathology, Division of Pathology-Lab Medicine, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA Hugh S. Taylor  Department of Obstetrics, Gynecology and Reproductive Science, Yale University School of Medicine, New Haven, CT, USA Mary Ann Thomas Departments of Medical Genetics and Pediatrics, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada Alessandro Vaisfeld  Medical Genetics Unit, IRCCS Azienda OspedalieroUniversitaria di Bologna, Bologna, Italy; Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy

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Stanislav A. Vasilyev  Laboratory of Genomic Tools, Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation Qin Wang Medical Genetic Center, Shenzhen Maternity and Child Healthcare Hospital, Shenzhen, Guangdong, China Xianfu Wang  Specialty Laboratory, Department of Pediatrics, College of Medicine, Oklahoma University Health Science Center, Oklahoma City, OK, USA Allison Watson  Ring20 UK, Warley, Brentwood, UK Sainan Wei  Department of Pathology and Laboratory Medicine, University of Kentucky College of Medicine, Kentucky, USA Jiadi Wen  Laboratory of Clinical Cytogenetics, Department of Genetics, Yale University School of Medicine, New Haven, CT, USA Weiqing Wu Medical Genetic Center, Shenzhen Maternity and Child Healthcare Hospital, Shenzhen, Guangdong, China Sarah Wynn  Unique, The Stables, Surrey, UK Jingwei Yu  Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA Hui Zhang  Department of Genetics, Yale University School of Medicine, New Haven, CT, USA Hui Zong  Department of Microbiology, Immunology, and Cancer Biology, University of Virginia Health System, Charlottesville, VA, USA Ying S. Zou  The Johns Hopkins Cytogenomics Laboratory, Department of Pathology, Division of Molecular Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

Contributors

Abbreviations

aCGH Array comparative genomic hybridization ACMG American College of Medical Genetics and Genomics ADHD Attention deficit hyperactivity disorder ALL Acute lymphoblastic leukemia ALT Atypical lipomatous tumors AMA Advanced maternal age AMKL Acute megakaryoblastic leukemia AML Acute myeloid leukemia aRC Acquired ring chromosome ARSA Arylsulfatase A AS Angelman syndrome ASD Atrial septal defect ASD Autism spectrum disorder ATRT Atypical teratoid rhabdoid tumors BAC Bacterial artificial chromosome BBS Birk-barel syndrome BFB Breakage-fusion-bridge C18CRC Chromosome 18 clinical research center CdCs Cri-du-Chat syndrome CDH Congenital diaphragmatic hernia CES Cat eye syndrome CKO Conditional knockout ClinGen Clinical genome resource CLL Chronic lymphocytic leukemia CMA Chromosome microarray analysis CML Chronic myeloid leukemia CNS Central nervous system CNV Copy number variant CRISPR Clustered regularly interspaced short palindromic repeats CT Computerized tomography CVS Chorionic villus sampling DAPI 4',6-diamidino-2-phenylindole DCM Dilated cardiomyopathy DD Developmental delay DDLPS Dedifferentiated liposarcomas DECIPHER Database of chromosomal imbalance and phenotype in humans using ensemble resource xix

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DEE Developmental and epileptic encephalopathy DFSP Dermatofibrosarcoma protuberans DGS DiGeorge syndrome DGV Database of genomic variants DMR Differentially methylated regions DNA Deoxyribonucleic acid DOR Diminished ovarian reserve DSB Double strand breaks DSCR Down syndrome critical region ECG Electrocardiogram EEG Electroencephalogram ESC Embryonic stem cells FISH Fluorescence in situ hybridization GERD Gastroesophageal reflux disease GFP Green fluorescent protein GI Gastrointestinal GTG G-band by trypsin using Giemsa IBS Irritable bowel syndrome ICHRC International consortium for human ring chromosome ICSI Intercytoplasmic sperm injection ICU Intensive care unit ID Intellectual disability iPSC Induced pluripotent stem cells IQ Intelligence quotient ISCN International System for Human Cytogenomic Nomenclature IUGR Intrauterine growth restriction/retardation IVF In vitro fertilization Kb Kilobase lncRNA Long nocoding ribonucleic acid LOH Loss of heterozygosity MADM Mosaic analysis with double markers Mb Megabase MDLS Miller-Dieker lissencephaly syndrome MDS Myelodysplastic syndrome MLD Metachromatic leukodystrophy MLPA Multiplex ligation-dependent probe amplification MRI Magnetic resonance imaging NAHR Non-allelic homologue recombination NF2 Neurofibromatosis type 2 NGS Next-generation sequencing NICU Neonatal intensive care unit NIPT Non-invasive prenatal testing NOR Nucleolar organizing regions NORD National organization for rare disorders OCD Obsessive-compulsive disorder OGM Optical genome mapping OMIM Online Mendelian inheritance in man OSMD Osteosclerotic metaphyseal dysplasia PA Pleomorphic adenomas

Abbreviations

Abbreviations

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PAO PCC PCN PCR PDA PGT PMS PWS RC RD RFP SCE SEM SKY SNP SNV sSMC sSRC STR SV TAD TOP UPD VSD WDLPS WES WGS WHS WSCR YAC

Patient advocate organization Premature chromosome condensation Plasma cell neoplasms Polymerase chain reaction Patent ductus arterosus Preimplantation genetic testing Phelan-McDermid syndrome Prader-Willi syndrome Ring chromosome Rare disease Red fluorescent protein Sister chromatid exchange Scanning electron microscopy Spectral karyotyping Single nucleotide polymorphism Single nucleotide variant Small supernumerary marker chromosome Small supernumerary ring chromosome Short tandem repeat Structural variant Topological associated domain Termination of pregnancy Uniparental disomy Ventricular septal defect Well-differentiated liposarcomas Whole exome sequencing Whole genome sequencing Wolf-Hirschhorn syndrome Williams syndrome critical region Yeast artificial chromosome

Part I

Introduction

1

Historical Perspective of Human Ring Chromosomes Peining Li and Thomas Liehr

Abstract

Human ring chromosomes (RCs) are a rare type of chromosomal structural abnormality. Current cytogenomic analysis revealed heterogeneous genomic rearrangements in the ring structures, variable levels of dynamic mosaicism, and selective karyotype evolution in various tissues. This cytogenomic heterogeneity is likely correlated with variable clinical heterogeneity ranging from generalized features of ‘ring syndrome’, chromosomespecific and segmental aneuploidy related phenotypes, to risks of infertility and various types of cancers. Better understanding of the molecular mechanisms governing RC formation and its mitotic behavior can contribute toward best practice in comprehensive cytogenomic analyses and evidence-based treatment and management for affected patients of RCs. Collaborative efforts for systematic

evidence review on cases of specific RCs, close interaction with patient advocate organization (PAO), and an online registry of ring chromosome cases are undertaken by an International Consortium of Human Ring Chromosomes (ICHRC). These efforts are aimed to develop chromosome-specific guidelines and recommendations in laboratory diagnosis and genetic counseling and provide more reliable clinico-cytogenomic correlations for clinical management and treatment for patients of RCs.

Keywords

Ring chromosome (RC) · Dynamic mosaicism · Cytogenomic heterogeneity · Ring syndrome · Clinical heterogeneity · Laboratory standards and guidelines

1.1 An Overview of Human Ring Chromosomes P. Li (*)  Laboratory of Clinical Cytogenetics, Department of Genetics, Yale School of Medicine, New Haven, CT, USA e-mail: [email protected] T. Liehr  Jena University Hospital, Institute of Human Genetics, Friedrich Schiller University, Am Klinikum 1, 07747 Jena, Germany e-mail: [email protected]

Chromosomes carry genetic material of deoxyribonucleic acid (DNA) inside the nuclei of all living cells. The DNA for a human genome, consisting of three billion nucleotides, is organized into 22 pairs of autosomes numerically named from 1 to 22 and one pair of sex chromosomes with XX for female and XY for male. An individual human chromosome has

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_1

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a rod shape structure with a short and a long arm joined at a centromere at the proximal end and capped by a telomere at the distal end. The DNA is packed in histone and non-histone proteins and presented as interphase chromatins and metaphase chromosomes in a multi-stage process during cell cycles. Recent studies provided deep insights into DNA-folding and chromosome structure being organized as millions of multilayers along a chromosome arm (Daban 2021). Chromosomes facilitate the replication and repair of DNA, the spatial coordination of chromatin domains and epigenetic interactions to regulate gene functions, and the transmission of DNA in somatic cells by mitosis and in germline cells by meiosis. Errors in replicating and repairing DNA could result in structural chromosomal abnormalities and pathogenic copy number variants (pCNVs). Errors in segregating chromosomes through cell cycles of mitosis and meiosis could cause numerical chromosomal abnormalities and derivative chromosome variants. Chromosomal abnormalities occur in 1 out of 154 live births; pCNVs including deletions, duplications, and compound deletion and/or duplication occur in approximately 1 out of 300 live births. Taken together, chromosomal abnormalities and pCNVs are seen in approximately 1% of newborns (Chai et al. 2019a; Nussbaum et al. 2001). Human ring chromosomes (RCs) are a unique type of structural chromosomal abnormality, in which the rod shape chromosome is changed to a ring structure by a fusion at both ends. This is a very rare chromosomal abnormality with an estimated incidence of 1 in 50,000 newborns. Given an average global birth rate of 1.8% on a total population of 8 billion as of year 2022, it is estimated that there are about 2800 newborns carrying a RC annually (Li et al. 2022). In 1962, the first case of a RC derived from sex chromosome X was reported and followed by several cases showing clinical association with some characteristics of Turner syndrome (Lindsten and Tillinger 1962; Rowley et al. 1964; Turner et al. 1962; Wang et al. 1962). Cells showing loss of the RC, a

P. Li and T. Liehr

double-sized RC, an interlocked RC, and other ring variants were noted from these cases (Hoo et al. 1974). From 1962 to 1970, solid staining of metaphase chromosomes detected more than 30 cases with a RC involving chromosomes X, 1, 2, 3, 4, 5, 13, 16, 18, and other undefined chromosomes. Figure 1.1 shows the RCs and their variations in size and shape observed under a microscope from these earlier studies. Over the past 60 years, banding cytogenetics, fluorescence in situ hybridization (FISH), chromosome microarray analysis (CMA), and whole genome sequencing (WGS) have been used to detect RCs and to characterize their cellular behavior and genomic imbalances. A recent review on over 1000 cases of RCs collected in PubMed (https://pubmed.ncbi.nlm.nih. gov/) revealed the occurrence, cytogenomic findings, and correlated clinical manifestations for all autosomes and sex chromosomes (Li et al. 2022). Firstly, there are uneven occurrences of RCs with relative frequencies of 10–12% for chromosomes 18, 20, and X, 5–9% for chromosomes 13, 14, 15, 21, 22 and Y, and less than 4% for the remaining chromosomes. The least frequently seen RCs with a relative frequency less than 1% raised a question of ‘RC intolerance’ for chromosomes 1, 8, 12, 16, and 19. Figure 1.2 shows the relative frequencies of RCs and estimated annual cases for a specifical chromosomes globally based on the newborn incidence. Second, genomic imbalances were detected in approximately 90% of cases analyzed by CMA or WGS. This cytogenomic heterogeneity of RCs could reflect different molecular mechanisms in ring formation. Third, variable clinical manifestations of developmental delay, dysmorphic facial features, intellectual disability, microcephaly, and hypotonia were noted in most autosomal RCs. This clinical heterogeneity of RCs with impacts on disability, inheritance, and cancer predisposition need to be evaluated by a chromosome-specific approach on a case-bycase basis (Li et al. 2022). Current diagnostic and clinical practice on patients with a RC require a better understanding on the ‘laws of the ring’. The ‘biologic

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Fig. 1.1  RCs and variants observed in earlier cytologic analysis. a RCX detected in a 22-year-old woman with short stature. Reproduced with permission from Lindsten and Tillinger (1962). b Various configurations of RC13.

The formation of an interlocked ring and a dicentric ring is shown in the bottom left and right panels, respectively. Reproduced with permission from Hoo et al. (1974)

laws’ governing RC formation and its meiotic and mitotic segregations could contribute to the ‘diagnostic laws’ toward a comprehensive genetic analysis for precisive interpretation of clinico-cytogenomic correlations, and the ‘clinical laws’ of evidence-based treatment and management for patients with a RC (Wei et al. 2013).

1.2 Mechanisms in Ring Formation and Cellular Behaviors The study of chromosomal and genomic structures from cases with a constitutional RC proposed four mechanisms for ring chromosome formation (Guilherme et al. 2011; Pristyazhnyuk

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Fig. 1.2  a Relative frequencies of RCs 1 to 22, X and Y based on 1020 reported cases of RCs. b Estimated annual newborn cases for specific RCs globally per incidence of 1/50,000. (RC, ring chromosome)

and Menzorov 2018). The first mechanism is the fusion of telomeric or subtelomeric regions at the distal short arm and long arm from a

chromosome, which forms a so-called complete ring chromosome without any loss and gain of genetic material (Guilherme et al. 2011). For

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example, telomeric and subtelomeric fusions forming complete rings were confirmed in a RC4 and a RC17 (Burgemeister et al. 2017; Surace et al. 2014). The second mechanism is the fusion of either a double-strand break at one end and telomere at the other or double-strand breaks at both ends of a chromosome to form an ‘incomplete ring’ with loss of genetic material as reported in a RC6 and a RC21 (Zhang et al. 2004, 2012). WGS on a RC9 indicated an alternative end joining mechanism involving firstly inverted repeats induced intra-stand fold back and then microhomology-mediated DNA synthesis and ligation to form a ring with distal deletions and an interstitial duplication (Chai et al. 2020); this intra-strand repairing could explain the formation of other RCs with distal deletions and an interstitial duplication (Zhang et al. 2012). The third mechanism involves multiple genomic rearrangements from an inverted duplication/deletion rearrangement to chromothripsis to form a complex ring, a supernumerary ring, a neocentromeric ring, or an acentric marker chromosome. The inverted duplication deletion mechanism likely resulted in an intra-/inter-chromosomal small supernumerary marker chromosome and RC (sSMC/sSRC) and other acentric or neocentromeric marker chromosomes (Pristyazhnyuk and Menzorov 2018; Wang et al. 2015). The observation of a small RC3 and a supernumerary acrocentric fragment of distal portions of 3p and 3q suggested a novel mechanism for the origin of sSRCs (Maraschio et al. 1996). A rare incidence of chromothripsis involving multiple segments in a RC22 was characterized by WGS (Kurtas et al. 2018). It is hypothesized that some sSMCs and sSRCs are the residual chromosomal component from the rescue of mostly trisomy and occasionally large RCs; however, the presence of residues indicated that the ‘rescue’ was incomplete. sSRCs as the rescue resultant of RCs were seen more frequently in chromosomes X, Y, and large autosomes (Callen et al. 1991; Chen et al. 1995). The fourth mechanism is likely due to the impact of nucleolar organizing region (NOR) association for chromosomes 13, 14, 15, 21, and 22. RC13, RC15,

and RC21 were noted in offspring from parental carriers of a Robertsonian translocation (Ki et al. 2003; Ledbetter et al. 1980; Stetten et al. 1990; de Almeida et al. 1983). Spatial closeness and functional interaction in NOR association of these chromosomes induced the formation of these RCs in a relatively high frequency of occurrence. A transcriptome involving multiple genes from different chromosomal loci could result in giant RCs in liposarcoma (Chai et al. 2022). All these observations indicated that there are multiple mechanisms involving replication, repairing, transcription, and epigenetic regulation in forming RCs. Figure 1.3 shows the complete and incomplete RCs observed from chromosome and FISH analyses. Cytogenomic analysis performed on 163 autosomal RCs showed that 8% had a complete ring, 71% had an incomplete ring with simple deletions at one or both ends of the chromosome, and 21% had a complex ring with genomic rearrangements from concomitant segmental deletions/duplications to chromothripsis (Li et al. 2022). These results indicated that less than 10% of constitutional autosomal rings were formed by a telomeric or subtelomeric fusion and 90% involved the repairing of at least two double-strand breaks in a chromosome. Integrated chromosome FISH, CMA, and WGS should be recommended and performed for all patients with a RC. Cells with a RC experience a breakagefusion-bridge cycle during mitosis to generate RC variants or derivative chromosomes (Hu et al. 2018; Pristyazhnyuk and Menzorov 2018). A RC replicated in the S phase with none, one (or odd number), or two (or even number) sister chromatid exchanges will generate an intact ring, a  dicentric ring, or interlocked rings, respectively. Through mitosis, the dicentric or interlocked RCs require a breakage event and thus show lagging at anaphase and nondisjunction into telophase. A dicentric chromosome can persist through mitosis and cytokinesis by forming a long chromatin bridge coated with nuclear membrane between the two daughter cells; this bridge resolves into single stranded DNA by the cytoplasmic 3’ repair exonuclease

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Fig. 1.3  RC formation mechanisms. a A complete RC4 showing the presence of subtelomeric sequence by FISH. (Reproduced with permission from Burgemeister et al. 2017). b In left panel, an incomplete RC6 showing loss of distal segments by FISH; in right panel, a derivative

chromosome 6 from the broken of RC6 and positive for FISH using probes for 6p25.1 and 6q27 (arrow points to the RC6 and the derivative chromosome 6. (Reproduced with permission from Zhang et al. 2004)

1 (TREX1) and induces nuclear envelope rupture (Maciejowski et al. 2015). Mis-segregated dicentric or interlocked RCs are captured in a micronucleus and experience further breakfusion rearrangement to form RC variants or to loss the RC. The initial RC and RC variants and derivatives may experience cellular selection for a karyotypic evolution. For example, the break-fusion-bridge cycle induced chromothripsis within chromosome 21 in pediatric B-lineage acute lymphoblastic leukemia and segmental rearrangements at a distal deletion of 5p (Li et al. 2014; Chai et al. 2019b). In 1977, McDermott et al. discussed this RC instability and suggested the concept of ‘dynamic mosaicism’ from a case of a RC4 (McDermott et al. 1977): “Their instability is reflected in the numbers of rings present, variation in the size of the rings, variation in the number of centromeres they possess, the occurrence of interlocked rings, and the presence of chromosome fragments. Therefore, the consequence of the irregular but persistent generation of genetically different cells, promoted by the behavioral

peculiarities of a ring chromosome, may be described as a dynamic mosaicism.” It was hypothesized that the initial breakfusion event for RC formation and the breakfusion-bridge cycle in mitotic segregation vary for different RCs, and thus present differences in RC structural variability and mitotic stability. More studies on larger case series of RCs are needed to fully understand the underlying mechanisms involving the cell cycle arrest by anaphase lagging, cell death by resultant segmental or whole chromosome aneuploids, and the selection process for karyotypic evolution. It was speculated that the formation of a RC could induce epigenetic changes by spreading of heterochromatinization and dysregulate the gene expression (Surace et al. 2014; Zollino et al. 2012). Furthermore, the RC structure and associated epigenetic changes may lead to rescue of cells by losing the RC and replicating the normal chromosome as isodisomy (Kim et al. 2014). Loss of the RC followed by monosomy compensatory rescue for a mosaic pattern consisting of cells carrying the RC and cells with an

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apparently normal karyotype has been observed in patients of RC8 and RC21; this mechanism resulted in uniparental isodisomy of these rescued normal chromosomes (Gradek et al. 2006; Petersen et al. 1992). Molecular analysis should be introduced to differentiate cells with isodisomy due to monosomy compensatory rescue in dynamic mosaicism from cells with heterodisomy of true mosaicism. The risk of uniparental disomy (UPD)-related disorders should be taken into consideration (Liehr 2022).

with clinical findings. A jointed effort from peer experts and professional organizations is needed to generate consensus and up-to-date standards toward best practice on diagnostic procedures and result interpretation. Inherited RCs in familial cases were reviewed from earlier reported cases. Maternal transmission was noted in 21 families involving RC11, RC13, RC14, RC15, RC18, RC20, RC21, RC22, and RCX; paternal transmission was noted in only two families of RC17 and RC22. Approximately, 50% of maternal transmission involved RC21, and the proportion of cases with inherited rings, among all patients with a ring, was calculated to be 5.6% as an upper limit. Further analysis on the outcome of 58 pregnancies from 24 women with a RC noted that approximately 51% had an offspring with the RC, 21% ended in a spontaneous abortion, 21% had a normal child, and 7% had an offspring with a chromosome anomaly other than ring (Kosztolanyi et al. 1991). A recent review of follow up parental study performed on 325 families showed approximately 88% de novo, 11% of a maternal carrier, and less than 1% of a paternal carrier. This estimation could be biased since 73% of familial cases with a maternal carrier were in RC15, RC18, RC20, and RC21 (Li et al. 2022). The infertility and inheritability of RCs should be evaluated on individual chromosomes. As Kosztololanyi et al. stated that “because of a probable difference in survival and fertility between individuals with transmitted and de novo rings, and because of the preferential publication of cases involving inherited rings (and thus a publication bias), the proportion of inherited rings should in reality be no more than 1%” (Kosztolanyi et al. 1991). Cell lines from patients with RCs have been preserved in cell depository for research purposes (Kistenmacher et al. 1975; Nikitina et al. 2018). Reprograming these patient-derived cell lines to induced pluripotent stem cells (iPSCs) offers unprecedented opportunities of in vitro cellular models for studies of human development, regenerative medicine, drug screening, and cell therapy (Takahashi et al. 2007). Reprograming human fibroblasts containing

1.3 Cytogenomic Diagnosis and Genetic Research Cell-based karyotyping and FISH testing are routinely used to detect RCs and to define their dynamic mosaicism; DNA-based CMA and WGS have been introduced to characterize the genomic structure and imbalances in the RCs (Chai et al. 2020; Zhang et al. 2004, 2012). Current cytogenetic laboratory guidelines have a general principle for analyzing mosaic chromosomal findings (Hook 1977), but are not specific for analyzing RCs and quantifying dynamic mosaicism for chromosome instability. In the analysis of the relationship between growth failure and ring instability, a ring was regarded as ‘stable’ when secondary aberrations were found in 0–5% of the mitoses and ‘instable’ or ‘labile’ when aberrations occurred in more than 5% of the mitoses counted (Kosztolanyi 1987b). This definition of stable vs instable rings has not yet been validated and applied in RC analysis. For all constitutional RCs, an integrated cell-based and DNA-based cytogenomic approach should be implemented to (1) differentiate complete rings from incomplete or partial ones, (2) delineate genomic structure and imbalances in the RCs, (3) define levels and nature of RC instability for dynamic mosaicism, and 4) recommend follow up parental study. Outlines for diagnostic recommendations and guidelines for specific RCs have been proposed (Hu et al. 2018; Zhang et al. 2012). Still, there is a need for systematic evidence review to have consensus on defining levels of RC instability and their correlation

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RC13 and RC17 to iPSC found the correction of RC through a compensatory UPD mechanism (Bershteyn et al. 2014). This cell-autonomous correction involved first the loss of the RC and then the replication of the homolog normal chromosome in five to ten cell culture passages; the correction ratio varied from different iPSC clones. A potential strategy for chromosome therapy to correct RC or other chromosome abnormalities was proposed (Plona et al. 2016). Cells containing a RC could be reprogramed in in vitro cell cultures to induce ring loss and trigger compensatory UPD, and the corrected cells could be used in cell therapy. However, there are technical limitations, procedure risks, and ethical considerations in this strategy. The cytogenetic results in most RC cases did not observe in vivo cell-autonomous correction, probably as somatic cells in human do not undergo as many divisions as in (immortal) cell lines. However, trisomy rescue, monosomy compensatory, and resultant UPD were known from prenatal findings of fetoplacental discrepancy and confined placenta mosaicism. Cellular reprogramming to iPSC may be a necessary step to trigger compensatory UPD. Further study to understand the mechanisms of RC loss and compensatory UPD is needed for practical chromosome therapy.

1.4 Evidence-Based Clinical Management and Treatment In 1981, Cote et al. reported a case of RC2 and proposed the term ‘ring syndrome’ to explain shared features of severe growth failure likely resulted from the continuous loss of cells from dynamic mosaicism, Cote et al. stated that “their elimination in vivo implies a very high cellular death rate and an enormous waste of metabolism that should have the same phenotypic effects no matter what chromosome is involved. These phenotypic anomalies form a ring syndrome that can be clinically recognized and consists of severe growth failure, mental retardation, and a pleasant personality. The syndrome is usually masked by the more severe abnormalities produced by the deletions

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present in most cases of ring chromosomes” (Cote et al. 1981). Decreased cell viability and reduced cloning efficiency in cultured fibroblasts from patients with a RC4 and a RC15 suggested that continuous production of hypomodal cells by the RC was responsible for the severe somatic retardation observed in patients with a RC (Kosztolanyi 1987a). Further analysis of 207 case reports on patients with ring autosomes and their clinical groups by major malformations and body height showed that: “(1) Forty patients, a fifth of the total, had extreme growth failure together with an otherwise almost-normal appearance, viz. no major malformation, no specific deletion syndrome, no or only a few unspecific minor anomalies. This phenotype may be regarded as the “ring syndrome” since it is independent of what chromosome is involved. (2) Severe growth failure, the sole major physical abnormality in the “ring syndrome”, was seen significantly more often among patients with ring of larger chromosomes than among patients with a smaller ring, indicating that the greater the chromosome involved in ring formation, the higher is the probability of severe growth failure. (3) Larger ring chromosomes showed significantly more often instability than smaller rings, suggesting that there may be a correlation between ring instability and the size of the chromosome involved. (4) Growth failure was present in significantly more patients with a “labile” ring than with a “stable” ring, indicating that a correlation may exist between ring instability and growth failure. It is suggested that the “ring syndrome” observed in many cases with ring autosome may result from end-to-end fusion of chromosome ends, an event not involving deletion in the genetic sense” (Kosztolanyi 1987b). A spectrum of phenotypes ranging from relatively healthy to variable manifestations of developmental delay, dysmorphic facial features, intellectual disability, microcephaly, and hypotonia was noted in autosomal RCs (Hu et al. 2018; Yip 2015). Syndromic or non-syndromic phenotypes from distal and interstitial deletions or duplication have been reported in RC cases. Specific phenotypes like epilepsy

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correlating with RC14 and RC20, infertility in patients with RCX, RCY, RC21 and RC22, and Turner syndrome in RCX were well documented (Li et al. 2022). For example, clinical phenotypes from patients with a RC21 were classified into three groups: (1) relatively normal phenotype with reproduction problems, (2) Down syndrome like phenotype due to the ring duplication, and (3) abnormal phenotypes from RC instability and segmental deletions and duplications (Zhang et al. 2012). All these findings indicated that the clinical heterogeneity of cases with a RC most likely correlated with the cytogenomic heterogeneity. Cytogenomic mapping and bioinformatic mining could define critical regions and putative candidate genes (Xie et al. 2022; Xu et al. 2014). The interpretation and prediction of clinic-cytogenomic correlations should be based on the characterized genomic structure, segmental imbalances, and dynamic mosaicism of a RC. In additional to congenital anomalies, risks for specific cancers have been reported in patients with different RCs (Li et al. 2022). RC7 was associated with skin lesions and malignant melanoma. Patients of RC11 associated with Wilms tumor, RC13 with retinoblastoma, RC17 with neurofibromatosis, RC21 with acute myeloid leukemia, and RC22 with neurofibromatosis, meningiomas, vestibular schwannoma have been reported. Changes in skin pigmentation and café au lait spot likely relating to dynamic mosaicism have been reported in patients of different RCs. Dynamic mosaicism and dysfunction of harbored tumor suppressor genes in these constitutional RCs likely mediated the predisposition to cancer (Tommerup and Lothe 1992). Cancer surveillance should be considered for patients carrying these RCs. RCs as acquired chromosomal abnormalities in various types of human neoplasia have also been reviewed and summarized (Gebhart 2008) and are included in Chaps. 31 and 32. Treatment of specific symptoms such as anticonvulsive therapy for epilepsy in RC14 and RC21 and growth hormone supplement for RC18 has been effective. Recently, evidencebased guideline recommendations for clinical

diagnosis and management of RC14 syndrome have been proposed by an ad hoc task force (Rinaldi et al. 2017). The major symptoms in patients with a RC 14 include epilepsy, hypotonia, recurrent infections, vision and hearing complications, respiratory complications, and communication and language disorders. The recommendations outlined general management and specific treatments for each symptom. Advice for taking care of a child with this rare and complex syndrome was offered to parents. This chromosome-specific and symptom-based disease management and treatment could be used as a model for other RCs.

1.5 Patient Advocacy Organization and Collaborative Efforts Cases of RCs reported in the literature are less than 1% of its occurrence; the cytogenomic and clinical findings from this small portion of cases could be biased for more severe phenotypes and thus missed a large portion of mild and subclinical cases (Li et al. 2022). The observed cytogenomic and clinical heterogeneity of RCs demonstrated the necessity of an organized effort on a large case series for accurate cliniccytogenomic correlations and evidence-based genetic counseling and clinical management. Ring 14 International (R14I) is a patient advocacy organization (PAO) founded in 2012 as a non-profit organization to help affected people and their caregivers and to promote and support scientific research projects. Recently, R14I managed an ad hoc task force to publish the first report on recommended guidelines for diagnosis and clinical management of Ring 14 syndrome (Rinaldi et al. 2017). According with those guidelines, children with neuro-psychological alterations and drug-resistant epilepsy need to have CMA as the first diagnostic step and all subjects for whom a 14q terminal deletion is identified should also have a standard karyotype to assess for the presence of a ring. Another PAO is the British ring 20 research. This organization presents real life stories from patients

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and supports patient-led approaches to assess the role of ketogenic dietary therapy in reducing seizure frequency and preserving cognition for affected patients (Gordon et al. 2020; Watson et al. 2015). UNIQUE (https://rarechromo.org/), an overarching family support group for all chromosomal aberrations, is an internationally active group which is in contact with many families of RC carriers. An International Consortium for Human Ring Chromosomes (ICHRC) was launched in 2021 at the European Cytogenomic Conference (ECC) and organized a concurrent session in the 2022 annual meeting of the American College of Medical Genetics and Genomics (ACMG). The goals of the ICHRC are: (1) developing laboratory standards and guidelines for analyzing RCs, (2) reanalyzing, reviewing, and registering RC cases into the Human Ring Chromosome Registry as an online database; and (3) performing further genomic characterization and functional analysis of RC structure and behavior. The development of this online Ring Chromosome Registry has provided an interactive platform to compile and curate cytogenomic and clinical findings for RC cases (Hu et al. 2018). For registering cases into this registry, a task force by clinical cytogeneticists and geneticists will be organized to develop diagnostic standards and registering criteria. Accumulation of more RC cases with defined genomic structures and dynamic mosaicism and detailed clinical manifestations will provide better clinco-cytogenomic correlations to develop chromosome-specific guidelines and recommendations for genetic counseling and clinical treatment. There are dedicated working groups for RCs comprised of clinical and molecular cytogeneticists, clinical geneticists, and genetic researchers. Systematic evidence reviews will be performed by each working group on a specific RC following the evidence-based practice guideline proposed by the American College of Medical Genetics and Genomics (ACMG). Working together with the ACMG laboratory quality assurance committee, laboratory standards and guidelines for human RCs will include

P. Li and T. Liehr

technical details of karyotyping, FISH, CMA, and WGS for patients with a RC, diagnostic definitions of complete vs. incomplete and stable vs. instable RCs, clinical interpretation for associated phenotypes, and recommendations for follow up parental studies and clinical management. A program on acquired RCs in various tumors is also considered. Furthermore, collaborative studies are needed for better understanding of the biologic, diagnostic, and clinical laws for human constitutional RCs. The dynamic mosaicism of RCs provides an in vivo system to monitor cellular function affected by RC variants, derivatives, and loss. The cell-autonomous correction of RCs in iPSC cells provides insight into a potential therapeutic approach. Joint efforts for indepth research on the governing mechanisms, standardized diagnostic practice, and better treatment approach will benefit patients with a RC.

1.6 Perspective for Clinicians, Geneticists, and Patients This book represents joint efforts from clinical geneticists, cytogeneticists, molecular geneticists, genetic counselors, genetic researchers, and PAO representatives to present a systematic record and summary of the known literature for constitutional and somatic RCs. The volume is divided into four parts of introduction, constitutional RCs, acquired RCs, and RC research. The part I covers the historical perspective, current diagnostic methods, online resources for cytogenomic analysis, and advocate activities by PAOs. For part II, we have arranged chapters for the 22 autosomes and sex chromosomes X and Y, and sSRCs with detailed review and summary of cytogenomic and clinical findings for these constitutional RCs. More importantly, we have arranged a chapter for RCs from a patient’s perspective, which present real life experience for affected children and their families. For part III, acquired RCs in tumors of hematopoietic and lymphoid tissues and solid tumors are summarized in designated chapters. The part IV covers

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current research activities and progress related to human RCs. The study of human RCs is a continuous and dynamic process involving the adaptation of novel genomic, genetic, and epigenetic technologies. The application of various cytogenomic technologies has further characterized the chromosomal structure, dynamic mosaicism, and genomic rearrangements and imbalances in the human RCs, which facilitate the systematic evidence reviews of clinico-cytogenomic correlations and evidence-based clinical management and treatment. Collaborative efforts from PAOs and ICHRC will contribute to a better understanding of the molecular mechanisms governing ring chromosome formation and mitotic segregation, and thus develop laboratory standards and guidelines toward best practice in technology-driven genetic analyses and evidence-based treatment of RC disorders. These will enhance our knowledge on the unique traits of human chromosomes and have significant impacts on RC disorders and beyond. We hope that this effort could provide resources for continuous updating of clinical cases, laboratory results, and research progress from supporting researchers. We also hope that book will be an indispensable reference on human RCs for clinicians, diagnostic geneticists, genetic counselors, and patients and their families.

Characterization by molecular genetics. Am J Hum Genet 48(4):769–782 Chai H, DiAdamo A, Grommisch B, Xu F, Zhou Q, Wen J, Mahoney M, Bale A, McGrath J, Spencer-Manzon M, Li P, Zhang H (2019a) A retrospective analysis of 10-year data assessed the diagnostic accuracy and efficacy of cytogenomic abnormalities in current prenatal and pediatric settings. Front Genet 10:1162. https://doi.org/10.3389/fgene.2019.01162 Chai H, Grommisch B, DiAdamo A, Wen J, Hui P, Li P (2019b) Inverted duplication, triplication and quintuplication through sequential breakage-fusion-bridge events induced by a terminal deletion at 5p in a case of spontaneous abortion. Mol Genet Gen Med 7(10):e00965. https://doi.org/10.1002/mgg3.965 Chai H, Ji W, Wen J, DiAdamo A, Grommisch B, Hu Q, Szekely AM, Li P (2020) Ring chromosome formation by intra-strand repairing of subtelomeric double stand breaks and clinico-cytogenomic correlations for ring chromosome 9. Am J Med Genet A 182(12):3023–3028. https://doi.org/10.1002/ ajmg.a.61890 Chai H, Xu F, DiAdamo A, Grommisch B, Mao H, Li P (2022) Cytogenomic characterization of giant ring or rod marker chromosome in four cases of well-differentiated and dedifferentiated liposarcoma. Case Rep Genet 2022:6341207. https://doi. org/10.1155/2022/6341207 Chen H, Tuck-Muller CM, Batista DA, Wertelecki W (1995) Identification of supernumerary ring chromosome 1 mosaicism using fluorescence in situ hybridization. Am J Med Genet 56(2):219–233. https://doi. org/10.1002/ajmg.1320560221 Cote GB, Katsantoni A, Deligeorgis D (1981) The cytogenetic and clinical implications of a ring chromosome 2. Ann Genet 24(4):231–235 Daban JR (2021) Soft-matter properties of multilayer chromosomes. Phys Biol 18(5). https://doi. org/10.1088/1478-3975/ac0aff de Almeida JC, Llerena JC Jr, Gomes DM, Martins RR, Pereira ET (1983) Ring 13 in an adult male with a 13:13 translocation mother. Ann Genet 26(2):112–115 Gebhart E (2008) Ring chromosomes in human neoplasias. Cytogenet Genome Res 121(3–4):149–173. https://doi.org/10.1159/000138881 Gordon D, Watson A, Desurkar A, Cowley L, Hiemstra TF (2020) Assessing the role of ketogenic dietary therapy in ring chromosome 20 syndrome: A patientled approach. Epilepsia Open 5(2):295–300. https:// doi.org/10.1002/epi4.12387 Gradek GA, Kvistad PH, Houge G (2006) Monosomy 8 rescue gave cells with a normal karyotype in a mildly affected man with 46, XY, r(8) mosaicism. Eur J Med Genet 49(4):292–297. https://doi.org/10.1016/j. ejmg.2005.08.004 Guilherme RS, Meloni VF, Kim CA, Pellegrino R, Takeno SS, Spinner NB, Conlin LK, Christofolini DM, Kulikowski LD, Melaragno MI (2011) Mechanisms of

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Pristyazhnyuk IE, Menzorov AG (2018) Ring chromosomes: From formation to clinical potential. Protoplasma 255(2):439–449. https://doi. org/10.1007/s00709-017-1165-1 Rinaldi B, Vaisfeld A, Amarri S, Baldo C, Gobbi G, Magini P, Melli E, Neri G, Novara F, Pippucci T, Rizzi R, Soresina A, Zampini L, Zuffardi O, Crimi M (2017) Guideline recommendations for diagnosis and clinical management of Ring14 syndrome-first report of an ad hoc task force. Orphanet J Rare Dis 12(1):69. https://doi.org/10.1186/s13023-017-0606-4 Rowley J, Muldal S, Lindsten J, Gilbert CW (1964) H3-thymidine uptake by a ring X chromosome in a human female. Proc Natl Acad Sci U S A 51(5):779– 786. https://doi.org/10.1073/pnas.51.5.779 Stetten G, Tuck-Muller CM, Blakemore KJ, Wong C, Kazazian HH Jr, Antonarakis SE (1990) Evidence for involvement of a Robertsonian translocation 13 chromosome in formation of a ring chromosome 13. Mol Biol Med 7(6):479–484 Surace C, Berardinelli F, Masotti A, Roberti MC, Da Sacco L, D’Elia G, Sirleto P, Digilio MC, Cusmai R, Grotta S, Petrocchi S, Hachem ME, Pisaneschi E, Ciocca L, Russo S, Lepri FR, Sgura A, Angioni A (2014) Telomere shortening and telomere position effect in mild ring 17 syndrome. Epigenetics Chromatin 7(1):1. https://doi. org/10.1186/1756-8935-7-1 Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872. https://doi. org/10.1016/j.cell.2007.11.019 Tommerup N, Lothe R (1992) Constitutional ring chromosomes and tumour suppressor genes. J Med Genet 29(12):879–882. https://doi.org/10.1136/ jmg.29.12.879 Turner B, Jennings AN, den DG, Stapleton T (1962) A self-perpetuating ring chromosome. Med J Aust 49(2):56–58 Wang HC, Melnyk J, Mc DL, Uchida IA, Carr DH, Goldberg B (1962) Ring chromosomes in

human beings. Nature 195:733–734. https://doi. org/10.1038/195733a0 Wang Q, Wu W, Xu Z, Luo F, Zhou Q, Li P, Xie J (2015) Copy number changes and methylation patterns in an isodicentric and a ring chromosome of 15q11q13: Report of two cases and review of literature. Mol Cytogenet 8(1):97. https://doi.org/10.1186/ s13039-015-0198-4 Watson A, Watson D, Taylor C (2015) Life with r(20)Ring chromosome 20 syndrome. Epilepsia 56(3):356358. https://doi.org/10.1111/epi.12729 Wei Y, Xu F, Li P (2013) Technology-driven and evidence-based genomic analysis for integrated pediatric and prenatal genetics evaluation. J Genet Genomics 40(1):1–14. https://doi.org/10.1016/j.jgg.2012.12.004 Xie X, Chai H, DiAdamo A, Grommisch B, Wen J, Zhang H, Li P (2022) Genotype-phenotype correlations for putative haploinsufficient genes in deletions of 6q26-q27: Report of eight patients and review of literature. Glob Med Genet 9(2):166–174. https://doi. org/10.1055/s-0042-1743568 Xu F, Li L, Schulz VP, Gallagher PG, Xiang B, Zhao H, Li P (2014) Cytogenomic mapping and bioinformatic mining reveal interacting brain expressed genes for intellectual disability. Mol Cytogenet 7(1):4. https:// doi.org/10.1186/1755-8166-7-4 Yip MY (2015) Autosomal ring chromosomes in human genetic disorders. Transl Pediatr 4(2):164–174. https://doi.org/10.3978/j.issn.2224-4336.2015.03.04 Zhang HZ, Li P, Wang D, Huff S, Nimmakayalu M, Qumsiyeh M, Pober BR (2004) FOXC1 gene deletion is associated with eye anomalies in ring chromosome 6. Am J Med Genet A 124A(3):280–287. https://doi. org/10.1002/ajmg.a.20413 Zhang HZ, Xu F, Seashore M, Li P (2012) Unique genomic structure and distinct mitotic behavior of ring chromosome 21 in two unrelated cases. Cytogenet Genome Res 136(3):180–187. https://doi. org/10.1159/000336978 Zollino M, Ponzi E, Gobbi G, Neri G (2012) The ring 14 syndrome. Eur J Med Genet 55(5):374–380. https:// doi.org/10.1016/j.ejmg.2012.03.009

2

Diagnostic Methods for Ring Chromosomes Benjamin Hilton and Barbara R. DuPont

Abstract

The molecular characterization of ring chromosome (RC) structural aberrations requires the use of cytogenomic techniques. The first RCs were characterized by the analysis of non-banded chromosomes and quinacrine fluorescent dye. G-banding, fluorescence in situ hybridization (FISH), and chromosome microarray analysis (CMA) have been used to identify the chromosome origin of the ring, the degree of mosaicism, the regions deleted and/ or gained from the RC, and the dynamic mosaicism present in each patient. RCs in CMA often appear as one chromosome with terminal deletions or additional material around the pericentromeric region of a chromosome. Other molecular techniques have been used to distinguish which chromosome is involved in the ring and which regions are present or missing from the RC. Next-generation sequencing (NGS) will define the joint sequences from both the short arms and long arms to reveal ring formation mechanisms. Optical genome mapping (OGM), a new technology not yet standard in many cytogenomic laboratories, B. Hilton · B. R. DuPont (*)  Cytogenomics Laboratory, Greenwood Genetic Center, Greenwood, SC, USA e-mail: [email protected] B. Hilton e-mail: [email protected]

has been used to identify the chromosome of origin, the ring structure and the sequences lost and gained from the ring, and the mosaicism present in a single analysis. Integrated cytogenomic analyses should be recommended and performed to define the chromosomal structure, cellular dynamic mosaicism, genomic imbalance, and rearrangement in RCs in current diagnostic practice.

Keywords

Ring chromosome (RC) · Chromosome G-band analysis · Fluorescence in situ hybridization (FISH) · Chromosome microarray analysis (CMA) · Next-generation sequencing (NGS) · Optical genome mapping (OGM)

2.1 Introduction Ring chromosomes (RCs) and/or small supernumerary marker or ring chromosome (sSMC/ sSRC) are diagnosed in a number of ways. Historically, they were first reported in the early 60s using solid chromosome staining analysis with one of the earliest reports published in 1962 with patients with Turner-like phenotypes. (Lindsten and Tillinger 1962). This was followed by a report of Bain et al. on a patient with ring X in a patient with dwarfism (Bain

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_2

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et al. 1965). Other reports followed including patients with characteristics of 5p deletion with a ring from a B-group chromosome (Rohde and Tompkins 1965; Steele et al. 2004). These early reports of ring chromosomes with solid banding were followed by reports using quinicrine mustard (Q-banding) staining for the characterization and chromosome identification of ring chromosomes (Atkins et al. 1972; Miller et al. 1971; Moore et al. 1973; Verma et al. 1983). Moore et al. (1973) used Q-banding to identify a RC6 (Moore et al. 1973). This banding was followed by early G-banding which was also important in identifying the chromosome involved in the RCs. Zackai and Breg used both Q- and G-banding to show similar banding patterns of RC7s found in two males with very different phenotypes (Zackai and Breg 1973). Lansky et al. identified a ring originating from chromosome 10 in a patient with mild developmental delay, short stature, widely spaced nipples, and pectus excavatum (Lansky et al. 1977). Ledbetter et al. used both Q- and G-banding to identify a RC15 and then did further characterization of the ring using silver staining to identify nucleolus organizing regions (NOR) (Ledbetter et al. 1980). Now with widespread use of chromosome microarray analysis (CMA) as the first line testing and standard of care, the finding of a structural RC has become somewhat more difficult as CMA reveals losses and gains of DNA copy number but does not reveal structural information of an aberration or on any balanced rearrangement that might be present (South et al. 2013). Large RCs may appear as a complete or nearly complete chromosome with terminal deletion(s). These terminal losses may be at one or both telomeric ends of the short and/ or long chromosome arms or they may be more complex such as a duplication next to the deleted terminal region. sSMC/sSRC will usually appear in CMA as small duplications around the centromere with small sections of both the p-arm and the q-arm, or may be comprised of just DNA on either the p-arm or the q-arm around the centromere of the chromosome. These too may be more complicated. If

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the aberration observed by CMA is suggestive of a RC or sSRC, G-banded chromosome analysis would need to be performed for confirmation of the structural formation of a RC. Next-generation sequencing (NGS), including whole exome sequencing (WES), whole genome sequencing (WGS), and long read sequencing (LRS), has been introduced for clinical diagnosis. Results from NGS will have fragments with sequences from both the short (p) and the long (q) arms (Ji et al. 2015). Again, definitive confirmation of the RC would require standard karyotype analysis. Optical genome mapping (OGM) is a new molecular technology which has been shown to detect copy number variations, structural rearrangements, both balanced and unbalanced, and mosaicism in a single assay (Mantere et al. 2021). This technology could eliminate the need for multiple testing strategies, such as C-banding and FISH, to elucidate both the size, structure and complexity of the RC, as well as the presence of mosaicism and the percentage of mosaicism often present with RCs.

2.2 Chromosome Analysis 2.2.1 Chromosome Preparation For all these different applications of chromosome analysis, the basic approach is the same. The cells are arrested in metaphase or prometaphase, treated and stained to obtain readable metaphase spreads, and then counted, karyotyped, and analyzed. Banding is important to identify individual chromosomes by their specific banding patterns. These chromosome specific banding patterns are used to determine which chromosomes are involved in chromosome aberrations. The number of cells to be examined is determined by the nature of the tissue and the study being undertaken. For most RC analyses, a minimum of 30 cells should be analyzed to make sure a low-level mosaic pattern is not missed. Ring instability should be ascertained by analyzing 200–300 metaphases or more, looking for cells with ring variants of

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di-/tri-/tetracentric rings and extra rings, rearranged derivatives from the broken of rings, and monosomy due to loss of the ring. Additional special staining may be necessary to define the aberration(s) present in the RC. The special staining may include one or more of the following: C-banding, NOR-silver staining, FISH analysis using centromeric, pericentromeric, and subtelomeric probes. Karyotype analysis on different tissue types from the same patient (if available) would also be warranted to look for different percentages of RCs in different tissues, as well as, RC instability. Some reports have shown higher frequencies of metaphase cells with monosomy and no RC in fibroblast cultures when compared to lymphocyte cultures (Moore et al. 1973; Neveling et al. 2021; Peeden et al. 1983). However, other reports by Ledbetter et al. and Manouvrier-Hanu et al. found higher abnormal metaphases in fibroblast cultures compared to lymphocyte cultures (Ledbetter et al. 1980; Manouvrier-Hanu et al. 1988). The same differences have been found with amniotic fluid cultures and lymphocyte cultures (Grass et al. 2000). Chromosome analysis from peripheral blood cultures is the simplest method for obtaining a large quantity of metaphase cells. Only a small volume of blood is necessary for these cultures, which makes these studies and those on family members straightforward. Blood is obtained in sodium heparin Vacutainer tubes (5–10ml peripheral blood; 1–2ml from newborn infants). Small blood specimens (0.5–1ml) are added to 10 ml culture medium, which contains fetal calf serum, L-Glutamine, Penicillin/Streptomycin and sodium biocarbonate. Phytohemagglutinin (PHA), a mitogen, is added to stimulate lymphocyte cell division. These cultures are incubated at 37 °C for 48, 72 or 96 h (Costa et al. 1998). Blood samples from newborns are treated similarly; however, fetal lymphocytes have a shorter cell cycle time and are usually only cultured for 48 h. Chromosome harvest begins with the addition of colcemid or colchicine to block mitotic

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spindle formation. The culture is incubated with colcemid for a short duration, usually 20 min, to allow a number of cells to accumulate at metaphase. This is followed by removing the supernatant and adding a hypotonic solution of KCl which causes the cells to expand and allows better visualization of the chromosomes. This is followed by fixation of cells with fresh 3:1 methanol:acetic acid fixative. In order to get chromosomes with good morphology and banding, long chromosomes are needed. Proper techniques for harvesting dividing cells are critical for attaining the quality needed for correct karyotype analysis. There are several methods used to obtain high-resolution chromosomes. Each method has several steps which are important in obtaining a cell pellet with quality metaphase and prometaphase spreads. Using these methods, the chromosomes are less condensed than in routine metaphase analysis and the number of identifiable bands is expanded, allowing a more sensitive analysis of the karyotype. The simplest method is to treat the culture with ethidium bromide which elongates the chromosome by binding proteins involved in chromosome condensation and DNA and blocks chromosome condensation (Ikeuchi 1984). After the incubation of the cells in ethidium bromide, the harvest is initiated by the addition of colcemid and then completed. The other widely used method for long chromosomes involves synchronization of the cell cultures with the use of amethopterin (methotrexate) and BrDU. In this method, amethopterin is added to the cultures to block cells at the S phase of the cell cycle. This way dividing cells are accumulated at S phase. These cells are then released from the block at S phase by the addition of BrDU, an analog of thymidine which also inhibits chromosome condensation (Lawce and Brown 2017). Harvest of these cultures is then completed with the addition of colchicine, followed by the standard harvesting procedure. Slides are then made and metaphases are ready for staining.

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2.2.2 G-Bands by Trypsin Using Giemsa (GTG) G-banding is a rapid method of banding chromosomes using trypsin pretreatment followed by Giemsa staining. G-banding was a major advance for the field of cytogenetics by producing high-resolution banding patterns which were unique for each chromosome (Seabright 1971; Sumner et al. 1971). This method induces specific and reproducible changes in the proteins of the mitotic chromosomes producing a pattern of light and dark bands along the length of the chromosome, much like a bar code. Analysis of the specific banding pattern for each chromosome allows the detection of changes in the structure of the chromosome and the identification of chromosome markers and rings. Figure 2.1 shows a GTG banded karyotype of a RC14 with the CMA identifying the amount of material deleted from the long arm of the chromosome. There are a number of different variations of this banding method, but together they are the most widely used staining method in cytogenetics. Wright’s stain is frequently used instead of Giemsa for staining chromosome preparations as it gives sharper resolution and reveals finer bands in high-resolution analysis. In this method, slides are immersed in trypsin for a short period of time (20–35 s) followed by immersion in Giemsa stain or Wright’s stain. Each laboratory must determine the length of trypsinization and time in stain. These times will vary depending on the following: which stain is being used, method of slide preparation, the length of the chromosomes, the temperature, and humidity of the room (Lawce 2017). Analysis of a patient’s metaphases stained by G-banding should begin with 20 metaphases. If a RC is identified in the original 20 cell count, the analysis should then be extended by an additional 80 cells, for a total of 100 metaphase cells analyzed. The analysis of 20 cells provides exclusion of 14% mosaicism or above at 95% confidence when all cells analyzed have an identical karyotype. Proceeding to a 50 cell analysis allows for exclusion of 6% mosaicism and a 100 cell analysis allows for exclusion of

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3% mosaicism, at 95% confidence (Hook 1977; Kozma et al. 1988). These cutoffs also apply for RCs with different ring variants (dic r, tetra r, +r) and ring derivatives (ring broken back to rod shape). Related to this, many RCs are unstable which is considered to be when a secondary aberration is present in greater than 5% of cells analyzed. Conversely, a RC is considered stable when secondary aberrations are present in 0–5% of cells analyzed (Kozma et al. 1988). Early studies by Moore et al. and Zackai and Breg looked at 60–250 metaphase cells to determine instability in a RC6 and a RC7, respectively (Moore et al. 1973; Zackai and Breg 1973). McDermott et al. screened 300 metaphase cells looking for cells with absent RCs, monocentric and dicentric RCs. In their analysis, 83% had a single monocentric RC, 8.3% had 2 monocentric RCs, 5.3% had a single dicentric RC, 0.7% had two dicentric RCs and 2.7% had 45 chromosomes with no RCs (McDermott et al. 1977). Pezzolo et al. analyzed chromosomes derived from blood lymphocytes from a young male with short stature and hypospadias using G- and Q-banding and FISH (Pezzolo et al. 1993). They analyzed 200 metaphases and found 12 different karyotypes with 40.8% having two RCYs, each a different size. More recently, Sodre et al. studied RC instability in six patients with RC4, RC14, RC15, and RC18 by analyzing 600 metaphase cells each, 300 from 48-h cultures and 300 from 72-h cultures (Sodre et al. 2010). Ciuladaite et al. analyzed 30 high-resolution metaphase cells to determine initial breakpoints of a patient with a RC10 and followed this by screening an additional 370 cells for RC instability; further studies using a Human ISCA CGH 180 K microarray refined the breakpoints and defined the 10p loss as a 3.68 Mb and the 10q loss a 4.62 Mb (Ciuladaite et al. 2015).

2.2.3 Special Stains Special stains are often necessary to ascertain the presence of complex chromosome rearrangements which may be present in cells with RCs. RCs may be unstable and present cells

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Fig. 2.1  a Karyotype of RC14 patient using GTG banding. b Microarray from RC14 patient with loss of 14q32. (Courtesy of Greenwood Genetic Center)

with double RCs, broken RCs, RC fragments. In order to determine the presence of the results of RC instability; i.e., one or more centromeres

in double RCs, lack of centromere sequences in a RC fragment, presence of nucleolar organizing regions (NOR) within the RC, different

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special staining should be used. Ledbetter et al. reported on a RC15 with multiple variations of the RC by using special stains (Ledbetter et al. 1980). In this report, G-banding analysis revealed a practically full length RC15 in a 15 month old girl with severe developmental delay and acral skeletal hypoplasia. In order to determine the position and number of centromeres in single RCs, C-banding was used and revealed a single centromere. Double RCs were positive for two centromeres. Ag-NOR staining revealed the presence of an active nucleolus organizing region within the single RC and two NOR regions in those cells with double RCs. C-banding C-banding is used to stain the constitutive heterochromatin found at the pericentromeric region of all chromosomes and Yq. This method has been used to identify polymorphic size variants of constitutive heterochromatin, especially those of chromosomes 1, 9, 16 and Yq, as well as pericentric inversions, and translocations involving the Y chromosome. It has also been used to identify the presence and position of a centromere in a RC as well as the number of RCs in larger dicentric and tricentric RCs and interlocked RCs. Cantu et al. used G- and C-banding, and Ag-NOR staining to characterize the karyotype and level of mosaicism found in a Turner syndrome patient with intellectual disability and unusually short stature (Cantu et al. 1995). The analysis revealed a sSRCX with a functional centromere and no evidence of a nucleolus organizer region. This method uses barium hydroxide to denature less condensed chromatin which leaves the constitutive heterochromatin to be stained (Lawce 2017; Steele et al. 2004). There are multiple variations of this method being used including the method listed here. Slides are incubated first in HCL solution followed by incubation in Ba(OH)2 solution and then hot sodium citrate solution to denature the chromosomes and remove much of the histone proteins. Slides are then stained with Giemsa or Wright’s stain to produce chromosomes with

B. Hilton and B. R. DuPont

dark centromeres and other regions of constitutive heterochromatin. NOR staining The nucleolar organizer regions (NORs) are found on the end of the short arm of the satellited D- and G-group chromosomes 13, 14, 15, 21, and 22. This stalk region contains the genes for 18s and 28s rRNA and is very basophilic. The number of each gene per NOR is not constant and this results in NORs with variable sizes. Early studies by Goodpasture et al. observed that not all 10 NORs will stain in each metaphase (Goodpasture et al. 1976). It was reported by Verma et al. that only the active NORs would stain positive with silver staining (Verma et al. 1983). Silver staining has been used to study polymorphisms of the NORs and can be used on ring chromosomes to determine if the NOR region of an acrocentric chromosome is included in a ring. It can also be used for the identification of a specific acrocentric chromosome if there is a polymorphism of the NOR. Small rings may be identified as acrocentric if an NOR is present. NOR staining using silver allows the NOR to be labeled with silver. In a report by Ledbetter et al., Ag-NOR staining was used to show the RC was silver stain positive, that the dicentric ring chromosomes had two active silver stain positive regions and that micronuclei, when present, had one or two active silver staining regions (Ledbetter et al. 1980).

2.3 Molecular Techniques The use of molecular techniques to compliment traditional cytogenetic analysis has been instrumental in the characterization of RC structure and the elucidation of the mechanisms leading to RC formation. Early studies employed the use of FISH, Southern blotting, polymerase chain reaction (PCR), and multiplex ligationdependent probe amplification (MLPA) to identify breakpoints and genomic content of RCs,

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specifically sSRCs for which traditional methods could not reach the resolution necessary for characterization. More recently, genomic methods including CMA and NGS have been used with higher resolution and the added benefit of being able to assess the genome as a whole. Finally, innovative methods such as optical genome mapping (OGM) which can not only assess copy number variation across the genome but can also identify balanced rearrangements associated with ring chromosome formation are emerging.

2.3.1  Fluorescence In Situ Hybridization (FISH) FISH is a molecular cytogenetic technique, where known-sequence pieces of DNA are fluorescently labeled with fluorochromes and used to hybridize to interphase nuclei or metaphase spreads and visualized by fluorescent microscopy. FISH has been routinely used in clinical cytogenetics for many years to confirm chromosome and microarray aberrations. In one of the first reports using FISH, Kozma et al. used a Y-specific probe in a male patient to identify a small RC as having a Y chromosome origin (Kozma et al. 1988). That same year, a female patient with a mosaic 45, X/46, X, r(?) was determined to have a RCX using FISH with X- and Ycentromere-specific alphoid repetitive sequence probes (Crolla and Llerena 1988). Callen et al. characterized 10 sSMCs/sSRCs using pericentric repeat probes (Callen et al. 1991). They were able to identify markers from chromosomes 1, 3, 6, 12, 14, 16, 18, 20, 13 or 21, and the X. One patient had two markers, one derived from chromosome 6 the other from the X chromosome. There are a number of different types of FISH probes, which can be employed to help discriminate various aspects of the ring and ring instability. Whole chromosome paints can be used to determine the chromosome of origin of the ring or if more than one chromosome is involved in the RC structure. Aalfs et al. used

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centromere probes and other chromosome specific probes to identify two sSRCs each from a different chromosome in a boy with minor dysmorphic features and mild developmental delay (Aalfs et al. 1996). The karyotype was determined to be 46, XY[17%]/47, XY, r(6)[24%]/47, XY, r(9)[20%]/48, XY, r(6), r(9)[39%]. Corolla et al. used chromosome paints and centromere probes to examine 26 patients with autosomal sSMCs, five of which were RCs from chromosomes 3, 6 (2 cases), 20 and 21 (Crolla et al. 1998). Guediche et al. used chromosome paint probes specific for chromosome 20, paints to the long and short arm of chromosome 20, centromere-specific probes and BAC probes for specific regions on both arms of chromosome 20 to characterize the sSMCs detected in two prenatal cases. Conventional G-banding revealed 47, XX, + mar in 54% of 77 metaphase cells in patient 1 and 47, XX, + mar in 43% of 175 metaphase cells in patient 2 (Guediche et al. 2010). Chromosome 20 specific paints and centromere probe identified the sSMCs as originating from chromosome 20 and also that both arms of chromosome 20 were contained in the marker. The series of BAC probes were used to determine the approximate breakpoints in both the long and short arms. Centromere-specific probes can be used to determine the chromosome of origin of the ring as well as determine the number and placement of centromeres within the ring (Guilherme et al. 2011a). These probes can also help determine if there are double rings or ring fragments. Subtelomere-specific probes can be used to determine if the RC has a deletion of the subtelomeric sequences of a chromosome. Brandt et al. used FISH with telomere-specific probes for chromosome 20 to prove the absence of the telomere-specific sequences on both the long and short arms (Brandt et al. 1993). Most probes can be commercially obtained from a number of different companies. FISH studies should be done along with G-banding analysis. These studies can be done simultaneously to confirm CMA results and to look for ring instability.

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2.3.2 Southern Blotting Southern blot analysis aides in the identification of structural rearrangements by determining DNA identity, size, and abundance using labeled sequence-specific probes for a target region in the genome. The early identification of RCs and ultimately the mechanisms leading to the formation of ring structures were elucidated via Southern blot analysis. This was achieved by pairing high-resolution chromosome analysis with Southern blotting with sequences targeting the regions suspected to be involved in the copy number variants associated with the RC. Wong et al. characterized a RC21 formed by a complex sequence of rearrangements: duplication of the centromere and long arm of chromosome 21 followed by proximal and telomeric breakage and reunion resulting in the RC21 (Wong et al. 1989). In this study, Southern blotting was used in the mapping of sequences at the proximal and telomeric breakpoints involved in the formation of the RC. Subsequently, Falik-Borenstein et al. used a similar approach to identify and characterize a RC21 segregating in a 4-generation family. Southern blot analysis of 5 random DNA sequences and 4 expressed genes allowed for the mapping of the regions involved in the formation of this RC21 (Falik-Borenstein et al. 1992). Southern blotting was also crucial in the initial characterization of RC structures involving the sex chromosomes, which are often small in size and difficult to identify by conventional cytogenetics alone. Guttenbach et al. identified a mosaic RCX and excluded the possibility of Y chromosome involvement using Southern blotting with Y-specific DNA probes (Guttenbach et al. 1991). In the case of a small RC found along with one normal X chromosome, Southern blotting identified the small RC as originating from Y chromosome material but lacking the entire heterochromatic portion of the long arm of chromosome Y (Pezzolo et al. 1993). In these cases, Southern blotting allowed for the characterization of small RCs that were otherwise unidentifiable by conventional and high-resolution cytogenetics techniques.

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2.3.3 Polymerase Chain Reaction (PCR) PCR using primers to amplify specific loci of a RC can define the copy number variation and breakpoints involved in RC structure. When used to compliment conventional cytogenetics, PCR can be useful to characterize RCs with added sensitivity, especially in the recognition of mosaicism. Much like Southern blotting, PCR has historically been used in the identification of small RCs that were otherwise unidentifiable by conventional methods alone. Henegariu et al. employed PCR along with FISH to characterize a RCY (Henegariu et al. 1997). Primers designed to amplify regions in the short and long arm of chromosome Y in addition to primers near the centromere and pseudoautosomal regions showed that the marker Y chromosome was a complex RC with breakpoints proximal to the centromere and within the terminal pseudoautosomal region. Recently, Barbosa et al. used PCR to assess the presence of Y material in a cohort of patients diagnosed with Turner syndrome (Barbosa et al. 2020). This study identified the presence of Y chromosome material in approximately 10% of patients with Turner syndrome and no previous indication of Y chromosome involvement. When the origin of the RC has been determined, PCR can be used to compliment conventional methods in the further characterization of RC structure with high sensitivity. Additionally, PCR may be used in microsatellite genotyping targeting the polymorphic regions allowing for the identification of parent of origin. Zhang et al. determined the parent of origin using PCR for microsatellite genotyping showing that a RC6 originated from paternal as only the maternal polymorphic polyglycine tracts were inherited in the normal chromosome 6 (Zhang et al. 2016). Given the sensitivity of PCR analysis, this method can be informative in the analysis of sSMCs that are mosaic in a low percent of cells. van der Veken et al. identified and characterized a sSRC18 composed of six copies of the 18q11 region. Microsatellite

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markers within the pericentromeric region on chromosome 18 were used to examine the maternal and paternal alleles. The microsatellite analysis of the sSRC18 compared against the parental alleles demonstrated that the sSRC18 was paternally derived and both normal copies showed biparental disomy (van der Veken et al. 2010). These studies demonstrate the utility of PCR targeting the repetitive sequences making up the microsatellite regions in the determination of parental origin of RCs.

2.3.4 Multiplex LigationDependent Probe Amplification (MLPA) MLPA is a PCR-based technique that utilizes custom-designed probes to specific regions in the genome to detect DNA copy number changes. In the context of RC characterization, MLPA is a useful tool in the analysis of subtelomeric regions in the RC. Guilherme et al. applied MLPA in the determination of the mechanisms leading to the formation of the RCs in their cohort (Guilherme et al. 2011b). Such as in the Guilherme study, MLPA may be used to complement other high-resolution testing, such as CMA, at a higher resolution over chromosome analysis alone. Furthermore, due to the repetitive nature of the DNA sequence at the telomere, MLPA can be useful in determining the involvement of subtelomeric regions of a chromosome that microarray cannot resolve. MLPA typically targets multiple loci across the subtelomere (approximately 50 per assay), compared to subtelomere FISH testing which typically only allows for the visualization of one loci per assay. Hermsen et al. molecularly characterized a RC19 with a combination of MLPA and microarray testing (Hermsen et al. 2005). By using MLPA targeting, the subtelomeric region of chromosome 19 this study was able to show that the RC19 did not involve the eurchromatic region of chromosome 19 and was confined to the heterochromatin at the telomere. This determination aided the genotype–phenotype correlation in the patient that

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presented with hypopigmentation along the lines of Blaschko but no additional abnormalities in development.

2.4 Genomic Analysis 2.4.1 Chromosome Microarray Analysis (CMA) As CMA has emerged as the first-tier test for genomic imbalances associated with developmental delay and congenital malformations, the identification of RCs has become more complicated as microarrays show gains and losses of DNA copy number aberrations but do not show any structural information for an aberration (South et al. 2013). Large RCs usually appear as a chromosome with terminal losses on both the p- and q-arm of the same chromosome. Some rings may be more complex with a duplicated region adjacent to the terminal deleted region. sSRCs will appear as copy number gain regions on both the p- and q-arms spanning the centromere. They also may be observed as duplicated regions on one or the other of the p- or q-arms near the centromere. Other more complex combinations of gains or losses are also possible. In cases for which traditional cytogenetics is performed first, CMA should be considered as follow-up in order to determine the breakpoints of the chromosome gains and/or losses as well as the genomic content of the duplication/deletion. Guilherme et al. characterized 14 novel RCs (Guilherme et al. 2011b). The characterization of each RC included G-banded chromosome analysis of 200 cells, FISH analysis of 100 cells, MLPA subtelomere analysis (discussed above), and CMA. Using the combination of these techniques, the molecular mechanism by which each RC formed was determined and was variable from case to case. The mechanisms identified included: breaks in both chromosome arms followed by end-to-end reunion; a break in one chromosome arm followed by fusion with the subtelomeric region of the other; a break in

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one chromosome arm followed by fusion with the opposite telomeric region; fusion of two subtelomeric regions; and telomere-telomere fusion. In a majority of cases, the RC was found to be unstable, detected by loss of the RC in G-banded chromosome analysis and presence of secondary aberrations in microarray and MLPA analyses. Clinical correlation also was performed and appeared to be related to a variety of factors. Gene haploin sufficiency and triplo sensitivity as well as RC instability were the factors most closely related to observed clinical presentations. However, epigenetic factors due to the circular architecture of RCs also must be considered given the presence of clinical features in individuals identified with complete RCs. For this case series, the use of CMA was instrumental in the determination of genomic content and ultimately the genotype–phenotype correlations in this collection of cases (Guilherme et al. 2011b). Therefore, if an aberration observed by CMA is suggestive of a RC, additional analyses should be performed to confirm the presence of a RC. These data can then be analyzed in combination to draw conclusions about the potential impact of the genomic imbalances for the patient as well as recurrence risk for their family. Additionally, for cases in which molecular studies have not been performed previously, CMA or sequencing-based techniques should be performed to determine breakpoints and the extent of the genomic content involved in the deletion and/or duplication. On CMA, sSRCs often appear as a duplication that spans the centromere of a chromosome or a duplication that resides in the p- or q-arm next to a centromere (Fig. 2.2). Additionally, sSRCs are often found in the mosaic state and due to the inherent instability may be difficult to detect in some analyses. CMA on DNA extracted directly from the patient specimen may prove informative in these cases. However, the resolution of the microarray platform in the detection of low-level mosaic RCs may become a challenge. Bi et al. compared microarray and traditional chromosome analysis in 3710 unrelated patients (Bi et al. 2013). Of the cases with

B. Hilton and B. R. DuPont

an abnormal microarray result, 33% were found to have a marker or RC. In a small percentage of these cases, CMA alone was not sufficient to detect the mosaic structural abnormality. This included one case for which there was mosaicism for two abnormal cell lines, resulting in a balanced net effect and a normal chromosomal microarray analysis. These data suggest that although microarray analysis can potentially clarify the genomic content of a RC, in some instances where the RC is found in the mosaic state, other methods including chromosome or FISH analysis, may be necessary in the initial detection of the abnormality. In contrast, in cases of cytogenetically similar findings, CMA can be invaluable in the determination of size and genomic content. Glass et al. characterized two cytogenetically similar RC15s in two unrelated patients with drastically dissimilar clinical presentations (Glass et al. 2006). Previous analysis of these patients included G-banded chromosome analysis and 15 subtelomeric FISH analysis. Chromosome analysis showed a RC15 and FISH analysis showed loss of the 15 subtelomeric region in both patients. However, upon further characterization by CMA, patient 1 who presented with a relatively severe phenotype including extremely small stature, heart defects, and developmental delay was found to have a 6 Mb deletion of distal 15q, while patient 2 who presented with short stature and lived independently was found to have a small terminal deletion of 15qter. These cases illustrate the utility of CMA in genotype–phenotype correlation for cases where cytogenetic analysis was suggestive of a similar outcome.

2.4.2 Optical Genome Mapping (OGM) The use of an emerging technology, OGM, in clinical cytogenomics laboratories may allow a single test to elucidate structural variation by replacing karyotype, FISH, and microarray analysis. OGM has the capability of detecting both balanced and unbalanced rearrangements, the

2  Diagnostic Methods for Ring Chromosomes

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Fig. 2.2  CMA showing small supernumerary ring chromosome 3 (a) and full ring chromosome 18 (b). Courtesy of Greenwood Genetic Center

orientation of segments, the location of inserted segments, all at a resolution of several kilobases. In this technology, very long linear single DNA molecules (median size > 250 kb) are labeled at specific sequence motifs sites across the genome. The labeled DNA molecules are loaded into specialized chips for linearization and imaging in nanochannel arrays. The unique patterns observed are used to assemble the patient’s genome and identify structural variants via the Bionano software. OGM has recently been approved for clinical diagnosis in the detection of aberrations in leukemia (Neveling et al. 2021).

2.4.3 Next-Generation Sequencing (NGS) NGS, or massively parallel sequencing, is a high throughput technology for determining sequence and more recently copy number variants contained within genomic DNA. As the cost of NGS testing has continued to decrease the application of this technology in the elucidation of genomic variants has rapidly increased. This remains true for the characterization of RCs for which NGS is proving to be a viable alternative to more traditional techniques. NGS has an

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advantage over traditional methods in that it can determine breakpoints at a higher resolution and can potentially identify all copy number variants at the base pair level in a single assay. However, the limitations related to genomic architecture remain, for which alternative methods must be considered. Ji et  al. implemented low-coverage NGS to characterize two cases of RC18 (Ji et al. 2015). The results from NGS testing were compared against results from CMA which confirmed the breakpoints of the deletions observed by NGS. The resolution of the breakpoints in the low-coverage NGS was estimated to be approximately 100 base pairs and one breakpoint was found within a gene which may be contributing to the patient’s clinical outcome. Similarly, Zhang et al. characterized a novel RC6 using NGS (Zhang et al. 2004, 2016). The NGS was performed concurrently with CMA and identified an additional copy number change. Both assays detected a terminal deletion on the short arm of chromosome 6 and an interstitial deletion in the long arm of chromosome 6; however, the microarray did not identify a terminal deletion on the long arm of chromosome 6 where NGS did. These data suggest that NGS could be used as an alternative method to CMA at a higher resolution while maintaining the ability to access additional copy number variants across the genome. Although these findings are promising, future studies demonstrating the utility of NGS testing in cases of rare structural variants are necessary to determine the limitations of this testing. As with alternative sequencing-based methodologies, traditional analyses remain a value tool in visual confirmation of these findings.

2.5 Considerations and Recommendations Chromosome analysis, FISH, molecular studies, and CMA should be performed to define the RC structure, dynamic mosaicism, and genomic imbalance for patients with RCs. Each of these methods provides one piece of the puzzle and should be used in an integrated fashion to fully characterize a RC of interest. Genomic studies

B. Hilton and B. R. DuPont

by CMA and NGS provide the highest resolution and are instrumental in the elucidation of genomic content involved in the losses, gains, and/or breakpoints of a RC. These methods can provide the most extensive investigation of the genes that are involved and ultimately can suggest potential clinical outcomes. Although genomic methods provide information related to genomic content they must be used in conjunction with more traditional cytogenetic methods to confirm the structure associated with the genomic imbalances. Chromosome analysis is essential to confirm visually the existence of a RC structure, while FISH analysis can be especially useful in the characterization of sSMC for which mosaicism is often a feature. Proper description of the events related to RCs is of utmost importance as the nomenclature is often complex, can be difficult for those outside of cytogenetics to understand, and will be documented in the patient chart for the duration of clinical care. Therefore, attention to detail is necessary in the documentation of RC abnormalities.

References Aalfs CM, Jacobs ME, Nieste-Otter MA, Hennekam RC, Hoovers JM (1996) Two supernumerary marker chromosomes, derived from chromosome 6 and 9, in a boy with mild developmental delay. Clin Genet 49(1):42–45. https://doi. org/10.1111/j.1399-0004.1996.tb04323.x Atkins L, Miller WL, Salam M (1972) A ring-20 chromosome. J Med Genet 9(3):377–380. https://doi. org/10.1136/jmg.9.3.377 Bain AD, Gauld IK, Farquhar JW (1965) A ring X chromosome in dwarfism. Lancet 1(7389):820. https:// doi.org/10.1016/s0140-6736(65)92992-2 Barbosa LG, Souza MA, Siviero-Miachon AA, Dias-daSilva MR, Spinola-Castro AM (2020) Y chromosome sequences in Turner syndrome: Multiplex PCR, a new method for diagnosis. J Genet Gen Res 7(1):4. https://doi.org/10.23937/2378-3648/1410052 Bi W, Borgan C, Pursley AN, Hixson P, Shaw CA, Bacino CA, Lalani SR, Patel A, Stankiewicz P, Lupski JR, Beaudet AL, Cheung SW (2013) Comparison of chromosome analysis and chromosomal microarray analysis: What is the value of chromosome analysis in today’s genomic array era? Genet Med 15(6):450–457. https://doi.org/10.1038/ gim.2012.152

2  Diagnostic Methods for Ring Chromosomes Brandt CA, Kierkegaard O, Hindkjaer J, Jensen PK, Pedersen S, Therkelsen AJ (1993) Ring chromosome 20 with loss of telomeric sequences detected by multicolour PRINS. Clin Genet 44(1):26–31. https://doi. org/10.1111/j.1399-0004.1993.tb03837.x Callen DF, Eyre HJ, Ringenbergs ML, Freemantle CJ, Woodroffe P, Haan EA (1991) Chromosomal origin of small ring marker chromosomes in man: Characterization by molecular genetics. Am J Hum Genet 48(4):769–782 Cantu ES, Jacobs DF, Pai GS (1995) An atypical Turner syndrome patient with ring X chromosome mosaicism. Ann Clin Lab Sci 25(1):60–65 Ciuladaite Z, Burnyte B, Vanseviciute D, Dagyte E, Kucinskas V, Utkus A (2015) Clinical, cytogenetic and molecular study of a case of ring chromosome 10. Mol Cytogenet 8(1):29. https://doi.org/10.1186/ s13039-015-0124-9 Costa D, Borrell A, Soler A, Carrio A, Margarit E, Ballesta F, Puerto B, Caballin MR, Fortuny A (1998) Cytogenetic studies in fetal blood. Fetal Diagn Ther 13(3):169–175. https://doi.org/10.1159/000020832 Crolla JA, Long F, Rivera H, Dennis NR (1998) FISH and molecular study of autosomal supernumerary marker chromosomes excluding those derived from chromosomes 15 and 22: I. Results of 26 new cases. Am J Med Genet 75(4):355–366. https://doi. org/10.1002/(sici)1096-8628(19980203)75:43.0.co;2-p Crolla JA, Llerena JC Jr (1988) A mosaic 45, X/46, X, r(?) karyotype investigated with X and Y centromerespecific probes using a non-autoradiographic in situ hybridization technique. Hum Genet 81(1):81–84. https://doi.org/10.1007/BF00283735 Falik-Borenstein TC, Pribyl TM, Pulst SM, Van Dyke DL, Weiss L, Chu ML, Kraus J, Marshak D, Korenberg JR (1992) Stable ring chromosome 21: Molecular and clinical definition of the lesion. Am J Med Genet 42(1):22–28. https://doi.org/10.1002/ ajmg.1320420107 Glass IA, Rauen KA, Chen E, Parkes J, Alberston DG, Pinkel D, Cotter PD (2006) Ring chromosome 15: Characterization by array CGH. Hum Genet 118(5):611–617. https://doi.org/10.1007/ s00439-005-0030-z Goodpasture C, Bloom SE, Hsu TC, Arrighi FE (1976) Human nucleolus organizers: The satellites or the stalks? Am J Hum Genet 28(6):559–566 Grass FS, Brown CA, Backeljauw PF, Lucas A, Brasington C, Gazak JM, Nakano S, Ostrowski RS, Spence JE (2000) Novel ring chromosome composed of X- and Y-derived material in a girl with manifestations of Ullrich-Turner syndrome. Am J Med Genet 93(5):343–348.  https://doi. org/10.1002/1096-8628(20000828)93:53.0.CO;2-0 Guediche N, Brisset S, Benichou JJ, Guerin N, Mabboux P, Maurin ML, Bas C, Laroudie M, Picone O, Goldszmidt D, Prevot S, Labrune P, Tachdjian G (2010) Chromosomal breakpoints characterization of two supernumerary ring chromosomes 20. Am J Med

29 Genet 152A(2):464–471. https://doi.org/10.1002/ ajmg.a.33250 Guilherme RS, Bragagnolo S, Pellegrino R, Christofolini DM, Takeno SS, Carvolheira GM, Kulikowski LD, Melaragno MI (2011a) Clinical, cytogenetic and molecular study in a case of r(3) with 3p deletion and review of the literature. Cytogenet Genome Res 134(4):325–330. https://doi.org/10.1159/000329478 Guilherme RS, Meloni VF, Kim CA, Pellegrino R, Takeno SS, Spinner NB, Conlin LK, Christofolini DM, Kulikowski LD, Melaragno MI (2011b) Mechanisms of ring chromosome formation, ring instability and clinical consequences. BMC Med Genet 12:171. https://doi. org/10.1186/1471-2350-12-171 Guttenbach M, Kohler J, Schmid M (1991) Cytogenetic and molecular characterization of a small ring chromosome in the complex karyotype of a girl with Turner syndrome. Hum Genet 87(6):680–684. https:// doi.org/10.1007/BF00201725 Henegariu O, Kernek S, Keating MA, Palmer CG, Heerema NA (1997) PCR and FISH analysis of a ring Y chromosome. Am J Med Genet 69(2):171–176. https://doi.org/10.1002/(sici)10968628(19970317)69:23.0.co;2-i Hermsen MA, Tijssen M, Acero IH, Meijer GA, Ylstra B, Toral JF (2005) High resolution microarray CGH and MLPA analysis for improved genotype/phenotype evaluation of two childhood genetic disorder cases: Ring chromosome 19 and partial duplication 2q. Eur J Med Genet 48(3):310–318. https://doi. org/10.1016/j.ejmg.2005.04.009 Hook EB (1977) Exclusion of chromosomal mosaicism: Tables of 90%, 95% and 99% confidence limits and comments on use. Am J Hum Genet 29(1):94–97 Ikeuchi T (1984) Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high-resolution chromosome banding. Cytogenet Cell Genet 38(1):56–61. https://doi. org/10.1159/000132030 Ji X, Liang D, Sun R, Liu C, Ma D, Wang Y, Hu P, Xu Z (2015) Molecular characterization of ring chromosome 18 by low-coverage next generation sequencing. BMC Med Genet 16:57. https://doi.org/10.1186/ s12881-015-0206-x Kozma R, Fear C, Adinolfi M (1988) Fluorescence in  situ hybridization and Y ring chromosome. Hum Genet 80(1):95–96. https://doi.org/10.1007/ BF00451465 Lansky S, Daniel W, Fleiszar K (1977) Physical retardation is associated with ring chromosome mosaicism: 46, XX, r(10)/45, XX,10 minus. J Med Genet 14(1):61–63. https://doi.org/10.1136/jmg.14.1.61 Lawce H, Brown MG (2017) Cytogenetics: An overview. In: Arsham MS, Barch MJ, Lawce H (eds) The AGT cytogenetics laboratory manual, 4th edn. Wiley & Sons Inc., Hoboken, New Jersey, pp 25–84 Lawce H (2017) Chromosome stains. In: Arsham MS, Barch MJ, Lawce HJ (eds) The AGT cytogenetics laboratory manual, 4th edn. Wiley & Sons, Inc., Hoboken, New Jersey, pp 213–299. 9781119061281

30 Ledbetter DH, Riccardi VM, Au WW, Wilson DP, Holmquist GP (1980) Ring chromosome 15: Phenotype, Ag-NOR analysis, secondary aneuploidy, and associated chromosome instability. Cytogenet Cell Genet 27(2–3):111–122. https://doi. org/10.1159/000131472 Lindsten J, Tillinger K-G (1962) Self-perpetuating ring chromosome in a patient with gonadal dysgenesis. Lancet 279:593–594.  https://doi.org/10.1016/ S0140-6736(63)92780-6 Manouvrier-Hanu S, Turck D, Gottrand F, Savary JB, Loeuille GA, Deminatti MM, Farriaux JP (1988) [Ring chromosome 9. Case report and review of the literature]. Ann Genet 31(4):250–253 Mantere T, Neveling K, Pebrel-Richard C, Benoist M, van der Zande G, Kater-Baats E, Baatout I, van Beek R, Yammine T, Oorsprong M, Hsoumi F, OldeWeghuis D, Majdali W, Vermeulen S, Pauper M, Lebbar A, Stevens-Kroef M, Sanlaville D, Dupont JM, Smeets D, Hoischen A, Schluth-Bolard C, El Khattabi L (2021) Optical genome mapping enables constitutional chromosomal aberration detection. Am J Hum Genet 108(8):1409–1422. https://doi. org/10.1016/j.ajhg.2021.05.012 McDermott A, Voyce MA, Romain D (1977) Ring chromosome 4. J Med Genet 14(3):228–232. https://doi. org/10.1136/jmg.14.3.228 Miller DA, Allderdice PW, Miller OJ, Breg WR (1971) Quinacrine fluorescence patterns of human D group chromosomes. Nature 232(5305):24–27. https://doi. org/10.1038/232024a0 Moore CM, Heller RH, Thomas GH (1973) Developmental abnormalities associated with a ring chromosome 6. J Med Genet 10(3):299–303. https:// doi.org/10.1136/jmg.10.3.299 Neveling K, Mantere T, Vermeulen S, Oorsprong M, van Beek R, Kater-Baats E, Pauper M, van der Zande G, Smeets D, Weghuis DO, Stevens-Kroef M, Hoischen A (2021) Next-generation cytogenetics: Comprehensive assessment of 52 hematological malignancy genomes by optical genome mapping. Am J Hum Genet 108(8):1423–1435. https://doi. org/10.1016/j.ajhg.2021.06.001 Peeden JN, Scarbrough P, Taysi K, Wilroy RS, Finley S, Luthardt F, Martens P, Howard-Peebles PN (1983) Ring chromosome 6: Variability in phenotypic expression. Am J Med Genet 16(4):563–573. https:// doi.org/10.1002/ajmg.1320160413 Pezzolo A, Perroni L, Gimelli G, Arslanian A, Porta S, Gandullia P, Gandullia E (1993) Identification of ring Y chromosome: Cytogenetic analysis, Southern blot and fluorescent in situ hybridization. Ann Genet 36(2):121–125 Rohde RA, Tompkins R (1965) Cri du Chat” due to a ring-B chromosome. Lancet 2(7421):1075–1076. https://doi.org/10.1016/s0140-6736(65)90609-4

B. Hilton and B. R. DuPont Seabright M (1971) A rapid banding technique for human chromosomes. Lancet 2(7731):971–972. https://doi.org/10.1016/s0140-6736(71)90287-x Sodre CP, Guilherme RS, Meloni VF, Brunoni D, Juliano Y, Andrade JA, Belangero SI, Christofolini DM, Kulikowski LD, Melaragno MI (2010) Ring chromosome instability evaluation in six patients with autosomal rings. Genet Mol Res 9(1):134–143. https:// doi.org/10.4238/vol9-1gmr707 South ST, Lee C, Lamb AN, Higgins AW, Kearney HM (2013) ACMG Standards and Guidelines for constitutional cytogenomic microarray analysis, including postnatal and prenatal applications: Revision 2013. Genet Med 15(11):901–909. https://doi.org/10.1038/ gim.2013.129 Steele MW, Breg WR, Eidelman AI, Lion DT, Terzakis TA (2004) A B-group ring chromosome with mosaicism in a newborn with cri du chat syndrome. Cytogenet Genome Res 5(6):419–429. https://doi. org/10.1159/000129917 Sumner AT, Evans HJ, Buckland RA (1971) New technique for distinguishing between human chromosomes. Nat New Biol 232(27):31–32. https://doi. org/10.1038/newbio232031a0 van der Veken LT, Dieleman MM, Douben H, van de Brug JC, van de Graaf R, Hoogeboom AJ, Poddighe PJ, de Klein A (2010) Low grade mosaic for a complex supernumerary ring chromosome 18 in an adult patient with multiple congenital anomalies. Mol Cytogenet 3(1):13. https://doi.org/10.1186/1755-8166-3-13 Verma RS, Rodriguez J, Shah JV, Dosik H (1983) Preferential association of nucleolar organizing human chromosomes as revealed by silver staining technique at mitosis. Mol Gen Genet 190(2):352– 354. https://doi.org/10.1007/BF00330664 Wong C, Kazazian HH Jr, Stetten G, Earnshaw WC, Van Keuren ML, Antonarakis SE (1989) Molecular mechanism in the formation of a human ring chromosome 21. Proc Natl Acad Sci U S A 86(6):1914–1918. https://doi.org/10.1073/pnas.86.6.1914 Zackai EH, Breg WR (1973) Ring chromosome 7 with variable phenotypic expression. Cytogenet Cell Genet 12(1):40–48. https://doi.org/10.1159/000130436 Zhang HZ, Li P, Wang D, Huff S, Nimmakayalu M, Qumsiyeh M, Pober BR (2004) FOXC1 gene deletion is associated with eye anomalies in ring chromosome 6. Am J Med Genet A 124A(3):280–287. https://doi. org/10.1002/ajmg.a.20413 Zhang R, Chen X, Li P, Lu X, Liu Y, Li Y, Zhang L, Xu M, Cram DS (2016) Molecular characterization of a novel ring 6 chromosome using next generation sequencing. Mol Cytogenet 9(1):33. https://doi. org/10.1186/s13039-016-0245-9

3

Genetic Databases and Online Ring Chromosome Registry Qiping Hu, Deqiong Ma, Peining Li and Thomas Liehr

Abstract

The rapid adaptations of genomic technologies into genetic testing require knowledge-based genetic databases and disease registries in various capacities. General Web resources for clinical and diagnostic genetics include the UCSC (University of California, Santa Cruz) Genome Browser, Online Mendelian Inheritance in Man (OMIM), Clinical Genome Resource (ClinGen), database of chromosomal imbalance and phenotype in humans using ensemble resource (DECIPHER), and Database of Genomic Variants (DGV). Clinical cytogenetics and ring chromosome related web resources include the ChromoSomics database for small supernumerary marker and ring chromosomes (sSMC/sSRC), atlas of genetics and cytogenetics in oncology and hematology (AGCOH), patient advocate organization Q. Hu (*)  Department of Cell Biology and Genetics, School of Basic Medical Sciences, Guangxi Medical University, Nanning, Guangxi, China e-mail: [email protected] D. Ma · P. Li  Department of Genetics, Yale School of Medicine, New Haven, CT, USA T. Liehr  Jena University Hospital, Institute of Human Genetics, Friedrich Schiller University, Am Klinikum 1, D-07747 Jena, Germany

(PAO) developed supporting and networking groups for specific ring chromosomes, and an online human ring chromosome registry. This registry represents efforts from the international consortium for human ring chromosome (ICHRC) to compile and curate cytogenomic and clinical findings for better diagnostic interpretation and clinical management for patients affected by ring chromosomes.

Keywords

Web resources · Genetic databases · Patient advocate organization · Ring chromosome registry

3.1 Introduction The completion of Human Genome Project has prompted rapid applications of various genomic technologies such as chromosome microarray analysis (CMA), exome sequencing (ES), and whole genome sequencing (WGS) onto clinical diagnosis of genetic defects (Choi et al. 2009; Lionel et al. 2018; Shaffer et al. 2007). This genome-wide genotyping-first approach has significantly improved the analytic resolution and diagnostic accuracy on detecting genetic defects from copy number variants (CNVs) to single nucleotide variants (SNVs). The analysis

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_3

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of these variants and the interpretation of their clinical significance required genetic databases and disease registries in various capacities. This chapter is aimed to introduce Web-based resources for general clinical and diagnostic genetics such as Human Genome Browser (https://genome.ucsc.edu/), Online Mendelian Inheritance in Man (OMIM, https://www. omim.org/), and Clinical Genome Resource (ClinGen, https://clinicalgenome.org/), and for more disease-specific online databases of clinical cytogenetics and chromosomal abnormalities such as the Database of Genomic Variants (DGV, http://dgv.tcag.ca/), the Atlas of Genetics and Cytogenetics in Oncology and Hematology (AGCOH, https://atlasgeneticsoncology.org/), and the ChromoSomics database (https://cs-tl. de/DB.html) including collections on small supernumerary marker and ring chromosomes (sSMC/sSRC), chromosomal heteromorphisms, chromosomal breakpoints, and chromosomespecific uniparental disomy (UPD). These Web resources are regularly updated to ensure that they remain comprehensive and up-to-date resources on related topics. They are widely used by clinical and laboratory geneticists, genetic counselors, healthcare professionals, patients and affected families, and genetic researchers. Furthermore, patient advocate organization (PAO) developed websites for specific ring chromosomes are presented, and an online interactive human ring chromosome registry is introduced for registering, compiling, and curating clinical and cytogenomic findings from ring chromosome cases for better diagnostic practice and clinical management (Hu et al. 2018). All these Web resources are useful tools in interpretating cytogenomic findings and evaluating potential clinical consequence of specific ring chromosomes.

Q. Hu et al.

3.2 General Online Resources for Clinical and Diagnostic Genetics 3.2.1 The University of California Santa Cruz (UCSC) Genome Browser The UCSC Genome Browser provides convenient access to human genome sequence, annotation data, and bioinformatic tools along with a dozen vertebrate species and major model organisms. With over 20 years of continuous effort in renovation and integration, the Genome Browser has been an omics data consolidator, graphic viewer, and general bioinformatics resources to support genomics community (Karolchik et al. 2003; Nassar et al. 2023). For analyzing variants in the human genome, it serves as a data aggregator for annotating and visualizing regions of interest across publicly available or custom-built data sets. The heavily annotated human genome data can be displayed graphically as ‘tracks’ align to the genomic sequence and grouped according to specific features, such as mapping and sequencing, variation, genes and gene predication, phenotype, and literature. It allows users to add and view any given piece of genome at any scale and any type of annotation. The nomenclature of pathogenic and likely pathogenic CNVs and chromosomal rearrangements in cytogenomic analysis follows the genomic coordinates in the human genome assembly (GRCh37/hg19) from the Genome Browser (International Standing Committee on Human Cytogenomic Nomenclature et al. 2020). Still, as recently highlighted, chromosome idiograms in UCSC and other genome browsers do not consider ISCN standards and invent new or ignore well-established chromosomal subbands (Liehr 2021a).

3  Genetic Databases and Online Ring Chromosome Registry

3.2.2 Online Mendelian Inheritance in Man (OMIM) The OMIM is a comprehensive and authoritative compendium of human genes and genetic phenotypes that is freely available and updated daily. This database was initiated in the early 1960s by Dr. Victor A. McKusick as a catalog of Mendelian traits and disorders (Hamosh et al. 2021). OMIM has designed unique and separate entries for genes and phenotypes. As of April 2023, there are 19,979 entries for genes and 6602 entries for phenotypes with known molecular basis. However, number of genes may vary between different databases for gene-coding ones between 19,962 and 20,916 and for genes overall between 54,644 and 270,168 (Liehr 2021b). The OMIM has been an indispensable resource to understand the genetics of the inherited diseases, spectrum of disease-causing variants, genotype–phenotype correlations, and related literature. For example, ring chromosome 14 syndrome has a designated OMIM phenotype entry #616606 with summarized, text, description, clinical features, and references.

3.2.3 Clinical Genome Resource (ClinGen) The ClinGen is a National Institutes of Health (NIH) funded authoritative central resource that aims to establish the clinical relevance of genomic variants and genes to facilitate accurate interpretation of genomic data in a clinical setting. It has organized working groups and expert panels for many curation activities such as gene–disease validity, variant pathogenicity, clinical actionability, dosage sensitivity, somatic cancer variant, and baseline annotation. It also serves as a platform for collaboration among researchers, clinicians, and industry partners to improve the understanding of the clinical significance of genetic variation. It provides a lot of information, including disease–gene relationships, gene dosage map, variant-level classifications, phenotype-based classification

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refinement, and evidence-based gene-disease validations. This information can be used to support the interpretation consistency of genomic variants in clinical practice. Technical standards for the interpretation and reporting of constitutional CNVs was developed by a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the ClinGen (Riggs et al. 2020).

3.2.4 DatabasE of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER) The DECIPHER (https://www.deciphergenomics.org/) is an interactive Web-based database that collects and curates information on genomic structural variations and their associated phenotypic effects in humans and incorporates a suite of tools designed to aid the interpretation of genomic variants (Firth et al. 2009). The database includes information on a wide range of genomic structural variations, including those that cause well-known genetic disorders, as well as those that are less well-understood. The DECIPHER can be used to (1) search for consented patients sharing a defined chromosomal location, (2) navigate regions of interest using in-house visualization tools and the Ensembl genome browser, (3) analyze affected genes and prioritize them according to their likelihood of haploin sufficiency, (4) upload patient aberrations and phenotypes, and (5) create printouts at different levels of detail (Corpas et al. 2012).

3.2.5 Database of Genomic Variants (DGV) The DGV has provided a publicly accessible, comprehensive curated catalog of structural variation (SV) found in the genomes of control individuals from worldwide populations (MacDonald et al. 2014; Zhang et al. 2006). Current summary statistics in the DGV contains

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983,845 CNVs and 4083 inversions from 75 studies. From this large collection of CNVs, population incidence of benign and likely benign CNVs in certain loci can be estimated and pathogenicity of recurrent CNVs within a given genomic coordinate could be ruled out. However, the CNVs being identified were through different platforms on different samples from different studies. It is recommended to use DGV along with other clinical-oriented databases such as ClinGen and DECIPHER to avoid errors in the interpretation of CNVs (BastidaLertxundi et al. 2014).

3.2.6 Orphan Disease European Reference Portal (Orphanet) The Orphanet (https://www.orpha.net/) is a centralized resource gathering knowledge on rare disease to improve the diagnosis, care, and treatment of patients with rare diseases (Singh 2013). The Orphanet consortium is organized by European experts in the field of rare diseases and provide information on over 7000 rare diseases. For each rare disease such as ring chromosomes, brief descriptions of the diseases, their symptoms, diagnosis, and possible treatments could be found; as in OMIM, Orphanet provides identifiers like ORPHA:1440 for ring chromosome 14. The Orphanet consortium provides also information on orphan drugs, which are drugs developed specifically for the treatment of rare diseases. This resource helps to raise awareness of rare diseases and the challenges faced by those affected by them by also listing patient support groups. Besides, specialists, institutions, expert centers, and diagnostic facilities for rare diseases are listed on Orphanet web page. A patient support group UNIQUE (https:// rarechromo.org/), also listed in Orphanet, provides support and networking to affected families for understanding rare chromosome and gene disorders. Brief descriptions of rare diseases, their symptoms, diagnosis, and possible treatments are provided by UNIQUE in different languages. UNIQUE serves as an overarching

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family support group for all chromosomal aberrations and has an internationally active group that is in contact with many patients and families including such affected by constitutional ring chromosomes. UNIQUE is also connected with national patient support groups, such as LEONA e.V. (https://www.leona-ev.de/start/) in Germany.

3.2.7 The National Organization for Rare Disorders (NORD) The NORD (https://rarediseases.org/) is a nonprofit organization that provides medical information and support for people with rare diseases and their families. It maintains a database of information on over 1200 rare diseases and disorders. The database also includes information on patient support organizations and resources for financial and emotional support. In addition, the NORD also provides advocacy and education services, and works to increase awareness of rare diseases and the challenges faced by those affected by them. NORD also provides support for research in rare diseases, here as an overarching organization for smaller patient support groups, preliminarily from USA. NORD's mission is to improve the lives of people with rare diseases by promoting understanding, advocacy, research, and education.

3.2.8 The DGV-DECIPHER-ClinGenOMIM Route for CNVs The existence of CNVs as a major cause of structural variations in the human genome was evidenced by clinical application CMA since the earlier reports two decades ago (Iafrate et al. 2004; Sebat et al. 2004). The presence of these CNVs likely reflect errors from DNA recombination and replication machineries. Pathogenic CNVs could cause phenotypes by gene dosage effects of haploinsufficiency and triple-sensitivity, loss of function by gene disruption, gain of function by gene fusion, and position effects by epigenetic regulation; they

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are bound to have vital role in Mendelian diseases, sporadic diseases, complex diseases, disease susceptibility, and drug response (Almal and Padh 2012; Mikhail 2014). The ACMG Practice Guidelines have recommended microarray as the first-tier test for patients with developmental delay and intellectual disability, congenital anomalies, and dysmorphic features (South et al. 2013). The American College of Obstetricians and Gynecologists Committee on Genetics recommended microarray analysis to replace karyotype for patients with a fetus with one or more major structural abnormalities identified on ultrasonographic examination and who are undergoing invasive prenatal diagnosis (American College of and Gynecologists Committee on 2013). The above-mentioned online databases facilitate the classification, interpretation, and reporting of CNVs in clinical cytogenetics laboratories. A retrospective analysis of 10-year data indicated that pathogenic CNVs accounted for 15% and 55% of cytogenomic abnormalities detected by CMA in current prenatal and pediatric practice (Chai et al. 2019). Cytogenomic mapping using Genome Browser, searching for recurrent CNVs in patients using DGV and DECIPHER, and looking for candidate genes and genotype–phenotype correlations using ClinGen and OMIM have been a standardized routine in analyzing and reporting pathogenic CNVs (Hao et al. 2022; Xie et al. 2022).

3.3 Web Resources Related to Clinical Cytogenetics 3.3.1 ChromoSomics Databases The freely available ChromoSomics databases (https://cs-tl.de/DB.html in English) provide information on small supernumerary marker and ring chromosomes (sSMC/sSRC), constitutional chromosomal uniparental disomy (UPD), chromosomal heteromorphisms, and constitutional chromosomal breakpoints.

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3.3.1.1 sSMC/sSRC Database sSMC or sSRC are structurally abnormal additional chromosomes detectable by banding cytogenetics, but their chromosomal origin and gene content remain uncharacterized due to their small size (Liehr et al. 2004). Further molecular characterization of an sSMC using FISH and/ or CMA is required for interpreting its clinical significance (Reddy et al. 2013). This molecular cytogenetic approaches on a large prenatal case series defined the chromosomal distribution and occurrence of de novo sSMCs (Malvestiti et al. 2014). The database on small supernumerary marker chromosomes (https://cs-tl.de/DB/ CA/sSMC/0-Start.html) is with more than 7130 sSMC cases collected. The aims of the database are to collect all available sSMC case reports, define critical regions for partial trisomy or tetrasomy due to the presence of sSMC, and provide information for patients and clinicians. The online sSMC database has chromosome-specific pages with cases classified by four categories: cases without clinical findings, cases with clinical findings, cases with unclear clinical correlation, and cases with neocentromeres. At the beginning of each chromosome-specific page, there are schematic drawings describing the presently known dosage sensitive centromerenear regions. For all sSMC cases, detailed cytogenetic information, clinical symptoms, and related references are provided. 3.3.1.2 Constitutional Chromosomal Uniparental Disomy (UPD) Database The database on cases with uniparental disomy (https://cs-tl.de/DB/CA/UPD/0-Start.html) includes > 5150 constitutional chromosomal UPD cases. It is similarly organized and has comparable goals as the sSMC subpage mentioned before. As UPD is present in 3–5% of de novo sSMC cases, this page was initially set up. It lists all constitutional UPD cases of maternal, paternal, and not clarified origin from the literature. Also, a short subpage on imprinting disorders has been recently added.

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3.3.1.3 Database on Chromosomal Heteromorphisms/ Cytogenetically Visible Copy Number Variants As 70% of sSMC cases have no clinical impact, the page on cases with heteromorphisms (https:// cs-tl.de/DB/CA/HCM/0-Start.html) was established in the ChromoSomics databases in 2017. Here, ~250 different heteromorphism variants of heterochromatin, and ~250 gene-containing regions of gains or losses of (molecular) cytogenetic visible euchromatin variants are listed. In contrast to the aforementioned two databases, here types of variants are listed—and not single cases. Thus, here two kinds of genomic regions are listed: (1) such regions which do contain no genes (heterochromatin) and thus, can create all kinds of huge, cytogenetically visible gains or losses known as chromosomal heteromorphisms; and (2) euchromatic genomic regions which only contain dosage insensitive genes. 3.3.1.4 Constitutional Chromosomal Breakpoint Database The database most recent part of the ChromoSomics databases is a collection of constitutional chromosomal breakpoints (https:// cs-tl.de/DB/CA/BPs/0-Start.html), characterized during last 25 years in the laboratory of Thomas Liehr, Jena, Germany. Here, the goal is to identify among the overwhelming majority of private rearrangements yet overlooked recurrent chromosomal inversions and translocations.

3.3.2 Atlas of Genetics and Cytogenetics in Oncology and Hematology The correlation of somatic clonal chromosomal abnormalities with different types of human malignancies has been widely recognized. To catalog recurrent chromosomal abnormalities in cancer in a systematic and concise way, the Atlas of Genetics and Cytogenetics in Oncology and Hematology has been created as a peerreviewed online database through collaborative

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efforts of cytogeneticists, molecular biologists, and clinicians in oncology, hematology, and pathology (Huret et al. 2013). This Web-based resource maintains sections of chromosomes, diseases, genes, and education. The section of chromosomes provides chromosome-specific search to involving recurrent chromosomal rearrangements. The section of diseases provides links to hematologic neoplasms, solid tumors, cancer-prone diseases, and ‘case reports’ for rare cytogenetic anomalies in various malignancies. The section of genes provides links to gene reviews, gene fusions, and gene mutations. The education section contains ‘deep insights’ with review articles focusing on specific cancerrelated topics such as chromothripsis, centrosome, autophagy, and so forth. For example, a deep insight article on ring chromosomes by Dr. Davis Gisselsson summarized the mechanisms of ring formation and ring chromosome prevalence in human tumors. The information in this website helps cytogeneticists and oncologists to define cancer classification, disease prognosis, and treatment recommendation based on chromosomal findings. In addition, The Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (http://cgap.nci.nih.gov/ Chromosomes/Mitelman) is also a useful tool to search for recurrent chromosome aberrations in cancer and related literature.

3.4 Patient Advocate Organization (PAO) Developed Websites and Online Registry for Ring Chromosomes 3.4.1 PAO Developed Websites for Specific Ring Chromosomes The PAO for specific chromosomal disorders plays important roles in supporting, networking, and educating affected patients and their families and in organizing and participating in patient-centered management and clinical trials.

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Ring 14 International (R14I, http://www.ring14. org/) is a PAO founded in 2012 as a nonprofit organization to help affected patients and their caregivers and to promote and support scientific research projects. Recently, R14I managed an ad hoc task force to publish the first report on recommended guidelines for diagnosis and clinical management of Ring 14 syndrome (Rinaldi et al. 2017). According to these guidelines, children with neuro-psychological alterations and drugresistant epilepsy need to have CMA as the first diagnostic step, and all subjects for whom a 14q terminal deletion is identified should also have a standard karyotype to assess for the presence of a ring. Another PAO is the British Ring 20 research (https://ring20researchsupport.co.uk/). This organization presents real-life stories from patients and supports patient-led approaches to assess the role of ketogenic dietary therapy in reducing seizure frequency and preserve cognition for affected patients (Gordon et al. 2020; Watson et al. 2014). Other websites and social groups for ring chromosomes include ring chromosome 18 registry and research society (https://www.chromosome18.org/), ring chromosome 22 central (http://www.c22c.org/). A great review on ring chromosome associated PAOs can be found at UNIQUE (https://rarediseases. org/non-member-patient/unique-rare-chromosome-disorder-support-group/) including RCs 4, 6, 9, 14, 15, 18, and 22. However, also information on all other RCs can also be found on UNIQUE website—best to access via Orphanet (https://www.orpha.net) search function for each individual ring chromosome.

3.4.2 Online Registry for Human Ring Chromosomes To compile and curate cytogenomic and clinical findings of these rare disorders, a Web-based interactive ‘human ring chromosome registry’ using Microsoft access based relational database was developed and loaded online as a website (https://www.yybio.tech/HRC/home. asp) (Hu et al. 2018). This registry includes modules of administrative and submission,

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chromosome-specific cases, resources, and glossary. Two search formats, a smart search and an item-specific search, are also available on the registry home page and each module. The chromosome-specific case module includes demographic data of an assigned identification number, gender, age, and country, laboratory data of test specimens, analytic methods, and cytogenomic results, and clinical findings including physical observations, dysmorphic features, congenital anomalies, cancer risk, reproduction, and family history. The module of resources provides a list of references with links to the PubMed (https://pubmed.ncbi.nlm. nih.gov/) or WANGFANG DATA (https://g. wanfangdata.com.cn/index.html, for Chinese articles). The glossary module contains genetic and clinical terms used in genetic diagnostic and clinical description. The administrative and submission module is used for case registration and database management. To validate the modular relations and functions in this online ring chromosome registry, the ring chromosome cases in the Chinese population was collected as the initial testing data set. Of the 113 Chinese cases with a constitutional ring chromosome, 89 cases (77 autosomal and 12 sex chromosomal rings) were assessed in perinatal or pediatric clinics, 14 cases (13 autosomal and one sex chromosome rings) were diagnosed prenatally, and 10 cases (six autosomal and four sex chromosome rings) were noted in infertility or reproductive clinics. The most common findings were developmental delay (65%), dysmorphic facial features (52%), intellectual disability (41%), microcephaly (34%), and hypotonia (23%). These findings are considered as features of so-called ‘ring chromosome syndrome,’ but variable clinical manifestations are obvious. Chromosomal-specific syndromic phenotypes included Wolf–Hirschhorn syndrome in a ring chromosome 4, cri-du-chat syndrome in a ring chromosome 5, epilepsy in ring chromosomes 14 and 20, Turner syndrome in ring chromosome X, and infertility in ring chromosomes 13, 21, 22, and Y. The clinical indications for prenatal diagnosis of ring chromosome cases included ultrasound findings of intrauterine growth restriction,

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oligohydramnios, microcephaly, lissencephaly, anencephaly, ventricular septal defect, and increased nuchal fold as well as abnormal prenatal screening results for increased risk of Down syndrome. Termination of pregnancy had been an option for fetus with severe anomalies and defined ring chromosome abnormalities. Excluding cases with parental denial or no follow up tests, parental studies performed in 60 families with a proband

carrying a ring chromosome documented normal karyotypes for both parents. This result indicated that almost all ring chromosomes are de novo in origin. The clinical presentations from prenatal and postnatal cases with a ring chromosome and their family history could be helpful for genetic counseling. The common clinical findings, age, and gender distribution of these Chinese cases of ring chromosomes are shown in Fig. 3.1.

Fig. 3.1  Cytogenomic and clinical results of ring chromosome cases in the Chinese population. a The frequency of most common clinical features in Chinese patients of ring chromosomes. b The distribution of age, gender, and number of cases for each specific ring

chromosomes. Each dot represents one case (blue for male and orange for female). Prenatal cases are placed at age zero, while pediatric and adult cases are separated by a red dash line at age 20. Reproduced from an OA article by Hu et al. (2018) with license CC-BY 4.0

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Table 3.1  Criteria for ring chromosome case registration Patient demographic data Age, gender, ethnic background Laboratory analysis Chromosome analysis: Ring chromosome structure, ring variants and derivatives, dynamic mosaicism Fluorescence in situ hybridization (FISH) Chromosome microarray analysis (CMA) Genomic sequencing (GS) Gene function and other molecular analyses Epigenetic analysis Prenatal cases Indications for prenatal testing: Invasive procedures: CVS, amniocentesis, PUBS Prenatal clinics Sonographic abnormalities Fetal growth and structural anomalies Placental and cervical complications Pregnancy outcome Prenatal genetic counseling and informative decision Spontaneous abortion (20 w.o.g.) Mode and gestational weeks of delivery Fetal/neonatal Presence of organ/system abnormalities suspected or not suspected by fetal ultrasound ICU admission, indications and treatment Major or minor interventions during neonate Pediatric and adult cases Pediatric clinics Ring syndrome features: severe growth retardation, mild to moderate intellectual disability, minor congenital anomalies Syndromic presentations: abnormalities in organs and systems Reproduction outcome Cancer predisposition Patient follow-up and family history Genetic counseling Family history, life expectation, and causes of deceases Clinico-cytogenomic correlation Case registration with cytogenomic and clinical findings Summary of clinico-cytogenomic correlations for laboratory and clinical guidelines

CVS, chorionic villus sampling; ICU, intensive care unit; PUBS, percutaneous umbilical blood sampling; w.o.g., weeks of gestation

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This registry for human ring chromosomes is part of the effort of International Consortium for Human Ring Chromosomes (ICHRC) (Li et al. 2022). The criteria for case registration, as listed in Table 3.1, including comprehensive cytogenomic results, detailed clinical findings at different developmental stages, and defined clinic-cytogenomic correlations. The abovementioned Web resources for cytogenomic mapping of ring chromosomes will be presented. Additional bioinformatic and biostatistics tools for data mining and systematic evidence review will be introduced (Xu et al. 2014). Contributors to chapters of specific ring chromosome can lead a working group to submit cases of ring chromosomes meeting the criteria. This registry will also serve as a resource for interpretating ring chromosome results, promoting patientcenter education and networking, and supporting translational research projects.

3.5 Conclusions and Future Directions The Web resource for general clinical and diagnostic genetics, clinical cytogenetics, and specifically ring chromosome abnormalities are summarized in this chapter. It is by no means a complete list of all related online resources but the most reliable and relevant to routine use in current cytogenomic diagnosis and clinical management for human ring chromosomes. The rarity of ring chromosome cases demands organized efforts to compile and curate findings from ring chromosome cases for better diagnostic interpretation, phenotypic classification, and clinical management. The PAO-developed websites provide support and networking for affected patients and their families. The implemented human ring chromosome registry can play important roles in not only facilitating genetic diagnosis and clinical management by providing references for geneticists, counselors, patients, and their families, but also promoting and developing translational research through international collaboration.

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4

Advocate Activities and PatientCentred Approaches Marco Crimi and Allison Watson

Abstract

Keywords

Patient advocacy organisations (PAOs) are increasingly involved in biomedical science and pharmaceutical research and development because of scientific advances and growing attention to rare diseases. They have traditionally focused on advocating for their patient community and providing practical support to individuals and families as they cope with everyday life with their rare disease. PAOs can also make valuable contributions to research, such as formulating study designs, setting research priorities, stimulating biological sampling/data collection, and fund-raising. Here, we highlight the two most active PAOs for people living with ring chromosome (RC) disorders: Ring14 and Ring20 are driven by passion and a desire to improve quality of life and treatment outcomes for all affected children, their families, and caregivers.

Patient advocacy organisations (PAOs) · Research and treatment engagement · Rare diseases (RDs) · Ring chromosome (RC)

M. Crimi (*)  Ring14 International, Via Flavio Gioia 5, 42124 Reggio Emilia, Italy e-mail: [email protected] Kaleidos SCS, Scientific Office, Via Andrea Moretti, 24121 Bergamo, Italy A. Watson  Ring20 UK, 26 Headley Chase, Warley, Brentwood CM14 5BN, UK

4.1 Background Patient advocacy organisations (PAOs) are emerging to be an integral part of biomedical science and pharmaceutical research and development. Advances in scientific knowledge, such as genetic sequencing technologies, and the increasing general attention towards rare diseases (RD) have enabled PAOs to become even more involved in the field of research. Consequently, interactions between PAOs and scientists are evolving through various partnerships and collaborations. These relationships allow PAOs to promote science not just by increasing public recognition of RD or seed financing studies directly, but they can also take part in the conceptualisation and implementation of biomedical research, even research meant to create diagnostic tests and therapeutic products. PAOs strive to empower patients, giving them a platform to be heard by those in power. For the last 30 years, activist organizations have been increasingly involved in biomedical research, making major contributions to design and implementation of new diagnostic tools and

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_4

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therapeutic strategies for RD. There is no shortage of cases where patient advocacy has been influential. PAOs significantly impact the connections between researchers and those who are part of their studies. Researchers, clinicians, patients and families, and organisations have multiple ways of teaming up. Numerous examples into such undertakings already exist within the greater realms of healthcare organisations and civil life.

4.2 Patient Advocacy Organisations and Scientific Partnerships PAOs by definition have traditionally had a primary focus on advocating for their patient community and providing practical support for individuals and families to cope with their rare condition in everyday life, supplementing information available through stretched national health systems and social support services. Research into the causes and possible therapies of their diseases of interest has become an important vision for many patient groups, especially RD organisations. For these organisations, it is not always easy to interact with scientists engaged in the biomedicine field, especially academic researchers. It is even harder to establish relationships with biopharmaceutical companies (Stein et al. 2018). However, PAOs have been viewed as playing a crucial role in the development of research and therapies for rare genetic conditions since the 1980s. This has recently seen an increase in public–private partnerships between PAO and researchers, which has only grown with the rise of interest in such conditions during this century (Koay and Sharp 2013). Much of the work that scientists have undertaken has been to understand these diseases more thoroughly— describing them in greater detail and seeking to comprehend the various interpretations held by those who are advocating for patients. Other focus is dedicated to making collaborations between stakeholders more effective. Scientists and patient advocates have observed that genetic

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research can be a sensitive issue for individuals who are struggling with genetic conditions, as well as the organisations that assist them. During the 1990s, when biotechnology and the human genome project were rapidly developing and the Internet was gaining traction, many new PAOs were created. The latter concentrated on issues regarding strengthening’ patient autonomy and challenging expert advice; now citizens’ engagement in biomedical studies is resulting in an alteration from “empowerment” to “collaboration” discourses (Koay and Sharp 2013). Such advancements have been instrumental in fostering collaboration between PAOs, academics, and companies. Lastly, there is widespread recognition that PAOs can make a valuable contribution throughout all phases and components of research and not simply in patient recruitment, including formulating study design, setting research priorities, biological testing/data gathering, and finance procurement. Patient advocates from PAOs have to become experts in their own rare disease—through personal lived experience and sharing of experiences within their patient community; however, this real-world evidence may often be debunked as merely anecdotal. Advocates have to learn fast, typically coming from a non-medical or scientific background; they have to be able to understand and interpret complex medical information and be able to communicate as peers with esteemed professionals. Their drive to research the available information and findings on best practices in diagnosis, treatment, and care to corroborate or challenge as necessary aims to increase understanding from a practical perspective, as opposed to a theoretical basis adopted by many researchers, clinicians, and academics. This is not the fault of the latter, merely a symptom of rare diseases, where knowledge is scarce and competition to find the next breakthrough high. PAOs are not driven by egos or targets; they frequently volunteer their time out of passion and a desire to improve quality of life and outcomes for all. PAOs committed to furthering research have to deal with numerous tensions, such as pinpointing their focus on diagnostics or

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Fig. 4.1  The timeline of Ring14 International

therapeutics, basic or clinical/translational research, studying genetic or non-genetic sources of illness, plus striving to advance investigations whilst simultaneously maintaining fundamental services for their members (Nguyen et al. 2022a, b). PAOs balance these ever-increasing demands on tight budgets, with limited funding and resources. There are very few patient groups dedicated to supporting those affected by ring chromosome (RC) disorders. Here, we describe two PAOs active in this field.

coordinating independent national chapters around the world. With these expansions, countries such as France, UK, Spain, and the Netherlands have joined up under the umbrella of Ring14 (Fig. 4.1). In the future, it is expected that many more chapters will follow suit. The overarching goal is for Ring14 to become an advocacy hub for any person affected by a chromosomal rearrangement on the 14th chromosome—even if they are affected by one of the rarest or nameless syndromes out there.

4.2.1 Ring14

4.2.2 Ring20 Research and Support UK CIO

In 2002, a group of families with children suffering from the rare genetic disorder RC14 syndrome (OMIM#616606) established Ring14 Italy, a non-profit in Reggio Emilia (Azzali et al. 2015). This syndrome is caused by a circularisation (ring) of one chromosome 14 and the loss of chromosome distal ends and fusion of remaining portions (Vaisfeld et al. 2021). For the past decade, Ring14 Italy has been striving to improve the lives of those afflicted by this anomaly. In 2014, Ring14 USA combined forces with their Italian counterparts to form Ring14— an organisation dedicated to promoting and

Ring20 Research and Support UK CIO [Ring20] was established in 2014 to promote research, education, and continuous support to end undiagnosed and misdiagnosed RC20 epilepsy. RC20 syndrome is a very rare condition in which one of the two copies of chromosome 20 has formed a ring rather than the typical linear chromosome structure. In the majority of cases, where only a percentage of chromosome 20s has formed a ring (mosaicism), there is no apparent loss of DNA. The consequence of the ring formation is difficult to control epilepsy, typically with onset in

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early childhood and associated with intellectual disability and behaviour problems which may be progressive. It is believed to be underdiagnosed or misdiagnosed in some people with epilepsy. The ring formation is deemed to be a symptom and not the cause of the epilepsy and other comorbidities, but more research is needed. Ring20’s mission is to provide support for individuals, families, and healthcare professionals who are affected by, or who come into contact with RC20 syndrome or r(20)—a rare epilepsy syndrome. Ring20 are a UK-based charity operating from the UK with a global reach. As the only patient group in the world for RC20 syndrome, at the time of writing, Ring20 supports 60% of the 200 cases of RC20 syndrome reported in the medical literature across the globe (Fig. 4.2). A team of Ring20 Champions representing various countries including Spain, Portugal, Germany, France, Belgium, Italy, the Netherlands, and Japan help to connect non-native English-speaking families locally; we also have Champions for the USA/ Canada and Australia. Ring20’s remit includes supporting patients with small supernumerary

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marker chromosomes (sSMCs) on the chromosome 20. In 2020, recognising the power of collaboration, Ring20 founded an informal network of UK-based patient groups representing those impacted by rare and complex epilepsies—UK Rare Epilepsies Together [UKRET].

4.3 Biobanking and Data Collection The limited numbers and spread of people with RD can create numerous challenges for research. Patient groups collaborate in many ways to facilitate progress in this area including obtaining data, creating, and sustaining registries, and “building” biobanks (stores of biological specimens). There are plentiful registers and biobanks created by disease-specific organisations that are invaluable when it comes to gathering data/samples (Nguyen et al. 2022a, b). The contribution of PAOs in the collection of samples/data assists researchers and policymakers in setting and agreeing research objectives, whilst considering the appropriate research budget.

Fig. 4.2  Worldwide distribution of cases of RC20 syndrome reported in the medical literature

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Patient registries driven by PAOs are known to yield larger cohorts and increased genetic and clinical diversity. On the flip side, those initiatives might struggle with internal validity issues and not meet national or international standards. Logistical complications posed by accessing such data offer plenty of scope for PAO participation. Academics widely agree that joint researcher–PAO involvement can generate insight into natural history and inform study designs. Registries are a fundamental prerequisite to RD research; however, the cost of creating and maintaining such a resource (time, funding, and staff/volunteers) and the onus to abide by ethics and full data protection regulations is often too much for many PAOs to consider on their own. Initiatives promoted by individual PAOs may be open to be adopted by a wider community, presenting an area where umbrella organisations, such as EURORDIS, have an important part to play, being able to bring individual RD PAOs together and encourage advocacy for setting up national or international RD registries and alliances. A collaborative registry could tackle issues related to consistency, information platforms, and governance/consent protocols whilst applying FAIR (findability, accessibility, interoperability, and reusability) principles: (i) standardising language; (ii) promoting meta-analyses; (iii) leading to improved interoperability. Moreover, qualitative evidence and data harmonisation encourage sustainability, a key factor for developing a RD registry. It is also becoming increasingly important to consider the transferability of biological and digital resources (data sharing). As a result of “silo mentalities”, data sharing is complicated by commercial conflicts of interest and concerns about losing professional advantages gained from data ownership. It is important to note, however, that limitations in data sharing inhibit reciprocal operations between institutions. Whilst a PAO may support a single RD, collaborative efforts are crucial to maintaining RD data sharing at the heart of research.

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Ring14 effort Ring14 Italy signed an agreement with the Telethon Network of Genetic Biobanks (TNGB) in December 2009, and as a result, the TNGB’s Biobank now contains over 300 biological samples for international use (Mora et al. 2015). Furthermore, Ring14 manages a database of patient information to ascertain which symptoms are associated with these syndromes, aiding translational research, and listed in various international programmes. This database can be accessed through submitting a letter of intent/ scientific project. Ring20 effort There is currently no patient registry for RC20 syndrome; however, Ring20 are seeking partners to try to address this fundamental gap in basic research. In the absence of a patient registry and very limited medical publications comprising many single case studies and research studies based on a maximum of 25 patients, Ring20 are working towards on an international patient families survey with the support of Newcastle University to uncover the true patient experience of living with r(20). Using patient-reported data, in 2022, Ring20 plotted decline in diagnostic rates against the advances in genetic screening, contrasting with the rapid rise in genetic diagnoses of many rare diseases patients, as part of UNRAVEL—making genomic understanding of ring chromosomes possible, in collaboration with Illumina Inc. Phase II of UNRAVEL commenced in early 2023 with a pilot study by Illumina Inc. utilising samples from individuals with a confirmed diagnosis of RC20 syndrome, to see whether sequencing techniques can be used to “see” the genomic change(s) that cause the chromosome to make a ring formation (Fig. 4.3). In 2022, Ring20 received approval from the NIHR BioResource for the creation of a new rare disease research cohort for RC disorders to be established in the UK. It is anticipated

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Fig. 4.3  Employment of NGS techniques to detect genomic change(s) causing a ring chromosome formation

that patient recruitment will commence in 2023 including the collection of blood samples and phenotypic data for wider research use. Ring20 will collaborate with Unique – Rare Chromosome Disorder Group to broaden patient recruitment across multiple RCs.

4.4 Priorities and Grant Funding for Research Projects Throughout history, the relationship between scientists and advocacy leaders has been strained by conflicting research priorities. Researchers have voiced concerns that basic research may be devalued, whilst some advocacy groups might be too focused on finding a cure. On the other hand, closely working with researchers can give PAOs the power to alter the direction of scientific progress. From its role in counteracting marginalisation to aiding the success of clinical protocols, there are many

reasons why RD advocacy groups can align their “agenda” with that of researchers for a mutual benefit (Landy et al. 2012). It is possible for PAOs to improve their efficiency and standardisation in order to set and address research priorities effectively. Individual PAOs may also be limited in their ability to offer a wide range of research and support services if they are financially unsustainable. However, it is possible to strengthen patient empowerment and long-term goals through cross-collaboration, with appropriate government endorsements and an international collaborative infrastructure. Indeed, umbrella organisations can assist by pooling resources and training patient advocates at national levels, i.e. UNIAMO in Italy and Beacon in UK, and regional levels, such as EURORDIS, REN, and others (Mavris and Cam 2012). By integrating multinational and multidisciplinary teams, new diagnostic tools and therapeutic innovation are possible; those, combined

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with transnationally curated registry data, may maximise global research expenditures’ collective impact. In parallel with the proliferation of biomedical technologies, academia has turned to patient-centred practices, with PAOs becoming increasingly acknowledged as active participants with public patient involvement (PPI) or public patient engagement (PPE) often now a prerequisite for research study funding applications. More work needs to be done, however, to truly include “patients as equal partners” in research, recognising the added value that PAOs bring— the patient perspective on the impact of and living with the rare condition. There should not be an expectation that PAO representatives should always give their time, knowledge, and experience voluntarily. If contributions from PAOs are deemed a mandatory contribution to effective research, then patient representatives should be appropriately reimbursed for their time (not just their expenses), and PAO costs should be included within research budgets as for all other collaborators. In this way, we help to ensure the sustainability of PAOs—an equally vital research resource. Some philanthropic models have been adopted by PAOs in the field of biomedical venture. Venture philanthropy offers small funds to underfunded stages of therapeutic development, incentivizes research, and reduces the risks inherent in new therapy development when industry funding is lacking. Private funding can promote siloed research and limit research capacity and collaboration, but it may also encounter long-term sustainability and efficiency challenges. Issues with the allocation of research funds by PAOs may include a lack of informed grants assignment, decisions based on “lay expertise”, and inadequate dialogue between scientists and patient representatives on expectations. Together, such elements can complicate the evaluation of research investments for their clinical benefit to individuals with RD. This can also reveal governance weaknesses in PAOs that would require accountability from beneficiaries who gain access to PAO grants or ad hoc funding. By making available specific RD research funding, public initiatives can be

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assured of financial support without compromising the rigour of grant submission reviews. In order to inform future public funding decisions, further research into methods of assessing RD treatment affordability and sustainability is required. Ring14 effort More than 50 K Euros of funding have been allocated to specific programmes for a centralised biological resource of r(14) samples and the development of a clinical data repository. In addition, Ring14 extends targeted and urgent calls for research, ensuring funding for research into its objectives and stimulating research or research capacity in focused health and medical science fields. Scientific projects have been sustained and supported by Ring14 since its founding. Indeed, Ring14 has sponsored ambitious basic science projects, including the creation of r(14) cellular (iPS cells) and animal models (Kim et al. 2017), gene expression profiling of immortalised r(14) cells, as well as phenotypic and language development studies (Zampini and Zanchi 2014), those ones especially supported by Ring14 Italy, the Italian branch of Ring14 (Table 4.1). However, scientists funded by Ring14 faced that the genetic instability (RCs are prone to further breakage) made it difficult to create stable cellular models using cells with RCs, as the cells may not behave consistently over time. Additionally, the presence of a RC can affect the expression of genes located on that chromosome, further complicating the use of these cells for modelling disease or testing therapies. Ring20 effort A total of £77,000 of funding has been raised by member families to fund an International Natural History and Biomarker Study for RC20 syndrome, including the creation of a patient registry, hoped to commence 2023. This will be the first research grant offered by Ring20. Ring20 is a small PAO formed of circa 120 families worldwide; being a UK-based charity,

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Table 4.1  Scientific projects supported by Ring14 Principal investigator (host institution)

Topic

Years (notes)

Zampini L. (University of Bicocca in Milan, Italy)

Communicative skills assessment in Ring14 children

2007–2015 and 2016–2018 (funded by Ring14 Italy)

Spinner N. (Philadelphia Hospital, USA)

Gene expression analysis in Ring14 tissues and neurons

2012–2014 and 2015

Neri G. (Catholic University of Rome, Italy)

Genetic basis of the ring chromosome 14 syndrome

2004–2012 (funded by Ring14 Italy)

Herault Y. (University of Strasbourg, France)

Generation of a murine model of 2012–2014 Ring14 syndrome

Zuffardi O. (University of Pavia, Italy)

Genotype–phenotype correlation 2013–2015 (funded by in chr-14 syndromes Fondazione Telethon in Italy)

Garozzo D. (CNR Catania, Italy)

Serum N-glycome studies in Ring14 patients

2018-ongoing (pro-bono project)

Wynshaw-Boris A. (Cleveland CCM, USA)

Ring chromosome loss during iPSC reprogramming

2015

Vezzani A. (Mario Negri Institute in Milan, Italy)

Testing anti-convulsive drugs in 2017–2018 a Ring14 rat model

fund-raising primarily comes from within the UK only, so the opportunity to raise grant funding is significantly more limited than Ring14.

4.5 Study Recruitment and Facilitating Access to Therapies Disorders caused by RCs are untreatable so far. When medicine does not yet have the answers, practical support can be invaluable for families affected by rare genetic disorders. This support can take many forms, such as: educating families about the disorder and its symptoms, advocating for services, bringing families together through support groups or online communities, and raising awareness to help reduce stigma (Smith et al. 2021). By providing resources, advocacy, connections, and awareness, PAOs can help families navigate the challenges of daily life and build a stronger community of support. Generally speaking, the partnership between researchers and PAOs in the effort to find cures is tangible. PAO contributions can be vital to successful study recruitment, retention, and

therapeutic access since PAOs have direct and trusted connections with their patient communities. PAOs have become increasingly proactive in recruitment for studies, mobilising specialists, pharma, biotech, and industry representatives to stimulate research interest in their disease area. The financial, physical, emotional, mental, and psycho-social costs of caregiving have a profound impact on each aspect of family life, from adjustments to careers to psychological anguish. Many caregivers praise the intervention of PAOs for granting them access to necessary funds, equipment, and social aid. The remit of PAOs can vary from providing financial support, promoting educational programmes, organising support services, encouraging community cohesion, and furnishing disease-specific information to medical professionals. This support not only facilitates the introduction of new treatments but also enables optimised outcomes through patient education, even with digital resources (Cazzaniga et al. 2022), family advice, and communal events. Even with challenging situations for their PAO volunteers/staff members to manage these objectives remain attainable when dealing with neglected patient requirements due to the passion and drive behind PAO

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leaders. Participation of consumer and PAOs in formal regulatory and reimbursement processes is increasing. Cost–benefit analyses, subsidisation, and reimbursement may be informed by patient perception of therapeutic benefits with the inclusion of patient-reported outcome measures (PROMs) in study design, which may differ from clinical trial endpoints measured and may be equally valid in measurements for success. In conventional value assessments, they have not yet been considered. The role PAOs may play in facilitating therapeutic access remains a matter of active academic debate. Besides their meaningful contributions to approval and reimbursement processes, PAOs can advise pharmaceutical companies on managed access programmes, enabling treatment prior to local approval, subsidising treatment, and advocating on behalf of patients. In the future studies, PAO perceptions of their lobbying roles in drug marketing and equitable patient access should be clarified and explored. The Ring14 International facilitating access to therapies for patients with RC14 syndrome is challenging, as there are currently no approved therapies for them. However, there are several strategies that Ring14 employs to facilitate access to possible therapies:

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• Clinicians and healthcare providers who treat patients with RC14 syndrome can be a valuable resource for identifying potential study participants and referring them to the study. Ring20 efforts are yet to be involved in a research study hence why Ring20 are leading the way in promoting opportunities for and seed funding collaborative research, to stimulate new interest in r(20). There has never been a clinical trial for RC20, and given the size of the known cohort, it is postulated unlikely that any clinical trials would be conducted in RC20 syndrome alone. However, Ring20 are continuously advocating for more innovative clinical trial designs to be employed such as basket trials, observational trials, or N-1 trials, much more suited to the inclusion of participants from an ultra-rare community. Ring20 actively attend conferences and events to raise awareness and network to seek opportunities for collaboration and support to stimulate interest in researching RC20 and RCs generally.

4.6 Challenges for PAOs Engaged in Science Resources and funding

• Encouraging and supporting the development of clinical trials for potential therapies for RC14 syndrome can provide affected individuals with access to new treatments. • Expanded access programmes, also known as compassionate use programmes, can provide access to investigational therapies for individuals with serious or life-threatening conditions who are unable to participate in clinical trials. • Ring14 advocates for increased funding and research for RC14 syndrome, as well as for policies that facilitate access to therapies for rare genetic disorders affecting the chromosome 14 (Rinaldi et al. 2017). • Researchers can leverage existing genetic testing databases to identify individuals with RC14 syndrome and contact them about potential participation in the study.

The invaluable contribution of patient representatives is often on a voluntary basis, or at best through offers of reimbursement of travel expenses alone. This model devalues the recognition of the skills, lived experience, and unique knowledge that patient representatives bring as equal partners in research as experts in their own disease and despite many patient advocates having transferable professional skills. This is compounded by the fact that health budgets traditionally focused on addressing diseases, rather than involving people in tackling their determinants, treatments, and management. PAO funding is often largely attributable to donations and fund-raising from the patients themselves, i.e. from within very small communities; such personal contributions to improve one’s own health outcomes only add to the burden of living with

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a rare disease. As a result, PAOs sometimes find themselves struggling to reach their full potential due to a lack of resources, people, and funds, both at the international and local level. Without explicitly earmarked funds devoted towards systematic involvement of individual patients and patient groups, it is going to be tricky for them to overcome these barriers. RD PAOs would benefit from adopting models of collaborative research and support, e.g. across all/many RCs, where funding and resources can be shared to the wider benefit of the many not the few. Such collaborative models may be more attractive to grant funders, researchers, and pharma/industry, but there is a fear amongst some PAOs that such an approach would dilute benefit to their patient community creating an environment for unhealthy competition. Legislative gaps A clear gap in legislation regarding meaningful patient involvement and systematic collaboration goes beyond individual clinical or medical issues, making it difficult for PAOs to effectively operate. Despite various legislative initiatives in place at a national level across Europe (and beyond), there has yet to be a comprehensive overview of the current situation. Involvement of patients with health policy and decisionmaking has been gradually increasing, yet this area is still rather fragmented with plenty of work needed before patients are able to enjoy complete autonomy. This includes ensuring references to patients in legislation that regulates health and research budgets and priorities, as well as transparency and rights to assure a uniform access to health facilities and services. Credibility PAOs often struggle to gain formal recognition as trusted stakeholders and reliable partners in both health and non-health policy debates. Patients’ perspectives are sometimes disregarded as being too closely associated with the views of the pharmaceutical industry or lacking broader insight beyond their illness/ailment (Gadd

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2012). EMA acknowledges that professionalising may put representativeness in danger. It is thought that when organisations focus more on influencing than building membership, they can become detached from those they represent. PAOs must seek ways to benefit from greater professionalism without jeopardising their primordial role of representation. With decreased resources due to budget cuts, PAOs rely on private funding. Although some turn down any form of support by industries, many acquire financial aid either for projects or for operational activities coming from such sources—something which could lead to a decrease in public confidence and impact negatively their credibility (House et al. 2019). This is partially owing to external entities not fully understanding how PAOs collaborate with industry counterparts whilst preserving autonomy and integrity free from conflicts of interest. As PAOs have become more professionalised, awareness about transparency and ethical practices has grown within these communities, although additional measures are being taken to reduce apparent contradictions: Many have implemented protocols for transparency and ethics along with engagement rules.

4.7 Conclusions and Key Message PAOs are evolving the “traditional” divide between scientists and patients, resulting in an ever-shifting interpretation of their role in scientific study. Consequently, PAOs must remain cognisant of their foundational ideals: representing, mobilising, and empowering patients as well as defending their rights. To further illustrate their significance to external stakeholders, PAOs need to consistently remind others on what they do, why and how they partner with corporate entities, and the additional value PAOs bring. Drawing upon advances in communication technology, research and development can empower PAOs with enhanced connections, attendance, and endurance in a fluctuating landscape. Indeed, any stakeholder has a noteworthy

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responsibility in this process. Revolutionising new and innovative ways of co-creating science has a unique social relevance when conducted in a patient-centred or patient-led manner. As a result of contextual background, community development, and empowerment of patients, they can provide more qualitative empirical data to enrich the political and practical narrative within the health research area. PAOs are best represented in research consortia where they provide added value through identifying patient priorities, bringing the patient perspective on the reality of living with the condition and the impact of different treatments (real-world evidence obtained through lived experience), and using their extensive networks to ensure patient involvement at all levels. Patients should be at the centre of research for their condition; patient-centred outcomes being equally valid as clinical outcomes in determining measures for success. To quote EURORDIS “nothing about us, without us”. Including PAOs as equal partners in research is the gold standard to which we should all aspire, for improved outcomes for all.

References Azzali S, DeWoody Y, Rinaldi B, Crimi M (2015) Ring14 international: Development of a nationalbased patient association towards a “global” network initiative to fight a chromosomal disorder. J Genet Disor Genet Rep 4:2. https://doi. org/10.4172/2327-5790.1000124 Cazzaniga A, Plebani M, Crimi M (2022) Genome access and other web-based IT solutions: Genetic counseling in the digital era. Front Public Health 10:1035316. https://doi.org/10.3389/fpubh.2022.1035316 Gadd C (2012) European Medicines Agency, Professionalisation and representativeness among civil society representatives. Available at: http:// www.ema.europa.eu/docs/en_GB/document_library/ Presentation/2012/05/WC500127916.pdf House T, O’Donnell K, Saich R, Di Pietro F, Broekgaarden R, Muir A, Schaller T (2019) The role of patient advocacy organizations in shaping medical research: The Pompe model. Ann Transl Med 7(13):293. https://doi.org/10.21037/atm.2019.04.28 Kim T, Plona K, Wynshaw-Boris A (2017) A novel system for correcting large-scale chromosomal

53 aberrations: Ring chromosome correction via reprogramming into induced pluripotent stem cell (iPSC). Chromosoma 126(4):457–463. https://doi. org/10.1007/s00412-016-0621-6 Koay PP, Sharp RR (2013) The role of patient advocacy organizations in shaping genomic science. Annu Rev Genomics Hum Genet 14:579–595. https://doi. org/10.1146/annurev-genom-091212-153525 Landy DC, Brinich MA, Colten ME, Horn EJ, Terry SF, Sharp RR (2012) How disease advocacy organizations participate in clinical research: A survey of genetic organizations. Genet Med 14(2):223–228. https://doi.org/10.1038/gim.0b013e3182310ba0 Mavris M, Le Cam Y (2012) Involvement of patient organisations in research and development of orphan drugs for rare diseases in Europe. Mol Syndromol 3(5):237–243. https://doi.org/10.1159/000342758 Mora M, Angelini C, Bignami F, Bodin AM, Crimi M, Di Donato JH, Felice A, Jaeger C, Karcagi V, LeCam Y, Lynn S, Meznaric M, Moggio M, Monaco L, Politano L, de la Paz MP, Saker S, Schneiderat P, Ensini M, Garavaglia B, Gurwitz D, Johnson D, Muntoni F, Puymirat J, Reza M, Voit T, Baldo C, Bricarelli FD, Goldwurm S, Merla G, Pegoraro E, Renieri A, Zatloukal K, Filocamo M, Lochmüller H (2015) The EuroBioBank Network: 10 years of hands-on experience of collaborative, transnational biobanking for rare diseases. Eur J Hum Genet 23(9):1116–1123. https://doi.org/10.1038/ ejhg.2014.272 Nguyen CQ, Alba-Concepcion K, Palmer EE, Scully JL, Millis N, Farrar MA (2022a) The involvement of rare disease patient organisations in therapeutic innovation across rare paediatric neurological conditions: A narrative review. Orphanet J Rare Dis 17(1):167. https://doi.org/10.1186/s13023-022-02317-6 Nguyen CQ, Kariyawasam D, Alba-Concepcion K, Grattan S, Hetherington K, Wakefield CE, Woolfenden S, Dale RC, Palmer EE, Farrar MA (2022b) Advocacy groups are the connectors’: Experiences and contributions of rare disease patient organization leaders in advanced neurotherapeutics. Health Expect 25(6):3175–3191. https://doi. org/10.1111/hex.13625 Rinaldi B, Vaisfeld A, Amarri S, Baldo C, Gobbi G, Magini P, Melli E, Neri G, Novara F, Pippucci T, Rizzi R, Soresina A, Zampini L, Zuffardi O, Crimi M (2017) Guideline recommendations for diagnosis and clinical management of Ring14 syndrome-first report of an ad hoc task force. Orphanet J Rare Dis 12(1):69. https://doi.org/10.1186/s13023-017-0606-4 Smith J, Damm K, Hover G, Chien J (2021) Lessons from an experiential approach to patient community engagement in rare disease. Clin Ther 43(2):421–429. https://doi.org/10.1016/j.clinthera.2020.12.002 Stein S, Bogard E, Boice N, Fernandez V, Field T, Gilstrap A, Kahn SR, Larkindale J, Mathieson T (2018) Principles for interactions with

54 biopharmaceutical companies: The development of guidelines for patient advocacy organizations in the field of rare diseases. Orphanet J Rare Dis 13(1):18. https://doi.org/10.1186/s13023-018-0761-2 Vaisfeld A, Spartano S, Gobbi G, Vezzani A, Neri G (2021) Chromosome 14 deletions, rings, and epilepsy genes: A riddle wrapped in a mystery inside

M. Crimi and A. Watson an enigma. Epilepsia 62(1):25–40. https://doi. org/10.1111/epi.16754 Zampini L, Zanchi P, D’Odorica L (2014) Developing with ring 14 syndrome: A survey in different countries. Clin Linguist Phon 28(11):844–856. https://doi. org/10.3109/02699206.2014.911963

Part II

Constitutional Ring Chromosomes

5

Ring Chromosome 1 Sainan Wei and Sheila Saliganan

Abstract

Keywords

Human chromosome 1 is the largest chromosome and comprises ~249 million base pairs (Mb). Constitutional ring chromosome 1 (RC1) is an ultra-rare disorder with less than ten cases described in the scientific literature since the 1960s, when conventional karyotyping was incorporated into clinical practice. This chapter summarizes the clinical and cytogenetic data of eight published cases with RC1. The major phenotype includes severe growth retardation, congenital microcephaly, and global developmental delay. Dysmorphic features are generally non-specific, and congenital anomalies are highly variable. There is a potential predisposition to malignancy. Comprehensive cytogenomic analysis should be performed to define the dynamic mosaicism and genomic structure for RC1. Genetic counseling and symptomrelated medical management need to be provided to patients and families with RC1.

Ring chromosome 1 (RC1) · Ring syndrome · Malignancy risk · Genetic counseling

S. Wei (*)  Department of Pathology and Laboratory Medicine, University of Kentucky College of Medicine, Kentucky, USA e-mail: [email protected] S. Saliganan  Ambry Genetics Corporation, Aliso Viejo, California, USA

5.1 Introduction Human chromosome 1 is the largest chromosome in the human genome. It spans about 249 million base pairs (Mb) and represents approximately 8% of the total human DNA; it is six times longer than the smallest chromosome 21 and one of the most gene-rich chromosomes (Gregory et al. 2006). More than 350 human diseases including both congenital and somatic conditions are associated with changes in the sequences and/or chromosome segments of chromosome 1 (Gajecka et  al. 2007; Shah et al. 2017). Trisomy 1 is extremely rarely found in human embryos, and none in term pregnancy (Banzai et al. 2004), which is at least partly due to its large size. However, genomic disorders resulting from chromosomal structural aberrations such as 1p36 microdeletions (OMIM#607872, Gajecka et  al. 2007) and 1q43q44 microdeletions (OMIM#612337, Depienne et al. 2017) that are related to human chromosome 1 are frequently reported. Somatic structural aberrations involving loss on the short arm (p) or gain on the long arm (q) of chromosome 1 have been reported in neuroblastoma,

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_5

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Wilms tumor, Burkitt lymphoma, and multiple myeloma (Ambros et al. 2001; Roug et al. 2014; Shah et al. 2017; Tamimi et al. 2007). Thus, human chromosome 1 is medically important. Human ring chromosomes (RCs) are circular DNA molecules and a specific type of chromosome structural aberrations which occur rarely in the human genome. About 47% of cases of human RC formation arise from acrocentric chromosomes (Pristyazhnyuk and Menzorov 2018). Constitutional human RC1 is one of the least frequently reported RC with a relative frequency of less than 1% compared to other types of RC. Structurally there are two types of RCs: One is complete RC in which there is no loss of genetic material formed by telomere-to-telomere fusion, and the other is incomplete RC with distal or interstitial deletion and/or duplication by one or multiple fusion events (Li et al. 2022). In addition, RC can be presented as a small supernumerary marker chromosome (sSMC) or small supernumerary ring chromosome (sSRC). A RC1 replacing one of the normal homologs is designated as a karyotype of 46, XX/XY, (r) (1) (Yip 2015). A sSRC1 is designated in the karyotype as 47, XX/XY,  +  r(1) (McGowanJordan et al. 2020). 116 sSRC1 cases have been reported in prenatal and postnatal cases (Liehr 2023). This chapter will focus on cytogenetic and clinical findings from eight cases of constitutional RC1 (excluding sSMC/sSRC) from PubMed and Google Scholar and briefly discuss the possibility of sSRC1 as residues of RC1 rescue.

5.2 Demographic Data Seven females and one male have been reported with a constitutional RC1 and are designated herein as RC1-1 (Gordon and Cooke 1964; Cooke and Gordon 1965), RC1-2 (Wolf et al. 1967), RC1-3 (Bobrow et  al. 1973), RC1-4 (Kjessler et al. 1978), RC1-5 (Gardner et al. 1984), RC1-6 (Hu et al. 2018; Jiang 1994), RC1-7 (Cutenese et  al. 2000), and RC1-8 (Saliganan et al. 2016). The case of RC1 by Maltby and Suvarna (2007) lacked sufficient

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details for inclusion; however, this meeting abstract provided long-term follow-up data on one case that is presumed to be RC1-1, although this could not be definitively confirmed (personal communication). RC1-6 was Chinese, and RC1-8 was mixed Northern European and Caucasian. Ethnicity was not reported for the remaining six cases. Reported cases ranged in age from birth to 12.5 years old at the time of initial publication. Table 5.1 provides a summary of key laboratory and clinical features for these eight cases.

5.3 Laboratory Results Conventional karyotyping with solid stain in the early 1960s (RC1-1) originally identified the first clinical case of RC1. Since that time, laboratory assays and standards of practice have evolved. Thus, cytogenetic results are reported with varying levels of detail for these eight cases with RC1. Laboratory analyses were performed using solid stain and/or G-banding karyotype in all cases, as well as fluorescence in situ hybridization (FISH) (RC1-7, RC1-8) and chromosomal microarray analysis (CMA) (RC1-8) in a subset of cases. All cases had testing performed on peripheral blood lymphocytes, and four cases had testing performed in more than one specimen type including skin fibroblasts (RC1-3, RC1-4), umbilical cord fibroblasts (RC1-7), bone marrow (RC1-3), and amniocytes (RC1-8). Karyotype showed RC1 in the majority of cells in all cases. Six cases (RC1-1, RC1-3, RC1-4, RC1-5, RC1-7, RC1-8) had various combinations of derivative karyotypes—including tetraploidy (most likely cultural artifacts), monosomy chromosome 1 by loss of RC1, derivative dicentric chromosome, two monocentric RC1, and/or normal karyotype—in a minority of cell lines consistent with dynamic mosaicism due to chromosomal instability. In RC1-7, a complex rearrangement was observed in 90% of umbilical cord fibroblasts suggestive of clonal tissue artifact. In RC1-3, cultured skin fibroblasts showed the same karyotype as lymphocytes in only approximately 25% of cells, which was presumably due to unsatisfactory

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Table 5.1  Summary of laboratory and clinical details in eight published RC1 cases Case ID

RC1-1

RC1-2

RC1-3

References

Gordon and Cooke (1964)

Wolf et al. (1967)

Bobrow Kjessler Gardner Jiang (1994), Cutenese et al. (2000) et al. (1973) et al. (1978) et al. (1984) Hu et al. (2018)

RC1-4

RC1-5

RC1-6

RC1-7

RC1-8 Saliganan et al. (2016)

Age at initial report

5 years

35 months

9 years

10 months

27 months

12.5 years

2 years

36 months

Sex

F

F

F

F

F

F

F

M

Genomic imbalance

+

N.R.

–

–

–

N.R.

+

+

Dynamic mosaicism

+

–

+

+

+

N.R.

+

+

Parental testing

N.R.

N.R.

de novo

de novo

de novo

N.R.

N.R.

de novo

Delayed development

+

+

+

+

+

+

+

+

Cheerful personality

+

+

+

–

–

+

–

–

Congenital microcephaly +

+

+

+

+

N.R.

+

+

Growth retardation

+

+

+

+

+

+

+

+

Congenital anomalies

–

–

+

+

+

+

+

+

Malignancy/tumor

+ (NHL, leiomyoma)a

N.R.

+ (AML)

N.R.

N.R.

N.R.

N.R.

+ (brain tumor)

Abbreviations + present, – absent, N.R. not reported, AML acute myeloid leukemia, NHL non-Hodgkin's lymphoma a Additional details in Maltby and Suvarna (2007), which is presumed to be the same patient as originally published by Gordon and Cooke (1964) [personal communication]

and prolonged culture. Bobrow et al. (1973) also demonstrated the finding of chromatin bridge with or without colchicine in lymphocytes, thereby ruling out possible artifact of RC1 in case RC1-3. Testing in bone marrow in RC1-3 initially showed RC1 in majority of cells; repeat testing in bone marrow revealed the RC1 in normal appearing cells, but leukemic appearing cells showed loss of the ring structure and presence of a derivative chromosome 1 and additional marker chromosome. RC1-2 was the only case to show the RC1 in 100% of cells with a stable 46-count karyotype using classical/solid stain from cultured peripheral blood. Limited detail was provided for RC1-6. Three cases had documented evidence of deletion of chromosome material. In RC1-1, arm length measurements showed that approximately 10% of the normal chromosome length was deleted. In RC1-7, telomeric FISH showed apparent deletion of the terminal bands of the long and short arms. RC1-8 was the only case to be characterized by chromosomal microarray analysis (CMA), which showed a 6 Mb deletion at 1q43q44, although the p-arm could not be adequately assessed due to paucity of

probes within the region. Three cases had RC1 of apparently normal size and/or in which no loss or gain of chromosomal material could be detected (RC1-3, RC1-4, RC1-5). Data on potentially deleted material were not assessed or not available in RC1-2 or RC1-6. Parental chromosome testing when performed showed normal chromosomes consistent with de novo formation (RC1-3, RC1-4, RC1-5, RC1-8).

5.4 Prenatal Findings Prenatal history was available in four cases. RC1-8 was diagnosed prenatally via amniocentesis after the pregnancy was complicated by intrauterine growth retardation (IUGR) and suboptimal brain and cardiac anatomy identified on second trimester fetal ultrasound. RC1-3 was complicated by an episode of bleeding at 6 weeks’ gestation and RC1-4 by recurrent episodes of bleeding in the third and fourth months of pregnancy, uterine contractions at 28 weeks’ gestation, and premature birth at 35 weeks’ gestation. RC1-7 also showed IUGR and had two

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vessel cord identified on third trimester fetal ultrasound. In the remaining cases, prenatal history was not significant or not reported.

5.5 Pediatric and Adult Cases The general phenotype for cases with RC1 is characterized by severe growth retardation since birth, congenital microcephaly, and global developmental delay (Table 5.1; Figs. 5.1, 5.2 and 5.3). The developmental outcome is variable. Dysmorphic features are generally

S. Wei and S. Saliganan

non-specific. Major congenital anomalies have been reported in six cases, but these are highly variable with no penetrant features. The central nervous system was involved in all cases as indicated by global developmental delay. Three cases were reported to walk independently between 2 and 4 years of age (RC1-2, RC1-3, RC1-6), and at least two cases attained some verbal language ability (RC1-2, RC1-3), albeit delayed. Four cases were felt to function at the level of a 2, 9, 11, or 12 months old at a chronological age of approximately 2 years, indicating consistent but variable global delays

Fig. 5.1  Height/length measurements from reported RC1 cases compared to United States CDC growth charts (Kuczmarski et al. 2002; Center for Disease Control and Prevention 2023)

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Fig. 5.2  Weight measurements from reported RC1 cases compared to United States CDC growth charts Kuczmarski et al. 2002; Center for Disease Control and Prevention 2023)

(RC1-2, RC1-5, RC1-7, and RC1-8). RC1-4 had essentially a lack of developmental milestones at the time of report at 10 months of age, whereas for RC1-1, the developmental delays reportedly became more obvious with age. Four cases have been reported to have a cheerful or lively personality (RC1-1, RC1-2, RC1-3, and RC1-6). Seven cases had congenital microcephaly, although RC1-4 was later reported to have increased head circumference with tense anterior fontanelle and sunset phenomenon at two months of age. Head circumference was not reported in RC1-6. Two cases had

corpus callosum dysgenesis (RC1-7 and RC1-8). Additional neurological features each reported in a single case include tremulousness (RC12), primitive sulci and gyral pattern (RC1-8), and abnormal EEG with slow waves and diffuse encephalopathy (RC1-8). The musculoskeletal system was involved in all cases as indicated by growth retardation. Additional shared musculoskeletal features include clinodactyly (RC1-3, RC1-4, RC1-7), abnormal flexion creases (RC1-4, RC1-7, RC18), congenital hip dislocation (RC1-3, RC1-4), rocker bottom feet (RC1-3, RC1-7), and brisk

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Fig. 5.3  Head circumference measurements from reported RC1 cases compared to United States CDC growth charts (Kuczmarski et al. 2002; Center for Disease Control and Prevention 2023)

reflexes (RC1-2, RC1-3). Musculoskeletal features each reported in a single case include diminished muscle mass or thin extremities (RC1-2), diminished strength (RC1-2), delayed bone age (RC1-2), tapered fingers (RC1-3), talipes cavus (RC1-3), cerebral palsy (RC1-3), lower extremity hypertonia (RC1-3), generalized hypotonia (RC1-4), hypoactive reflexes (RC14), short extremities (RC1-4), short fingers (RC1-4), retroposition of the fourth toe (RC1-4), short sternum (RC1-4), split vertebral anomaly (RC1-4), median cleft palate (RC1-4), radial ray

defect (RC1-5), and possible lack of interosseous muscles in the fingers (RC1-8). Dysmorphic facial features have been reported in seven cases, but there is no common facial gestalt recognized. Shared dysmorphic facial features include small anterior fontanelle (RC1, RC1-4, RC1-8), prominent nasal root or high nasal bridge (RC1-3, RC1-7, RC1-8), micro- or retrognathia (RC1-4, RC1-5, RC1-7), large ears (RC1-2, RC1-4, RC1-8), upward slanting palpebral fissures (RC1-3, RC1-4, RC1-7), small palpebral fissures (RC1-4, RC1-7), low-set ears

5  Ring Chromosome 1

(RC1-2, RC1-7), sparse scalp hair (RC1-2, RC14), and thin lips (RC1-5, RC1-8). Dysmorphic facial features that have each been reported in a single case include elfin face (RC1-2), upturned corners of the mouth (RC1-2), small mouth (RC1-2), absent maxillary lateral incisors (RC12), bilateral preauricular sinuses (RC1-3), dysplastic ears (RC1-4), protruding occiput (RC1-4), small nose (RC1-4), anteverted nares (RC1-4), depressed nasal bridge (RC1-4), long philtrum (RC1-4), epicanthal folds (RC1-4), sloping forehead (RC1-5), prominent nose (RC1-5), simple ears (RC1-7), high broad forehead (RC1-7), sagittal ridging (RC1-8), high arched palate (RC1-8), lateral flaring of the eyebrows (RC1-8), and glabellar hemangioma (RC1-8). The cardiovascular system was involved in three cases. Two cases had atrial septal defect (RC1-7, RC1-8). Additional cardiovascular features each reported in a single case include right-sided cardiac hypertrophy (RC1-4), persistent left superior vena cava (RC1-7), tricuspid regurgitation (RC1-7), and patent ductus arteriosus (RC1-8). The genitourinary system was involved in three cases with no shared features. Genitourinary features each reported in a single case include ambiguous genitalia (RC1-6), small dysplastic kidneys (RC1-7), vesicoureteral reflux (RC1-7), renal cyst (RC1-7), cryptorchidism (RC1-8), and penile phimosis (RC1-8). The gastrointestinal system was involved in two cases with no shared features. Gastrointestinal features each reported in a single case include hepatosplenomegaly (RC1-4), elevated liver enzymes (RC1-4), feeding difficulties necessitating tube feeding (RC1-4), recurrent vomiting (RC1-4), inguinal hernia (RC1-8), and anteriorly placed anus (RC1-8). The endocrine system was involved in two cases with no shared features. Endocrine features each reported in a single case include abnormal growth hormone response (RC1-2) and congenital hypothyroidism (RC1-8). Other reported features include edema (RC1-4, RC18), history of metabolic acidosis during illness (RC1-2), transient neonatal thrombocytopenia (RC1-8), mild left hearing loss that resolved

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(RC1-8), sacral pit (RC1-8), and suprasternal sinus (RC1-3). Three cases were reported with malignancy or tumor (RC1-1, RC1-3, RC1-8). Two of these cases are known to be deceased at the ages of 10 years (RC1-3) and 41 years old (RC1-1). RC1-3 developed anemia shortly before 8 years old which progressed to pancytopenia, bone marrow hypoplasia, and acute myeloid leukemia resulting in death at age 10. Another patient (presumed to be RC1-1) died at age 41 due to cirrhotic liver disease following a history of gastric marginal zone B cell non-Hodgkin's lymphoma, in addition to leiomyoma, absent parathyroids, heart disease, esophageal varices, osteoporosis, and diabetes mellitus (Maltby and Suvarna 2007). RC1-8 developed a brain tumor at 23 months old, although the pathology had not been determined. Follow-up data are not available for the remaining cases. Family history was significant for Down syndrome, juvenile diabetes, and thyroid dysfunction in three cases (RC1-4, RC1-7, and RC1-8, respectively). Two cases were reported with negative family history (RC1-3, RC1-6). Family history was unknown or not reported in the remaining cases.

5.6 Conclusions and Recommendations Constitutional RC1 is exceedingly rare. There are only eight published cases spanning a period of more than five decades. There is no apparent ethnic preference as cases have been published in various regions around the world, but a possible sex preference cannot be excluded as most reported cases were biologically female (7:1 female-to-male ratio). The rare incidence of RC1 may be attributed to its large size and biological importance. Deletion and/or duplication of genetic fragments on chromosome 1 may contain more genes, which, in turn, may cause lethal effect. The higher rate of crossover in sister chromatid exchange (SCE) during mitosis, resulting in more mitotic instability and abnormal

64

segregation, may further jeopardize the survival of individuals with RC1 (Nikitina et al. 2021). A possible rescue mechanism for the severe mitotic disturbance associated with large RC has been theorized, which may result in loss of the RC with sSMC formation and/or uniparental disomy of the normal chromosomes (Li et al. 2022). A possible gender effect for survival, rescue mechanism, or a combination of these factors in such a small cohort cannot be determined. The natural history of RC1 is not completely elucidated due to the small number of cases reported. In addition, there is variability in these case reports in terms of the clinical history recorded and laboratory investigations performed, which limits direct comparison. While growth failure and developmental delay have been reported in all cases with RC1, there is high variability in other organ systems involved. Importantly, growth failure and developmental delay are also shared features with other ring chromosomes (Li et al. 2022). This shared phenotype is thought to constitute a “ring syndrome” attributed to mitotic instability and dynamic mosaicism of the ring structure itself; the syndrome may then be compounded by genomic imbalances resulting in more systemic involvement (Li et al. 2022), although it may not always be possible to make this distinction. Indeed, RC1-8 had overlapping features of both RC1 and 1q43q44 microdeletion syndrome, and while some features may be better explained by the deletion of a candidate gene (i.e., ZNF238 for corpus callosum dysgenesis), other overlapping features such as developmental delay or microcephaly are non-specific and seen in both disorders (Saliganan et al. 2016). There are limited long-term follow-up data available for cases with RC1, but at least one case indicates that survival into adulthood is possible, although several comorbidities were present (Maltby and Suvarna 2007). An important consideration is the report of various tumors and cancers in cases with RC1—a finding that has also been reported in cases with other RCs (Li et al. 2022). It has been suggested that a number of tumor suppressor

S. Wei and S. Saliganan

genes mapped to terminal chromosome 1p36, and deletions of this region have been reported in various malignancies (Bagchi and Mills 2008). In addition, recurrent somatic deletions of chromosome terminal 1p region have been associated with adverse progression in cases with multiple myeloma (Boyd et al. 2011). Loss or inactivation of this region might be a common initiating event for several malignancies or a critical event in tumorigenesis. Whether there is a predisposition to malignancy that may be attributed to ring instability, dynamic mosaicism, genomic imbalance (i.e., loss of tumor suppressor genes), both, or neither is not fully understood, and there are insufficient data to provide specific cancer risks at this time. Ultra-rare or orphan diseases present unique challenges for practitioners, families, and patients (Boulanger et al. 2020; Crowe et al. 2020; Dharssi et al. 2017; Kerr et al. 2023; McMullan et al. 2020; van Eeghen et al. 2022). There are no disease-specific treatment recommendations or evidence-based management guidelines for RC1, although guidelines for other RC may serve as a model in future (Rinaldi et al. 2017). In addition, patient registries can provide an opportunity to advance care and policy for rare or orphan diseases (Boulanger et al. 2020; Lacaze et al. 2017; Li et al. 2022; McMullan et al. 2020). Generally, clinical management for RC should include a comprehensive assessment, genetic counseling, and treatment of medically actionable symptoms (Hu et al. 2018). Likewise, there are currently no standardized laboratory guidelines for RC. Comprehensive cytogenetic testing is useful to distinguish the type of RC (complete or incomplete), determine the extent of deleted material, detect mosaicism and/or ring instability, and provide information about inheritance and recurrence risk (Hu et al. 2018). This may be accomplished utilizing a combination of conventional karyotyping, FISH, array comparative genomic hybridization (aCGH) or single-nucleotide polymorphism (SNP) microarray, or next-generation genomic testing such as whole genome sequencing (WGS) and/or optical genome mapping (OGM), which may provide better resolution

5  Ring Chromosome 1

(Cook et al. 2021; Hu et al. 2018; King et al. 2017; Mantere et al. 2021; Sahajpal et al. 2022). With better resolution of newer technologies, there is the potential to correlate the genomic results more accurately with clinical phenotypes including prognosis or systemic involvement depending on the extent of ring instability or deleted genes. Previously reported cases with RC1 have been sporadic, and recurrence risk is therefore low; however, negative parental testing cannot rule out the possibility of parental germline mosaicism, and parental transmission has been reported in other RCs (Li et al. 2022). In summary, there must be an individualized and holistic approach to care for patients and families with RC1, and a comprehensive approach to testing can facilitate a timely and accurate diagnosis, genetic counseling, and risk assessment. Acknowledgements  The authors of this chapter would like to acknowledge Daryl Saliganan, MBA, for creation of Figs. 5.1, 5.2 and 5.3.

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66 Gordon RR, Cooke P (1964) Ring-1 chromosome and microcephalic dwarfism. Lancet 2(7371):1212–1213. https://doi.org/10.1016/s0140-6736(64)91045-1 Gregory SG, Barlow KF, McLay KE, Kaul R, Swarbreck D, Dunham A, Scott CE, Howe KL, Woodfine K, Spencer CC, Jones MC, Gillson C, Searle S, Zhou Y, Kokocinski F, McDonald L, Evans R, Phillips K, Atkinson A, Cooper R, Jones C, Hall RE, Andrews TD, Lloyd C, Ainscough R, Almeida JP, Ambrose KD, Anderson F, Andrew RW, Ashwell RI, Aubin K, Babbage AK, Bagguley CL, Bailey J, Beasley H, Bethel G, Bird CP, Bray-Allen S, Brown JY, Brown AJ, Buckley D, Burton J, Bye J, Carder C, Chapman JC, Clark SY, Clarke G, Clee C, Cobley V, Collier RE, Corby N, Coville GJ, Davies J, Deadman R, Dunn M, Earthrowl M, Ellington AG, Errington H, Frankish A, Frankland J, French L, Garner P, Garnett J, Gay L, Ghori MR, Gibson R, Gilby LM, Gillett W, Glithero RJ, Grafham DV, Griffiths C, GriffithsJones S, Grocock R, Hammond S, Harrison ES, Hart E, Haugen E, Heath PD, Holmes S, Holt K, Howden PJ, Hunt AR, Hunt SE, Hunter G, Isherwood J, James R, Johnson C, Johnson D, Joy A, Kay M, Kershaw JK, Kibukawa M, Kimberley AM, King A, Knights AJ, Lad H, Laird G, Lawlor S, Leongamornlert DA, Lloyd DM, Loveland J, Lovell J, Lush MJ, Lyne R, Martin S, Mashreghi-Mohammadi M, Matthews L, Matthews NS, McLaren S, Milne S, Mistry S, Moore MJ, Nickerson T, O’Dell CN, Oliver K, Palmeiri A, Palmer SA, Parker A, Patel D, Pearce AV, Peck AI, Pelan S, Phelps K, Phillimore BJ, Plumb R, Rajan J, Raymond C, Rouse G, Saenphimmachak C, Sehra HK, Sheridan E, Shownkeen R, Sims S, Skuce CD, Smith M, Steward C, Subramanian S, Sycamore N, Tracey A, Tromans A, Van Helmond Z, Wall M, Wallis JM, White S, Whitehead SL, Wilkinson JE, Willey DL, Williams H, Wilming L, Wray PW, Wu Z, Coulson A, Vaudin M, Sulston JE, Durbin R, Hubbard T, Wooster R, Dunham I, Carter NP, McVean G, Ross MT, Harrow J, Olson MV, Beck S, Rogers J, Bentley DR, Banerjee R, Bryant SP, Burford DC, Burrill WD, Clegg SM, Dhami P, Dovey O, Faulkner LM, Gribble SM, Langford CF, Pandian RD, Porter KM, Prigmore E (2006) The DNA sequence and biological annotation of human chromosome 1. Nature 441(7091):315–321. https://doi. org/10.1038/nature04727 Hu Q, Chai H, Shu W, Li P (2018) Human ring chromosome registry for cases in the Chinese population: Re-emphasizing cytogenomic and clinical heterogeneity and reviewing diagnostic and treatment strategies. Mol Cytogenet 11(1):19. https://doi. org/10.1186/s13039-018-0367-3 Jiang Y (1994) A case with ring chromosome 1 syndrome. J Chinese Med Genet 11(2):71. n.a. Kerr AM, Bereitschaft C, Duty KM, Sisk BA (2023) Navigating care for rare diseases: Caregiver and patient advice for families and clinicians managing

S. Wei and S. Saliganan care for vascular malformations. Patient Educ Couns 107(1):107569. https://doi.org/10.1016/j. pec.2022.11.011 King DA, Sifrim A, Fitzgerald TW, Rahbari R, Hobson E, Homfray T, Mansour S, Mehta SG, Shehla M, Tomkins SE, Vasudevan PC, Hurles ME, Deciphering Developmental Disorders Study (2017) Detection of structural mosaicism from targeted and wholegenome sequencing data. Genome Res 27(10):1704– 1714. https://doi.org/10.1101/gr.212373.116 Kjessler B, Gustavson KH, Wigertz A (1978) Apparently non-deleted ring-1 chromosome and extreme growth failure in a mentally retarded girl. Clin Genet 14(1):8–15. https://doi.org/10.1111/j.1399-0004.1978. tb02054.x Kuczmarski RJ, Ogden CL, Guo SS, Grummer-Strawn LM, Flegal KM, Mei Z, Wei R, Curtin LR, Roche AF, Johnson CL (2002) 2000 CDC growth charts for the United States: Methods and development. Vital Health Stat 11(246):1–190. Lacaze P, Millis N, Fookes M, Zurynski Y, Jaffe A, Bellgard M, Winship I, McNeil J, Bittles AH (2017) Rare disease registries: A call to action. Intern Med J 47(9):1075–1079. https://doi.org/10.1111/imj.13528 Li P, Dupont B, Hu Q, Crimi M, Shen Y, Lebedev I, Liehr T (2022) The past, present, and future for constitutional ring chromosomes: A report of the international consortium for human ring chromosomes. HGG Adv 3(4):100139. https://doi.org/10.1016/j. xhgg.2022.100139 Liehr T (2023) Small supernumerary marker chromosomes. https://cs-tl.de/DB/CA/sSMC/0-Start.html. Accessed on 25 Apr 2023 Maltby E, Suvarna K (2007) Constitutional ring 1 chromosome: A case study of the genetic and pathological implications. In: Abstracts of the 6th European cytogenetics conference, Istanbul, Turkey, 7–10 July 2007 Mantere T, Neveling K, Pebrel-Richard C, Benoist M, van der Zande G, Kater-Baats E, Baatout I, van Beek R, Yammine T, Oorsprong M, Hsoumi F, OldeWeghuis D, Majdali W, Vermeulen S, Pauper M, Lebbar A, Stevens-Kroef M, Sanlaville D, Dupont JM, Smeets D, Hoischen A, Schluth-Bolard C, El Khattabi L (2021) Optical genome mapping enables constitutional chromosomal aberration detection. Am J Hum Genet 108(8):1409–1422. https://doi. org/10.1016/j.ajhg.2021.05.012 McGowan-Jordan J, Hastings RJ, Moore S (2020) ISCN 2020: An International System For Human Cytogenomic Nomenclature. S. Karger, Basel, Switzerland McMullan J, Crowe AL, Bailie C, Moore K, McMullan LS, Shamandi N, McAneney H, McKnight AJ (2020) Improvements needed to support people living and working with a rare disease in Northern Ireland: current rare disease support perceived as inadequate. Orphanet J Rare Dis 15(1):315. https://doi. org/10.1186/s13023-020-01559-6

5  Ring Chromosome 1 Nikitina TV, Kashevarova AA, Gridina MM, Lopatkina ME, Khabarova AA, Yakovleva YS, Menzorov AG, Minina YA, Pristyazhnyuk IE, Vasilyev SA, Fedotov DA, Serov OL, Lebedev IN (2021) Complex biology of constitutional ring chromosomes structure and (in)stability revealed by somatic cell reprogramming. Sci Rep 11(1):4325. https://doi.org/10.1038/ s41598-021-83399-3 Online Mendelian Inheritance in Man, OMIM®. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD. https:// omim.org/. Accessed 29 Jan 2023 Pristyazhnyuk IE, Menzorov AG (2018) Ring chromosomes: From formation to clinical potential. Protoplasma 255(2):439–449. https://doi. org/10.1007/s00709-017-1165-1 Rinaldi B, Vaisfeld A, Amarri S, Baldo C, Gobbi G, Magini P, Melli E, Neri G, Novara F, Pippucci T, Rizzi R, Soresina A, Zampini L, Zuffardi O, Crimi M (2017) Guideline recommendations for diagnosis and clinical management of Ring14 syndrome-first report of an ad hoc task force. Orphanet J Rare Dis 12(1):69. https://doi.org/10.1186/s13023-017-0606-4 Roug AS, Wendtland P, Bendix K, Kjeldsen E (2014) Supernumerary isochromosome 1, idic(1)(p12), leading to tetrasomy 1q in Burkitt lymphoma. Cytogenet Genome Res 142(1):7–13. https://doi. org/10.1159/000355985 Sahajpal NS, Mondal AK, Tvrdik T, Hauenstein J, Shi H, Deeb KK, Saxe D, Hastie AR, Chaubey A, Savage NM, Kota V, Kolhe R (2022) Clinical validation and diagnostic utility of optical genome mapping for enhanced cytogenomic analysis of hematological

67 neoplasms. J Mol Diagn 24(12):1279–1291. https:// doi.org/10.1016/j.jmoldx.2022.09.009 Saliganan S, Lee J, Wei S (2016) A patient with constitutional ring 1 chromosome characterized by SNP array CGH. Clin Case Rep 4(4):442–448. https://doi. org/10.1002/ccr3.522 Shah GL, Landau H, Londono D, Devlin SM, Kosuri S, Lesokhin AM, Lendvai N, Hassoun H, Chung DJ, Koehne G, Jhanwar SC, Landgren O, Levine R, Giralt SA (2017) Gain of chromosome 1q portends worse prognosis in multiple myeloma despite novel agent-based induction regimens and autologous transplantation. Leuk Lymphoma 58(8):1823–1831. https://doi.org/10.1080/10428194.2016.1260126 Tamimi Y, Ziebart K, Desaulniers N, Dietrich K, Grundy P (2007) Identification of a minimal region of loss on the short arm of chromosome 1 in Wilms tumor. Genes Chromosomes Cancer 46(4):327–335. https:// doi.org/10.1002/gcc.20413 van Eeghen AM, Bruining H, Wolf NI, Bergen AA, Houtkooper RH, van Haelst MM, van Karnebeek CD (2022) Personalized medicine for rare neurogenetic disorders: Can we make it happen? Cold Spring Harb Mol Case Stud 8(2):a006200. https://doi.org/10.1101/ mcs.a006200 Wolf CB, Peterson JA, LoGrippo GA, Weiss L (1967) Ring 1 chromosome and dwarfism–a possible syndrome. J Pediatr 71(5):719–722. https://doi. org/10.1016/s0022-3476(67)80211-7 Yip MY (2015) Autosomal ring chromosomes in human genetic disorders. Transl Pediatr 4(2):164–174. https://doi.org/10.3978/j.issn.2224-4336.2015.03.04

6

Ring Chromosome 2 Jaclyn B. Murry and Ying S. Zou

Abstract

Ring chromosome 2 (RC2) in the context of a karyotype with a modal number of 46 chromosomes is an infrequently documented chromosomal structural abnormality. However, fourteen unique RC2 cases are reviewed here as the sole aberration. Males and females are equally represented, with one prenatal-only ascertainment. Notably, one individual had a natural history of up to 34 years. No normal cells were identified in at least seven individuals who displayed the monocentric ring as an isolated event with variable ring structures observed in a subset of cells. At least eleven individuals were de novo for RC2. Recurring break points at cytobands 2p25 and 2q37 are noted in the reported cases. Excessive cell death and genetic imbalance resulting in growth deficiency might depend on the degree of aneuploidy or mosaicism. Overall, mitotic instability of the ring is observed. An

J. B. Murry (*) · Y. S. Zou  The Johns Hopkins Cytogenomics Laboratory, Department of Pathology, Division of Molecular Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA e-mail: [email protected] Y. S. Zou e-mail: [email protected]

associated ring syndrome phenotype may be appreciated, which appears to differ from the features observed in individuals affected by pure terminal copy number changes. Cytogenomic follow-up testing is needed to improve accurate genotype–phenotype correlations. Beyond these initial reports, there is a paucity of longitudinal studies.

Keywords

Ring chromosome 2 (RC2) · Dynamic mosaicism · Mitotic instability · Cytogenomic heterogeneity · Ring syndrome · Clinical heterogeneity

6.1 Brief Historical Review Seminal ring chromosome 2 (RC2) cases were reported in tandem in the late 1970s, corresponding with the introduction of G-banded metaphase analysis enabling definitive identification. The first report of RC2 was described in 1973 in an Italian publication utilizing only solid-staining karyotype (Garau et al. 1973). The first definitive description of RC2 with approximate break points was reported in 1978 in a single individual (Sutherland and Carter 1978). At that time, ring stability was attributed to size

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_6

69

70

(Kistenmacher and Punnett 1970). Cases of RCs were noted with irregular phenotypic expression (Zdansky et al. 1975). As a result, it was suggested that the instability of RC2 might contribute to cell division dysregulation with more significant influence than that conferred with the consequential losses that might coincide at both ends of the RC. A potential phenotypic overlap for additional patients affected with RC2 with apparently similar break points was beginning to emerge. However, whether the phenotype was due to the resultant loss of genomic material secondary to the ring formation or a consequence of subsequent ring behavior was uncertain. The term “ring syndrome” was introduced to explain how individuals bearing RCs shared associated phenotypes irrespective of the chromosome involved. Another possibility is that surviving aneuploid cells might contribute to the phenotype via instability at cell division leading to high cellular death rates and growth retardation. Under the assumption that little, if any, material was lost, telomere-to-telomere fusions were provided as a possible mechanism for ring formation in the absence of distal genomic loss. Other derivatives secondary to RC2 might also influence the chromosomal imbalance, evidenced by rod structures. The advent of fluorescence in situ hybridization (FISH) in the early 2000s enabled the refinement of gross karyotypic findings involving RC2 for three additional cases, which shared no specific common subtelomeric event. One RC2 case reported in 2001 bore a subtelomeric 2p deletion (Dee et al. 2001). Two other RC2 cases were reported in 2005: One lacked subtelomeric loss of either arm (Kosho et al. 2005), while the other harbored a subtelomeric 2q loss (Alkuraya et al. 2005). With the introduction of array comparative genomic hybridization (aCGH) and single-nucleotide polymorphism (SNP) array, also called chromosomal microarray analysis (CMA), cases affected with RC2 underwent multiple complementary cytogenetic approaches to clarify their break points and perturbed gene content. With increased access to emerging

J. B. Murry and Y. S. Zou

genomic technologies, there is a viable opportunity for follow-up for these rare cases to establish the genotype–phenotype spectrum for RC2 syndrome and inform genetic counseling.

6.2 Demographic Data Instances of RC2 are exceedingly rare, with an estimated relative frequency of 1.4% in human RCs, based on recent curation (Li et al. 2022). All fourteen reported cases of RC2 are reviewed here and are designated numerically based on the time of publication from the earliest to the most recent. Seven of the fourteen cases reviewed here are male, indicating no gender bias. Where available, nine out of fourteen cases are documented to be white, and two are described as Asian (see Table 6.1). This first case of RC2, defined as RC21, was identified in a male child who notably passed away due to cardiac failure at 10.5 months of age (Garau et al. 1973); the same individual is described in a subsequent 1979 publication and is referenced there as case 3 (Maraschio et al. 1979). RC2-2 was identified in 1978 in a severely affected white young girl with a presumed de novo RC2 in mosaic form (Sutherland and Carter 1978). RC2-3 was characterized in 1979 in a 15-month-old male who was an single child (Maraschio et al. 1979), where he was referenced as case 1. RC2-4 was a ten-month-old white female (Maraschio et al. 1979 referred to as case 2). RC2-5 (Vigfusson et al. 1980), an Asian female infant, had a de novo RC2 in mosaic; she was studied twice in her early life. Her mild affectation was attributed to the presence of the normal line and the likelihood that the mosaic RC2 arose in a post-zygotic event. In a 1.5-year-old white female, RC2-6 was first identified in 1981 as de novo (Cote et al. 1981). Subsequent complementary cytogenomic studies in adulthood (20 and 34 years of age) were reported in 2015 (Sarri et al. 2015) in this oldest documented adult RC2 patient. RC2-7 was published in 1982 (Jansen et al. 1982) in a four-year-old white female who underwent conventional

NA

NA

De novo (Presumed De novo De novo

+

+

+

+

+

+

+

+

Break points

Level of normal

Inheritance

IUGR

Postnatal growth retardation

Psychomotor retardation

Microcephaly

Flat occiput

Upward slant of palpebral fissures

Epicanthus

Flat nasal bridge

+

+

Micrognathia

Low−set ears

Long philtrum +

6 mo

Age ascertained

+

+

+

+

+

+

+

+

50–14%

p25;q37

6 days; 7 weeks; 7 yo

white

+

+

−

+

+

+

−

+

+

+

+

None

p25;q37

15 mo

white

M

NA

F

M

Ethnicity

RC2-3

Sex

RC2-2

RC2-1

Case

+

+

+

−

+

−

+

+

+

+

+

t(2;6) (p15;q15) dpat

None

2q37;6q27

10 mo

white

F

RC2-4

white

F

RC2-7

None

p25;q37

+

+

+

+

+

+

+

+

+

+

+

+

Mild

−

+

Birth

white

F

RC2-9

−

+

+

+

+

+

+

+

+

+

13 mo

Asian

F

RC2-10

−

+

−

−

+

−

−

+

−

+

+

De novo

+

+

+

+

+

+

+

+

−

−

+

+

+

De novo De novo

None

p25;q37.3 p25;q37 p25.3;q37.3 2nd study

Birth; 10 yo

white

M

RC2-8

Single cell Single cell None

p25;q37

1.5 yo.; 4 yo 20 yo; 34 yo

white

F

RC2-6

De novo De novo De novo

16%

p25;q37

Infant

Asian

F

RC2-5

Table 6.1  Clinical characteristics of patients affected by RC2

None

p25.3;q37.3

Fetus; TOP

NA

M

RC2-12

−

+

+

+

+

−

+

+

+

+

+

+

+

+

+

−

+

NA

NA

+

(Presumed) De novo De novo

None

p25;q37

Birth

white

M

RC2-11

+

+

+

+

−

+

+

+

+

+

De novo

One cell

p25;q37

10 mo

NA

M

RC2-13

+

+

+

+

+

+

8/14

8/14

8/14

7/14

14/14

6/14

5/14

13/14

10/14

11/14

14/14

9/14 white; 2/14 Asian

7/14

Frequencies

(continued)

De novo

Eight cells

p25.3; q37.3

Birth

white

M

RC2-14

6  Ring Chromosome 2 71

−

+

+

+

+

+

+

+

RC2-3

−

−

−

−

−

+

−

−

RC2-4

−

+

+

RC2-5

−

+

+

+

RC2-6

+

+

+

−

RC2-7

+ self-resolved

−

−

−

+

+

−

−

RC2-8

+ resolved

+

+

+

RC2-9

+ self-resolved

+

RC2-10

−

−

+

+

−

+

RC2-12

+ surgically + repaired

+

+

+

−

±

+

RC2-11

−

+

+

−

+

−

−

−

RC2-13

2/14

6/14

1/14

5/14

10/14

7/14

3/14

8/14

Frequencies

+ self-re- 4/14 solved

+

+

+

RC2-14

Abbreviations: M male; F female; ±borderline; TOP termination of pregnancy; NA not available; mo months old; yo years old; IUGR intrauterine growth restriction; dpat derived from chromosome abnormality of paternal origin; + present; - absent

+heart failure

Cardiac

−

Cutis laxa

−

+

Foot deformities

Skin pigmentation

−

Clinodactyly

+

−

+

Hypogenitalism

−

+

Widely spaced + nipples

Skeletal anomalies

+

+

Short neck

RC2-2

RC2-1

Case

Table 6.1  (continued)

72 J. B. Murry and Y. S. Zou

6  Ring Chromosome 2

cytogenetic testing twice using peripheral blood. RC2-8 was seen at birth in 1999 in a white male child who was ascertained once more at ten years of age (Lacassie et al. 1999). RC2-9 was reported in 2001 in a newborn white female (Dee et al. 2001) who was followed until ten months of age. RC2-10 was found in a 13-month-old Asian female who was followed up clinically to 19 months of age (Kosho et al. 2005). Also in 2005, RC2-11 was characterized in a white male at birth (Alkuraya et al. 2005). RC2-12 was identified in a male fetus ascertained at 23 weeks’ gestation that was terminated and reported in 2013 (Chen et al. 2013); a de novo RC2 was identified in this first prenatal report. Case RC2-13 was found in a ten-monthold male in peripheral blood (López-Uriarte et al. 2013). RC2-14 has been ascertained in a white male at birth as de novo and in mosaic form (Severino et al. 2015).

6.3 Laboratory and Clinical Findings This section reviews the additional pertinent case-level laboratory data and clinical findings for the fourteen reported cases of RC2 (see Table 6.1). Recurrent break points at 2p25 and 2q37 are shared across these cases that have been predominantly identified by karyotype alone. Peripheral blood is the most interrogated specimen due to ease of access. Where available, cases demonstrated a de novo origin; one individual’s ring arose on the maternally derived chromosome 2. The presence of normal cell levels varied among cases; overall, the number of cells evaluated varied greatly. Ring variability and mitotic instability were also observed. Subsequent complementary molecular cytogenomic studies have identified subtle copy number losses at either one or two of the break points in a subset. The following cases were only studied by solid staining or GTG-banding:

73

RC2-1 (Garau et al. 1973; Maraschio et al. 1979, Case 3) This subject was identified using solid-stained metaphase preparations in 1973. Therefore, approximate break points could not be determined in this peripheral blood study. This individual, initially ascertained in childhood due to multiple cyanotic episodes, passed away due to heart failure in childhood. During his early life, he presented with intrauterine growth retardation (IUGR), postnatal growth retardation, psychomotor retardation, microcephaly, flat occiput, the upward slant of palpebral fissures, epicanthus, flat nasal bridge, a long philtrum, micrognathia, low-set ears, a short neck, widely spaced nipples, hypogenitalism, foot deformities, and cardiac malformations, which included ventricular septal defect, a hypoplastic pulmonary artery with right ventricular hypertrophy, and displacement of the aortic route to the right. RC2-2 (Sutherland and Carter 1978) G-banded karyotype identified break points at cytobands 2p25 and 2q37. Based on her first chromosome analysis, 50% of diploid cells contained a monocentric RC2, thereby representing the first mosaic case of RC2 with normal cells. A subsequent karyotype study at seven years of age showed a decrease in the frequency of normal diploid cells from 50 to 14%. The authors estimated the involvement of minimal terminal deletion of 2p/2q, if any, by karyotype alone. No familial studies are described, though a prior healthy child was born to her parents. This individual was ascertained at birth and was reported to have IUGR, postnatal growth retardation, psychomotor retardation, microcephaly, a flat occiput, epicanthus, flat nasal bridge, low-set ears, a short neck, widely spaced nipples, and skeletal anomalies. She was the second child born to her 42-year-old parents, whose previous child was typically presenting.

74

RC2-3 (Maraschio et al. 1979, Case 1) The de novo RC2 with break points at cytobands 2p25 and 2q37 was present in 93% of cells examined. No dicentric rings were noted. Six cells examined lost the ring with a modal number of 45, while the remaining three cells displayed two rings with a modal number of 47. A subset of diploid cells had ring variation, and no normal cells were seen, implicating an early meiotic event. The authors estimated the involvement of minimal terminal deletion of 2p and 2q, if any, by karyotype alone. The child who initially presented at 2.5 months because of diet-resistant diarrhea was found to be affected with IUGR, postnatal growth retardation, psychomotor retardation, microcephaly, an upward slant of palpebral fissures, epicanthus, a flat nasal bridge, micrognathia, low-set ears, a short neck, widely spaced nipples, hypogenitalism, clinodactyly, foot deformities, cutis laxa, and skeletal anomalies. The chromosomes of the parents were normal. RC2-4 (Maraschio et al. 1979, Case 2) Her initial karyotypic studies were carried out in peripheral blood, but findings were subsequently confirmed in skin fibroblasts. Her ring was demonstrated to have arisen secondary to an unbalanced translocation involving chromosomes 2 and 6. The paternally derived familial balanced translocation was identified in a karyotype with a modal number of 46 chromosomes. Break points involved cytobands 2q37 and 6q27. The net consequences implicated in her ring included partial terminal deletion of 2q and 6q. While the identified RC2 involves chromosomes 2 and 6 and may not best be compared to other cases of pure and complete RC2, the event itself may represent a unique mechanism and is included in this review. A subset of double rings were observed but no dicentrics or normal cells were identified, implicating an early meiotic event. This child presented at ten months of age due to an identified left inguinal hernia and was subsequently found to have IUGR, postnatal growth retardation, psychomotor retardation,

J. B. Murry and Y. S. Zou

microcephaly, a flat occiput, epicanthus, a long philtrum, micrognathia, low-set ears, and hypogenitalism. RC2-5 (Vigfusson et al. 1980) Her initial G-banded karyotype identified a predominant finding of the conventional de novo RC2 with break points at cytobands 2p25 and 2q37. The net consequence estimated by the authors included minimal terminal deletion of 2p and 2q, if any, by karyotype alone. Combined G-banding studies identified that the de novo ring was present in 77.8% of diploid cells; 16% were normal. The lack of expression of all features was attributed to her likely post-zygotic mosaicism, given the presence of a normal diploid line. The mildly affected female infant born to parents that were 25 and 26 years of age had IUGR, postnatal growth retardation, psychomotor retardation, microcephaly, epicanthus, low-set ears, hypogenitalism, and clinodactyly. Her older male sibling was typically presenting and cytogenetically normal. In early life, she was noted to be a poor eater and presented with reduced size and poor growth. Karyotypes for her parents and her sibling were normal. RC2-6 (Cote et al. 1981; Sarri et al. 2015) G-banding from peripheral blood identified RC2, and 90% contained one or two rings (Cote et al. 1981)—see Table 6.1. No normal cells were identified. A total of two cells were monosomic for the ring, and the remaining cells had secondary formations of chromosome 2 or variations in ring size. In 1981, it was thought there was no apparent deletion in the de novo ring. Both parents had normal karyotypes. Remarkably, a 35-year-later follow-up that included a molecular cytogenetics study of blood and skin fibroblasts was provided, confirming the prior RC2 result (Sarri et al. 2015) with aCGH (BlueGnome), microsatellite markers as well as subtelomeric multiplex ligationdependent probe amplification (MLPA) and FISH analysis (TelVysion, Vysis). Again no

6  Ring Chromosome 2

normal cells were observed. A terminal deletion at cytoband 2q37.3-2qter was identified and confirmed using multiple methods. A 1.57 megabase (Mb) deletion (chr2:240,996,485– 242,565,565; hg19) was identified by aCGH. The deletion arose on the maternally derived chromosome 2 based on informative results from subtelomeric MLPA testing. The individual had IUGR, mild psychomotor retardation, microcephaly, the upward slant of the palpebral fissures, epicanthus, a long philtrum, micrognathia, low-set ears, a short neck, clinodactyly, and skeletal anomalies. In early life, she was noted to have a small weight and height for her age which persisted into five years of age at last ascertainment. Due to no health concerns, the patient was subsequently lost to follow-up until adolescence. Subsequent evaluations determined that she underwent normal puberty and later developed scoliosis. Her smaller presentation persisted well into adulthood at 29 years of age, and she displayed signs of developmental delay and moderate intellectual disability. No evidence of autism was appreciated. At last evaluation at 35 years old revealed that she could communicate with her father via computer assistance. RC2-7 (Jansen et al. 1982) G-banded chromosome analysis studies on peripheral blood in this individual identified the RC2 in 78 of the 169 metaphases analyzed from both studies combined. The de novo RC2 had break points at 2p25 and 2q37; a single normal cell was found. Her cells demonstrated mitotic instability; a high level of aneuploid cells displayed single, double, or quadruple ring structures and hypermodal and hypomodal cells. The child was born to her 22- and 25-yearold parents and was clinically evaluated upon recognition of poor growth and weight gain in childhood, microcephaly, and delayed developmental milestones. She was ultimately found to have IUGR, postnatal growth retardation, psychomotor retardation, microcephaly, a flat occiput, upward slant of palpebral fissures, epicanthus, a flat nasal bridge, micrognathia, hypogenitalism, clinodactyly, and foot deformities. The last

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clinical ascertainment was carried out at four years of age with improved growth measures but was still under the mean of typically presenting children. Her mother had two additional younger children, and both were normal. RC2-8 (Lacassie et al. 1999) Cytogenetic studies were carried out on peripheral blood seven years apart; initial studies did not indicate instability in this de novo RC2. However, the subsequent study indicated ring persistence at 65%, along with ring variation, and loss of the ring in 27% of cells examined. A single normal cell was observed. The final clarification of cytogenetic break points at 2p25 and 2q37.3 was determined based on the latter study. The child born to his 30- and 31-year-old parents had IUGR, postnatal growth retardation, microcephaly, epicanthus, micrognathia, hypogenitalism, clinodactyly, and a self-resolving cardiac malformation that was noted to be a ventricular septal defect. His most striking features included being small for gestational age, short stature, and microcephaly without gross delay, among other anomalies. Based on his initial physical presentation, there was a clinical concern for Silver–Russell syndrome. These noted physical features were followed up on ten years later. The patient underwent surgical repair of his unilateral inguinal hernia. The patient had two older healthy male siblings. This child experienced significantly improvement with early interventions like speech therapy. Both parents had normal chromosomes. As molecular cytogenetics emerged in the early 2000s and introduced the fluorescence in situ hybridization (FISH) method, this technological advancement confirmed subtle gains or losses involving subtelomeric sequences. Thus, three additional RC2 cases with shared break points at 2p25 and 2q37 were identified. RC2-9 (Dee et al. 2001) RC2 was found to be de novo and present in 79% of cells by postnatal blood karyotyping.

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The RC2 had break points at cytogenetic bands 2p25 and 2q37. Ring variation was described in the absence of normal cells. A subset of double rings was observed. Subtelomeric FISH probes for 2p (TelVysion, Vysis) identified a subtelomeric deletion within 300 kb of the end of the chromosome. The child was born to their 22- and 30-yearold parents and was reported to have IUGR, postnatal growth retardation, psychomotor retardation, microcephaly, the upward slant of the palpebral fissures, epicanthus, a flat nasal bridge, a long philtrum, a short neck, skeletal anomalies, skin pigmentation (café au lait spots), and a self-resolving cardiac malformation (multiple muscular ventricular septal defects). Her developmental delay was appreciated at the last ascertainment at ten months of age. Other salient features identified in this individual included a bifid thumb and a high-pitched cry. Both parents had normal karyotypes.

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for this presumed de novo RC2 case. Mitotic instability was noted in the absence of normal cells. The individual exhibited a terminal 2q deletion (subtelomeric 2q deletion via FISH). The child born to his 30-year-old mother had IUGR, postnatal growth retardation, psychomotor retardation, microcephaly, the upward slant of the palpebral fissures, epicanthus, a long philtrum, micrognathia, a short neck, widely spaced nipples, clinodactyly, skeletal anomalies, and a cardiac malformation that was surgically repaired. Initial findings at birth included significantly below third percentiles for body length, weight, and head circumference. After the surgical repair of the heart malformation, the child was discharged from the hospital with a feeding tube two weeks after birth. In the mid-2000s, CMA was introduced. Four additional cases of RC2 were reported that also had concurrent G-banded chromosome analysis, FISH, and CMA, one of which (RC2-6) was a follow-up from an earlier report.

RC2-10 (Kosho et al. 2005) RC2-12 (Chen et al. 2013) This de novo RC2 was identified in 66 cells from peripheral blood with break points in 2p25.3 and 2q37.3. No normal cells were identified, and mitotic instability was noted in the remaining 34 cells. The individual displayed partial terminal deletion of 2p and 2q (distal to subtelomeric regions, which were intact via FISH) since at least no significant material was deleted at the subtelomeric level by FISH (Oncor). The child was born to her 23-year-old parents and presented with IUGR, epicanthus, a flat nasal bridge, clinodactyly, and a self-resolved cardiac malformation (ventricular septal defect). Notably, she lacked significant malformations. Early life features included poor suck and growth retardation and other dysmorphic features. However, her development was considered normal at the last ascertainment at 19 months old. Her parents’ chromosomes were normal. RC2-11 (Alkuraya et al. 2005) Mosaicism for four different cell lines was noted by karyotype in leukocytes and skin fibroblasts

aCGH performed on uncultured amniocytes identified two distal microdeletions of 3.3 megabases (Mb) at 2p25.3 and 4.4 Mb at 2q37.3. Specifically, the karyotype revealed an RC2 in a single amniocyte, 20 cord blood cells, and 40 placental villi cells. No normal cells were observed. Metaphase FISH (BAC probes) performed on cultured placental cells supported the loss identified by aCGH. This is the first prenatal case of RC2 diagnosed in a male fetus from a 29-year-old primigravid female. At 23 weeks of gestation, prenatal ultrasound identified no parietal–occipital fissure, no calcarine fissure, and a shallow Sylvian fissure and brain MRI findings. The parents elected to terminate the pregnancy. All findings noted in the conceptus are seen in association with postnatal presentations of RC2 (see Table 6.1): microcephaly epicanthus, hypertelorism, a flat nasal bridge, micrognathia, low-set ears, a long philtrum, a short neck, clinodactyly, and hypogenitalism. The distinctive features of lissencephaly were only identified

6  Ring Chromosome 2

in this particular case of RC2; oligohydramnios was not noted. Both parents had normal chromosomes; the inversion 9 was maternally inherited. RC2-13 (López-Uriarte et al. 2013). The mosaic ring was de novo in origin and present in 80% of the studied peripheral blood cells. Also a subset of cells with two rings and modal number of 47 was observed along with a single normal cell. FISH probes (TotelVysion, Vysis) for the subtelomeric regions of the short and long arms of chromosome 2 indicated that any material loss may have been distal to the FISH probe sequence as these regions were intact via subtelomeric FISH. CMA identified a terminal deletion of 139 kilobases (Kb) at 2p25.3 and a 147 Kb deletion at 2q37.3. The child had IUGR, postnatal growth retardation, psychomotor retardation, microcephaly, a flat occiput, epicanthus, a long philtrum, micrognathia, low-set ears, clinodactyly, skeletal anomalies, and skin pigmentation findings of café au lait spots. The heart was structurally normal via echocardiogram. The parents’ karyotypes were normal. RC2-14 (Severino et al. 2015) This mosaic de novo RC2 was found in 92% of peripheral blood cells. The remaining eight cells were normal. Additional cytogenetic workup included metaphase FISH analysis using subtelomeric probes for the long and short arms of chromosome 2. Absence of either signal for the subtelomeric regions was demonstrated on the RC; aCGH also demonstrated terminal deletions of 439 Kb at 2p25.3 and 3.4 Mb at 2q37.3. The child born to his 28-year-old mother had IUGR, postnatal growth retardation, psychomotor retardation, microcephaly, epicanthus, a long philtrum, a short neck, clinodactyly, foot deformities, and a self-resolved cardiac malformation (patent ductus arteriosus). Additional features included an L-shaped kidney; brain malformations identified via MRI and diffusion tensor imaging (DTI) findings. He initially presented with low growth measurements with the

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achievement of typical milestones. The most salient features included failure to thrive, minor dysmorphic features, and microcephaly. He was last followed clinically at two years of age. Furthermore, he was found to have brain anomalies, particularly a corpus callosum malformation that had previously not been associated with RC2 or 2q37.3 syndrome. Karyotypes of the parents were normal.

6.4 Prenatal Findings With varying degrees of information, prenatal phenotypes are provided for seven cases, with only fetal findings for the first reported prenatal case RC2-12. All fourteen cases affected with RC2 are reported to have experienced IUGR (see Table 6.1). Five of seven cases with prenatal information reported oligohydramnios, which can arise due to multiple factors. The following postnatally ascertained individuals displayed prenatal features associated with RC2: RC2-8 had oligohydramnios, decreased fetal activity, and a breech position (Lacassie et al. 1999). RC2-9 presented with an abnormal prenatal ultrasound with findings of a two-vessel cord and ventriculomegaly. A second ultrasound revealed oligohydramnios and no fetal breathing movements. A prenatal karyotype on amniocytes was attempted but failed (Dee et al. 2001). RC2-10 and RC2-11 also presented oligohydramnios and were delivered at 33 weeks’ six days and 41 weeks’ gestational age, respectively (Kosho et al. 2005; Alkuraya et al. 2005). In conclusion, the paucity of information for prenatal diagnosis for RC2 highlights the need and value of updated cytogenomic characterization with detailed information on genotype–phenotype for improved genetic counseling in the prenatal setting.

6.5 Adult and Pediatric Findings Thirteen of the fourteen cases described herein presented in the early postnatal setting. A commonality of IUGR (100%), epicanthus (100%),

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microcephaly (93%), postnatal growth retardation (79%), psychomotor retardation (71%), clinodactyly (71%), low-set ears (57%), micrognathia (57%), short neck (57%), long philtrum (57%), flat nasal bridge (50%), hypogenitalism (50%), the upward slant of palpebral fissures (43%), skeletal anomalies (43%), foot deformities (36%), flat occiput (36%), cardiac malformations (29%), widely spaced nipples (21%), and in some cases, skin pigmentation (14%) or cutis laxa (7%) has emerged (see Table 6.1). The severity of these features may depend on the level of ring mosaicism. Life history is limited, as it is available for up to ten years in one individual and up to 34 years for another. There has been a report of early death in one case with noted heart failure; however, the exact break points could not be determined in this case predating G-band karyotyping methods. Four individuals experienced spontaneously self-resolving cardiac malformations like ventricular septal defects or patent ductus arteriosus, and one child’s cardiac malformations were successfully surgically repaired. The rest of the individuals affected with cardiac malformations were compatible with life. One individual had a structurally normal heart. It has been suggested that growth concerns may persist due to mitotically unstable cells that cannot survive.

6.6 Genotype–Phenotype Correlations

Fig. 6.1   OMIM-disease-associated genes within the deletions detected by array technologies. A screenshot of the UCSC Genome Browser of chromosome 2p and

2q (GRCh37/hg19) indicates the relevant tracks for comparison of loss of potentially significant genes identified by array-based methods for four cases with RC2

Four cases affected with RC2 underwent multiple complementary cytogenetic approaches to clarify their break points, enabling more accurate genotype–phenotype correlations (Fig. 6.1). Importantly, no single overlapping genomic gain or loss event was shared across all four cases affected with RC2 who underwent chromosomal  microarray testing. The two RC2s described in 2013 involved subtelomeric losses of both arms: a fetal-only ascertainment (Chen et al. 2013) and a postnatal-only case (LópezUriarte et al. 2013). One RC2 case described in 2015 involved a follow-up study of the child initially reported by Cote et al. in 1981 (Sarri et al. 2015), who was found to bear only a subtelomeric 2q deletion. The other case reported in 2015 was also found to bear subtelomeric deletions involving both arms (Severino et al. 2015). Here, we review the genotype–phenotype correlations for the fourteen individuals with RC2, as far as available. In RC2-1, it was speculated that fatal cardiac failure may have been due to a lack of normal cells. In RC2-2, RC27, RC2-9, and RC2-10, observed ring instability and its influence on cell division were discussed as most likely contributing to symptoms. In

6  Ring Chromosome 2

RC2-3, RC2-8, RC2-9, and RC2-11, partial monosomies were discussed to influence the phenotypes. However, as far as available, the sizes of deletions were different in each case. Still, three out of the other four RC2 cases with CMA detecting loss of 2q shared loss of KIF1A, CAPN10, and AGXT; the additional loss of HDAC4, a neighboring gene also on 2q, was common in only two. The KIF1A-encoded protein is a kinesin-related protein, and the gene is associated with multiple dominant and recessive forms of KIF1A-neuropathy-related disorders (MIM*601255). To date, various pathogenic point mutations and truncating variants have been described in association with KIF1Arelated disorders. Prior curation of this gene indicates no evidence of haploinsufficiency in ClinGen. However, KIF1A achieves a pHaplo score of 0.99 which may support a prediction of haploinsufficiency in an in silico manner in DECIPHER. CAPN10 may be associated with susceptibility to diabetes mellitus, non-insulindependent (MIM*601283), and AGXT is associated with recessive forms of hyperoxaluria, primary, type 1 (MIM*259900). An association between RC2 features and KIF1A-neuropathyrelated disorders has been raised as  a possible contributory factor for intellectual disability, but in the absence of brain malformations, likely has not yet been formally explored (Severino et al. 2015). For comparison, pure terminal chromosome 2q37 deletion syndrome is associated with features such as short stature, obesity, hypotonia, facial dysmorphism, autism, joint hypermobility, scoliosis, seizures, neuronal ocular, and gastrointestinal cardiac and renal abnormalities (MIM*600430). Additionally, individuals may present with brachymetaphalangy. In the two RC2 cases with overlapping 2q losses inclusive of HDAC4, one is a conceptus, and the other is identified in a liveborn individual; however, strikingly different unique features are noted. Studies of microdeletions involving the 2q37 locus implicate that loss of the HDAC4 gene in some individuals is associated with incomplete penetrance and is a primary contributing genetic factor to

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the syndrome (Le et al. 2019). The HDAC4 gene encodes histone deacetylase 4. Heterozygous de novo missense variants in this gene are associated with an autosomal dominant neurodevelopmental disorder with central hypotonia and dysmorphic features (MIM*619797). While no genes in this loss are established as haploinsufficient, HDAC4 is fully contained and historically has been curated to have a score of 1 out of a maximum of 3, indicating little evidence for haploinsufficiency in ClinGen. Given the absence of phenotypic overlap of brachydactyly type E between HDAC4-related disorders and RC2, no association has been extensively explored; however, loss of HDAC4 may contribute to intellectual disability in part (Severino et al. 2015). Additionally, these two patients share complete gene deletion of TWIST2, a gene with conflicting predictions for haploinsufficiency in an in silico manner in ClinGen and DECIPHER.

6.7 Conclusions and Recommendations Multiple complementary cytogenetic and genomic methods to detect chromosomal structural rearrangement events, like RC2, may still be required until technology improves so that a single method can capture its unique properties. Another laboratory testing consideration may be using research-level emerging methods like optical genome mapping and or long-read genomic sequencing methods to provide probe-independent evidence for the intactness of the terminal regions of 2p and 2q. Recent improvements in the reference human genome sequence that now includes highly repetitive regions like telomeres and centromeres (Nurk et al. 2022) may aid the future study of chromosomal rearrangements, like rings. For patient care, it is clear that multidisciplinary teams are necessary, and additional longitudinal studies are needed to understand care management for individuals affected by RC2. Based on available reports of children and adults, physical, occupational, and speech therapy might improve aspects of psychomotor

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delay. Specifically, understanding successful strategies to assist with managing developmental and behavioral needs is an area for improvement. Collaborative efforts with other groups like family and patient-focused groups interested in connecting individuals with RC2 will likely inform our understanding of this rare chromosomal finding.

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J. B. Murry and Y. S. Zou Le TN, Williams SR, Alaimo JT, Elsea SH (2019) Genotype and phenotype correlation in 103 individuals with 2q37 deletion syndrome reveals incomplete penetrance and supports HDAC4 as the primary genetic contributor. Am J Med Genet 179A(5):782– 791. https://doi.org/10.1002/ajmg.a.61089 Li P, Dupont B, Hu Q, Crimi M, Shen Y, Lebedev I, Liehr T (2022) The past, present, and future for constitutional ring chromosomes: A report of the international consortium for human ring chromosomes. HGG Adv 3(4):100139. https://doi.org/10.1016/j. xhgg.2022.100139 López-Uriarte A, Quintero-Rivera F, de la Fuente CB, Puente VG, Campos MDRV, de Villarreal LEM (2013) Ring 2 chromosome associated with failure to thrive, microcephaly and dysmorphic facial features. Gene 529(1):65–68. https://doi.org/10.1016/j. gene.2013.06.056 Maraschio P, Danesino C, Garau A, Saputo V, Vigi V, Volpato S (1979) Three cases of ring chromosome 2, one derived from a paternal 2/6 translocation. Hum Genet 48(2):157–167. https://doi.org/10.1007/ BF00286899 Nurk S, Koren S, Rhie A, Rautiainen M, Bzikadze AV, Mikheenko A, Vollger MR, Altemose N, Uralsky L, Gershman A, Aganezov S, Hoyt SJ, Diekhans M, Logsdon GA, Alonge M, Antonarakis SE, Borchers M, Bouffard GG, Brooks SY, Caldas GV, Chen NC, Cheng H, Chin CS, Chow W, de Lima LG, Dishuck PC, Durbin R, Dvorkina T, Fiddes IT, Formenti G, Fulton RS, Fungtammasan A, Garrison E, Grady PGS, Graves-Lindsay TA, Hall IM, Hansen NF, Hartley GA, Haukness M, Howe K, Hunkapiller MW, Jain C, Jain M, Jarvis ED, Kerpedjiev P, Kirsche M, Kolmogorov M, Korlach J, Kremitzki M, Li H, Maduro VV, Marschall T, McCartney AM, McDaniel J, Miller DE, Mullikin JC, Myers EW, Olson ND, Paten B, Peluso P, Pevzner PA, Porubsky D, Potapova T, Rogaev EI, Rosenfeld JA, Salzberg SL, Schneider VA, Sedlazeck FJ, Shafin K, Shew CJ, Shumate A, Sims Y, Smit AFA, Soto DC, Sović I, Storer JM, Streets A, Sullivan BA, Thibaud-Nissen F, Torrance J, Wagner J, Walenz BP, Wenger A, Wood JMD, Xiao C, Yan SM, Young AC, Zarate S, Surti U, McCoy RC, Dennis MY, Alexandrov IA, Gerton JL, O’Neill RJ, Timp W, Zook JM, Schatz MC, Eichler EE, Miga KH, Phillippy AM (2022) The complete sequence of a human genome. Science 376(6588):44-53.  https:// doi.org/10.1126/science.abj6987 Sarri C, Douzgou S, Kontos H, Anagnostopoulou K, Tümer Z, Grigoriadou M, Petersen MB, Kokotas H, Merou K, Pandelia E, Giouroukou E, Papanikolaou K, Côté GB, Gyftodimou Y (2015) 35-year followup of a case of ring chromosome 2: Array-CGH analysis and literature review of the ring syndrome. Cytogenet Genome Res 145(1):6–13. https://doi. org/10.1159/000382046

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Ring Chromosome 3 Maria Isabel Melaragno and Bruna Burssed

Abstract

Ring chromosome 3 (RC3) is an ultra-rare constitutional chromosomal abnormality and has been described in only 13 live-born patients since 1966. Cytogenetic analysis detected the short-arm and long-arm breakpoints in RC3 with different banding resolutions. Chromosome microarray analysis defined genomic imbalances in two patients of RC3. The most frequently seen clinical features for patients of RC3 include growth retardation, intellectual disability, microcephaly, and facial dysmorphism. Genotype–phenotype correlations for genes in the distal deletions of 3p have been suggested. Comprehensive cytogenomic analysis should be performed to define the ring structure, dynamic mosaicism, and genomic imbalance. Proper genetic counseling and clinical management should be recommended to patients and their families.

Keywords

Ring chromosome 3 (RC3) · Clinical findings · Ring instability · Ring syndrome · Genotype–phenotype correlations

7.1 Introduction Constitutional ring chromosome 3 (RC3) is an ultra-rare chromosome abnormality and accounts for approximately 1% of all RCs. The first patient reported with a RC3 was a 2-yearold girl with growth retardation and developmental delay. She was described in 1966 by Mukerjee and Burdette, who visualized the RC in the karyotype from peripheral blood culture. Interestingly, the first patient with RC3 presented a translocation between chromosome 3 and X-chromosome. In 1996, fluorescence in situ hybridization (FISH) was first performed in a case of RC3 using a whole chromosome painting, subtelomeric, and centromeric probes (Yip et al. 1996), which enabled the characterization of a non-reciprocal translocation between short arms of chromosomes 3 and 6, resulting in the following karyotype: 46,XY,r(3) (p23q29),der(6)t(3;6)(p23;p25.3). In 2011, chromosome microarray analysis (CMA) and multiplex ligation-dependent probe amplification (MLPA) were performed to define the genomic imbalance and breakpoints in a RC3 (Guilherme et al. 2011a). A review of the literature for the past 60 years has revealed a collection of 13 cases of RC3.

M. I. Melaragno (*) · B. Burssed  Genetics Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_7

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7.2 Demographics and Family History The 13 cases of RC3 were designated as RC3-1 (Mukerjee and Burdette 1966), RC3-2 (Picciano et al. 1972), RC3-3 (Witkowski et al. 1978), RC3-4 (Wilson et al. 1982), RC3-5 (Kitatani et al. 1984), RC3-6 (Narahara et  al. 1990), RC3-7 (Lakshminarayana and Nallasivam, 1990), RC3-8 (McKinley et al. 1991), RC3-9 (Teyssier et al. 1991), RC3-10 (Yip et  al. 1996), RC3-11 (Barajas-Barajas et al. 2001), RC3-12 (Guilherme et al. 2011a), and RC3-13 (Zhang et al. 2016). Table 7.1 presents all 13 patients with their gender, age, and laboratory results. Among the 13 live-born patients with RC3 reported so far, seven are male and six are female, thus showing no influence of gender in the occurrence of RC3. Regarding the patient’s ages, three were 1 year old or less, two were 2 years old, two were 3–5 years old, one was 11 years old, one was 16 years old, one was 18 years old, and the oldest patient reported was 46 years old. Gender also had no influence on the patient’s age. The RC3 patients were reported in different regions, and no ethnic preference is noted. Most patients were referred to genetic studies due to growth and developmental delay as well as dysmorphic features. Regarding family history, one patient, with a phenotype corresponding to Cornelia de Lange syndrome, had consanguineous parents who were first cousins (Lakshminarayana and Nallasivam 1990), while the remaining patients’ parents were all non-consanguineous and healthy. The mothers’ ages range from 21 to 36 years and the fathers’ ages from 21 to 43 years. All the patients’ siblings were normal and healthy. Given the presented family history, we can infer that all RC3 were de novo since no family history of genetic alterations was detected in the parents or siblings. It is important to note that not all parents were submitted to genetic testing and not all studies provided a detailed family history.

M. I. Melaragno and B. Burssed

7.3 Laboratory Results All studies identified the RC using G-banding karyotype. The breakpoints of some RCs were not identified (Mukerjee and Burdette 1966; Picciano et al. 1972; Witkowski et al. 1978; Lakshminarayana and Nallasivam 1990). The remaining cases had their breakpoints defined with different levels of chromosome resolution, as visible in Fig. 7.1, which summarizes the deleted regions in the RC3 patients according to McGowan-Jordan et  al. 2020. Some authors (Wilson et al. 1982; Kitatani et al. 1984; Barajas-Barajas et al. 2001) described patients with the 46,XY,r(3)(p26q29) karyotype and similar breakpoints. Narahara et al. (1990) reported a patient with a 46,XX,r(3)(p26.1q29) karyotype and McKinley et al. (1991) presented a patient with a 46,XX,r(3)(p26.2q29) karyotype. Yip et al. (1996) and Guilherme et al. (2011a) performed FISH using a chromosome 3 painting probe as well as telomeric and centromeric probes. The use of this technique allowed to discover a non-reciprocal translocation between chromosome 3 and chromosome 6, resulting in the following karyotype: 46,XY,r(3) (p23q29),der(6)t(3;6)(p23;p25.3). To refine the breakpoint, Guilherme et al. (2011a) performed CMA and MLPA and Zhang et al. (2016) performed CMA to better characterize the RC3. Both patients presented deletions only in the short arm of chromosome 3 with the following genomic results: 46,XY,r(3) (p26.1q29).arr[GRCh37] 3p26.3p26.1(307,82 0_6,045,520) ×  1 and 46,XX,r(3)(p25.3q29). arr[GRCH37] 3p26.3p25.3(61,891_9,979,408)  × 1, respectively.

7.3.1 Ring Instability and Dynamic Mosaicism of RC3 The phenomenon of RC instability, in which secondary alterations are seen in more than 5% of the analyzed cells, is more frequently seen in larger RCs (Kosztolányi, 1987; Guilherme

3p

+

High nasal bridge

Broad nasal bridge

Bushy eyebrows

Epicanthic folds

Hypertelorism

Ptosis

Short palpebral fissures

+

+

+

+ +

+

+

+

+

+

+

+

+

+

3q29

+ +

4 m

M

+

+

+

+

+

NA

46 y

M

+

+

+

+

3q29

+

+

+

+

3q29

3p26.2 3p26

18 y

F

+

+

+

3q29

3p23

11 y

M

+

+

+

+

3q29

3p26

NA

F

+

+

+

+

+

+

3q29

6 Mb

3p26.1

16 y

M

+

+

+

+

+

+

+

+

+

+

+

+

+

+

3q29

9 Mb

3p25.3

1y

F

(continued)

23% (3/13)

38% (5/13)

23% (3/13)

46% (6/13)

15% (2/13)

23% (3/13)

38% (5/13)

61% (8/13)

84% (11/13)

30% (4/13)

23% (3/13)

69% (9/13)

30% (4/13)

92% (12/13)

38% (5/13)

7M, 6F

RC3-7 RC3-8 RC3-9 RC3-10 RC3-11 RC3-12 RC3-13 Total

3p25.3-3p26.1 NA

5y

+

+

+

+

+

+

3q29

3p26

10 m

+

+

+

+

Triangular face

+

+

+

Microcephaly

+

+ +

+

Hypotonia

Feeding difficulties Facial characteristics

+

+

+

+

Failure to thrive

+

Intellectual disability

+

+

3q29

+

NA

3p26

3.5 y

+

NA

NA

2y

Postnatal growth retardation

NA

NA

NA

Prenatal growth retardation

3q breakpoint Neuromuscular development

deletiona

NA

F

2y

F

3p breakpoint

M

Age

M

F

Gender

M

RC3-1 RC3-2 RC3-3 RC3-4 RC3-5 RC3-6

Patient

Table 7.1  Genetic data and clinical features of RC3 patients

7  Ring Chromosome 3 85

Long philtrum

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

30% (4/13)

23% (3/13)

46% (6/13)

38% (5/13)

61% (8/13)

23% (3/13)

30% (4/13)

38% (5/13)

46% (6/13)

38% (5/13)

23% (3/13)

23% (3/13)

Patient’s references: RC3-1: Mukerjee and Burdette (1966), RC3-2: Picciano et al. (1972), RC-3: Witkowski et al. (1978), RC-4: Wilson et al. (1982), RC-5: Kitatani et al. (1984), RC-6: Narahara et al. (1990), RC-7: Lakshminarayana and Nallasivam (1990), RC-8: McKinley et al. (1991), RC-9: Teyssier et al. (1991), RC-10: Yip et al. (1996), RC-11: Barajas-Barajas et al. (2001), RC-12: Guilherme et al. (2011a), RC-13: Zhang et al. (2016). Abbreviations: a Deletion size according to chromosome microarray; y: years; m: months; F: female; M: male; +: positive finding; NA: not available

Articular limitation

+

+

+ +

Clinodactyly

Brachydactyly

Hypoplastic thumbs

+

+

+

+

+ +

+

+

+

Microretrognathia Limbs and trunk features

+

Thin upper lip

High-arched palate

+

+

+

Downturned corners of the mouth

+

+

+

+ +

+

+

RC3-7 RC3-8 RC3-9 RC3-10 RC3-11 RC3-12 RC3-13 Total

Full nasal tip

+

RC3-1 RC3-2 RC3-3 RC3-4 RC3-5 RC3-6

Hypoplastic alae nasi

Anteverted nostrils

Patient

Table 7.1  (continued)

86 M. I. Melaragno and B. Burssed

7  Ring Chromosome 3

87

Fig. 7.1  Scheme of the deleted regions in the RC3 of the patients from the literature. On the left, ISCN (2020) chromosome 3 idiograms at the level of resolution of approximately 400- and 850-bands. On the right, the question marks show the cases with no breakpoint

specification, the empty boxes represent the deleted region reported without microarray determination, and the two black boxes show the deleted region according to the chromosomal microarray results. Patients are shown according to the chronological order of publication

et al. 2011a). Since RC3 is one of the largest RCs given the size of the involved chromosome, instability is expected to be found. In fact, Witkowski et al. (1978) were the only authors who found a same-sized monocentric ring chromosome in all analyzed cells. The remaining authors found mosaics in all their patients. The reported percentage of cells with a single monocentric RC3 replacing the normal chromosome 3 was 75% (Picciano et al. 1972), 92% (Wilson et al. 1982), 77.4% (Kitatani et al. 1984), 87% (Narahara et al. 1990; Guilherme et al. 2011a), 84% (McKinley et al. 1991), 93% (Teyssier et al. 1991), 83% (Yip et al. 1996),

and 90% (Zhang et al. 2016). The remaining cells presented a variety of RCs such as dicentric ring, rod-shaped ring, tetracentric ring, two monocentric rings, and interlocked rings. Figure 7.2 shows examples of RC instability seen in RC3-12 (Guilherme et al. 2011a). Kitatani et  al. (1984) described the first case of RC3 aneuploidy. Cells missing a chromosome have a greater imbalance of genetic material and, therefore, are less likely to survive. This reduction in cell number may lead to growth retardation in the prenatal and postnatal stages of life. This phenotype is characteristic of the ‘ring syndrome’ (McKinley et al. 1991).

88

M. I. Melaragno and B. Burssed

Fig. 7.2  Cytogenetic data from RC3-12 (Guilherme et al. 2011a). a partial karyotype in G-banding from different cells showing one normal chromosome 3 and one monocentric ring, two separate rings, ring loss, and

interlocked rings. b FISH with centromeric chromosome 3 probe showing one monocentric and one dicentric RC3, and c FISH with pantelomeric probe showing no signal in the RC3 (white arrow)

7.3.2 Ring Formation Mechanisms

3p23 to 3pter segment was then involved in a translocation with 6p. The use of FISH with subtelomeric probes showed that the der(6) presents the chromosome 6 subtelomeres as well as the 3p subtelomeres, confirming that the whole chromosome 6 is present and, therefore, the translocation is non-reciprocal. Given that the patient presents no deletion of 3p, the authors propose that the 3p23 break had the potential to determine the patient’s phenotype due to loss of the gene function, deletion/mutation at the breakpoint, or by unmasking of recessive genes at the breakpoint. It is important to note that the definition of the mechanisms of RC formation relies on the precise determination of their breakpoints, which should be sequenced in order to be analyzed at the nucleotide level (Burssed et al. 2022).

The mechanisms of ring chromosome formation involve the fusion of both chromosome arms, which can have terminal deletions or not (Burssed et al. 2022). The patients with RC3 have different sizes of deletions in 3p and 3q, with the latter probably being subtelomeric in most of them. These breaks in both chromosome arms fused together to form the RC3 in the patients. Two patients, however, present non-reciprocal translocations involving the RC. Mukerjee and Burdette (1966) reported a patient with a translocation between chromosomes 3 and X. The authors proposed that the ring was formed in a similar way to the other patients, with deletions in both arms followed by fusion of the broken ends. One deleted fragment of the chromosome 3 then fused with Xp, which suffered a terminal deletion. As a result, the late replicating chromosome X presents a larger Xp, giving the chromosome a metacentric configuration. Yip et al. (1996) reported a patient with a translocation between chromosome 3 and chromosome 6. Similarly, the ring was suggested to be formed by a break at 3p23 followed by its fusion with the subtelomeric region of 3q. The displaced

7.4 Clinical Findings All reported pregnancies of patients with RC3 were normal, uncomplicated, and ranged between 37 and 43 weeks of gestation. Five patients showed prenatal growth retardation. Most reported deliveries were normal labor

7  Ring Chromosome 3

with vaginal delivery at term, with one baby in breech presentation (Narahara et al. 1990). One mother failed to progress in labor and had a caesarian section (Yip et al. 1996). Two families had a history of miscarriages and abortions (Kitatani et al. 1984; Zhang et al. 2016). Twelve of the patients presented postnatal growth retardation, with four of them also presenting prenatal growth retardation. The remaining patient only presented growth retardation in the prenatal stage. Intellectual disability was reported in nine patients. These features were the main reasons why most patients were referred to genetic studies. Table 7.1 displays detailed information of patient’s phenotypes. Regarding facial characteristics (Table 7.1), microcephaly was reported in eleven patients. Triangular face and microretrognathia were seen in eight patients each. Other characteristics seen in a significant number of patients include epicanthic folds, downturned corners of the mouth, short palpebral fissures, broad nasal bridge, full nasal tip, long philtrum, and high-arched palate. Nine patients presented with dysplastic ears (Picciano et al. 1972; Wilson et al. 1982; Lakshminarayana and Nallasivam 1990; Narahara et al. 1990; McKinley et al. 1991; Yip et al. 1996; Barajas-Barajas et al. 2001; Guilherme et al. 2011a; Zhang et al. 2016), with two of them presenting hearing loss (Narahara et al. 1990; Zhang et al. 2016). Regarding limbs and trunk features, clinodactyly, brachydactyly, and articular limitation were the most frequent features observed (Table 7.1). Genito-urinary system alterations were reported only in the male patients: out of seven, two presented cryptorchidism (Yip et al. 1996; Guilherme et al. 2011a), three had hypospadias (Picciano et al. 1972; Wilson et al. 1982; Lakshminarayana and Nallasivam 1990), and two showed renal anomalies (Picciano et al. 1972; Guilherme et al. 2011a). Some features were reported in only one patient with RC3, such as synophrys (Lakshminarayana and Nallasivam 1990), scoliosis (Guilherme et  al. 2011a), syndactyly (Zhang et al. 2016), widely spaced nipples

89

(Kitatani et al. 1984), and congenital heart defects (Zhang et al. 2016). Figure 7.3 presents updated pictures of RC3-12 (Guilherme et al. 2011a) at the age of 30 years old showing microcephaly (head circumference =  47.5  cm) and dysmorphisms. He also presented short stature and noticeable clinodactyly. Despite the intellectual disability, with difficulty mainly in mathematics, RC312 is independent and able to work on simple activities.

7.5 Genotype–Phenotype Correlations 7.5.1 Genes and Regions Impaired in RC3 Clinical features found in patients with RC3 are similar to the ones found in patients with 3p monosomy, such as microcephaly, ptosis, and dysplastic ears (Wilson et al. 1982). The extent of this deletion in the ring chromosome is probably what determines the patients’ phenotypes. Six genes in the distal region of 3p present a pLI score of 1.00 and low LOEUF scores: ITPR1, BRPF1, BHLHE40, SETD5, SRGAP3, and GRM7, with four of them being associated with OMIM phenotypes. The ITPR1 (inositol 1,4,5-trisphosphate receptor type 1, OMIM*147,265) gene is associated with Gillespie syndrome (OMIM #206,700) and spinocerebellar ataxia (OMIM #606,658 and #117,360), the BRPF1 (bromodomain and PHD finger containing 1, OMIM *602,410) gene with intellectual developmental disorder with dysmorphic facies and ptosis (OMIM#617,333), which are among the most frequent phenotypes seen in RC3 patients, the SETD5 (SET domain containing 5, OMIM*615,743) gene with intellectual developmental disorder (OMIM#615,761), and the GRM7 (glutamate metabotropic receptor 7, OMIM%604,101) gene with neurodevelopmental disorder with seizures, hypotonia, and brain abnormalities (OMIM #618,922).

90

M. I. Melaragno and B. Burssed

Fig. 7.3  Updated photograph of RC3-12 (Guilherme et al. 2011a) at the age of 30 years old. a face and b and c lateral view of the patient, showing microcephaly, triangular face

with narrow forehead, short palpebral fissures, broad and high nasal bridge, anteverted nostrils, long philtrum, higharched palate, thin upper lip, and dysplastic ears

The loss of 3q29 does not appear to affect the patients’ phenotypes (McKinley et al. 1991).

Inconsistencies in the phenotypes are also found and may be related to differences in the extent of the deletions involved in the RC formation, rather than to the degree of the RCs mitotic instability (Narahara et  al. 1990; Guilherme et al. 2011b). McKinley et al. (1991) presented the patient with the most distal breakpoint in the short arm of chromosome 3, at 3p26.2, and with the mildest phenotype. Her main clinical findings include growth retardation and mild facial dysmorphisms, which could be explained by the ring syndrome as they are not associated with the loss of a specific genetic material. As mentioned previously, the ring syndrome is common to all RC cases and is due to the loss

7.5.2 Ring Syndrome The definition of a genotype–phenotype correlation for patients with RC3 is challenging given that there is a limited number of reported patients, most of whom do not have their breakpoints and genetic imbalance properly defined. Similarity in phenotypes between patients with RC3 was noted, as detailed above. However, most of these clinical findings are not specific, as they can also be seen in other chromosome and RC disorders (Kitatani et al. 1984).

7  Ring Chromosome 3

of cells with great genetic imbalance, such as the loss of a RC (McKinley et al. 1991). The main phenotypes involved in the ring syndrome include growth retardation, mild dysmorphism, and variable intellectual disability, which are among the main clinical findings seen in the reported patients with RC3. Therefore, the ring syndrome can explain some of these patients’ phenotypes.

7.6 Conclusions and Recommendations Since 1966, thirteen patients with RC3 have been described in the literature. These patients present a wide range of phenotypes, with postnatal growth retardation, intellectual disability, microcephaly, triangular face, microretrognathia, dysplastic ears, and brachydactyly being the most frequent ones. Most of these patients presented 3p deletions, but only two of them had their breakpoints defined by CMA. The use of high-resolution techniques to evaluate patients of RC3 is vital to assist in the understanding of which regions and genes are responsible for their phenotypes and to better comprehend how the RCs are formed.

References Barajas-Barajas LO, Velarde-Félix S, Elizarrarás-Rivas J, Hernández-Zaragoza G, Vázquez-Herrera JA (2001) De novo ring chromosome 3 in a girl with hypoplastic thumb and coloboma of iris. Genet Couns 12(2):151–156 Burssed B, Zamariolli M, Bellucco FT, Melaragno MI (2022) Mechanisms of structural chromosomal rearrangement formation’. Mol Cytogenet 15(1):1–15. https://doi.org/10.1186/s13039-022-00600-6 Guilherme RS, Bragagnolo S, Pellegrino R, Christofolini DM, Takeno SS, Carvolheira GM, Kulikowski LD, Melaragno MI (2011a) Clinical, cytogenetic and molecular study in a case of r(3) with 3p deletion and review of the literature. Cytogenet Genome Res 134(4):325–330. https://doi.org/10.1159/000329478 Guilherme RS, Meloni VF, Kim CA, Pellegrino R, Takeno SS, Spinner NB, Conlin LK, Christofolini DM, Kulikowski LD, Melaragno MI (2011b) Mechanisms of ring chromosome formation, ring instability and clinical consequences.

91 BMC Med Genet 12(1):171. https://doi. org/10.1186/1471-2350-12-171 Kitatani M, Takahashi H, Yasuda J, Chen CC, Ida F, Shike S (1984) A case of ring chromosome 3, 46, XX,-3,+r(3)(p26q29). Jinrui Idengaku Zasshi 29(2):157–162. https://doi.org/10.1007/BF01873537 Kosztolányi G (1987) Does “ring syndrome” exist? An analysis of 207 case reports on patients with a ring autosome. Hum Genet 75(2):174–179. https://doi. org/10.1007/BF00591082 Lakshminarayana P, Nallasivam P (1990) Cornelia de Lange syndrome with ring chromosome 3. J Med Genet 27(6):405–406. https://doi.org/10.1136/ jmg.27.6.405 McGowan-Jordan J, Hastings RJ, Moore S (2020) ISCN 2020: An International System for Human Cytogenomic Nomenclature. Karger, Basel, Switzerland McKinley M, Colley A, Sinclair P, Donnai D, Andrews T (1991) De novo ring chromosome 3: A new case with a mild phenotype. J Med Genet 28(8):536–538. https://doi.org/10.1136/jmg.28.8.536 Mukerjee D, Burdette WJ (1966) Multiple congenital anomalies associated with a ring 3 chromosome and translocated 3/X chromosome. Nature 212(5058):153–155. https://doi. org/10.1038/212153a0 Narahara K, Kikkawa K, Murakami M, Hiramoto K, Namba H, Tsuji K, Yokoyama Y, Kimoto H (1990) Loss of the 3p25.3 band is critical in the manifestation of del(3p) syndrome: Karyotype-phenotype correlation in cases with deficiency of the distal portion of the short arm of chromosome 3. Am J Med Genet 35(2):269–273. https://doi.org/10.1002/ ajmg.1320350225 Picciano DJ, Berlin CM, Davenport SL, Jacobson CB (1972) Human ring chromosomes: A report of five cases. Ann Genet 15(4):241–247 Teyssier M, Piperno D, Charrin C (1991) Chromosome 3 en anneau chez un adulte nain et retardé mental [Ring chromosome 3 in a mentally retarded adult dwarf]. Ann Genet 34(1):33–36 Wilson GN, Pooley J, Parker J (1982) The phenotype of ring chromosome 3. J Med Genet 19(6):471–473. https://doi.org/10.1136/jmg.19.6.471 Witkowski R, Ullrich E, Piede U (1978) Ring chromosome 3 in a retarded boy. Hum Genet 42(3):345–348. https://doi.org/10.1007/BF00291318 Yip MY, MacKenzie H, Kovacic A, McIntosh A (1996) Chromosome 3p23 break with ring formation and translocation of displaced 3p23–>pter segment to 6pter. J Med Genet 33(9):789–792. https://doi. org/10.1136/jmg.33.9.789 Zhang K, Song F, Zhang D, Liu Y, Zhang H, Wang Y, Dong R, Zhang Y, Liu Y, Gai Z (2016) Chromosome r(3)(p25.3q29) in a patient with developmental delay and congenital heart defects: A case report and a brief literature review. Cytogenet Genome Res 148(1):6– 13. https://doi.org/10.1159/000445273

8

Ring Chromosome 4 Kathleen M. Bone, Judy Chernos and Mary Ann Thomas  

Abstract

We describe the cytogenomic and clinical characteristics of 47 individuals of ring chromosome 4 (RC4) published in the literature. In nine cases, mosaicism for a normal cell line was observed, but no other forms of constitutional mosaicism involving different stable anomalies of chromosome 4 were reported. All published RC4 were large with distal breakpoints most often in p15-p16 and q34-q35. Given the variability of genomic imbalance related to the different sizes of RC4, there is clinical variability. Individuals with a near complete RC4 often present with normal neurodevelopment, but demonstrate poor growth likely related to the “ring syndrome”. Patients with larger deletions that

K. M. Bone  Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI, USA e-mail: [email protected] J. Chernos  Department of Medical Genetics, University of Calgary, Calgary, AB, Canada M. A. Thomas (*)  Departments of Medical Genetics and Pediatrics, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada e-mail: [email protected]

encompass the 4p Wolf–Hirschhorn critical region present with features of this syndrome. Those with a larger 4q deletion present with variable anomalies. Of note, none of these cases have the characteristic features of the 4q deletion syndrome since the majority had breakpoints that were distal to the typical 4q31-q35 region. Based on this review, it is recommended to obtain imaging for heart and renal anomalies, as well as assessment of vision, hearing, and neurodevelopment. Genetic counseling for the possibility of gonadal mosaicism is also recommended as recurrence in a family has been reported.

Keywords

Ring chromosome 4 (RC4) · Ring syndrome · 4p deletion · 4q deletion · Wolf–Hirschhorn syndrome · Dynamic mosaicism · Short stature · Growth restriction

8.1 Brief Historic Review on Ring Chromosome 4 Chromosome 4 is a large submetacentric (B-group) chromosome comprising almost 6.5% of the total human genome, or ~190 Mb of DNA. The earliest clinical reports of constitutional ring chromosome 4 (RC4) predated the

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_8

93

94

development of banding techniques and were reliant on autoradiographic data to differentiate individual chromosomes. What is believed to be the first case report of a patient with a RC4 was published in 1969 (Carter et al. 1969). The patient was a newborn with multiple congenital anomalies who died at one month of age. Karyotype analysis from peripheral blood (PB) lymphocytes and sternal bone marrow (BM) cultures demonstrated the presence of a large ring replacing one B-group chromosome; however, assignment to chromosome 4 or 5 by autoradiography was precluded by the patient’s demise. Consequently, the origin of the ring was deduced to be likely of chromosome 4 origin by comparing the congenital anomalies of the patient to reported cases with deletions of B-group chromosomes. The phenotype did not resemble the recently characterized ‘cri-du-chat’ syndrome, associated with partial loss of the short arm of chromosome 5 (including one case with RC5), but rather the patient shared abnormalities with reported cases with deletions of the short arm of chromosome 4. The association of RCs with congenital anomalies and intellectual disability (ID) was recognized in the early days of human cytogenetics and attributed to partial monosomy for distal chromosome segments lost during ring formation. Prior to the advent of banding methods that allowed unequivocal assignment of the ring origin, several case reports of probable RC4 were published (Carter et al. 1969; Bobrow et al. 1971; Surana et al. 1971; Bofinger et al. 1973; Parker et al. 1974). The first positive identification of a RC4 by G-banding revealed a large ring with no discernable loss of material (Niss and Passarge 1975). Despite the suspected differences in composition of the RC4, a pattern of clinical features began to emerge from these early reports (Carter et al. 1969; Parker et al. 1974; Niss and Passarge 1975; McDermott et al. 1977). Low birth weight, small stature, microcephaly (often proportionate), and hypoplastic thumbs with or without radial ray defects were common features. In addition, some cases fit the clinical pattern of anomalies associated with

K. M. Bone et al.

the newly defined Wolf–Hirschhorn syndrome (WHS, OMIM#194190) (Hirschhorn et  al. 1965) associated with partial monosomy for the short arm of chromosome 4 (Pérez-Castillo and Abrisqueta 1977; del Mazo et al. 1978). In some patients with RC4, the genetic imbalance is minimal. In 1971, the first report of a RC in a patient with normal intelligence and no congenital anomalies was identified during investigations for proportional small stature (37 weeks) or late preterm (34–36 weeks) with 15 and 7 cases, respectively. Three were born moderate preterm between 32 and 34 weeks, and three were born post term at 42 weeks or later. The mode of delivery was not reported for most cases. Minimally, 6/47 were by C-section, two for breech presentation and one for fetal distress. There was one case with an abnormal maternal serum screen, which indicated a positive screen for Down syndrome (Akbas et al. 2013). The fetus presented with IUGR. First trimester or maternal screening results were not available for any other case in this group. There were no noted placental or cervical complications. Only four cases in this group had prenatal testing, one was for the abnormal maternal serum screen, and the others were for IUGR and/or congenital anomalies (Akbas et al. 2013; Chen et al. 2007; Kocks et al. 2002; Sherer et al. 1991).

8  Ring Chromosome 4

8.3.2 Neonatal, and Pediatric and Adult Characteristics Neonatally, most patients presented with delayed growth across all parameters, with body weight, height, and head circumference typically noted to be well below the 10% percentile. Detailed clinical information for all RC4 cases is outlined in Table 8.2. Severe growth delays and microcephaly, as well as failure to thrive, appear to be a common feature that typically persists into the pediatric and adult periods with 91% of reported cases described to have this finding. Dysmorphic facial features were noted in almost all cases where sufficient clinical information was provided (81%), with commonalities including abnormal ears, iris coloboma, hypertelorism, small mouth, small chin, short neck, and an abnormal nose. Cleft lip and or palate was also present in a number of cases (21% of described cases). Hand and foot anomalies were common (70% of cases with sufficient clinical information). Structural abnormalities were present, affecting multiple organ systems, most commonly cardiac, genitourinary (GU), and genitourinary (GI), as was developmental delay (DD) and/or intellectual disability (ID). Patients who had short-arm deletions including the WHS CR were more likely to present with clinical features overlapping with WHS. Only one patient was described with clinical features that were similar to 4q- microdeletion syndrome (Hou and Wang 1996). Early neonatal death was noted in seven cases. The first case died minutes post live birth (Sherer et al. 1991). This case has been extensively described in the prenatal section of this chapter. The second case presented at birth with tachycardia and intermittent tachypnea, as well as signs of congenital heart failure 3 days before death (Carter et al. 1969). This patient died at 4 weeks due to renal failure and terminal bronchopneumonia. At autopsy, multiple structural abnormalities were noted, including brain hypoplasia, patent foramen ovale, abnormal diaphragm structure, incomplete bowel rotation, and hypoplastic, cystic kidneys. The

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third patient contracted pneumonia and died at 94 days due to heart failure (Bofinger 1973). He was born with a J-shaped deformity of right forearm ending in two long, tapered digits with fused third and fourth metacarpals and missing the first and second metacarpals, and, post-mortem, all other bones were found to be hypoplastic. He was also noted to have an atrial septal defect (ASD) and an accessory spleen. Case four died of pneumonia at 5 days and no structural abnormalities noted (Pérez-Castillo and Abrisqueta 1977). The fifth case was born with metabolic acidosis and was deceased at day three of birth due to what was described as amniotic fluid aspiration (del Mazo at el. 1978); skeletal anomalies (agenesis of the radii, bilateral pes valgus), and interventricular communication with multiple bilateral pulmonary hemorrhages were present at autopsy. Case six was born after induction and C-section at 36 weeks due to fetal distress; resuscitation failed, and this patient died at 15 min post-live birth (Fryns et al. 1988). A chest X-ray revealed a small chest, bilateral pneumothoraces, and massive pneumopericardium. Bilateral renal agenesis and Potter sequence, which was suspected prenatally, were confirmed on ultrasound. The final case had a heart murmur and ductus arteriosus, and right-sided renal agenesis; this patient died at 10 weeks due to respiratory distress, cardiac failure, and renal impairment (Paththinige et al. 2016). Seven cases were noted with minor interventions after admission to the neonatal intensive care unit (NICU) or nursery due to small size (Surana et al. 1971; Parker et al. 1974; Lee et al. 2005), poor feeding and failure to gain weight (Finley et al. 1981; Gutkowska et al. 1985; Anderson et al. 1997), frequent episodes of vomiting, loose stools, and upper respiratory tract infections (Surana et al. 1971), as well as transient tachypnea of newborn, hyperbilirubinemia, and transient hyperglycemia (Phillips et al. 2021). One case had major neonatal interventions (Hou and Wang 1996). This case presented prenatally with a dilated bowel, suspicious for a bowel obstruction, which was confirmed at day

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two of life by laparotomy after frequent episodes of vomiting. This patient had significant structural anomalies noted, including dextrocardia with situs solitus, patent ductus arteriosus, incomplete patent foramen ovale with left to right shunt, complete absence of whole left upper extremity, short bowel with segmental dilatation below duodenum, and midgut malrotation. During the NICU course, an excision of an atrophic accessory spleen and resection of the dilated bowel loop with subsequent end-to-end anastomosis was performed surgically. Clinical information was available for the pediatric period in 39 of the reviewed cases (Table 8.2). Severe growth delays were reported in almost all cases, a common feature typically associated with ring syndrome. Short stature was also a shared feature. Significant dysmorphic features, similar to what was described in patients presenting in the neonatal period, were common, including hypertelorism, small chin/ micrognathia, and abnormal ears. Other clinical features, including limb anomalies, cleft lip and or palate, and structural abnormalities were shared with those cases presenting in the neonatal period. Developmental delay and/ or intellectual disability were also a frequently described features; however, normal or nearnormal neurocognitive development was present in 10 of the cases reviewed (Surana et al. 1971; Chavin-Colin et  al. 1977; Freyberger et al. 1991; Sigurdadottir et al. 1999; Sodré et al. 2010; Dominguez et  al. 2010; Burgemeister et al. 2017; Zhonghua et al. 2020; Phillips et al. 2021 (2 cases)). Seizures were noted in five cases (Bobrow et al. 1971; Parker et al. 1974; Anderson et al. 1997; Lee et al. 2005; Laleye et al. 2006). One patient died at age 6 years due to complications post-tonsillectomy (Young and Zalneraitis 1980). This patient was noted to have difficulty swallowing at 6 months of age, and, at 18 months, had persistent vomiting with malrotation of the bowel and spina bifida occulta. Across all ages and cases reviewed, significant structural abnormalities were noted, the most common being those involving three main systems: cardiac (seen in 13 cases), GU (19

K. M. Bone et al.

cases), and GI (7 cases). Skeletal anomalies were also often present, with various abnormalities of both the hands and feet described, the most common being fifth finger clinodactyly, which was present in 12 of the reported cases. There appeared to be no sex-specific of patients diagnosed with RC4, as 45% were female, and 55% were male. Terminal deletions of 4q, especially when the breakpoint is at q31 or q32, have been associated with recurring findings including developmental delay, cleft palate and occasionally cleft lip, small chin that has been described as Pierre Robin sequence, as well as abnormal fingers and toes, including a distinctive fifth finger nail, as well as overlapping toes (Lin et al. 1988). None of the cases had described clinical features consistent with these features and the majority of cases had distal breakpoints, especially at q35, which may account for that difference. Reproduction outcomes were available in two of the cases studied. The first case was a female who presented at 27 years with infertility and short stature (Lee et al. 2005). The patient had seizures of unknown etiology at 10 months, strabismus of the right eye that was corrected with surgery at 23 years and was noted to be both physically and developmentally delayed. The patient received genetic counseling and became spontaneously pregnant with a chromosomally normal male. The second case was a male who presented at 23 years with oligospermia and an elevated follicle stimulating hormone (Yao et al. 2014). He was noted on exam to have dysmorphisms similar to WHS, intellectual disability and his right scrotal testis was soft and small. A testicular biopsy showed a reduction in the primary spermatocytes, with reduced recombination present (45.9 foci/cell compared to 47.8 in the normal control), a defect in prophase 1 meiosis and synapse formation, leading to spermatogenic arrest. This suggests that RC4 may affect fertility; however, as most patients were not followed long-term into reproductive age, there is not enough data for a definitive link to be established. A cancer predisposition was not noted in any of the reported cases.

8  Ring Chromosome 4

In all reported cases, there was no evidence of transmission of a RC4 from an affected carrier parent. There is one report of recurrence of a RC4 in siblings, due to presumed maternal gonadal mosaicism (Phillips et al. 2021). This family received appropriate genetic counseling on reproductive risks. Seven cases reviewed required medical intervention: three patients were treated medically for seizures; the type of seizures were not described (Bobrow et al. 1971; Parker et al. 1974; Anderson et al. 1997); one patient was treated with alpha 1 methionine for possible renal metabolic acidosis (Surana et al. 1971); one patient presented at 15 years with type 2 diabetes, treated with 5 mg glipizide daily and developed subsequent hyperlipidemia with obesity at 22 years, treated with atorvastatin 10 mg daily followed by a change from glipizide to troglitazone and then pioglitazone 30 mg/ day, where HbA1c subsequently normalized (Blackett et al. 2005); and growth hormone was given to one case for delayed growth parameters (Burgemeister et al. 2017).

8.3.3 Genotype–Phenotype Correlations There were 13 reported cases with a CMA result, although not all cases provided sufficient details to review OMIM gene content, such as only providing BAC names or not including the human genome build (Table 8.1). There were ten cases with information, including one case with no copy number changes on microarray. Regarding the chromosome 4 short arm, the breakpoints were variable. The largest deletion was 8.6 Mb; however, that patient also had a 7 Mb deletion on the long arm. Most 4p deletions were under 1 Mb in size, and a review of the OMIM genes did not reveal a clear gene causative for the features. One case had a terminal deletion and an adjacent duplication, and two sisters with the same CMA result had a terminal 4p duplication. There were deletions of the long arm of chromosome 4 in a number of cases. The breakpoints also varied, but of note, most were

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smaller than 1–2 Mb, and there are no OMIM disease genes in this region. There were two cases with larger deletions, and the genes did not clearly explain the phenotype based on our current understanding. There were no reported duplications in these cases. At this time, it is difficult to summarize any genotype–phenotype correlations other than when the WHS CR is deleted on 4p. This limited number of cases highlights the importance of completing CMA in more cases to further understand genotype– phenotype correlations in RC4. It was noted a few times that cases with a RC4 that did not include loss or gain of OMIM genes often present with normal neurodevelopment, but demonstrate poor growth. The majority of cases with a CMA also had karyotypes that demonstrated more than one cell line with variable large chromosome 4 imbalances, including but not limited to an extra RC4 and monosomy 4 due to loss of the RC4, resulting from mitotic chromosome instability. It is likely that at least some of the clinical features, particularly prenatal and postnatal growth restriction, can be attributed to these larger imbalances related to chromosome instability, also referred to as general ring syndrome.

8.4 Summary This chapter reviews 47 constitutional cases of RC4 including cytogenetic and genomic results and clinical findings. Mitotic instability, resulting in ring loss and dicentric structures, is a common in vitro feature. While most cases have terminal deletions of both the short and long arms, others have no demonstrable losses, and some cases have both gains and losses suggesting differing mechanisms of ring formation. There appear to be no specific hot spots for rearrangement, although breakpoints are clustered in the distal ends resulting in large rings which are clinically more tolerated than smaller ones with larger imbalances. A significant proportion presents with features of 4p deletion and WHS. There are only rare cases with significant features of 4q

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deletion. The most common associated finding was prenatal or early onset growth restriction that persists to adulthood. Congenital anomalies were common, most frequently involving the limbs, heart, and genitourinary tract. Based on this review, it is recommended to obtain baseline imaging for heart and renal anomalies, as well as assessment of vision, hearing, and neurodevelopmental, with early intervention when needed. Given at least one family with recurrence in two siblings due to presumed maternal gonadal mosaicism, genetic counseling for this possibility and to review prenatal testing options would be recommended.

References Akbas H, Cine N, Erdemoglu M, Atay AE, Simsek S, Turkyilmaz A, Fidanboy M (2013) Prenatal diagnosis of 4p and 4q subtelomeric microdeletion in de novo ring chromosome 4. Case Rep Obstet Gynecol 2013(1):1–5. https://doi.org/10.1155/2013/248050 Anderson CE, Wallerstein R, Zamerowski ST, Witzleben C, Hoyer JR, Gibas L, Jackson LG (1997) Ring chromosome 4 mosaicism coincidence of oligomeganephronia and signs of Seckel syndrome. Am J Med Genet 72(3):281–285. https://doi.org/10.1002/ (sici)1096-8628(19971031)72:33.0.co;2-u Balcı S, Engiz Ö, Aktaş D, Vargel I, Beksaç MS, Mrasek K, Vermeesch J, Liehr T (2006) Ring chromosome 4 and Wolf-Hirschhorn syndrome (WHS) in a child with multiple anomalies. Am J Med Genet 140A(6):628–632. https://doi.org/10.1002/ ajmg.a.31131 Bartram CR (1977) Sister chromatid exchanges in a ring chromosome 4. Cytogenet Genome Res 18(4):238– 241. https://doi.org/10.1159/000130766 Blackett PR, Li S, Mulvihill JJ (2005) Ring chromosome 4 in a patient with early onset type 2 diabetes, deafness, and developmental delay. Am J Med Genet 137A(2):213–216. https://doi.org/10.1002/ajmg.a.20386 Burgemeister AL, Daumiller E, Dietze-Armana I, Klett C, Freiberg C, Stark W, Lingen M, Centonze I, Rettenberger G, Mehnert K (2017) Continuing role for classical cytogenetics: Case report of a boy with ring syndrome caused by complete ring chromosome 4 and review of literature. Am J Med Genet 173(3):727–732. https://doi.org/10.1002/ajmg.a.38063 Bobrow M, Joness LF, Clarke G (1971) A complex chromosomal rearrangement with formation of a ring 4. J Med Genet 8(2):235–239. https://doi.org/10.1136/ jmg.8.2.235

K. M. Bone et al. Bofinger MK (1973) Reduction malformations and chromosome anomalies. Arch Pediatr Adolesc Med 125(1):135–143. https://doi.org/10.1001/ archpedi.1973.04160010095024 Calabrese G, Giannotti A, Mingarelli R, Di Gilio MC, Piemontese MR, Palka G (1997) Case Report: Two newborns with chromosome 4 imbalances: Deletion 4q33 → q35 and ring r (4) (pterq35.2qter). Clin Gen 51(4):264–267. https://doi. org/10.1111/j.1399-0004.1997.tb02467.x Carter R, Baker E, Hayman D (1969) Congenital malformations associated with a ring 4 chromosome. J Med Genet 6(2):224–227. https://doi.org/10.1136/ jmg.6.2.224 Chavin-Colin F, Turleau C, Limal JM, de Grouchy J (1977) Anneau du chromosome 4 II.—Sans dysmorphie faciale [Ring of the chromosome 4. II. Without facial dysmorphism]. Ann Genet 20(2):105–109 Chen CP, Hsu CY, Tzen CY, Lee CC, Chen WL, Chen LF, Wang W (2007) Prenatal diagnosis of mosaic ring chromosome 4. Prenat Diagn 27(5):485–487. https:// doi.org/10.1002/pd.1717 Chen CP, Lin SP, Su YN, Chern SR, Tsai EJ, Wu PC, Lee CC, Wang W (2011) Mosaic ring chromosome 4 in a child with mild dysmorphisms, congenital heart defects and developmental delay. Genet Couns 22(3):321–326 Concolino D, Rossi E, Strisciuglio P, Iembo MA, Giorda R, Ciccone R, Tenconi R, Zuffardi O (2007) Deletion of a 760 kb region at 4p16 determines the prenatal and postnatal growth retardation characteristic of Wolf-Hirschhorn syndrome. J Med Genet 44(10):647–650. https://doi.org/10.1136/ jmg.2007.050963 del Mazo J, Abrisqueta JA, Pérez-Castillo A, Aller V, Lucas MAM, de Torres ML, Martín MJ (1978) Partial deletion of 4p16 band in a ring chromosome and Wolf syndrome. Hum Genet 44(1):105–108. https://doi.org/10.1007/BF00283580 Dominguez MG, Barros-Núñez P, González-Ramos IA, Rivera H (2010) Variegated-like mosaicism and ring syndrome in a r(4) boy. Appraisal of 38 patients with a fairly complete ring 4. Genet Couns 21(4):411–422 Finley WH, Finley S, Chonmaitree T, Koors J, Chandler W (1981) Ring 4 chromosome with terminal p and q deletions. Arch Pediatr Adolesc Med 135(8):729-731. https://doi.org/10.1001/ archpedi.1981.02130320043015 Fraisse J, Lauras B, Couturier J, Freycon F (1977) Ring of the chromosome 4. I – with 4p- phenotype. Ann Genet 20(2):101–104 Freyberger G, Wamsler C, Schmid M (1991) Ring chromosome 4 in a child with mild dysmorphic signs. 39(2):151–155. https://doi.org/10.1111/j.13990004.1991.tb03003.x Fryns JP, Kleczkowska A, Jaeken J, Van den Berghe H (1988) Ring chromosome 4 mosaicism and Potter sequence. Ann Genet 31(2):120–122

8  Ring Chromosome 4 Giuffrè L, Cammarata M, Corsello G, Benigno V, Graziano L, Roccella F, Balsamo V (1987) [Ring chromosome 4 in twins] Pediatr Med Chir 9(3):349–350 Guilherme RS, Meloni VF, Kim CA, Pellegrino R, Takeno SS, Spinner NB, Conlin LK, Christofolini DM, Kulikowski LD, Melaragno MI. Mechanisms of ring chromosome formation, ring instability and clinical consequences. BMC Med Genet 12(1):171. https://doi.org/10.1186/1471-2350-12-171 Gutkowska A, Krajewska-Walasek M, Wiśniewski L (1985) Ring chromosome 4: 46,XY,r (4)(p16q35) in a boy. Klin Padiatr 197(4):294–296. https://doi.org/10 .1055/s-2008-1033986 Halal F, Vekemans M (1990) Ring chromosome 4 in a child with duodenal atresia. Am J Med Genet 37(1):79–82. https://doi.org/10.1002/ ajmg.1320370118 Hirschhorn K, Herbert LC, Firschein IL (1965) Deletion of short arms of chromosome 4–5 in a child with defects of midline fusion. Hum Genet 1(5):479-482. https://doi.org/10.1007/BF00279124 Hou JW, Wang TR (1996) Amelia, dextrocardia, asplenia, and congenital short bowel in deleted ring chromosome 4. J Med Genet 33(10):879–881. https://doi. org/10.1136/jmg.33.10.879 Kim JH, Oh PS, Na HY, Kim S-H, Cho HC (2009) A case of mosaic ring chromosome 4 with subtelomeric 4p deletion. Ann Lab Med 29(1):77–81. https://doi. org/10.3343/kjlm.2009.29.1.77 Kishi Y, Ikeda H (2018) A case of thumb polydactyly which ulnar thumb has no active motion in ring chromosome 4. J Hand Surg Asian-Pac 23(4):566–570. https://doi.org/10.1142/s2424835518720311 Kocks A, Endele S, Heller R, Schröder B, Schäfer H, Städtler C, Makrigeorgi-Butera M, Winterpacht A (2002) Partial deletion of 4p and 4q in a fetus with ring chromosome 4: Phenotype and molecular mapping of the breakpoints. 39(5):23e–223. https://doi. org/10.1136/jmg.39.5.e23 Kosztolányi G (1985) Ring chromosome 4: Wolf syndrome and unspecific developmental anomalies. Acta Paediatr Hung 26(2):157–165 Kosztolányi G, Opitz JM, Reynolds JF (1987) Decreased cell viability of fibroblasts from two patients with a ring chromosome: An in vitro reflection of growth failure? Am J Med Genet 28(1):181–184. https://doi. org/10.1002/ajmg.1320280125 Laleye A, Alao MJ, Adjagba M, Hans C, Delneste D, Gnamey DK, Ayivi B, Darboux RB (2006) Wolf Hirshhorn syndrome in a case of ring chromosome 4: Phenotype and molecular cytogenetic findings. Genet Couns 17(1):35–40 Lee MH, Park SY, Kim YM, Kim JM, Yoo KJ, Lee HH, Ryu HM (2005) Molecular cytogenetic characterization of ring chromosome 4 in a female having a chromosomally normal child. Cytogenet Genome Res 111(2):175–178. https://doi.org/10.1159/000086389 Lin AE, Garver KL, Diggans G, Clemens M, Wenger SL, Steele MW, Jones MC, Israel J, Opitz JM, Reynolds

111 JF (1988) Interstitial and terminal deletions of the long arm of chromosome 4: Further delineation of phenotypes. Am J Med Genet 31(3):533–548. https:// doi.org/10.1002/ajmg.1320310308 Lyu Y, Song F, Zhang K, Gao M, Ma J, Wang D, Wan Y, Liu Y, Gai Z (2020) Analysis of clinical and genetic characteristics of a child with ring chromosome 4 syndrome. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 37(8):843–846. https://doi.org/10.3760/cma.j. issn.1003-9406.2020.08.009 McDermott A, Voyce MA, Romain D (1977) Ring chromosome 4. J Med Genet 14(3):228–232. https://doi. org/10.1136/jmg.14.3.228 Niss R, Passarge E (1975) Derivative chromosomal structures from a ring chromosome 4. Hum Genet 28(1):9–23. https://doi.org/10.1007/bf00272478 Parker CE, Alfi O, Derencsenyi A, Mavalwala J, Donnell G (1974) A child with a ring-4 chromosome (46, XX/46, XX, r 4). Arch Pediatr Adolesc Med 128(3):371–374. https://doi.org/10.1001/ archpedi.1974.02110280101015 Paththinige CS, Sirisena ND, Kariyawasam UGIU, Saman Kumara LPC, Dissanayake VHW (2016) Ring chromosome 4 in a child with multiple congenital abnormalities: A case report and review of the literature. Case Rep Genet 2016(1):1–7. https://doi. org/10.1155/2016/4645716 Paz-y-Miño C, Proaño A, Verdezoto SD, García JL, Hernández-Rivas JM, Leone PE (2019) Clinical, cytogenetic, and molecular findings in a patient with ring chromosome 4: Case report and literature review. BMC Med Genomics 12(1):167. https://doi. org/10.1186/s12920-019-0614-4 Perez-Castillo A, Abrisqueta JA (1977) Ring chromosome 4 and Wolf syndrome. Hum Genet 37(1):87–91. https://doi.org/10.1007/bf00293777 Phillips EA, Caluseriu O, Schlade-Bartusiak K, Chernos J, McLeod DR, Thomas MA (2021) Clinical and molecular characterization of an almost complete ring chromosome 4 in two sisters, with recurrence due to gonadal mosaicism. 30(4):173–176. https:// doi.org/10.1097/mcd.0000000000000382 Sherer D, Shah Y, Wang N, Metlay L, Woods J (1991) Prenatal diagnosis and subsequent management of a fetus with a 46XY r(4)(p15–q35) karyotype. Amer J Perinatol 8(1):53–55. https://doi. org/10.1055/s-2007-999342 Sigurdardottir S, Goodman BK, Rutberg J, Thomas GH, Jabs EW, Geraghty MT (1999) Clinical, cytogenetic, and fluorescence in situ hybridization findings in two cases of complete ring syndrome. Am J Med Genet 87(5):384–390. https://doi.org/10.1002/ (sici)1096-8628(19991222)87:53.0.co;2-r Sodré CP, Guilherme RS, Meloni VFA, Brunoni D, Juliano Y, Andrade JAD, Belangero SIN, Christofolini DM, Kulikowski LD, Melaragno MI (2010) Ring chromosome instability evaluation in six patients with autosomal rings. Genet Mol Res 9(1):134–143. https://doi.org/10.4238/vol9-1gmr707

112 South ST, Bleyl SB, Carey JC (2007) Two unique patients with novel microdeletions in 4p16.3 that exclude the WHS critical regions: Implications for critical region designation. Am J Med Genet 143A(18):2137–2142. https://doi.org/10.1002/ ajmg.a.31900 Soysal Y, Balci S, Hekimler K, Liehr T, Ewers E, Schoumans J, Bui TH, Içduygu FM, Kosyakova N, Imirzalioğlu N (2009) Characterization of double ring chromosome 4 mosaicism associated with bilateral hip dislocation, cortical dysgenesis, and epilepsy. Am J Med Genet 149A(12):2782–2787. https://doi. org/10.1002/ajmg.a.33069

K. M. Bone et al. Surana RB, Bailey JD, Conen PE (1971) A ring-4 chromosome in a patient with normal intelligence and short stature. J Med Genet 8(4):517–521. https://doi. org/10.1136/jmg.8.4.517 Yao Q, Wang L, Yao B, Gao H, Li W, Xia X, Shi Q, Cui Y (2014) Meiotic prophase I defects in an oligospermic man with Wolf-Hirschhorn syndrome with ring chromosome 4. Mol Cytogenet 7(1):45. https://doi. org/10.1186/1755-8166-7-45 Young RS, Zalneraitis EL (1980) Neurological and neuropathological findings in ring chromosome 4. J Med Genet 17(6):487–490. https://doi.org/10.1136/ jmg.17.6.487

9

Ring Chromosome 5 Jingwei Yu

Abstract

Human ring chromosome 5 (RC5) is rare, only accounts for approximately 1.3% of the reported human ring chromosomes (RCs). Cri-du-Chat syndrome and ring syndromeassociated phenotypes are the most common clinical findings in individuals with RC5 when the ring replaces a normal chromosome 5. Small supernumerary ring chromosome 5 (sSRC5) may result in different phenotypes. In addition, RC5-derived mosaicism is common due to ring instability. RC5 can be correctly identified by chromosome karyotyping and further analyzed by fluorescence in situ hybridization (FISH) using targeted locusspecific and chromosome painting probes on interphases and metaphases. The severity of the phenotypes resulting from a RC5 appears to be associated with the size and content of the deletion or duplication caused by the RC5. Therefore, detailed characterization using advanced genomic technologies to define breakpoints, genomic imbalance, and gene content in the RC5 for appropriate clinical assessment of affected individuals is recommended. Most patients of RC5 were J. Yu (*)  Department of Laboratory Medicine, University of California, San Francisco, 185 Berry Street, Suite 290, San Francisco, CA 94107, USA e-mail: [email protected]

de novo and postnatally diagnosed. However, inherited and prenatally diagnosed RC5 cases have been reported. Thus, parental studies and genetic counseling for newly diagnosed prenatal and postnatal RC5s are warranted.

Keywords

Ring chromosome 5 (RC5) · Cri-du-Chat syndrome (CdCs) · Phenotypic presentation · Karyotype–phenotype correlations · Cytogenomic analysis

9.1 Introduction Chromosome 5 is the fifth largest human chromosome that spans approximately 181 million base pairs of nucleotides, representing almost 6% of the total human genomic DNA. It carries approximately 900 protein coding and 1300 non-coding RNA genes, including many known disease-causing genes (Schmutz et al. 2004, Ensemble Human Map View 2023). However, the gene density of chromosome 5 is among the lowest within the human genome, partially due to numerous gene-poor regions carried by the chromosome. These regions display a remarkable degree of non-coding and syntenic conservation with non-mammalian vertebrates, suggesting they are functionally constrained (Schmutz et al. 2004).

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_9

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Ring chromosome 5 (RC5) is a rare chromosomal abnormality that may result in genomic imbalance and cause various health problems. Fewer than 30 cases of RC5 have been reported in English, only accounting for approximately 1.3% of the reported human ring chromosomes (RCs), disproportional to the genomic size of chromosome 5 in the human genome (Li et al. 2022). Cases of RC5 can be classified into two distinguishable groups based on their size and genomic dosage effect: large RC5s that contain 80% or more of chromosome 5 content and usually replace a normal chromosome 5 in affected cells, and small RC5s that usually occur in affected cells as an additional small supernumerary ring chromosome (sSRC). This chapter mainly discusses the large RC5s. Phenotypes associated with Cri-du-Chat syndrome (CdCs) (OMIM #123450) caused by deletions within the short arm of chromosome 5 were the most common phenotypic features reported in cases with a large RC5. Small supernumerary rings (including sSRC5) and their pathogenic impacts are described in Chap. 25 of this book and can be also found elsewhere (Liehr 2023). The first case of RC5 was reported in a child with multiple anomalies that represented clinical features of CdCs in 1965 prior to the development of chromosome banding technology (Rohde and Tompkins 1965). The origin of the ring was identified based on a missing chromosome 5 that was replaced by the ring and the patient’s clinical presentation of CdCs that was known to be associated with a deletion within the short arm of chromosome 5 (5p). Except for a deletion in 5p, the breakpoints and the size of the deletion(s) of chromosome 5 in the RC5 were not known. Similar approaches were used for detection and analysis of at least one more RC5 case (Steele et al. 1966). During 1970s and 1980s chromosome banding technology, including Q-, G- and R-banding, had been applied to analysis of RC5, making identification of the breakpoints involved in RC5 and karyotype– phenotype analysis possible (Nakagome et al. 1973; Suerinck et al. 1978; Kula et al. 1981; Flannery et al. 1988). Since 2002, advanced

J. Yu

cytogenetic technologies, including fluorescence in  situ hybridization (FISH), chromosome microarray analysis (CMA), and multiplex ligation-dependent probe amplification (MLPA), have been used to further characterize RC5 in detail for better clinical assessment (Sizonenko et al. 2002; Ohashi et al. 2010; Basinko et al. 2012; Nozawa et al. 2020). However, the knowledge regarding the genomics and pathogenesis of RC5 is still very limited due to lack of cases available for detailed genomic analysis.

9.2 Laboratory and Clinical Findings 9.2.1 Diagnosis of RC5 All reported cases of RC5 were diagnosed by chromosome banding analysis. Despite of continuous development of advanced genomic technologies, cytogenetic analyses, including chromosome banding and FISH using painting and specific probes on interphases and metaphases, are still the only effective methods to correctly identify and verify RCs. Eighteen reported cases of large RC5 were summarized in Table 9.1. Based on these data, the ratio of RC5 affected males to females is estimated to be 0.64 (7/11). Few data regarding the parental origins of RC5 have been reported. All cases of large RC5 appeared to have a simple ring structure that was formed by cyclization of a portion of breaking-off chromosome 5 without additional rearrangements, although complex rings were reported in other RCs. Dynamic mosaicism with ring-derived abnormalities, such as double-ring, broken-ring, additional ring(s), and loss of ring to monosomy, is common in individuals with RC5 due to the instability of RCs. It is important to distinguish constitutional true mosaicism of RC5 from dynamic mosaicism by instable RC5. Constitutional mosaic RC5, e.g., an individual with both RC5 and normal cell populations, has been reported (Hu et al. 2018).

9  Ring Chromosome 5

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Table 9.1  Reported 18 cases of RC5 Case

Age at the Testing diagnosis methods

Reported karyotype

CdCs

RC5-1

Infant

SS

46,XX,r(5)

RC5-2

18 m

SS

RC5-3

4 m

RC5-4

Additional phenotypes

Heritage

References

+

De novo

Rohdeand and Tompkins (1965)

46,XX,r(5)a

+

De novo

Steele et al. (1966)

G

46,XYq+,r(5)(p15q35)a

+

De novo

Nakagome et al. (1973)

Infant

R

46,XY,r(5)(p?15q?35)a

+

De novo

Suerinck et al. (1978, case 1)

RC5-5

6y

Q, R

46,XY,r(5)(p15.2q35.2)a

+

De novo

Suerinck et al. (1978, case 2)

RC5-6

7y

Q, G

46,XY,r(5)(p15q35)a

+

Dental ano- De malies novo

RC5-7

2.5 m

Q, G

46,XY,r(5)(p?14q?33)a

+

De novo

RC5-8

4y

Q, G, R, SEM

46,XX,r(5)(p15.33q35.3)mata

Ring syndrome phenotypes

Fami- MacDermot et al. (1990, lial, mater- case 1) nally inherited

RC5-9

30 y

Q, G, R, SEM

46,XX,r(5)(p15.33q35.3)a

Ring syndrome phenotypes

Fami- MacDermot et al. (1990, lial, passed case 2) to daughter

RC5-10

4 m

Q, G, R, SEM

46,XX,r(5)(p15.33q35.3)a

Ring syndrome phenotypes

De novo

MacDermot et al. (1990, case 3)

RC5-11

3y

Q, G, R

46,XX,r(5)(p15.3q35.3)a

Ring syndrome phenotypes

De novo

Migliori et al. (1994)

RC5-12

3y

G

46,XX,r(5)(p14q35)[80]/ 45,XX,-5[8]/ 47,XX,r(5),+r(5)[3]

+

Oculo-auri- De culo-verteb- novo ral spectrum (OAVS)

Caba et al. (2012, case 1)

RC5-13

Infant

Q, G, MLPA, FISH, CP

46,XY,r(5)(p13.2q35.3)[183]/ 45,XY,-5[30]

+

Cardiac abnormalities, hypoplasia of both kidneys

De novo

Basinko et al. (2012)

RC5-14

3y

G?

46,XY,r(5)

+

No testis

De novo

Hu et al. (2018, case 1)

RC5-15

18 m

G?

46,XX,r(5)

+

De novo

Hu et al. (2018, case 2)

RC5-16

28 m

G?

46,XX,r(5)

+

Not tested

Hu et al. (2018, case 3)

Kula et al. (1981) Flannery et al. (1988)

(continued)

116

J. Yu

Table 9.1  (continued) Case

Age at the Testing diagnosis methods

Reported karyotype

CdCs

RC5-17

6 m

G?

Mos 46,XX,r(5)/46,XX

+

RC5-18

17 y

G, CMA

46,XX,r(5)(p14.3q35.3)

+

Additional phenotypes

Heritage

References

De novo

Hu et al. (2018, case 4)

Refractory Not reporcytopeted nia with multilineage dysplasia

Nozawa et al. (2020)

Abbreviations used in age at the diagnosis: m—month, y—year; Testing methods: CMA—chromosome microarray; CP—chromosome painting; FISH—fluorescence in situ hybridization; G—G-bands; MLPA—multiplex ligation-dependent probe amplification; Q—Q-bands; R—R-bands; SEM—scanning electron microscopy; SS—solid staining. a Dynamic mosaicism derived from RC5 was observed but not described in the karyotype nomenclature in these cases

9.2.2 Characterization of RC5 Chromosome banding analysis was the only technology to characterize RC5 in early days until 1990 when MacDermont et al. characterized ring structure and integrity of three cases of RC5 using scanning electron microscopy (SEM); however, this technology would not be able to distinguish the origin and band pattern of the rings (MacDermot et al. 1990). In these three cases, chromosome analysis on 100 to 200 metaphase cells detected dynamic mosaicism in 11–26% of cells, in which 3–7% of cells showed no RC5 but with a marker present and 2–10% of cells showed loss of the RC5 to monosomy 5. Since 2002, other molecular and genomic technologies, such as FISH, MLPA, and CMA, have been used for characterization of RC5 (Sizonenko et al. 2002; Ohashi et al. 2010; Basinko et al. 2012; Nozawa et al. 2020). Now in this post Human Genome Project era, a variety of genomic technologies, from well-adapted CMA to various emerging methods of nextgeneration sequencing (NGS), can be used to characterize RC5 and any other RC to define the breakpoints, imbalance involving genomic/gene content, additional rearrangements, and other genomic alterations involved in the ring with high resolution and accuracy. However, these molecular and genomic technologies, except for chromosome analyses, are yet apparently insufficient to verify the ring structure.

9.2.3 Clinical Features of Individuals with RC5 RC5-associated abnormalities include a broad spectrum of constitutional abnormalities, such as neonatal mewing cry, severe developmental delay, intellectual disability, short stature, hypotonia, dysmorphic features (including microcephaly, facial asymmetry, hypertelorism, epicanthal folds, abnormal ears, micro/retrognathia), congenital cardiac anomalies (such as atrial and ventricular septal defect, tricuspid insufficiency, and hypoplastic aorta), skeletal abnormalities (e.g., hypoplastic thumbs, anomalous ulna/radius, dysplastic metacarpals, and phalanges), and hematologic neoplasms (Genetic and Rare Diseases Information Center 2023). These abnormalities usually show high phenotypic variability, most likely due to differences in size, structure, genomic dosage, and instability of RC5. It is noteworthy that most of these abnormalities apparently represent or are overlapped with CdCs-associated phenotype (Table 9.2). In addition, some of the abnormalities, including growth retardation, short stature, and mild facial dysmorphism, have been seen in individuals with other constitutional RCs, or in a condition so called “ring syndrome” (Kosztolányi 1987). Most reported cases of RC5 were de novo and postnatally identified. There was one familial case with a mother-to-daughter transmission

9  Ring Chromosome 5

117

Table 9.2  CdCs-associated phenotype presentations (National Center for Advancing Translational Sciences 2023) Very frequent features Abnormality of the voice Cat-like cry Epicanthus (epicanthal fold; epicanthal folds; epicanthic folds; eye folds; palpebronasal fold; plica palpebronasalis; prominent eye folds) High-pitched voice Hypotonia Intellectual disability, severe Low-set, posteriorly rotated ears Microcephaly Microretrognathia Round face Severe global developmental delay Wide nasal bridge Frequent features Down-slanted palpebral fissures High palate Hypertelorism Intrauterine growth retardation (IUGR) Scoliosis Short neck Short stature Small hand Occasional features Abnormality of bone mineral density Abnormality of cardiovascular system morphology Finger syndactyly Inguinal hernia Joint hyperflexibility Pre-auricular skin tag Recurrent fractures

of the RC5 (MacDermot et al. 1990). A prenatal diagnosis of RC5 was also reported, in which a fetus  was detected carrying an abnormal chromosome 5 with a 6.1 Mb distal duplication at 5p15.33p15.31 and a 15.3 Mb interstitial deletion at 5p14.4p13.2, and a sSRC5 with a pericentric duplication of 3.4 Mb at 5p12p11. The pregnancy was uneventful, and the boy delivered at 39 weeks of gestation showed normal physical, developmental, and neurological examinations from newborn to 1 year and 6 months (Ohashi et al. 2010).

Two individuals of RC5 were reported with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but there was no clear evidence that the RC5 was the pathogenic cause of the neoplasms in the reports (Nozawa et al. 2020; Huh et al. 2012). In those cases, the neoplastic cells carried a RC5, but the critical pathogenic region known for MDS and AML on chromosome 5 was not deleted in the cells. Further analysis on the epigenetic dysregulation on the critical region in the RC5 may explain the predisposing risk for MDS and AML.

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9.3 Karyotype–Phenotype Correlations Reported cases of RC5 appeared to share a few noticeable genomic features. They were all simple rings with deletions less than 20% of the chromosome 5 in size. The breakpoints involved in the RC5 appeared to be non-randomly distributed, and the long-arm breakpoints seemed to be more restricted than the short-arm breakpoints in genomic localization (Fig. 9.1, Table 9.1). Identified long-arm breakpoints of large RC5 appeared to be clustered within the 5q35 region in 11 of 12 (91.7%) of reported cases. Only one case had a breakpoint that was mapped to the 5q33 region (Flannery et al. 1988). Within the same group of RC5, eight cases (66.7%) showed short-arm breakpoints mapped within the 5p15 region. There was evidence suggesting

Fig. 9.1  Phenotypes associated with chromosome 5p deletion clusters and deletion breakpoints of 12 reported cases of RC5. From left to right: ideograms of normal chromosome 5 and breakpoints in the RC5 from 12 patients (RC5-3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 18), showing the phenotype clusters related to the 5p

J. Yu

that the long arm of chromosome 5 was “resistant” to loss of the telomeric portion, decreasing the likelihood of a 5q deletion in the formation of RC5 (Flannery et al. 1988). Studies indicated that clinical presentations associated with a RC5 was apparently depending on the size and content of the deletion carried by the ring, but the karyotype–phenotype correlation data are limited. In 1990, MacDermot et al. reported three individuals who carried a similar large RC5 without CdCs (MacDermot et al. 1990). The authors characterized the RC5 using SEM and found that these large RC5 appeared to only have a small deletion of less than 1 Mb of DNA from chromosome 5. These individuals did not show CdCs phenotypes; instead, they had relatively mild phenotypic features of ring syndrome.

deletions and the reported breakpoints in each RC5. The normal chromosome 5 ideogram was prepared based on ISCN2020 at the 550-band level (McGowan-Jordan et al. 2020). The phenotype clusters were summarized based on reported genotype–phenotype studies (Cerruti Mainardi 2006; Nevado et al. 2021)

9  Ring Chromosome 5

Partial deletion of 5p resulting from a RC5 appeared to be more critical than the 5q deletion for phenotypic presentations of an affected individual. It was suggested that RC5 symptomatology was simply a short-arm syndrome (Suerinck et al. 1978). CdCs-associated phenotypes appeared to be the major phenotypic findings reported in individuals of RC5. Some of these phenotypes have been correlated to four deletion clusters within 5p (Fig. 9.1) (Cerruti Mainardi 2006; Nevado et al. 2021). The proposed CdCs genotype–phenotype correlations appeared to be consistent with that seen in individuals of RC5 (Table 9.1, Fig. 9.1). Partial deletions of 5q resulting from RC5 seemed to have limited phenotypic effect, and 5q deletionrelated phenotypes were usually not reported in RC5 cases. Speculated explanations for this included (1) distinctive and readily recognized 5p deletion-associated phenotypes would tend to overshadow the more subtle features of coexisting but less phenotypically flamboyant 5q deletion, and (2) 5p and 5q deletions had overlapped phenotypes (Flannery et al. 1988; Basinko et al. 2012). The average size of 5q deletions were noticeably smaller than that of 5p deletions in RC5, which might also result in fewer and less severe phenotypes (Fig. 9.1). Basinko et al. characterized a RC5 in a newborn infant with multiple technologies, including G- and Q-banding, FISH, metaphase chromosome painting, and MLPA (Basinko et al. 2012). They were able to define terminal deletions of 34.61 Mb and a 2.44 Mb on 5p and 5q, respectively, within the ring, and performed genotype–phenotype correlation studies. However, no specific phenotypic characteristics, except for those associated with CdCs, could be identified. There were a few additional phenotypes reported in individuals with RC5 (Table 9.1), but whether these phenotypes were RC5-associated could not be determined due to limited data. In addition, dynamic mosaicism with ring-derived abnormalities could make karyotype–phenotype correlations of RC5 more challenging.

119

9.4 Conclusions and Recommendations RC5s are uncommon, only account for approximately 1.3% of the reported human RCs. Less than 20 large RC5s have been reported. The average size of 5q deletions carried by these RC5s were noticeably smaller than that of the 5p deletions in the rings. CdCs-associated phenotypes were the major phenotypic findings in individuals with RC5. The phenotype severity appears to be related to the deletion size and location within the ring. CdCs phenotype would not occur if the CdCs critical region was not deleted in a RC5, but the ring carrier could still be affected by ring syndrome phenotypes, including growth retardation, short stature, and mild facial dysmorphism. No 5q deletion-related specific phenotypes have been reported in individuals with RC5. Chromosome studies (karyotyping, chromosome painting, and/or metaphase FISH) are the recommended methods for identification and/or verification of RC5 and other RCs and sSRCs. Advanced technologies, such as PCR-based analysis, CMA, and NGS, can be used to further characterize the genomic imbalance and structural changes of RC5 and other RCs. The data regarding genomic pathogenesis and genotype–phenotype correlation of RC5s are limited. Most of reported cases of RC5 were de novo and diagnosed postnatally. However, a familial case of RC5 and a prenatally diagnosed case of sSRC5 have been reported. Parental studies and genetic counseling are recommended for appropriate assessment and management of RC5 cases.

References Basinko A, Uzielli MLG, Scarselli G, Priolo M, Timpani G, Braekeleer MD (2012) Clinical and molecular cytogenetic studies in ring chromosome 5: Report of a child with congenital abnormalities. Eur J Med Genet 55(2):112–116. https://doi.org/10.1016/j. ejmg.2011.11.005

120 Caba L, Rusu C, Plăiaşu, Gug G, Grămescu M, Bujoran C, Ochiană D, Voloşciuc M, Popescu R, Braha E, Pânzaru M, Butnariu L, Sireteanu A, Covic M, Gorduza E (2012) Ring autosomes: Some unexpected findings. Balkan J Med Genet 15(2):35–46. https:// doi.org/10.2478/bjmg-2013-0005 Cerruti Mainardi P (2006) Cri du Chat syndrome. Orphanet J Rare Dis 1(1):33. https://doi. org/10.1186/1750-1172-1-33 Ensemble Human Map View (2023) Chromosome 5. https://useast.ensembl.org/Homo_sapiens/Location/ Chromosome?chr=5;r=5:1-180915260 [Accessed on 6 June 2023]. Flannery DB, Rogers WG, Byrd JR (1988) Ring chromosome 5. Clin Genet 34(1):74–78. https://doi. org/10.1111/j.1399-0004.1988.tb02619.x Genetic and Rare Diseases Information Center (2023) Ring chromosome 5. https://rarediseases.info.nih. gov/diseases/10841/ring-chromosome-5 [Accessed on 6 June 2023]. Hu Q, Chai H, Shu W, Li P (2018) Human ring chromosome registry for cases in the Chinese population: Re-emphasizing cytogenomic and clinical heterogeneity and reviewing diagnostic and treatment strategies. Mol Cytogenet 11(1):19. https://doi. org/10.1186/s13039-018-0367-3 Huh J, Mun YC, Chung WS, Seong CM (2012) Ring chromosome 5 in acute myeloid leukemia defined by whole-genome single nucleotide polymorphism array. Ann Lab Med 32(4):307–311. https://doi. org/10.3343/alm.2012.32.4.307 Kosztolányi G (1987) Does “ring syndrome” exist? An analysis of 207 case reports on patients with a ring autosome. Hum Genet 75(2):174–179. https://doi. org/10.1007/BF00591082 Kula K, Patil S, Hanson J, Nowak A, Zellweger H (1981) Ring chromosome 5 with dental anomalies. Pediatr Dent 3(4):329–333 Li P, Dupont B, Hu Q, Crimi M, Shen Y, Lebedev I, Liehr T (2022) The past, present, and future for constitutional ring chromosomes: A report of the international consortium for human ring chromosomes. Human Genet Genom Adv 3(4):100139. https://doi. org/10.1016/j.xhgg.2022.100139 Liehr T (2023) Small supernumerary marker chromosomes. https://cs-tl.de/DB/CA/sSMC/0-Start.html [Accessed on 25 April 2023]. MacDermot KD, Jack E, Cooke A, Turleau C, Lindenbaum RH, Pearson J, Patel C, Barnes PM, Portch J, Crawfurd MD (1990) Investigation of three patients with the “ring syndrome”, including familial transmission of ring 5, and estimation of reproductive risks. Hum Genet 85(5):516–520. https://doi. org/10.1007/BF00194228 McGowan-Jordan J, Hastings RJ, Moore S (eds) (2020) An International System for Human Cytogenomic Nomenclature (2020). Karger, Basel

J. Yu Migliori MV, Cherubini V, Bartolotta E, Pettinari A, Pecora R (1994) Ring chromosome 5 associated with severe growth retardation as the sole major physical abnormality. Am J Med Genet 49(1):108–110. https:// doi.org/10.1002/ajmg.1320490121 Nakagome Y, Iinume K, Taniguchi K (1973) Points of exchange in a human no. 5 ring chromosome. Cytogenet Cell Genet 12(1):35–39. https://doi. org/10.1159/000130435 National Center for Advancing Translational Sciences (2023) Cri-du-Chat syndrome. https://rarediseases. info.nih.gov/diseases/6213/cri-du-chat-syndrome [Accessed on 6 June 2023] Nevado J, Bel-Fenellós C, Sandoval-Talamantes AK, Hernández A, Biencinto-López C, MartínezFernández ML, Barrúz P, Santos-Simarro F, MoriÁlvarez MÁ, Mansilla E, García-Santiago FA, Valcorba I, Sáenz-Rico B, Martínez-Frías ML, Lapunzina P (2021) Deep phenotyping and genetic characterization of a cohort of 70 individuals with 5p minus syndrome. Front Genet 12(1):645595. https:// doi.org/10.3389/fgene.2021.645595 Nozawa A, Ozeki M, Yasue S, Endo S, Kadowaki T, Ohnishi H, Muramatsu H, Hama A, Takahashi Y, Kojima S, Fukao T (2020) Myelodysplastic syndromes in a pediatric patient with Cri du Chat syndrome with a ring chromosome 5. Int J Hematol 112(5):728–733. https://doi.org/10.1007/s12185-020-02909-7 Ohashi H, Suzumori K, Chisaka Y, Sonta S, Kobayashi T, Aoki Y, Matsubara Y, Sone M, Shaffer LG (2010) Implications of prenatal diagnosis of the fetus with both interstitial deletion and a small marker ring originating from chromosome 5. Am J Med Genet 155A(1):192– 196. https://doi.org/10.1002/ajmg.a.33764 Rohde RA, Tompkins R (1965) “Cri Du Chat” due to a ring-B (5) chromosome. Lancet 2(7421):1075–1076. https://doi.org/10.1016/s0140-6736(65)90609-4 Schmutz J, Martin J, Terry A, Couronne O, Grimwood J, Lowry S, Gordon LA, Scott D, Xie G, Huang W, Hellsten U, Tran-Gyamfi M, She X, Prabhakar S, Aerts A, Altherr M, Bajorek E, Black S, Branscomb E, Caoile C, Challacombe JF, Chan YM, Denys M, Detter JC, Escobar J, Flowers D, Fotopulos D, Glavina T, Gomez M, Gonzales E, Goodstein D, Grigoriev I, Groza M, Hammon N, Hawkins T, Haydu L, Israni S, Jett J, Kadner K, Kimball H, Kobayashi A, Lopez F, Lou Y, Martinez D, Medina C, Morgan J, Nandkeshwar R, Noonan JP, Pitluck S, Pollard M, Predki P, Priest J, Ramirez L, Retterer J, Rodriguez A, Rogers S, Salamov A, Salazar A, Thayer N, Tice H, Tsai M, Ustaszewska A, Vo N, Wheeler J, Wu K, Yang J, Dickson M, Cheng JF, Eichler EE, Olsen A, Pennacchio LA, Rokhsar DS, Richardson P, Lucas SM, Myers RM, Rubin EM (2004) The DNA sequence and comparative analysis of human chromosome 5. Nature 16(7006):268–274. https://doi.org/10.1038/nature02919

9  Ring Chromosome 5 Sizonenko LD, Ng D, Oei P, Winship I (2002) Supernumerary marker chromosome 5: Confirmation of a critical region and resultant phenotype. Am J Med Genet 111(1):19–26. https://doi.org/10.1002/ ajmg.10459 Steele MW, Breg WR, Eidelman AI, Lion DT, Terzakis TA (1966) A B-group ring chromosome with

121 mosaicism in a newborn with cri du chat syndrome. Cytogenetics 5(6):419–429.  https://doi. org/10.1159/000129917 Suerinck E, Noël B, Rethore MO (1978) Ring chromosome 5 in two malformed boys with cri du chat syndrome. Clin Genet 14(3):125–129. https://doi. org/10.1111/j.1399-0004.1978.tb02116.x

Ring Chromosome 6

10

Frenny Sheth   , Jhanvi Shah and Harsh Sheth  

Abstract

Ring chromosome 6 (RC6) is a rare constitutional structural abnormality that generally occurs during meiosis or early post-zygotic mitosis. Most of RC6 cases occur de novo except for very rare cases having parental origin. Its preponderance is seen more in males compared to females with majority of RC6 cases identified postnatally with short stature, gross developmental delay, and variable dysmorphic features, whereas prenatal cases are often presented with intra-uterine growth retardation (IUGR) and hydrocephalus. RC6 can be identified primarily by conventional karyotype analysis followed by chromosome microarray analysis (CMA) or next-generation sequencing (NGS) to precisely identify the break points and genomic imbalances. Further identification of candidate genes within the genomic imbalances provides

F. Sheth (*) · J. Shah  Department of Cytogenetics and Molecular Cytogenetics, FRIGE’s Institute of Human Genetics, Ahmedabad, India e-mail: [email protected] J. Shah e-mail: [email protected] H. Sheth  Advanced Genomic Technologies Division, FRIGE’s Institute of Human Genetics, Ahmedabad, India e-mail: [email protected]

evidence for genotype–phenotype correlations that can be contributory to the variable degree of clinical features. Commonly observed short stature and microcephaly probably represent ‘ring syndrome’ phenotype due to dynamic somatic mosaicism; however, variable penetrance and expressivity caused by genomic imbalance for other clinical features are observed. Banding cytogenetics to define the ring structure and dynamic mosaicism followed by CMA and/or NGS to define genomic imbalances should be performed for patients with RC6, with the goals of exact breakpoint characterization and genotype–phenotype correlations.

Keywords

Ring chromosome 6 (RC6) · Dynamic mosaicism · Ring syndrome · Growth retardation · Microcephaly · Hydrocephalus · Facial dysmorphism · FOXC1 gene

10.1 Brief Historic Review Constitutional ring chromosomes (RCs) in humans have an incidence of approximately 1:50,000 live births, with most of them involving intra-chromosomal structural aberrations (Li et al. 2022). While the first constitutional RC in humans was detected in 1962, the first reported

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_10

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RC involving chromosome 6 (RC6) was identified in a 2-year 6 months female in 1973 (Moore et al. 1973). The girl showed global developmental delay and anthropometric parameters consistently below third centile along with facial dysmorphism that included microcephaly, microphthalmia, micrognathia, microstomia, bilateral epicanthal folds, large low-set ears, depressed nasal bridge, mildly high-arched palate, delayed dentition, stiff ankles, mild pes equinus, and mild hyperkeratosis of the soles (Moore et al. 1973). Since then, conventional chromosome G-banding, fluorescence in  situ hybridization (FISH), and genomic technologies have been introduced in the analysis of RC6. There were 43 cases of RC6 reported in the literature, and RC6 accounts for approximately 4% of all RC cases reported to date in the literature (Li et al. 2022).

10.2 Diagnostic Methods for RC6 Analysis 10.2.1 RC6 Assessed by Banding and Molecular Cytogenetics RC6 was first observed using quinacrine mustard (Q)-banding in lymphocytes from peripheral blood (Moore et al. 1973). Interestingly, while the major cell line consisted of monocentric RC6, in approximately 12% of the cells, RC6 differed in either or a combination of number, size, and morphology that included cells with a single large dicentric ring, two monocentric rings, both dicentric and monocentric rings and small interlocking rings. The proband’s parents and siblings had a normal chromosome composition suggesting the RC6 to be of de novo origin. Fibroblasts cell cultures of the same proband suggested more morphological variants of RC6 compared to leucocyte culture (Moore et al. 1973). RCs in the past were thought to be a result of terminal breaks followed by fusion of the two arms of a chromosome forming a continuous

F. Sheth et al.

ring. However, the establishment of various techniques and their incorporation to characterize these RCs has identified genomic rearrangements and copy number variations (CNVs) involving the RC along with dynamic mosaicism. Giemsa banding (G-banding) and various types of fluorescence in situ hybridization (FISH) allowed deeper insights into the structures of RCs, whereas chromosomal microarray (CMA) and next-generation sequencing (NGS) contributed to the identification of breakpoints and genes that contributed to the phenotype of an individual with a RC (Li et al. 2022). Banding cytogenetics enabled the detection of RCs and their variants but failed to distinguish between complete and incomplete rings due to the low resolution of the G-bands as shown in Fig. 10.1a. Nonetheless, banding also allowed the comparison of rings of the same chromosome along while studying cases with similar breakpoints. Like traditional banding technique, availability of high-resolution banding was unable to provide precise breakpoints let alone identification of microdeletions. However, such deletions were detected using FISH techniques such as reverse painting of microdissected RCs, or centromeric and locus-specific probes as shown in Fig. 10.1b to define the extent of deletions, which helped in genotype– phenotype correlation. FISH has been able to assess the presence of submicroscopic deletions in some patients with apparently complete ring while in the rest the absence of any subtelomeric deletion (Birnbacher et al. 2001; Zhang et al. 2004; Höckner et al. 2008; Kosztolányi 2009). The first case of RC6 with the breakpoint characterization being carried out at the molecular level was using a combination of FISH and microsatellite marker-based genotyping. The patient was identified with RC6 which consisted of a 6 Mb deletion on the distal 6p region and an approximately 2 megebase (Mb) deletion on the distal 6q region (Zhang et al. 2004). FISH with targeted probes on metaphase chromosomes revealed a deletion of the FOXC1 gene at 6p, and a paternal origin of the RC6 was indicated using intragenic polymorphic markers.

10  Ring Chromosome 6

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Fig. 10.1  a Partial G-banded karyotype showing various patterns of RC6 in multiple cell lines as mos 46,XY,r(6)(p25.3q27)[54]/45,XY,-6[13]/46,XY,r(6) ( : : p 2 5 . 3 → q 2 7 : : p 2 5 . 3 → q 2 7 : : ) [ 1 3 ] / 4 7 , X Y, r ( 6 ) (p25.3q27)×2[2]/46,XY[6]. (Reproduced with permission from Sheth et al. 2018) b FISH study showed three

signals (green, blue, and red) that confirm an intact chromosome 6 (left), single signal (blue) on r(6) confirm subtelomeric deletions at both the arms in the RC6 (middle), and two signals (blue) confirm two centromeres present in a dicentric RC6 (right). Reproduced with permission from Sheth et al. (2018)

10.2.2 RC6 Instability, Dynamic Mosaicism, and Periodic Follow-Up in Different Tissues

during cell division leads to mosaicism and continuously generates aberrant daughter cells leading to various cellular, genetic, and phenotypic consequences and is referred to as “dynamic mosaicism” (Kosztolányi 2009; Li et al. 2022). In 1975, it was postulated that the amount of genetic material lost during ring formation and the different type and degree of mosaicism gets reflected on the variable expression of the phenotype (Dawson et al. 1995). In the past, RC6 was assumed to be relatively more stable than ring structures of other chromosomes, and the variable expression was attributed either to the amount of genetic material lost or to the position of the breakpoints (Carnevale et al. 1979). However, rings of larger chromosomes tend to undergo multiple SCEs increasing the instability and accumulating multiple aberrations (Yip 2015). Dynamic mosaicism is likely to cause variation in the chromosomal makeup over time across different tissue types, making diagnosis challenging, especially in a prenatal setting (Kosztolányi 2009; Li et al. 2022). RC6 has

Mosaicism is a commonly observed phenomenon whereby derivatives of RC variants can be present in the form of a dicentric ring, an interlocked ring, a small ring or a loss of the entire RC in different somatic cell lines (Li et al. 2022). These variants are a consequence of anaphase separation following sister chromatid exchange (SCE) that occurs in the ‘S-phase’ of the cell cycle (Paz-y-Miño et al. 1990). The cells with large and complex unbalanced aberrations undergo apoptosis increasing growth failure and the ones that survive are accompanied by deletions and/or duplications that contribute toward the phenotypic manifestation (Yip 2015). Due to the inherent chromosomal structure instability, RCs are prone to additional deletions, duplications, and translocations that can also affect other chromosomes (Pristyazhnyuk and Menzorov 2018). This ring instability

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been detected as early as the prenatal period in amniotic fluid and cord blood samples. Interestingly, cytogenetic analysis from additional tissues like Achilles tendon cells, chondrocytes, and placental biopsies has shown loss of RC6 compared to blood cells that tend to preserve RC6 (Urban et al. 2002). The first case of RC6 detection in a prenatal sample was presented in 1995, whereby karyotype analysis in amniocytes from a fetus with isolated hydrocephalus revealed mosaicism for RC6 and a monosomy for chromosome 6 (45,XY,−6/45,XY,−6,+ace/46,XY,r(6) (p25q27)). Subsequently, postnatal cytogenetic analysis from peripheral blood cells of this proband was observed to have 46,XY,r(6) (p25q27) and showed no indication of mosaicism (Walker et al. 1996). Indeed, almost all cases that carried out cytogenetic analysis on amniocytes and blood cells have shown mosaicism in amniocytes but not in peripheral blood and have been attributed to mitotic instability of ring structures. In a postnatal case, karyotype analysis in a male proband was carried out at 5-year interval. The first analysis reported all cells with an RC6 that replaced a normal chromosome 6 except one cell that showed a normal chromosome constitution, and in addition, two cells of RC6 were replaced by a derived chromosome 6 with additional material on the p-arm. After a period of 5 years, karyotyping from blood cells showed 46 chromosomes with one normal chromosome 6 replaced by RC6 of different structural variations such as two rings or double rings, and only two cells were hypomodal lacking the RC6 (Wurster-Hill and Hoefnagel 1975). Such dynamic mosaicism could lead to formation of cell lineage where RC6 is absent as it is favorable for the survival of the cell. Indeed, in a rare case, a proband with RC6 was observed to have the proportion of cells from the same tissue with RC6 decrease from 93 to 61% over time (Fryns et al. 1990). However, it is currently unknown whether this could lead to phenotypic rescue or regression over the corresponding time.

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10.2.3 Cases of RC6 by CMA and NGS A total of nine cases with RC6 have been characterized using CMA to date. All reported RC6 cases prior to 2013 were only karyotyped with a few exceptions that used additional FISH or multiplexed ligation probe amplification (MLPA) techniques due to unavailability of advanced molecular cytogenetic tests. In 2013, the first case of RC6 was characterized by CMA which aided in genotype–phenotype correlations that the terminal deletions in opposite arms accompanying RC6 were causing (Ciocca et al. 2013). In this case, the karyotype revealed all cells to have RC6 that substituted normal chromosome 6. Further FISH analysis using whole chromosome paint (WCP) probes showed both the normal chromosome 6 and RC6 to be entirely painted following which a subtelomeric FISH reported deletions with possible breakpoints in the 6p25 and 6q27 regions. CMA was performed to confirm these findings and showed 1.3 Mb and 6.7 Mb terminal losses with breakpoints at 6p25.3 and 6q26-27. Since then, this cytogenomic assay helped in deciphering duplication adjacent to deletion in two cases (Sheth et al. 2018; Dong et al. 2022), deletion and duplication in opposite arms of the same chromosome (Pace et al. 2017), and several cases having unilateral CNV (Nishigaki et al. 2015; Corona‐Rivera et al. 2019; Sunkak et al. 2021; Dong et al. 2022), as shown in Fig. 10.2. Till date only two cases of RC6 have been further characterized by an NGS based study (Zhang et al. 2016; Dong et al. 2022). NGS using copy number variation sequencing (CNV-seq) was first used in a patient with RC6 identified with a large 1.78 Mb terminal deletion at 6p25.3-pter. Additionally, there was weak evidence pointing toward the presence of two small deletions, one intra-chromosomally at 6q22.31 and another at 6q27 terminal region. An additional CNV, a 0.66 Mb duplication at the 5q11-q12 locus, was also detected by CNV-seq in this case (Zhang et al. 2016). In contrast, CMA was able to detect only the terminal deletion at 6p that was within the detection limit of the assay as compared to

10  Ring Chromosome 6

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Fig. 10.2  Schematic representation of chromosome 6 with breakpoints (boxed) generally involved in individuals with RC6. Red and blue thick bars represent deletions and duplications characterized by chromosomal microarray, respectively (RC6-1, Ciocca et al. 2013; RC6-2, Nishigaki et al. 2015; RC6-3, Zhang et al. 2016; RC6-4, Pace et al. 2017; RC6-5, Liu et al. 2018; RC6-6, Sheth et al. 2018; RC6-7, Corona‐Rivera et al. 2019; RC6-8,

Sunkak et al. 2021; RC6-9, Dong et al. 2022). The black thick arrows correspond to the largest deletions on the p-arm and q-arm. White bars with interspersed dark blocks represent OMIM genes associated with a phenotype. Numerical at the end of the bars on the extreme right indicates reference numbers. *RC6 is inherited from the mother having the same break points as the child

the ones at 6q and failed to detect the small intrachromosomal deletions, as well. This suggested that the resolution of NGS was likely better than that of CMA allowing detection of small telomeric/subtelomeric deletions. The clinical and genomic description of all nine RC6 cases characterized by CMA and/or NGS are outlined below:

Case RC6-2 (Nishigaki et al. 2015)

Case RC6-1 (Ciocca et al. 2013) Cerebral ventriculomegaly was detected during antenatal study. At 5 months of age, she portrayed high nasal bridge, prominent lips, and protruding tongue. Mild motor delay was observed during follow-up study at 16 months. Cerebral MRI revealed enlarged lateral ventricles, atrial septal defect, and patent ductus arteriosus. Karyotyping showed RC6, i.e., 46,XX,r(6)(p25q27). Absence of 6pter and 6qter regions was confirmed using subtelomeric FISH probes. CMA further confirmed a 1.3 Mb deletion at 6p25.3 and a 6.7 Mb deletion at 6q26-27.

IUGR was detected under prenatal sonography. Recurrent febrile seizures, short stature, and developmental delay were documented at the age of 3 years. Cerebral MRI revealed periventricular heterotopia and white matter abnormalities in bilateral parietal and occipital lobes. Various structural rearrangements involving RC6 were detected on karyotyping, i.e., mos 46,XX,r(6) (p25q27)[67]/45,XX,-6[25]/46,XX,r(6) (::p25→ q27::p25→ q27::)[6]/47,XX,r(6) (p25q27)×2[2]. CMA confirmed a 1.5 Mb deletion at 6q27. The mean log2ratio of the aberration region was −0.780567, indicating mosaic loss. Case RC6-3 (Zhang et al. 2016, Fig. 10.3) The proband was born with low birth weight. Postnatal growth retardation, microcephaly, facial dysmorphism, congenital heart defect, global developmental delay along with hyperactivity and autism were observed on clinical

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a

b

c

d

Fig. 10.3  Facial features of the proband showing craniofacial abnormalities (a), teeth agenesis (b), ocular abnormalities (c), shortened and incurred small finger (d). Reproduced with permission from Zhang et al. (2016)

evaluation at the age of 6 years. RC6 and monosomy 6 were identified on karyotyping as 46,XX,r(6)(p25.3q27)[81]/45,XX,-6[7]. CMA detected a 1.78 Mb deletion at 6p25.3. NGS using copy number variation sequencing (CNVseq) confirmed the 1.78 Mb terminal deletion at 6p and identified additionally 0.26 Mb and 0.56 Mb deletions at 6q22.31 and 6q27qter, respectively. An additional CNV, a 0.66 Mb duplication at the 5q11-q12 locus, was also detected by CNV-seq in this case. Except for 6p25.3, all other CNVs were missed by CMA.

cognitive and developmental milestones were delayed. At the age of 10 years, he was diagnosed with bilateral hearing loss which worsened progressively. A CT scan of the brain performed at the age of 21 years, revealed the presence of a Dandy-Walker variant. RC6 was confirmed by molecular cytogenetic methods; CMA detected a 1.8 Mb deletion at 6p25 and a 2.5 Mb duplication at 6q27 that was further confirmed by FISH using various subtelomeric probes.

Case RC6-4 (Pace et al. 2017)

Global developmental delay, microcephaly, short stature, and facial dysmorphism were observed in an 11-year-old boy. In addition, he also had penile chordee and sacral dimple. His MRI brain showed demyelination in both frontal and parietal lobes. Mosaic RC6 with various structural and numerical aberrations was identified on karyotyping: mos 46,XY,r(6) (p25.3q27)[54]/45,XY,-6[13]/46,XY,r(6) (::p25.3→q27::p25.3→q27::)[13]/47,XY,r(6)

A 49-years male was referred to the genetic clinic during a hospital admission for a chest infection. Physical examination revealed moderate ID, short stature, microcephaly facial dysmorphism, a small atrial septal defect, mild scoliosis, brachydactyly, overlapping toes on left foot, chronic venous insufficiency, and hypertension. He was a slow feeder and early motor,

Case RC6-5 (Sheth et al. 2018)

10  Ring Chromosome 6

(p25.3q27)×2[2]/46,XY[6]. Single nucleotide polymorphism based CMA (SNP array) detected a 0.5 Mb deletion adjacent to a 1.3 Mb duplication at 6p25.3 and a 0.44 Mb deletion at 6q27: arr[GRCh37] 6pterp25.3(156,974_665, 234)×1,6p25.3(668,700_1,929,528)×3,6q27q ter(170,466,513_170,914,297)×1. Subtelomeric and centromeric FISH probes confirmed the presence of both the terminal deletions and dicentric nature of the ring, respectively.

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A boy that was followed-up for a decade from 2 years 2 months to 12-years of age showed global developmental delay, hyperactivity,

dysarthria, recurrent upper respiratory tract infections, microcephaly, short neck, macropenis, and facial dysmorphism. Ophthalmic evaluation revealed slight esotropia in the left eye and MRI brain reported gray matter heterotopia and brain dysplasia. Karyotype analysis reported: mos 45,XY,-6 [13]/46,XY,r(6)(::p25 → q27::p25 → q27::)[4]/47,XY,r(6)(p25q27) x2[5]/47,XY,r(6)(::p25→q27::p25→q27::) x2[2]. FISH using subtelomeric probes confirmed the terminal deletions on both arms, whereas whole chromosome paint probes ruled out cryptic translocation of chromosome 6 to other chromosomes. CMA further authenticated a 0.26 Mb deletion at 6p25.3 and a 2.03 Mb deletion at 6q26-q27.

Fig. 10.4  Clinical images and growth chart of the propositus. The photographs of the patient were taken at the age of 3 years and 8 months (a, b), 5 years and 2 months (c), and 12 years and 2 months (d, e), respectively. From the frontal and side view, microcephaly, short neck, hypertelorism, low frontal hairline, bilateral epicanthus,

broad nasal bridge with protruding nose, long philtrum, big mouth, prominent chin, micrognathia, and big protruding cupped ears are noted. f the growth chart indicates that the growth level of the patient is significantly lower than of male children of the same age. Reproduced with permission from Liu et al. (2018)

Case RC6-6 (Liu et al. 2018, Fig. 10.4)

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Case RC6-7 (Corona‐Rivera et al. 2019, Fig. 10.5) A newborn male baby showed facial dysmorphism and corneal clouding with large corneas in both the eyes. In addition, there was redundant periumbilical skin, phimosis, and micropenis. Anterior segment dysgenesis was noted on ophthalmic evaluation and included megalocornea, glaucoma, visual acuity light perception, corneal edema, posterior embryotoxon, irregular shaped displaced pupils (corectopia), and iris hypoplasia in both the eyes. Renal ultrasonogram showed a left hydronephrosis due to pelvi-ureteric junction obstruction. CT of the brain indicated a mild frontal lobe atrophy. Karyotype analysis revealed RC6 as a single cell line as 46,XY,r(6)(p25.3q27). Further analysis by subtelomeric MLPA revealed a deletion encompassing the IRF4 gene. CMA identified a 1.88 Mb deletion at 6p25.3 and described as arr[GRCh38] 6p25.3(156,974_2,036,857)×1.

Fig. 10.5  Facial dysmorphisms of the male patient at the age of 2 years. Postnatal day (a), eye anomalies including megalocornea and corneal clouding with irregular shaped displaced pupils (b, c), redundant

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Case RC6-8 (Sunkak et al. 2021) A 10-year-old girl with microcephaly, facial dysmorphism, clinodactyly in the right hand, and pes planus along with atrial septal defect and restrictive cardiomyopathy showed RC6 on karyotype study, i.e., 46,XX,r(6)(p25q27). SNP array precisely confirmed a 3.5 Mb deletion at 6p25.3 region: arr[GRCh38] 6p25 .3p25.2(204,409_3,716,890)×1. Case RC6-9 (Dong et al. 2022) A 23 years old primi gravida woman was admitted to fetal medical center at 24 weeks of gestation to rule out the cause of antenatal IUGR, absent nasal bone, and VSD. Additionally, foramen ovale was observed at 30 weeks and all the detected abnormalities vanished by 34 weeks. Cytogenetic analysis and SNP array were carried out after amniocentesis. Parents were also cytogenetically studied. After birth, karyotype and

periumbilical skin (d), micropenis (e), and progression of the glaucoma and the left eye after enucleation at age 3 years (f). Reproduced with permission from CoronaRivera et al. (2019)

10  Ring Chromosome 6

SNP array were performed again. Pre- and postnatal banding cytogenetics showed a karyotype of 46,XY,r(6)(p25q27)mat. Mother’s peripheral blood karyotype showed RC6 in mosaic pattern: mos 46,XX,r(6)(p25q27)[44]/47,XX,r(6)(p25q27) x2[2]/46,XX[15]. Father was karyotypically normal (46,XY). CMA results in the fetus (amnio and peripheral blood) showed arr[GRCH37] 6p25.3 (203,254_1,138,134)× 1,6p25.3p25.2 (1,153,042_4,172,096)×3, and in the mother findings were arr[GRCH37] 6p25.3(203,254_1, 138,134)× 1~2,6p25.3p25.2(1,153, 042_4,172,096)×2~3. Furthermore, whole exome sequencing (WES) confirmed the SNP array results as: seq[GRCh37] r(6)(p25.3qter):g.[pter _1127408del::1127408_4191151dup::4191151qter]. Except for growth retardation and congenital heart malformations, no phenotypes were observed in the newborn. This is the only case of maternally inherited RC6 without major clinical consequences.

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ring chromosome; also an isoUPD(6) mosaicism was found in 10% of the cells, explaining the 15 cells with normal karyotype 46,XX found in cytogenetics as being due to monosomic rescue (Dong et al. 2022). A young mother with short stature, normal intellect with no major malformation (Fig. 10.6) having RC6 gave a birth to a healthy son with normal chromosomal make-up; here RC6 was not transmitted. The fertility of the mother was not compromised, as the RC6 formed was a result of telomere–telomere or near-telomere fusion without significant loss of euchromatin material. In such cases, a child inheriting the same chromosome have a theoretical inheritance risk of 50%. The selection process and large difference in the number of cell divisions during spermatogenesis and oogenesis was suggested as a reason behind maternal inheritance being predominant over paternal (Höckner et al. 2008).

10.2.4 Parental and Familial Analysis

10.3 Clinical Observations of RC6 Cases

Large genomic aberrations such as RCs affect reproductive fitness; hence in majority of the cases RCs occur de novo. Approximately 1% of the cases familial transmission of RCs has been reported (Pristyazhnyuk and Menzorov 2018). RC6 is a rare constitutional structural abnormality that generally occurs de novo during meiosis or early post-zygotic mitosis (Kosztolányi 2009). All reported RC6 cases were de novo (n =  42; ~97.7%) except one case (~2.3%) where maternal inheritance was observed. No paternal transmission of RC6 has been documented yet. An apparently healthy mother without major clinical consequences was a carrier for mosaic RC6 (mos 46,XX,r(6)(p25q27)[44]/47,XX,r(6) (p25q27)x2[2]/46,XX[15]). This RC6 was transmitted to her son who showed multiple anomalies during antenatal and postnatal assessment. Pre- and postnatal investigations for karyotype and SNP array gave identical results. In addition, SNP array in the mother confirmed the mosaic state in 90% of the cells carrying the

RC6 has been reported across the globe, irrespective of race, ethnicity, and socio-economic status. There are a total of 43 cases published in the literature with a higher incidence in males (n = 27) compared to females (n = 15) and one case of unknown gender (Urban et al. 2002) with an approximate 2:1 male to female ratio. Children with RC6 were born to mothers of varying age, and an increased age was not associated with the abnormality (Andrieux et al. 2005). The maternal age at the time of pregnancy ranged from 15 to 34 years old and paternal was 22 years to 57 years. The age of onset and age at diagnosis ranged from prenatal to postnatal period to an apparently healthy individual being detected with an RC6 on parental karyotyping (Dong et al. 2022). Cases diagnosed in the prenatal period contributed to approximately 18.7% cases (8 of 43), whereas 81.3% cases (35 of 43) were diagnosed postnatally as given in Table 10.1. RC6 is observed to occur de novo in majority of the cases with only one reported case of a familial transmission (Dong et al. 2022).

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Fig. 10.6  Patient at 22 years with minor dysmorphisms including deep and irregular frontal hairline, slightly upslanting palpebral fissures, a deep-set columella, a broad philtrum, a small mouth, and a broad neck. Reproduced with permission from Höckner et al. (2008)

Table 10.1  Demographic data on cases identified with RC6 Variable

Cases (n = 43)

Gender, n (%) Males

27 (62.8)

Females

15 (34.9)

Unknown

1 (2.3)

Male–female ratio Age, years (SD)

2:1

Age at diagnosis of probands

Prenatal period 49 years

Maternal age at conception

25 ± 4

Paternal age at conception Cases, n (%)

29 ± 7

Prenatal

8 (18.6)

Postnatal Broad phenotypic classificationa n (%)

35 (81.4)

Mild

7 (16.3)

Moderate

8 (18.6)

Severe a Phenotypic

(2004)

25 (65.1) classification was provided by Zhang et al.

10.3.1 Prenatal Cases of RC6 All prenatal cases were detected with abnormal ultrasound findings during late second trimester and the most frequently observed clinical features included IUGR (Dawson et al. 1995; Walker et al. 1996; Urban et al. 2002; Andrieux et al. 2005) and hydrocephalus (4/8; 50%) (Dawson et al. 1995; Walker et al. 1996; Urban et al. 2002). Abnormalities of the central nervous system (CNS) were also commonly observed (5 of 8 cases; ~62%) that included isolated hydrocephalus, cerebellar and vermin hypoplasia, craniomegaly, partial agenesis of posterior corpus callosum, and deficient brain growth. Hence, fetal MRI was recommended in cases with cerebral involvement (Andrieux et al. 2005). Absent nasal bone (Dong et al. 2022), increased nuchal thickness (Dawson et al. 1995), and cardiac anomalies (Dong et al. 2022) were also seen. Additional findings included limited fetal movements (Dawson et al. 1995), ocular hypertelorism (Dawson et al. 1995; Andrieux et al. 2005), broad nasal bridge (Dawson et al.

10  Ring Chromosome 6

1995), abnormality of fifth fingers (Urban et al. 2002), rocker bottom feet (Dawson et al. 1995), and micropenis (Andrieux et al. 2005). Amniocentesis were the preferred approach of investigation. Precise pre- and post-test genetic counselling was offered to the families, and in four (50%) cases, the pregnancy was medically terminated, whereas remaining (50%) families chose to continue the pregnancy.

10.3.2 Pediatric and Adult Cases of RC6 Postnatal cases harboring RC6 are frequently observed with short stature, microcephaly, mental retardation, and facial dysmorphism including micrognathia, epicanthic folds, hypertelorism, flat or broad nasal bridge, low-set ears, and short neck (Birnbacher et al. 2001; Ahzad et al. 2010; Liu et al. 2018) as given in Table 10.2. Cases of RC6 were classed into three groups: mild, moderate, and severe based on the clinical findings (Zhang et al. 2004). The clinical manifestations in the mild cases mainly included growth retardation with minimal or no intellectual and physical anomalies. The cases with growth retardation, intellectual disability along with physical anomalies like microcephaly, micrognathia, depressed nasal bridge, epicanthal folds, low-set ears, high arched palate, and short webbed neck were features of moderate and severe cases with additional major congenital anomalies distinguishing severe cases from moderate ones. Other clinical findings in probands with RC6 include CNS involvement, ocular and auditory system abnormalities, congenital heart defects, and even vertebral segmentation defects (Zhang et al. 2004, 2016; Ciocca et al. 2013; Corona‐Rivera et al. 2019). Caution must be taken during differential diagnosis in the clinic since probands with RC6 harbor wide inter-individual variability, which is likely to be due to the genetic content covered by the RC. Postnatal death often occurred due to cardiac or nervous system complications and ranged from neonatal period to childhood. To the best of our knowledge, only three cases with RC6

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have succumbed to early death. A male baby expired at 6 months of age due to acute pneumonia (Salamanca-Gonez et al. 2008), cardiopulmonary arrest in a female neonate (Peeden et al. 1983), and a male baby died on the 5th day due to respiratory distress and seizures (Peeden et al. 1983). There is no detail information except for three individuals surviving until adulthood.

10.4 Genotype–Phenotype Correlations for RC6 Clinical presentations of RC6 vary considerably between cases making it difficult for genotype– phenotype correlations. Before the advent of CMA and NGS based assays, karyotype-based phenotype associations cannot define precise breakpoints for genomic imbalances and gene content that would aid in genotype–phenotype correlations for individuals with RC6. Distal deletions involving 6p25 subtelomeric region are known for distinct clinical features consisting of developmental delay, mental retardation, language impairment, hearing loss, and ophthalmologic, cardiac, and craniofacial abnormalities (Gould et al. 2004). A number of genes encompassing 6p25-pter region such as DUSP22, IRF4, EXOC2, HUS1B, FOXQ1, FOXF2, FOXCUT, FOXC1, and GMDS with IRF4 and FOXC1 are known human disease-causing genes. IRF4 (OMIM*601900) causes variation in skin/hair/eye pigmentation, (OMIM#611724), EXOC2 (OMIM*615329) neurodevelopmental disorder with dysmorphic facies and cerebellar hypoplasia (OMIM#619306) and FOXC1 (OMIM*601090) anterior segment dysgenesis 3, multiple subtypes (OMIM#601631), and Axenfeld–Rieger syndrome, type 3 (OMIM#602482). Of these, only FOXC1 gene is considered as a haplo-insufficient gene with a HI score of three and a HI index of 9.01% in the ClinGen database. In addition, the GMDS is also a haplo-insufficient gene with a HI index of 3.84% but is not associated with a phenotype in the OMIM database. The FOX gene family is a group of transcription factors that play an important synergistic

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Table 10.2  Phenotypic presentations in cases with RC6 Clinical features

Mild phenotype n = 7

Moderate phenotype n = 8

Severe phenotype n = 28

Total N = 43

n

%

n

%

n

%

n

%

Intra-uterine growth retardation/low birth weight

3/6

50

4/6

67

16/25

64

23/37

85

Postnatal growth retardation Craniofacial

6/6

100

8/8

100

22/25

88

34/39

87

Growth

Microcephaly

4/6

67

8/8

100

20/26

77

32/40

80

Prominent forehead

0/4

0

0/2

0

8/11

73

8/17

47

Low-set malformed ears

2/6

33

6/8

75

20/26

77

28/40

70

Hearing loss

0/1

0

0/3

0

6/9

67

6/13

46

Flat/broad nasal bridge

1/5

20

4/5

80

17/25

68

22/35

63

High-arched palate

0/3

0

5/6

83

10/17

59

15/26

58

Micrognathia

3/4

75

6/7

86

15/19

79

24/30

80

Short neck Eyes

1/5

20

4/5

80

10/19

53

15/29

52

Hypertelorism

1/6

17

3/6

50

19/27

70

23/39

59

Down-slanting palpebral fissures

1/5

20

0/5

0

10/19

53

11/29

38

Microphthalmia

0/5

0

3/7

43

9/19

47

12/31

39

Epicanthal folds

1/6

17

6/6

100

15/25

60

22/27

81

Strabismus

0/5

0

2/7

29

5/15

33

7/27

26

Nystagmus

0/4

0

0/6

0

2/13

15

2/23

9

Retinal anomalies

0/2

0

0/7

0

7/14

50

7/23

30

Anterior segment dysgenesis

0/2

0

0/6

0

8/15

53

8/23

35

Corneal clouding

0/6

0

0/7

0

5/15

33

5/28

18

Glaucoma/megalocornea Cardiovascular

0/2

0

0/6

0

6/26

23

6/34

18

Congenital heart defect

1/2

50

0/2

0

9/18

50

10/22

45

Atrial/ventricular septal defect

0/2

0

0/2

0

9/20

45

9/24

38

Valvular defects

0/2

0

0/2

0

5/19

26

5/23

22

Patent ductus arteriosus Abdomen/visceral

0/2

0

0/2

0

5/18

28

5/22

23

Umbilical hernia

1/3

33

1/3

33

2/8

25

4/14

29

Imperforate anus Genitourinary

0/6

0

0/8

0

3/25

12

3/39

8

Genital anomalies

1/6

17

3/7

43

6/26

23

10/39

26

Kidney anomalies Skeletal

1/1

100

0/1

0

5/15

33

6/17

35

Bone age retardation

3/3

100

1/3

33

4/4

100

8/10

80

(continued)

10  Ring Chromosome 6

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Table 10.2  (continued) Clinical features

Mild phenotype n = 7 n

%

Moderate phenotype n = 8 n

%

Severe phenotype n = 28 n

%

Total N = 43 n

%

Column Hemivertebra

0/2

0

0/4

0

3/12

25

3/18

17

Scoliosis Hands

1/2

50

1/4

25

2/12

17

4/18

22

Clinodactyly Feet

3/3

100

0/3

0

9/11

82

12/17

71

Pes/talipes equinovarus 0/4 Central nervous system (CNS)

0

1/4

25

3/13

23

4/21

19

Global developmental 2/5 delay/intellectual disability

40

8/8

100

24/24

100

34/37

92

Hypotonia

0/1

0

1/4

25

11/15

73

12/20

60

Seizures

1/6

17

2/8

25

10/25

40

13/39

33

Cerebellar vermis hypoplasia

0/2

0

0/4

0

4/18

22

4/24

17

Hydrocephalus/ventriculo- 0/2 megaly

0

0/4

0

15/19

79

15/25

60

Dandy–Walker malformation

0/2

0

0/4

0

1/18

6

1/24

4

Absent/hypoplastic corpus 0/2 callosum

0

0/4

0

6/18

33

6/24

25

role in embryonic development, tissue-specific gene expression, morphogenesis (Bieller et al. 2001), as well as cardiovascular development (Zhu 2016). Four members of the FOX gene cluster at the 6p25.3-pter locus includes FOXQ1, FOXF2, FOXCUT, and FOXC1 genes. FOXC1 and FOXC2 genes are expressed during cardiac development, and variants in these genes have been associated with a wide range of cardiac abnormalities (Seo and Kume 2006). Zhang et al. suggest that the congenital cardiac anomalies observed in RC6 cases is due to haplo-insufficiency of FOXC1 (Zhang et al. 2016). Contrarily, in patients whose 6p25 deletion does not disrupt FOXC1 gene locus, the phenotype is mild or moderate (Zhang et al. 2004; Nishigaki et al. 2015; Sheth et al. 2018) and does not include anterior segment dysgenesis (Zhang et al. 2004; Höckner et al. 2008; Nishigaki et al. 2015; Liu et al. 2018). Developmental delay and varying

degrees of neurological defects are consistent features in 6p25 deletion syndromes. FOXC1, FOXF2, and GMDS are involved in central nervous system development and function. Moreover, haplo-insufficiency of the FOXC1 gene has been associated with hydrocephalus in humans (Gould et al. 2004). Any deviation from normal FOXC1 gene dosage results in central nervous system vascular anomalies. FOXC1 and SERPINB6 genes are associated with hearing deficits and FOXF2 with teeth agenesis. (Zhang et al. 2004, 2016; Ciocca et al. 2013; Pace et al. 2017). A RC6 case having short stature where breakpoint identified is telomeric to the DUSP22 gene, without disrupting FOXC1 and GMDS coding sequences at 6p is said to be attributed to mitotic instability of the ring chromosome (Höckner et al. 2008). Mutation in IRF4 gene is also said to be associated with eye pigmentation anomalies (Han et al. 2008).

136

Within the 6q27 deletion interval, there are ten genes with TBP (OMIM*600075) and DLL1 (OMIM*606582) genes recognized as OMIM disease genes with susceptibility to Parkinson disease (OMIM#168600) and neurodevelopmental disorder with nonspecific brain abnormalities and with or without seizures (OMIM#618709), respectively. Both these genes are haplo-insufficient with TBP having an HI index of 6.48% and DLL1 having a HI index of 4.66% in the ClinGen database. The clinical presentation of the patients with RC6 involving both 6p and 6q terminal deletions exhibit a very similar phenotype to patients with RC6 involving only 6p terminal deletions (Ahzad et al. 2010), and the 6q terminal deletion is less likely to have significantly contributed to the severe phenotype leaving RC6 mosaicism as the most likely explanation (Zhang et al. 2016). On comparison of cases with RC6 without loss of significant euchromatin (“ring syndrome”), those with only 6p terminal deletions in the RC6 and those with only 6q terminal deletions in RC6 indicated clinical features caused by hemizygosity of genes on 6p and 6q while some features were attributed to the presence of the ring chromosomes. Postnatal growth retardation was present more often in cases with RC6 without significant loss of euchromatin material compared to cases with deletions on either of the arms. This supports the concept of “ring syndrome,” wherein the growth retardation is consistently observed and sometimes is the only manifestation of a ring chromosome (Zhang et al. 2004). This is attributed to the continuous generation of cells with dynamic and de novo secondary variants (numerical or structural) derived from the primary ring chromosome that causes increase in cellular mortality leading to poor growth. This dynamic nature of RC is caused by their instability during cell divisions. Hence, the presence of the RC6 rather than haplo-insufficiency due to monosomy of genetic material is likely the cause of growth retardation. Till date, only nine cases have been characterized by CMA or NGS-based tests, and all are showing incomplete RC6. For cases prior to the

F. Sheth et al.

advent of these technologies, it is difficult to comment on the possibility of complete rings. However, to the best of our knowledge, none of the cases characterized by FISH showed complete RCs. Like RCs observed with other chromosomes, the mosaicism observed in multiple cases points toward the association of varying levels of mosaicism on the overall phenotypic severity in individuals with RC6. A normal cell line was more often found in individuals with mild or moderate manifestations than in severely affected cases (6/7; 85%). The presence of normal cell lines in mildly affected individuals indicated a limited distribution among various tissues suggesting the formation of the RC6 later in the developmental period. The dynamic instability of the RC6 couple with somatic mosaicism makes precise genotype–phenotype correlations always challenging. With the availability of high-through molecular assays such as NGS, long read sequencing, and optical genome mapping, there is opportunity to asses RC6 with a single nucleotide resolution in individual cell lines to delineate the process of RC formation and the impact of complex genomic aberration on the cellular mechanisms in an unprecedented detail.

10.5 Conclusions and Recommendations RC6 cases identified postnatally with short stature, microcephaly, mental retardation, and facial dysmorphism, including micrognathia, epicanthic folds, hypertelorism, flat or broad nasal bridge, low-set ears, and short neck. Other clinical findings include CNS involvement, ocular and auditory system abnormalities, congenital heart defects, and even vertebral segmentation defects. However, prenatal cases often presented with IUGR and hydrocephalus. Primary detection of RC6 is advisable by conventional karyotype study to define the ring structure and dynamic mosaicism, followed by CMA and/or NGS to delineate precise break points and genomic imbalances. NGS can be used to identify the

10  Ring Chromosome 6

genes involved in the ring formation and to correlate genotype–phenotype features. Majority of RC6 are de novo in origin, maternal transmission has been reported in a single case, and normal child can be born to a carrier mother. Hence, parents need to be investigated for karyotyping to rule out mode of inheritance and delineating recurrence risk for subsequent pregnancies.

References Ahzad HA, Ramli SF, Loong TM, Salahshourifar I, Zilfalil BA, Yusoff NM (2010) De novo ring chromosome 6 in a child with multiple congenital anomalies. Kobe J Med Sci 56(2):E79–84 Andrieux J, Devisme L, Valat A-S, Robert Y, Frnka C, Savary JB (2005) Prenatal diagnosis of ring chromosome 6 in a fetus with cerebellar hypoplasia and partial agenesis of corpus callosum: Case report and review of the literature. Europ J Med Genet 48(2):199–206. https://doi.org/10.1016/j. ejmg.2005.01.028 Bieller A, Pasche B, Frank S, Gläser B, Kunz J, Witt K, Zoll B (2001) Isolation and characterization of the human forkhead gene FOXQ1. DNA Cell Biol 20(9):555–561. https://doi.org/10.1089/104454901317094963 Birnbacher R, Chudoba I, Pirc-Danoewinata H, König M, Kohlhauser C, Schnedl W, Haas OA (2001) Microdissection and reverse painting reveals a microdeletion 6(q26qter) in a de novo r(6) chromosome. Ann Génét 44(1):13–18. https://doi.org/10.1016/ S0003-3995(00)01033-9 Carnevale A, Blanco B, Castillo J, del Castillo V, Dominguez D (1979) Ring chromosome 6 in a child with minimal abnormalities. Am J Med Genet 4(3):271–277. https://doi.org/10.1002/ ajmg.1320040310 Ciocca L, Surace C, Digilio MC, Roberti MC, Sirleto P, Lombardo A, Russo S, Brizi V, Grotta S, Cini C, Angioni A (2013) Array-CGH characterization and genotype-phenotype analysis in a patient with a ring chromosome 6. BMC Med Genomics 6(1):3. https:// doi.org/10.1186/1755-8794-6-3 Corona-Rivera JR, Corona-Rivera A, Zepeda-Romero LC, Rios-Flores IM, Rivera-Vargas J, Orozco-Vela M, Santana-Bejarano UF, Torres-Anguiano E, PintoCardoso M, David D, Bobadilla-Morales L (2019) Ring chromosome 6 in a child with anterior segment dysgenesis and review of its overlap with other FOXC1 deletion phenotypes. Congenit Anom 59(5):174–178. https://doi.org/10.1111/cga.12309 Dawson AJ, Marles SL, Harman CR, Phillips S, Menticoglou S (1995) Prenatal diagnosis of ring chromosome 6. Prenat Diagn 15(9):872–874. https:// doi.org/10.1002/pd.1970150915

137 Dong Y, Li J, Zeng Z, Liang M, Yi H, Luo J, Li J (2022) Growth retardation and congenital heart disease in a boy with a ring chromosome 6 of maternal origin. Mol Cytogenet 15(1):9. https://doi.org/10.1186/ s13039-022-00586-1 Fryns JP, Kleczkowska A, van den Berghe H (1990) Ring chromosome 6: Twenty years follow-up. Ann Genet 33(3):179 Gould DB, Jaafar MS, Addison MK, Munier F, Ritch R, MacDonald IM, Walter MA (2004) Phenotypic and molecular assessment of seven patients with 6p25 deletion syndrome: Relevance to ocular dysgenesis and hearing impairment. BMC Med Genet 5(1):17. https://doi.org/10.1186/1471-2350-5-17 Han J, Kraft P, Nan H, Guo Q, Chen C, Qureshi A, Hankinson SE, Hu FB, Duffy DL, Zhao ZZ, Martin NG, Montgomery GW, Hayward NK, Thomas G, Hoover RN, Chanock S, Hunter DJ (2008) A genome-wide association study identifies novel alleles associated with hair color and skin pigmentation. PLoS Genet 4(5):e1000074. https://doi. org/10.1371/journal.pgen.1000074 Höckner M, Utermann B, Erdel M, Fauth C, Utermann G, Kotzot D (2008) Molecular characterization of a de novo ring chromosome 6 in a growth retarded but otherwise healthy woman. Am J Med Genet 146A(7):925–929. https://doi.org/10.1002/ ajmg.a.32251 Kosztolányi G (2009) The genetics and clinical characteristics of constitutional ring chromosomes. J Assoc Genet Technol 35(2):44–48 Li P, Dupont B, Hu Q, Crimi M, Shen Y, Lebedev I, Liehr T (2022) The past, present, and future for constitutional ring chromosomes: A report of the international consortium for human ring chromosomes. HGG Adv 3(4):100139. https://doi.org/10.1016/j. xhgg.2022.100139 Liu S, Wang Z, Wei S, Liang J, Chen N, OuYang H, Zeng W, Chen L, Xie X, Jiang J (2018) Gray matter heterotopia, mental retardation, developmental delay, microcephaly, and facial dysmorphisms in a boy with ring chromosome 6: a 10-year follow-up and literature review. Cytogenet Genome Res 154(4):201–208. https://doi.org/10.1159/000488692 Moore CM, Heller RH, Thomas GH (1973) Developmental abnormalities associated with a ring chromosome 6. J Med Genet 10(3):299–303. https:// doi.org/10.1136/jmg.10.3.299 Nishigaki S, Hamazaki T, Saito M, Yamamoto T, Seto T, Shintaku H (2015) Periventricular heterotopia and white matter abnormalities in a girl with mosaic ring chromosome 6. Mol Cytogenet 8(1):54. https://doi. org/10.1186/s13039-015-0162-3 Pace NP, Maggouta F, Twigden M, Borg I (2017) Molecular cytogenetic characterisation of a novel de novo ring chromosome 6 involving a terminal 6p deletion and terminal 6q duplication in the different arms of the same chromosome. Mol Cytogenet 10(1):9. https://doi.org/10.1186/s13039-017-0311-y

138 Paz-y-Miño C, Benítez J, Ayuso C, Sánchez-Cascos A (1990) Ring chromosome 6: Clinical and cytogenetic behaviour. Am J Med Genet 35(4):481–483. https:// doi.org/10.1002/ajmg.1320350407 Peeden JN, Scarbrough P, Taysi K, Wilroy RS, Finley S, Luthardt F, Martens P, Howard-Peebles PN (1983) Ring chromosome 6: Variability in phenotypic expression. Am J Med Genet 16(4):563–573. https:// doi.org/10.1002/ajmg.1320160413 Pristyazhnyuk IE, Menzorov AG (2018) Ring chromosomes: From formation to clinical potential. Protoplasma 255(2):439–449. https://doi. org/10.1007/s00709-017-1165-1 Salamanca-Gonez F, Nava S, Armendares S (2008) Ring chromosome 6 in a malformed boy. Clin Genet 8(5):370–375. https://doi.org/10.1111/j.1399-0004.1975. tb01516.x Seo S, Kume T (2006) Forkhead transcription factors, Foxc1 and Foxc2, are required for the morphogenesis of the cardiac outflow tract. Dev Biol 296(2):421– 436. https://doi.org/10.1016/j.ydbio.2006.06.012 Sheth F, Liehr T, Shah V, Shah H, Tewari S, Solanki D, Trivedi S, Sheth J (2018) A child with intellectual disability and dysmorphism due to complex ring chromosome 6: Identification of molecular mechanism with review of literature. Ital J Pediatr 44(1):114. https://doi.org/10.1186/s13052-018-0571-0 Sunkak S, Kiraz A, Argun M, Erdoğan İ (2021) Restrictive cardiomyopathy with ring chromosome 6 anomaly in a child. Anatol J Cardiol 25(10):745–746. https://doi.org/10.5152/AnatolJCardiol.2021.80820 Urban M, Bommer C, Tennstedt C, Lehmann K, Thiel G, Wegner RD, Bollmann R, Becker R, Schulzke

F. Sheth et al. I, Körner H (2002) Ring chromosome 6 in three fetuses: Case reports, literature review, and implications for prenatal diagnosis: Ring chromosome 6. Am J Med Genet 108(2):97–104. https://doi.org/10.1002/ ajmg.10215 Walker ME, Lynch-Salamon DA, Milatovich A, Saal HM (1996) Prenatal diagnosis of ring chromosome 6 in a fetus with hydrocephalus. Prenat Diagn 16(9):857–861. https://doi.org/10.1002/(SICI)10970223(199609)16:93.0.CO;2-J Wurster-Hill DH, Hoefnagel D (1975) Banding identification of chromosomal abnormalities in four patients: Ring (6), translocation (2q-;15q+), translocation (21q;21q) and deletion (22q-). J Ment Defic Res 19(2):145–150. https://doi. org/10.1111/j.1365-2788.1975.tb01267.x Yip M-Y (2015) Autosomal ring chromosomes in human genetic disorders. Transl Pediatr 4(2):164–174. https://doi.org/10.3978/j.issn.2224-4336.2015.03.04 Zhang HZ, Li P, Wang D, Huff S, Nimmakayalu M, Qumsiyeh M, Pober BR (2004) FOXC1 gene deletion is associated with eye anomalies in ring chromosome 6. Am J Med Genet 124A(3):280–287. https://doi. org/10.1002/ajmg.a.20413 Zhang R, Chen X, Li P, Lu X, Liu Y, Li Y, Zhang L, Xu M, Cram DS (2016) Molecular characterization of a novel ring 6 chromosome using next generation sequencing. Mol Cytogenet 9(1):33. https://doi. org/10.1186/s13039-016-0245-9 Zhu H (2016) Forkhead box transcription factors in embryonic heart development and congenital heart disease. Life Sci 144:194–201. https://doi. org/10.1016/j.lfs.2015.12.001

Ring Chromosome 7

11

Thomas Liehr

Abstract

Keywords

In general, ring chromosomes (RCs) are among the rarest constitutional chromosomal aberrations. Hereby, chromosome 7 comprises only 23 postnatal and one prenatal clinical case with RC formation. Reasons are not clear yet, even though it is noteworthy, that chromosome 7 is underlying imprinting, which may have a negative effect on viability of RC7 carriers. It is interesting that all yet reported 24 cases of RC7 have three typical clinical features growth retardation, pigmented skin nevi, and microcephaly commonly seen in 92%, 83%, and 67% of patients, respectively. Besides, due to the small number of available cases, it is hardly possible to distinguish effects of RC-induced genomic imbalances and the effects of the practically always present mosaic cell line with a monosomy 7. RC7 can be found as acquired chromosomal changes in tumors occasionally, enhanced risk for malignant melanoma has been reported in constitutional cases, as a result of the mentioned pigmented skin nevi.

Ring chromosome 7 (RC7) · Ring syndrome · Uniparental disomy · Imprinting · Cancer risk

T. Liehr (*)  Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, Jena, Germany e-mail: [email protected]

11.1 Introduction In the early times of chromosome analysis by solid staining between 1962 and 1972, there were reports of ring chromosomes (RCs) of C group chromosomes suspected to be ring chromosome 7 (RC7). However, these cannot be resolved or clarified in retrospective (Biesecker et al. 1991). The first two definite RC7 carriers were reported in 1973 (Zackai and Breg 1973). Already, this first report highlighted that phenotypes in the two initially spotted patients were markedly different, but also had three features in common: growth retardation, pigmented skin nevi, and microcephaly. After the initial report of these two RC7 cases by quinancrine fluorescence banding patterns, more patients with RC7 were reported after introduction of GTG-banding techniques. They show the typical cytogenetic features of RC patients in general: mosaicism with or without a normal cell line, with a monosomy 7 cell line and or a cell line with RC duplication—the latter may be due to intrinsic duplication or the presence of one

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_11

139

140

RC7 and a supernumerary RC7 (Li et al. 2022; Zackai and Breg 1973). It is well-known that mosaicism rates can alter during time and be variant in different tissues of the patient (SalasLabadía et al. 2014). In 1996, fluorescence in  situ hybridization (FISH) was applied  to characterize a sub-band deletion of 7q36.3 in a RC7 (Sawyer et al. 1996). In 2013, chromosome microarray analysis (CMA) was used to define the genomic imbalances at the distal short (p) and long (q) arms in a RC7 (Henderson et al. 2013). In 2021, next-generation sequencing (NGS) on targeted cancer gene panel was used first time in a RC7 case (Roy et al. 2021). There are 24 reported cases of constitutional RC7, which comprise about 2% of all reported RCs in the literature (Li et al. 2022). Besides, there are also RC7 cases reported as constitutional small supernumerary marker chromosomes (sSMCs)—according to Liehr (2023), 17 of 53 sSMCs reported by now have ring chromosome shape (see Chap. 29). Furthermore, RC7 is regularly observed as acquired chromosomal aberration in tumor cases, like leukemia (Tirado et al. 2021), lymphoma (Jain et al. 2018), and solid tumors (Lopez-Gines et al. 2006) (see Chaps. 31 and 32). Additionally, in 3 of the here reviewed 24 RC7 cases, melanomas developed from the skin nevi.

11.2 Laboratory Results and Clinical Observations Of the 24 cases of RC7, there is an equal gender ratio of 12 cases for male and female. Except for one prenatal case, age of diagnosis and clinical follow-up ranged from newborn to 22 years. As summarized in Table 11.1, all 24 cases were assigned a number from RC7-1 to RC7-24 following a chronological order of the case reports. All cases were studied by banding cytogenetics; seven cases (RC7-13, RC-15 to RC7-17, RC720, RC7-21, and RC7-23) were furthermore analyzed by FISH; CMA was used in two cases, RC7-21 and RC7-22; and NGS-based analysis only in case, RC7-23. Ethnicities, as far as identified in the papers, include European (n = 10),

T. Liehr

South American (n = 1), middle-American (n = 2), and Asian descent of Japanese/Chinese (n = 6), and Indian/Indonesian (n = 2).

11.2.1 Detailed Genetic and Clinical Data for the 24 Published RC7 Cases RC7-1 (Zackai and Breg 1973, Case 1) The case was studied by Quinacrine fluorescence (QF) bands; the karyotype was: mos 46,XY,r(7)[57]/46,XY,r(7;7)[2]/45,XY,7[2]/47,XY,r(7),+r(7)[1]/46,XY[1]. This 23-month-old boy was studied due to growth and developmental delay, short stature  three independent chromosomal abnormalities). Compared to MDS with complex-karyotypes and lacking aRCs, MDS with aRCs have a shorter leukemia-free survival (P = 0.016) and a trend toward shorter durations of overall survival (P = 0.10) (Rosenbaum et al. 2017). In general, aRCs in MDS are associated with an adverse prognosis due to complex karyotypes. This review summarizes reports on aRCs of identified origin in MDS (Table 31.2). Among aRCs with identified origins in MDS, over onequarter are attributed to aRC7 (26.4%), followed by aRC5 (18.4%), aRC11 (8.0%), aRC6 (5.7%), aRC12 (5.7%), aRC20 (5.7%), aRC17, aRC8, aRC18, aRC1 and aRC3 with frequencies ranging from 4.6% to 2.3%, and the remaining aRCs of chromosomes 2, 14, 15, 16, 22, X and Y with a frequency of 1.1% (Fig. 31.2b). aRCs with complex rearrangements have also been infrequently reported in MDS patients, all of which involve two nonhomologous chromosomes such as r(7;12), r(9;21), r(11;19), and r(21;22) (Fig. 31.2b). Most of these complex aRCs are revealed by SKY/M-FISH (Fleischman et al. 1999). Among aRCs with identified origins in MDS, most (82.8%) of these ring chromosomes are part of complex karyotypes, 9.2% are present along with one other chromosomal

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

abnormality, and 8.0% are present as a sole chromosome abnormality. aRC1, aRC3, aRC5, aRC6, aRC7, and aRC11 have been reported as a sole chromosome abnormality in MDS, with chromosome 7 being the most common. aRC7 usually loses chromosome 7 material (such as 7q deletion) during the formation of aRC and monosomy 7 due to RC instability. 7q deletion/ monosomy 7 is recurrent in MDS and constitutes the sole cytogenetic aberration in a small percentage (< 5%) of patients. In MDS, del(7q) as a sole aberration is usually associated with intermediate risk, while del(7q), in combination with a complex karyotype, is associated with an unfavorable prognosis (Schanz et al. 2012). MDS patients with del(7q) as a sole aberration can be further characterized by their accompanying somatic mutations into various risk groups (Hartmann et al. 2019).

31.3.3 aRCs in Chronic Myeloid Leukemia (CML) The presence of aRCs, in general, is not a common finding in chronic myeloid leukemia (CML), which is a myeloproliferative neoplasm caused by a reciprocal translocation t(9;22) (q34;q11.2) with fusion of ABL1 and BCR genes. The derivative chromosome 22 with BCR::ABL1 fusion is cytogenetically visible as the Philadelphia chromosome (Ph), which leads to the constitutive expression of active tyrosine kinase that activates multiple signaling pathways and plays a vital role in the pathogenesis of CML. During the initial chronic phase of CML (CP-CML), the myeloid cell compartment is expanded with maintained differentiation. During the relapse and lack of effective therapy with resistance, CP-CML may progress into a blast phase of CML (BP-CML) associated with the accumulation of secondary cytogenetic and/ or molecular aberrations. BP-CML is an aggressive disease with acute leukemia of myeloid or lymphoid phenotypes. Various aRCs have been reported in CML, and this review summarizes reports on identified aRCs in Table 31.3. Among aRCs with identified

435

origins in CML, aRC17 (20.8%) is the most common, followed by other aRCs (Fig. 31.2c), possibly associated with the loss of the TP53 gene during the ring formation. Although aRCs could occur in CP-CML, they occur more frequently during BP-CML (Fig.  31.2c, Table 31.3). For aRCs that occur during BP-CML, approximately one-third (6/17 reported cases) occur as a sole chromosomal abnormality in addition to an initial Philadelphia translocation, and the remaining two-thirds (11/17) present as part of a complex karyotype along with other secondary chromosomal abnormalities such as trisomy 8, trisomy 19, double Ph chromosome, monosomy 17/17p deletion, etc. (Table 31.3). The data on aRCs in CML further supports the association of rings with aggressive disease and with the progression of CP-CML into BP-CML (Lewis et al. 1988; Sadamori et al. 1985). The BP-CML may be refractory to current therapy and is thus associated with a shortened survival and poor prognosis. aRCs could also occur during relapse, and aRCs 18 and 20 have been observed in CML after successful bone marrow transplants (Calabrese et al. 1989). Variant Ph translocations such as three-way Ph translocations t(9;V;22) have been infrequently described in CML. One instance of an aRC involving a translocated chromosome 9, that is r(9)t(9;16;22), has been identified using FISH with whole-chromosome paint probes and BCR and ABL1 probes in conjunction with karyotype studies (Morel et al. 2003).

31.3.3.1 The Philadelphia Chromosome as an aRC in CML The Ph chromosome is the hallmark of CML, and a complex karyotype including an extra ringshaped Ph chromosome along with a Ph translocation, tetrasomy 8 and trisomy 19 has been reported in an unusual BP-CML case (Wafa et al. 2015; Borjas-Gutierrez and Gonzalez-Garcia 2016). The ring-shaped Ph chromosome was initially characterized as an inv dup(22)(q11.23) using conventional chromosome analysis, FISH, and reverse transcription polymerase chain reaction (Wafa et al. 2015). It was observed after the

436

Y. S. Zou et al.

Table 31.3  aRCs in chronic myelogenous leukemia (CML) Case

Chronic phase (Karyotype)

Accelerated phase (Karyotype)

References

aRC4

46,XY,t(9;22)(q34;q11.2)

46,XY,+r(4),t(9:22),−17

Tien et al. (1989)

aRC6

N/A

46,XX,r(6)(p2?4q2?6)

Deininger et al. (2007)

aRC7-1

46,XY,t(9;22)(q34;q11.2)

46,XY,r(7),t(9;22)

Terre et al. (2004)

aRC7-2

46,XY,t(9;22)(q34;q11.2)

46,XY,r(7),t(9;22),add(9)(q?)

Prigogina et al. (1978)

aRC7-3

N/A

45,XX,−7,t(9;22)(q34;q11)/46,XX,r(7) (p13q11),t(9;22)(q34;q11.2)

Hehlmann et al. (2020)

aRC7-4

N/A

46,XY,r(7)(p11q32),del(7)(q11q22),del(9) (p24p12),t(9;22)(q34;q11.2)

Hehlmann et al. (2020)

N/A

aRC9

46,XX,r(9)t(9q;16p;22q)

aRC11-1

46,XY,t(9;22)(q34;q11.2),r(11) N/A

Sadamori et al. (1980)

Morel et al. (2003)

aRC11-2

47,XY,+r(11)(:p11.2;q13.1::q14:)

N/A

Starke et al. (2001)

aRC15-1

46,XY,t(9;22)(q34;q11.2)

46,XY,t(9;22),r(15)

Movafagh et al. (2015)

aRC15-2

46,XY,t(8;11),t(9;22) (q34;q11.2)

46,XY,del(6)(p21→pter),t(8;11),t(9;22),r(15)

Lai et al. (1987)

aRC17-1

46,XX,t(9;22)(q34;q11.2)

46,XX,t(9;22),r(17)

Borgstrom et al. (1982)

aRC17-2

46,XX,t(9;22)(q34;q11.2)

46,XX,t(9;22)(q34;q11.2),r(17)

Lewis et al. (1988)

aRC17-3

45,X,−Y,t(9;22)(q34;q11.2)

45,X,−Y,t(9;22)(q34;q11.2),r(17)

Lewis et al. (1988)

aRC17-4

46,XX,t(9;22)(q34;q11.2)

44,X,−X,−5,add(5) (p?),t(9;22),r(17),−21,+mar

Sadamori et al. (1981)

aRC17-5

N/A

46,X,−X,−2,−2,−8,−9,t(9;22) (q34;q11.2),−12,−15,r(17),+7mar

Lewis et al. (1988)

aRC18-1

47,XY,+8,t(9;22) (q34;q11.2),r(18)

46,XY,t(9;22)(q34;q11.2),r(18)

Calabrese et al. (1989)

aRC18-2

N/A

44–45,XY,dic(5;17)(q11;p11),t(9;22) (q34;q11.2),r(18)

Hagemeijer et al. (1981)

aRC18-3

46,XX,t(9;22)(q34;q11.2)

46,XX,t(6;7)(q27:p11.2),t(9;22) (q34;q11.2),r(18)

Da Silva et al. (1988)

aRC18-4

46,XY,t(9;22)(q34;q11.2)

1st: 46,XY,t(9;22),r(18); 2nd:45,XY,−5,t(9;22)(q34;q11.2),+t(5;17) (p?;q?),−17,r(18)

Hagemeijer et al. (1981)

aRC19

46,XY,t(9;22)(q34;q11.2)

49,XY,add(1)(q?),+8,t(9;22)(q34;q11.2), +t(9;22),+17,r(19)

Carbonell et al. (1982)

aRC20

46,XY,t(9;22)(q34;q11.2)

46,XY,add(3)(q?),t(9;22)(q34;q11.2),r(20)

Calabrese et al. (1989)

aRC(22;9)

46,XY,t(9;22)(q34;q11.2)

48,XY,+8,+8,t(9;22)(q34.1;q11.2),der(22) r(22;9)(::22p13 → 22q11.2::9q34::9q34::22q11.2 → 22p13::)/49, idem,+19

Wafa et al. (2015) and Borjas-Gutierrez et al. (2016)

aRC(X)

N/A

46,XX,der(7)del(7)(p11)del(7)(q11),t(9;22) Hehlmann et al. (2020) (q34;q11.2)/45,idem,der(3;9;17),+der(9) ins(9;17)(p11.?;?),der(9;17)dic(9;17) (q11;p11)del(9)(p24p13),der(14)t(1;14) (q21;q21),+17,der(22)t(9;22)/45,X,r(X),der(3;9;17),der(7)del(7)(p11.?)del(7) (q11.?),+der(9)ins(9;17)(p11.?;?),t(9;22) (q34;q11.2),der(9;17)dic(9;17) (q11;p11)del(9)(p24p13),der(14)t(1;14) (q21;q21),+17,der(22)t(9;22)

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

patient experienced an interruption of successful Imatinib treatment for 16 months, and subsequently, the patient rapidly progressed toward disease (Wafa et al. 2015). The ring-shaped Ph chromosome, in this case, was proposed to be a duplicated aRC22, and ring chromosome instability leads to the risk of the emergence of clones containing more and more BCR::ABL1 gene copies, which resulted in worsening of the patient’s prognosis (Wafa et al. 2015).

31.3.3.2 Co-concurrent aRCs and Double Minutes (dmin) in CML Co-concurrent aRCs (small-ring D group, largering D group, double ring D group) and various-sized dmin with two extra Ph, one pair of dot-shaped fragments, and trisomies of B group, 6, and 19 have been described in a BP-CML case (Uehara et al. 1987). The dmin chromosomes are a form of gene amplification presenting as small spherical paired chromatin bodies, an infrequent finding in CML and have no clear definition of the chromosomes of their derivation in most reported cases (Uehara et al. 1987; Morel et al. 2003; Suciu et al. 1983). These dmin chromosomes or isochromosomes of the Ph chromosome could increase the copy number of the BCR::ABL1 gene (Koka et al. 2017). These dmin chromosomes have been proposed to be acentric or small ring-shaped rings originating from large ring chromosomes (Marinello et al. 1980). The well-known breakage-fusionbridge cycle causes unstable chromosomal changes such as the formation of various-sized rings, breakage, deletion, and duplication, which has been proposed to be one possible mechanism for generating the various-sized rings observed in this case with both aRC and dmin chromosomes. The relation of aRCs to the presence of dmin in CML is still unelucidated.

31.3.4 aRCs in Miscellaneous Myeloid Neoplasms Besides CML, myeloproliferative neoplasms include Polycythemia Vera (PV), Essential

437

Thrombocythemia (ET), Primary Myelofibrosis (PMF), Chronic Neutrophilic Leukemia (CNL), Chronic Eosinophilic Leukemia (CEL), Juvenile Myelomonocytic Leukemia (JMML), and myeloproliferative neoplasm, not otherwise specified (MPN-NOS). Myelodysplastic/MPNs contain Chronic Myelomonocytic Leukemia (CMML), MDS/MPN with neutrophils, MDS/ MPN with SF3B1 mutations and thrombocytosis, or MDS/MPN not otherwise specified (MDS/MPN-NOS). While aRCs are not typical in these MPNs/related neoplasms, aRC6, aRC7, and aRC13 have been reported previously. aRC6 has been described in a JMML (Passmore et al. 1995), an MPN-NOS (Sun and Cook 2010), and a PMF (Miller et al. 1985) (see cases aRC6-1 to aRC6-3 in Table 31.4). aRC7 has been described in an MPN-NOS (Gao et al. 2022), a CMML (Streubel et al. 1998), an ET (Slee et al. 1981), and three PMF cases with non-complex karyotypes (Gibbons et al. 1994; Temperani et al. 1989; Emilia et al. 1989) (see cases aRC7-1 to aRC7-4, aRC7-7, and aRC711 in Table 31.4). aRC7 was also reported in donor cell-derived myeloid leukemias after allogeneic hematopoietic stem cell transplant in two patients with lymphoma as their primary disease (Hamdi et al. 2014) (see cases aRC7-5 and aRC7-6 in Table 31.4). aRC11 and aRC13 have been reported in ET and PMF with complex karyotypes, respectively (Sato et al. 1995; Liozon et al. 1997) (see cases aRC11 and aRC13 in Table 31.4). Besides AML, MDS, and MPN, aRCs have been infrequently reported in other myeloid hematologic malignancies, such as bi-lineage/ bi-phenotypic leukemia, systemic mastocytosis, etc. For bi-lineage / bi-phenotypic leukemia, aRC7 has been described in three patients (Gibbons et al. 1994; Strehl et al. 2008; Park et al. 2009a, b), and aRC2 has been reported in a single patient (Kim et al. 2015) (see cases aRC2, aRC7-8 to aRC7-10 in Table 31.4). aRC12, as part of a complex karyotype, has been reported in systemic mastocytosis (Kluin-Nelemans et al. 2021; Naumann et al. 2018) (see cases aRC12-1 and aRC12-2 in Table 31.4).

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Y. S. Zou et al.

Table 31.4  aRCs in miscellaneous myeloid neoplasms Case

Disease

Karyotype

References

aRC2

BAL

46,XY,r(2)(p25q31)/47, idem,−7,+10,+12

Kim et al. (2015)

aRC6-1

Idiopathic MF

46,XY,der(2)t(2;6)(q3?7;q1?5),r(6) (p2?5q1?5)

Miller et al. (1985)

aRC6-2

JMML

46,XY,r(6)/46,XY,del(4)(q25q31)

Passmore et al. (1995)

aRC6-3

MDS/MPN-NOS

46,XX,del(5)(q15q33)/46,idem,r(6) (p22q27)

Sun and Cook (2010)

aRC7-1

MDS/MPN-NOS

46,XX,t(3;3)(q21;q26)/46,idem,r(7)/45,idem,−7

Gao et al. (2022)

aRC7-2

CMML

46,XY,r(7),del(12)(p13p11)/45,XY,−7, Streubel et al. (1998) del(12)(p13p11)

aRC7-3

ET

46,XX,r(7)

Slee et al. (1981)

aRC7-4

Primary MF

46,X,idic(X)(q13),r(7)/46,X,idic(X) (q13)

Emilia et al. (1989)

aRC7-5

FL →donor cell AML

46,XY,r(7)(p13q11.2),del(12)(p13)

Hamdi et al. (2014)

MCL →donor cell MDS

46,XX,r(7)(p21q11.2)

Hamdi et al. (2014)

Idiopathic MF

46,XX,r(7)

Gibbons et al. (1994)

aRC7-8

BAL

46,XX,del(5)(?q31q34),dup(7) (?q22q32),r(7)

Gibbons et al. (1994)

aRC7-9

BAL

46,XX,del(5)(q23q32),r(7),t(8;12) (q12;p13),der(16)t(1;16)(?;?q?)

Strehl et al. (2008)

aRC7-10

BAL

46,XX,r(7)(p22q22)

Park et al. (2009a, b)

aRC7-11

Idiopathic MF

46,X,idic(X)(q13)/46,idem,r(7)(p15q32) Temperani et al. (1989)

aRC11

ET

46,XY,del(5)(q13q34),del(7) (q22q31),add(11)(q23),dic(11;16) (q23;q23),+r(11)(p15q23),der(21) t(21;22)(p13;q10),−17,dmin

Liozon et al. (1997)

aRC12-1

SM

46,XX,del(8)(q21),der(10)t(8;10) (q22;q26),inv(12)(q21q24),r(12) (p12q15),der(16)t(12;16)(q21;q22)

Kluin-Nelemans et al. (2021)

aRC12-2

SM

46,XX,del(8)(q21),der(10)t(8;10) (q22;q26),r(12)(p12q15),der(16) t(12;16)(q21;q22)inv(12)(q21q24)

Naumann et al. (2018)

aRC13

Idiopathic MF

47~50,XY,−5,del(7)(q31q35),+8,+11,- Sato et al. (1995) der(12)t(12;15)(p13;q1?2),add(13) (p11),r(?13),−15,−15, add(21) (q22),+2~5mar

aRC7-6 aRC7-7

AML: acute myeloid leukemia; BAL: bilineage or biphenotypic leukemia; CMML: chronic myelomonocytic leukemia; ET: essential thrombocythemia; FL: follicular lymphoma; JMML: juvenile myelomonocytic leukemia; MCL: mantle cell lymphoma; MDS/MPN-NOS: myelodysplastic/myeloproliferative disease, NOS; MF: myelofibrosis; SM: systemic mastocytosis

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

31.4 aRCs in Lymphoid Hematologic Malignancies 31.4.1 aRCs in Acute Lymphoblastic Leukemias/ Lymphomas (ALL) Acute lymphoblastic leukemia (ALL) is a hematologic neoplasm characterized by the clonal proliferation of lymphoid progenitor cells in the bone marrow and extramedullary sites. Although ALL can occur in young children and adults, it is the most common childhood cancer, especially among children under 15. While it predominantly affects children, ALL represents around 20% of leukemia in adults; of these, B-cell lineage constitutes 75% of cases (B-ALL), and T-cell lineage constitutes 25% of cases (T-ALL). Recurrent chromosomal aberrations including hyperdiploidy and chromosomal translocations, define pediatric B-ALL. Common cytogenetic-molecular abnormalities that confer prognostic value in B-ALL include hyperdiploidy (favorable), translocation t(12;21) leading to the ETV6::RUNX1 gene fusion (favorable), Philadelphia chromosome (Ph)-positive ALL (unfavorable), Ph-like ALL (unfavorable), hypodiploidy with less than 40 chromosomes (unfavorable), KMT2A rearrangement (unfavorable), and the translocation t(1;19) leading to TCF3::PBX1 gene fusion (unfavorable). Hyperdiploidy (with a chromosome count greater than 50) is the largest genetic entity B-ALL in children and is usually associated with a good prognosis. The incidence of cases with aRCs is not wellestablished in ALL. This review summarizes reports on identified aRC in ALL (Table 31.5). Among aRCs with identified origins in ALL, nearly one-third (30.9%) are aRC21 (20.2% are somatic and 10.7% are constitutional), followed by aRC7 (11.9%), aRC9 (10.7%), aRC15 (9.5%), aRC1 (7.1%), aRC10 (3.6%), aRCs of chromosomes 2, 4, 5, 12, 13, and 19 (2.4%), and aRCs of chromosomes 3, 6, 8, 11, 20, 22, Y, r(1;9), r(9;13), and r(BCR::ABL1) (1.2%) (see Table 31.5, Fig. 31.2d). These aRCs are

439

commonly part of non-hyperdiploid karyotypes (96.4%) compared to hyperdiploid karyotypes (3.6%). The majority (53.6%) of aRCs have abnormal karyotypes with 46 chromosomes, followed by karyotypes with 47 (15.5%), with 45 (13.1%), with 48 (5.9%), with 44 or 49 (3.6%), and with 26, 56, 59–60, or 83–93 chromosomes (1.2%) (see Fig. 31.2d). While in most cases, they are present as part of a complex karyotype (66.7%), aRCs can also present as a sole chromosome abnormality (15.5%) and as part of one of two chromosome abnormalities (17.8%) in other cases (Table 31.5).

31.4.1.1 aRCs of 9;22 with BCR::ABL1 Gene Fusion in ALL aRC derived from BCR::ABL1 gene fusion has been described in ALL (Adachi 2012) (see case aRC(Ph) in Table 31.5). This 44-year-old B-ALL case had a karyotype of monosomy 9, 22 and gain of an aRC. FISH demonstrated multiple BCR::ABL1 fusion signals in an aRC, and a minor bcr (m-BCR) of BCR::ABL1 transcripts was revealed by qualitative reverse transcriptasepolymerase chain reaction. It is postulated that this unique aRC structure contributes to the clinical presentation of B-ALL. 31.4.1.2 Constitutional Robertsonian Translocation t(15;21) and iAMP21 in ALL Constitutional RC21 is relatively common among RCs with identified origin in ALL (Cabrol et al. 1983; Falchi et al. 1987; Melnyk et al. 1995; Harrison and Schwab 2016; Li et al. 2014) (Fig. 31.2d, Table 31.5). Constitutional structural chromosome 21 rearrangements have been proposed to confer the risk of developing a B-ALL (Harrison and Schwab 2016). Constitutional chromosome 21 abnormalities have been related to intrachromosomal amplification of chromosome 21 (iAMP21) leukemogenesis. iAMP21 is well established in B-cell precursor ALL, at an incidence of approximately 2% (Moorman et al. 2013) and characterized by complex morphology of one copy of chromosome 21, comprising multiple regions of gain, amplification, inversion, and deletion (Robinson

440

Y. S. Zou et al.

Table 31.5  aRCs in acute lymphocytic leukemia (ALL) Case

Karyotype

References

aRC1-1

46,XX,r(1)(p36q3?2)

Ludwig et al. (1989)

aRC1-2

59,XY,+X,+2,+4,+5,+6,add(9) (p?),+8,+10,+14,+15,+16,+18,+21,+22/60,idem,+1, −add(9)/60,idem,+r(1),−add(9)

Waghray et al. (1986)

aRC1-3

46,XY,r(1)(p36q42~44),add(7)(p21),del(10) (q22q25),t(10;14)(q26;q11),qdp(21)(q11q22)/46, idem,t(12;15)(p11;q13)

Rand et al. (2011) and Harrison et al. (2014)

aRC1-4

45,XX,der(2)t(2;16),del(9),der(12)t(2;12),der(12) t(12;16),−16,der(16)t(16;19),der(19)t(9;19)/45, idem,r(1)/45,idem,r(1),−13,+21

Elghezal et al. (2001)

aRC1-5

46,XY,r(1)(p36q42),t(4;11)(q21;q23),inv(14) (q11q32)/45,XY,t(4;11)(q21;q23),der(11)t(11;17) (p11;q11),inv(14)(q11q32),−17

Chervinsky et al. (1995)

aRC(1;9)

46,XX,r(1;9)(p12q44;p11q24),t(1;7;9)(p12;q11;p11)

Heerema et al. (1994)

aRC2-1

46,XX,del(9)(p22)/46,idem,i(17)(q10)/46,XX,r(2) (p23~25q33~37)

Martineau et al. (1996)

aRC2-2

46,XY,der(19)t(1;19)(q23;p13)/46,idem,r(2),del(13) (q14q32)/49,idem,+4,+5,+8

Kovacs et al. (1986)

aRC3

83–93 ,XX,r(3)(p25q29),r(3)dup/trp(p25q29),inc

Gindina et al. (2015)

aRC4-1

46,XX,r(4)

Chessells et al. (2002)

aRC4-2

46,XX,r(4)/47,XX,r(4),+18/48,XX,r(4),+18,+18

Shah et al. (2001)

aRC5-1

46,XY,t(2;11)(p16;q24)/46,idem,r(5)(p14q12)

Grossmann et al. (2013)

aRC5-2

45,XX,r(5)(p10q34),del(9)(p24),−21/46,XX,del(4)(q31), del(5)(q21)

Karst et al. (2006)

aRC6

45–47,XX,add(1)(p36),add(3)(p24),r(6),inv(7) (p15q34),+20,+21

Arniani et al. (2022)

aRC7-1

46,XY,r(7),inc

Cauwelier et al. (2007)

aRC7-2

47,XX,r(7)(p12q31),+9

Roberts et al. (2012)

aRC7-3

46,XX,del(5)(q13q33),r(7)(p22q36)

Chang et al. (2006)

aRC7-4

46,XX,r(7)(p22q36),t(9;22)(q34;q11.2)

Cuneo et al. (1994)

aRC7-5

48,XY,del(6)(q13q22),r(7),+8,+12

Le Noir et al. (2012)

aRC7-6

46,XX,de1(5)(?q31q34),r(7),add(7)(q32)

Gibbons et al. (1994)

aRC7-7

45,XY,der(1)t(1;9)(p36;q?),del(6)(q2),r(7),−9

Barber et al. (2004)

aRC7-8

46–48,XX,+del(1)(q32),+3,r(7),add(14)(q32),−20,+mar

Kristoffersson et al. (1985)

aRC7-9

46,XX,r(7)(p22q36)/85–91,XXXX,−7,r(7)(p22q36) x1~2,−14

Tirado et al. (2021)

aRC7-10

46,XY,der(2)t(2;7;5)(q37;q36;q34),r(7)(p21q31),del(9) (p24p21)/46,XY, der(2)t(2;7;5)(q37;q36;q34),i(7)(q10), del(9)(p24p21)

Jarosova et al. (2003)

aRC8

46–48,XX,+r(8)(p?23q?22~24)x2,der(9;22)(22pter→ 22q11.2::9q34.1→9q34.2::9p10::9q34.1::22q11.2→ 22qter)x1~2/46~48,idem,ins(2;8)(q13; p?23q?22~24)

Edelhäuser et al. (2000)

aRC9-1

46,XX,r(9)(q34q34)

Graux et al. (2004)

aRC9-2

46,XY,del(9)(p21p21),r(9)(q34q34)

Graux et al. (2004)

aRC9-3

46,XY,del(9)(p21p21)x2,r(9)(q34q34)

Graux et al. (2004) (continued)

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

441

Table 31.5  (continued) Case

Karyotype

References

aRC9-4

46,XY,r(9)(q34q34),del(12)(p13)

Graux et al. (2004)

aRC9-5

46,XY,r(9)/46,XY,i(9)(q10)

Pui et al. (1992)

aRC9-6

46,XY,t(6;12)(p22;q24.3),+r(9)

Chesselss et al. (2002)

aRC9-7

47,XY,del(6)(q21),+8,r(9)(q34q34)

Graux et al. (2004)

aRC9-8

46,Y,add(X)(p22),t(8;22)(p22;q12),del(9)(p21p21),r(9) (q34q34),del(13)(q14q22)

Graux et al. (2004)

aRC9-9

46,XY,der(1)t(1;9)(p36;?),t(2;7)(p21;p15),der(5)t(5;9) (p13;?),r(9),del(13) (q14q21),der(19)t(9;19)(?;p13)t(5;9) (p13;?)

Grossmann et al. (2013)

aRC(9;13)

46,XY,r[der(9)t(9;13)(q31;q14)],der(13)t(9;13)(q31;q14)

GFCH (1993)

aRC10-1

45,XX,−7,r(10)

Heerema et al. (1999)

aRC10-2

48,XX,r(10)(p15q26),+21c,+21

Mullighan et al. (2009)

aRC10-3

26,X,+10,+18,+21/26,idem,r(10)

Holmfeldt et al. (2013)

aRC11

44,XY,r(11)(p15q23),t(12;20) (p13;q11.2),−17,−20[4]/43,idem,−9,der(21),−22,+mar

Rossbach et al. (1998)

aRC12-1

46,XX,r(12)(p13q24)

Yamamoto et al. (2006)

aRC12-2

46XY,t(2;22)p25;q11),r(12)/46,XY,t(2;22)(p25;q11)

Latham et al. (1994)

aRC13-1

46,XX,t(1;19)(q23;p13),i(9)(q10),r(13)

Jonveaux et al. (1990)

aRC13-2

44,X,−Y,der(1)t(Y;1)(q12;q?21),der(3)t(1;3)(p11;q?),del(5)(q3?3q3?5),der(6)(6pter→6q22::1q?::8q?),der(12) t(1;12)(?;p11),r(13)

Leroux et al. (2002)

aRC15-1

47,XY,+r(15)

Abdi et al. (1990)

aRC15-2

49,XX,+X,+10,r(15)(p11q26),−21,+2mar

Cooley et al. (2007)

aRC16-1

46,XX,r(16)

Callera et. al. (2008)

aRC16-2

44,XY,−4,dic(9;17)(p11;p11),r(16)

Coyaud et al. (2010)

aRC16-3

46,XX,t(8;12)(q13;p13),r(16)(p13q24)

Schneider et al. (2000)

aRC16-4

46,XY,add(7)(q34),del(8)(q21),r(16)(p13q24),−18,−21, +2mar/43,idem,−Y,−8,−13,−14,add(15)(p11),+mar

Cooley et al. (2007)

aRC16-5

46,XY,t(8;12)(q13;p13),r(16)/52,idem,del(1)(p22), del(5)(p13),+6,i(7)(q10),t(7;10)(q34~35;q24),del(7) (p13~15),+10,+11,der(12)t(7;12)(q11;p11), inv(12) (p12q15~21),t(12;13)(p11~12;q12),+14,i(17) (q10),+18,+22

Schneider et al. (2000)

aRC19-1

46,XX,r(19)(p13q13),add(21)(q22)

Baughn et al. (2015)

aRC19-2

56,XX,+X,+4,+6,+10,+14,+add(17)(p11),+18,+18,r(19) (p13q13),+21,+21

Martin et al. (1996)

aRC20

45,X,−X,r(20)/46,X,i(X)(p10),r(20)

Adeyinka et al. (2007)

aRC21-1

46,XY,r(21)

Harrison et al. (2014)

aRC21-2

46,XY,der(21)r(21)(q?)dup(21)(q?)

Harewood et al. (2003)

aRC21-3

46,XY,t(1;14)(p34;q11),r(21)

Kaneko et al. (1989)

aRC21-4

47,XX,+X,der(21)r(21)(q?)dup(21)(q?)

Harewood et al. (2003)

aRC21-5

47,XX,add(8)(q24),r(21),+mar

Harrison et al. (2014)

aRC21-6

46,XX,add(7)(p22),add(16)(q12),r(21)

Harrison et al. (2014) (continued)

442

Y. S. Zou et al.

Table 31.5  (continued) Case

Karyotype

References

aRC21-7

47,XX,+X,del(20)(q13q13),r(21)

Harrison et al. (2014)

aRC21-8

46,XX,add(11)(p15),r(21),dup(21)(q22q22)

Johnson et al. (2015)

aRC21-9

48,XX,del(11)(q22),r(21),+2mar/46,XX,r(21)

Heim et al. (1990)

aRC21-10

45,XX,−7,r(21)/45,idem,+7,−9,add(12)(q24)

Harrison et al. (2014)

aRC21-11

47,XX,+X,add(9)(p21),i(9)(q10),r(21),+mar

Harrison et al. (2014)

aRC21-12

47,XX,add(7)(q2),+10,der(21)r(21)(q?)dup(21)(q?)/47, idem,del(12)(p13)

Harewood et al. (2003)

aRC21-13

45,XX,dup(8)(p?),−11,der(15)t(11;15)(?;q24),der(21)r(21) Harewood et al. (2003) (q?)dup(21)(q?)

aRC21-14

45,Y,t(X;15)(q2?1;q2?4),dic(12;17)(p1?;p1?),der(21)r(21) Harewood et al. (2003) (q?)dup(21)(q?)

aRC21-15

45,XX,der(9)add(9)(p21)t(9;22)(q34;q11.2),−11,r(21),der(22)t(9;22)(q34; q11.2)

Harrison et al. (2014)

aRC21-16

46,XY,add(2)(q13),t(7;15)(q32;q24),r(21)(q?),der(22) t(9;22)(q34;q11.2)add(22)(q11.2)

Harrison et al. (2014)

aRC21-17

46,XY,t(8;11)(p2?1;q21),del(11)(q21),der(21)r(21)(q?) dup(21)(q?)/ 47,idem,+X

Harewood et al. (2003)

RC21c-1

46,XY,r(21)c

Falchi et al. (2022)

RC21c-2

46,XX,r(21)c

Cabrol et al. (1983)

RC21c-3

46,XX,r(21)c

Werner-Favre et al. (1986)

RC21c-4

46,XX,r(21)(p11.2q22)c

Baloda et al. (2022)

RC21c-5

49,XX,+10,−21,r(21)c,+3r

Stern et al. (1979)

RC21c-6

45–47,XX,del(11)(q22),r(21)c,inc

Andreasson et al. (2000)

RC21c-7

48,XX,del(9)(p21p21),del(11)(q22),r(21)c,+2mar

Andreasson et al. (20000

RC21c-8

48,XX,del(11)(q22),r(21)c,+2mar/46,XX,r(21)c

Andreasson et al. (2000)

RC21c-9

46,XY,t(2;16)(p?11;p13),der(9)(1;19)(q21;p13.3), dic r(21)/ 50,idem,+X,,+X,+6,+13,+14,−der(19) t(1;19),+22/46,XY,r(21)c

Uckun et al. (1998)

aRC22

47,XX,+X,der(9)t(8;9)(q24;p24),der(12)hsr(12)(p13) t(12;22)(p13;q11.?2), der(14)t(8;14)(q24;q32),r(22) (p13q13)

Chae et al. (2010)

aRC(Ph)

45,XX,−9,−22,+r(9;22), ring has multiple BCR::ABL signals by FISH

Adachi (2012)

aRC(Y)

47,X,r(Y)(p11q11)c,+5,t(8;14)(q11;q32),der(21)t(1;21) (q12;q22)

Messinger et al. (2012)

et al. 2007; Rand et al. 2011; Strefford et al. 2006). Patients harboring constitutional Robertsonian translocations involving chromosomes 15 and 21, der(15;21)c, have a ∼2,700fold increased risk of developing iAMP21-ALL (Harrison et al. 2014, Harrison and Schwab 2016). Constitutional RC21 is significantly predisposed to a B-ALL subtype with iAMP21

(Falchi et al. 1987). A patient with Down syndrome who carried a constitutional isodicentric chromosome 21 [idic(21)] developed B-ALL, which has specific genomic profiles consistent with iAMP21 leading to leukemic transformation (Verdoni et  al. 2022; Putra et  al. 2017). Isodicentric chromosome 21 are dicentric, constitutional rings of chromosome 21 can

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

frequently generate double-ring chromosomes due to the RC instability, which are also dicentric. In the der(15;21)c cases, iAMP21 was initiated by chromothripsis, which occurs through one massive genomic rearrangement during a single catastrophic event, simultaneously involving both sister chromatids of the dicentric Robertsonian chromosome (Li et al. 2014). These findings imply that constitutional structural abnormalities are predisposed to leukemia through chromothripsis, likely related to their dicentric nature. Therefore, these constitutional structural chromosomes 21 are not genomically stable and may lead to breakage caused by the mechanical forces of the mitotic spindle during mitosis. Once random breakage occurs on the 21q, the RUNX1 gene on 21q22 may undergo amplification along with loss of surrounding chromosomal regions possibly via breakagefusion-bridge cycles followed by chromothripsis (Zhang et al. 2015). Other complex structural rearrangements of chromosome 21 underlie the mechanism giving rise to the observed iAMP21ALL genomic profile (Harrison and Schwab 2016).

31.4.2 aRCs in Chronic Lymphocytic Leukemia (CLL) Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults and has a highly variable course with its biological heterogeneity. Chromosomal abnormalities, including trisomy 12 and deletions of 11q, 13q, and 17p, are common in CLL with various clinical outcomes. Conventional chromosome analysis frequently yields a normal karyotype because of the lack of actively dividing CLL cancer cells in in vitro culture conditions. Therefore, FISH is used to screen for these abnormalities. 17p deletion (17p-) results in loss of the TP53 gene on 17p13.1, along with other genes located on 17p, and is usually associated with a lack of response to standard treatment, poor prognosis, and short progression-free and overall survival times in CLL. In a series of 195 cases with CLL and 17p-, 2% of 17p- resulted from a aRC17, and

443

the majority (70%) of 17p- resulted from unbalanced translocations, followed by deletion 17p (23%), monosomy 17 (8%), and isochromosome 17q, i(17q), (5%) (Chapiro et al. 2018). This review summarizes reports on identified aRCs in Table 31.6. Among aRCs with identified origins in CLL, one-third (33.3%) are aRC17, followed by aRC15 (16.7%), aRC18 (11.1%), and aRCs of chromosomes X, 4, 7, 8, 11, 12, and 19 (5.6%) (see Table 31.6). Except for a case with aRC7 and a case with possible aRC19, in most cases, the aRCs in CLL are part of a complex karyotype (89%, Table 31.6). A complex karyotype, especially with aRC17 with consequent loss of the TP53 gene on 17p, is usually associated with a poor prognosis in CLL.

31.4.3 aRCs in Plasma Cell Neoplasms (PCN) Myeloma is a malignant neoplasm of plasma cells and is the second most common hematologic malignancy, comprising approximately 10–15% of hematologic neoplasms. Diagnosis and risk stratification requires the integration of histology, radiology, serology, and genetic/ genomic data because of heterogeneous pathogenesis. According to recurrent cytogenetic abnormalities, myeloma has traditionally been divided into hyperdiploid and non-hyperdiploid types, which compose 60% and 40% of multiple myeloma (MM), respectively. Hyperdiploidy (with 48 to 74 chromosomes) is characterized by trisomies of odd-numbered chromosomes 3, 5, 7, 9, 11, 15, 19, and/or 21; while nonhyperdiploidy is characterized by hypodiploidy (up to 44/45 chromosomes), pseudodiploidy (46/47), near-tetraploidy (74–91), hyper-haploidy (24–34 near haploid), tetraploidy (92), and translocations involving chromosome 14. The incidence of cases with aRCs is generally relatively low in plasma cell leukemia. Among those studies focused on MM with aRCs mentioned, they range from 0.9% to 2.6% of the evaluated cases (Rack et al. 2016; Rajkumar et al. 1999; Tiong et al. 2013; Wu et al. 2007). aRCs range from 0.7 to 6.3% of MM cases with

444

Y. S. Zou et al.

Table 31.6  aRCs in chronic lymphocytic leukemia (CLL) Case

Karyotype

aRC4

46,XX,r(4)(p16q31),t(4;6)(q31;q26),−10,t(10;12)(q24;q22),add(12) Speaks et al. (1992) (p13),del(14)(q21),+mar

aRC7

46,XY,r(7)

Yunis (1982)

aRC8

46,Y,der(X)t(X;2)(q26;p15)/46,XY,der(2)t(2;2)(p24;p15)/46,XY,der(5)t(2;5)(p15;q35),r(8)(p11q24),der(11)t(5;11)(?;q24)t(2;5) (p15;?)/46,XY,der(6)t(2;6)(p15;q27)

Ramos-Campoy et al. (2022)

aRC11

44,XY,r(11),−13,add(14)(p13),add(16)(q?),−17,−18,+mar

Ramos-Campoy et al. (2022)

aRC12

44~45,XY,r(12)(p13q24),del(13)(q14),del(17)(p13p11),inc

Kruzova et al. (2019)

aRC15-1

47,XY,+12,t(14;18),r(15),+mar

Lishner et al. (1995)

aRC15-2

47,XY,del(6)(q15),+12,r(15)

Lewin et al. (1988)

aRC15-3

46,XY,t(14;18)(q32;q21)/46,XY,r(15)/47,XY,+12

Amiel et al. (1994)

aRC17-1

46,XY,t(11;14)(q13;q32),r(?17),add(21)(p?)

Rimokh et al. (1993)

aRC17-2

47,XY,t(12;22)(p13;q11),+der(12)t(12;22)(p13;q11),r(17)

Costa et al. (2022)

aRC17-3 to 6

4 cases (2% in this study had ring chromosomes 17)

Chapiro et al. (2018)

aRC18-1

43,X,der(X)t(X;18)(p21;q12),der(7)t(7;10)(p21;q23),i(8)(q10),der(10)t(10;15)(q23;q11),−13,−15,der(17)t(13;17)(q12;p11), r(18) (p11q12)/ 43,XX,der(4)t(4;8)(q35;q22),−13,der(15;15)(q10;q10),der(16)t(13;16)(q34;q24)add(13)(q?),-17

Wawrzyniak et al. (2014)

aRC18-2

43,XX,−13,der(15;15)(q10;q10),der(16)t(13;16)(q34;q24)add(13) (q?),−17/43, idem,der(4)t(4;8)(q35;q22)/ 43,X,der(X)t(X;18) (p21;q12), der(7)t(7;10)(p21;q23), i(8)(q10),der(10)t(10;15) (q23;q11),−13,−15,der(17)t(13;17)(q12;p11),r(18)(p11q12)

Wawrzyniak et al. (2014)

r(19)

46,X?,r(?19)

Van Den Neste et al. (2007)

r(X)

46,Y,r(X),t(3;13)(p25;q11),−4,+t(4;?12)(q35;?q11),t(11;14) (q13;q32),+18/46,XY,add(2)(p?)

Huret et al. (1989)

abnormal karyotypes. A study on 138 MM cases with complex karyotypic aberrations detected aRCs in one (0.7% of the study) (Smadja et al. 2001). Of 120 cases of MM with abnormal karyotypes, two showed aRCs, one with a hypodiploid karyotype and an aRC17 and the other with an aRC of unidentified origin (1.7% of the study) (Mohamed et al. 2007). Among a consecutive series of 112 MM cases, 34 had abnormal karyotypes, and one case with an aRC was found (Rack et al. 2016). In another series of 66 MM cases with abnormal karyotypes, three cases had aRCs of unidentified origin (4.5% of the study) (García et al. 2018). In a small series of 38 MM, sixteen had abnormal karyotypes, and one had an aRC (6.3% of abnormal

References

karyotypes in this study) (Wu et al. 2007). Studies with smaller patient series of MM with abnormal karyotypes may have a higher incidence of cases with aRCs due to a potential selection bias. For example, a study on 13 MM cases with an abnormal karyotype detected aRCs in one case (Stella et al. 2011), and a study on six cases with plasma cell leukemia detected aRCs in two cases (Jonveaux and Berger 1992). This review summarizes rings with identified origins in plasma cell neoplasms, mainly in MM (see Table 31.7). In all cases, the aRCs are part of a complex karyotype. Most ring cases (81.8%) have hypodiploid karyotypes with chromosomal structural and numerical abnormalities, except two cases (18.2%) had hyperdiploid

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

karyotypes (see cases aRC3-1 and aRC6 in Table 31.7). Three cases (27.3%) also had dmin and aRCs (see cases aRC3-2, aRC5, and aRC18 in Table 31.7). Among aRCs with identified origins in MM, two cases (18.2%) have aRC3 or aRC9, and aRCs 5, 6, 12, 16, 17, 18, and X are described in one case (see Table 31.7). aRCs present as part of hypodiploidy with complex chromosomal abnormalities are usually associated with a poor prognosis in MM.

31.4.4 aRCs in Miscellaneous Lymphoid Neoplasms

445

B- and T-cell neoplasms (Fig. 31.2e). Other aRCs have been reported in B-cell neoplasms (Fig. 31.2e). aRC7 is the most common in both B- and T-cell neoplasms. Infrequently, aRC7 may also contain non-homologous chromosomal material. For example, chromosome 1 material has been reported in an aRC7 of a B-cell lymphoma by FISH analyses (Dascalescu et al. 1999) (see case aRC7-6 in Table 31.8). Besides, complex aRC of 1;7 and aRC of 14;18 have also been infrequently reported (see cases aRC(1;7) and aRC(14;18) in Table 31.8).

31.5 Conclusions aRCs have also been reported in mature B-cell and Perspectives neoplasms (such as hairy cell leukemia, marfor Hematologic Patients ginal zone B-cell lymphoma, lymphoplasmawith aRCs cytic lymphoma, follicular lymphoma, large B-cell lymphoma, lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, and mature B-cell neoplasm-NOS), in T-cell lymphoid neoplasms (such as peripheral T-zone lymphoma, T-prolymphocytic leukemia, Sezary syndrome and hepatosplenic T-cell lymphoma), and dendritic cell neoplasms (Table 31.8). Among these, approximately three-quarters of cases (76%) are associated with B-cell neoplasms, one-fifth of cases (21%) are associated with T-cell neoplasms, and the remaining cases (3%) are associated with stroma-derived neoplasms of lymphoid tissues (see Table 31.8, Fig. 31.2e). Among aRCs with identified origins, over one-eighth (15.5%) are aRC7, followed by aRCX (9.9%), aRC1 and aRC13 (8.5%), aRC17 (7.0%), aRCs #6, #22, and #Y (5.6%), aRCs of #8, #12, #16 and #19 (4.2%), aRC3 and aRC20 (2.8%), and aRCs of #2, #4, #10, #11, #15 and #18 (1.4%) (see Fig. 31.2e). Except for some cases with aRC7, aRC8, aRC19, and aRCX as a sole chromosome abnormality (7.0% of cases), in the majority of the cases (84.5%) they are part of a complex karyotype, and the remaining cases (8.5%) have a karyotype carrying an aRC along with another chromosomal abnormality (Table 31.8). aRC1, aRC7, aRC8, aRC16, aRC17, aRC22, and aRCX have been reported in both

aRCs with various chromosome origins have been occasionally reported in diverse myeloid and lymphoid neoplasms by banding cytogenetics. They occur more commonly in myeloid neoplasms than lymphoid neoplasms. They usually appear in complex karyotypes. They are generally associated with an unfavorable prognosis due to being part of complex karyotypes. In CML, aRCs are commonly present during the accelerated and blast phases. In MDS, they are associated with adverse prognosis and disease progression. Among aRCs with identified chromosome origins, aRC7 is the most common, followed by aRC11, aRC5, and aRC6. Other aRCs with various chromosome origins have a frequency of less than 5% in hematologic malignancies. Specific aRCs can be recurrently observed in a specific type of leukemia or lymphoma. For example, aRC21 is typical in ALL, aRC17 is present in one-third of CLL cases with identified aRC, aRC7 is common in AML and MDS, and aRC11 as part of complex karyotypes is common in AML. Constitutional RC21 play a role in in developing AML and ALL. Complex aRC involving multiple non-homologs or homologous chromosomes are confirmed by various FISH methods and/or chromosome microarray analysis. aRCs of the Philadelphia chromosome,

446

Y. S. Zou et al.

Table 31.7  aRCs in plasma cell neoplasms (PCN) Case

Karyotype

References

aRC3-1

At diagnosis: 56,XY,+Y,+r(3)(p26q29),+5,+7,+8,+9,+11,+15,+17,+19

Calasanz et al. (1997)

After treatment: 55,XY,+Y,+r(3)(p26q29),+5,+8,+9,+15,+17,+19,+mar After treatment: 55,XY,+Y,+r(3)(p26q29),+4,+dup(7) (p21),+9,+11,+15,+17,+19 aRC3-2

41,X,−Y,add(1)(p34),add(2)(q31),r(3)(p?q?),der(7)t(1;7)(q12;q36),add(11) Smadja et al. (2003) (q13),−13,−13,−14,add(16)(p1?1),−21, add(22)(q13),dmin/41,idem,r(3) (p?;q?),del(3)(p22)/40,idem,−1,r(3)(p?q?)[3]/75~89 ,idemx2

aRC5

43~44,XX,add(7)(p21),add(13)(p11),+mar,1~2dmin/43,XX,−2,−5,add(7) (p21),−9,add(13)(p11),−18,der(22)t(9;22) (p13;q11),+mar,1~2d min/44~46,XX,−2,+5,r(5)x2,add(7)(p21),−9,add(13)(p11),−18,+21, der(22)t(9;22)(p13;q11)

Smadja and Grange (1998)

aRC6

48~49,X,−X,add(1)(q21),+5,−6,r(6),add(7)(q21),+del(7)(q32),add(8) (p11),+del(9)(q13q33)x2,+t(11;19)(p11;q13),−13,+15,+mar

Kaufmann et al. (2003)

aRC9-1

41,X,−X,−8,−9,−13,−16,−17,−19,−20,−21,−21,del(1)(p13p31),+del(1) Yip et al. (1990) (p21),+?r(9),+der(9)t(7;9)(q21;p12),+t(12;14;14) (q22;q32;q23),+der(17) t(17;21)(p11.2;q11.2),+dic(16;19)(q11.2;q13.4)

aRC9-2

38,der(X)t(X;8)(q28;?),−Y,der(1)t(1;13)(q11;q12),der(5)t(5;13) (p11;q12),der(6)t(6;18)(q13;?),− 8,−8,der(9)t(Y::8::9p?→ 9q34::8),+r(9),−11,−11,−12,der(13)t(21::12::5p15?5p11::13p11? 13q32::9),−14,−14,der(15)t(19::15::7q3?→7p11::15p11? 15qter), der(17) t(8qter?8q12~q13::1::17p13?→17qter),−18,der(20)t(Yqter?Yq11::12:: 14q32?→14q11::20p?→20qter), dic(18;21)(p11;p11),del(22)(q11.?)

Nordgren et al. (2000)

aRC12

42,X,del(X)(q27),del(1)(p22p21),der(6)t(6;21)(q11;q11),der(8)t(8;16) (q22;p11),r(12),−13,der(15)t(15;16)(p?13;q11),−16, der(18)t(1;18) (q11;q23),−20,−21,del(22)(q13)

Sawyer et al. (1998)

aRC16

42,X,−X,i(1)(q10),t(4;18)(q2?3;p11),der(5)t(X;5)(?q25;p15),dup(12) (q?),−13,−14,r(16),−22

Marzin et al. (2006)

aRC17

42~44,X,−X,der(1;5)(q10;q10),del(6)(q13),−12,−13,−17,r(17)(p13;q25)

Mohamed et al. (2007)

aRC18

43~46,XX,−3,−5,−7,+8,r(18),+20,dmin

Sawyer et al. (1995)

aRC(X)

45,?r(X),Y,del(1)(p11p34),add(5)(q21),del(6)(q24),dup(7)(q21q22),del(10) Lai et al. (1998) (p12),t(11;14)(q13;q32),−16,del(20)(q12)

Table 31.8  aRCs in miscellaneous lymphoid neoplasms Case

Disease

Karyotype

References

aRC1-4

DLBCL

50,XY,+3,+7,+13,+18/50,idem,r(1)(p36q4?3)

Ferti et al. (2004)

aRC1-3

FL

48,XY,+del(1)(p22)x2,r(1)(p36q?44),add(2) (p23),t(14;18)(q32;q21)

Horsman et al. (2001)

aRC1-5

DLBCL

47,X,t(X;12)(p11;p13),r(1)(p13q44),t(3;14) (p13;q32),add(4)(q31),+12

Wlodarska et al. (2005)

aRC1-1

NMZL

48~51,X,−X,r(1)(q?),+3,+3,del(5)(q31),+der(7) Itoyama et al. (2002) t(1;7)(p13;q11),+13,+18,+22,inc

aRC1-6

PTCL

49,XX,r(?1),+3,−?7,+18,+del(3)(p14), +mar/49,XX,+3,+18,del(1)(q32),+del(3)(p14)

Lakkala-Paranko et al. (1987)

aRC1-2

NMZL

49~52,XY,−1,der(1)del(1)(p31) add(1)(q31),der(1)add(1)(p34)dup(1) (q21q31),r(1),+3,+del(3)(p13), del(6) (q15),+7,+10,−12,+13,+18,+mar

Dierlamm et al. (1996)

(continued)

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

447

Table 31.8  (continued) Case

Disease

Karyotype

References

aRC(1;7)

LL

46,XX,add(6)(q22),r(7)[3]/47,XX,idem+12[1]

Dascalescu et al. (1999)

aRC2

FL

80~92,XXX,del(X)(?q?),r(2)(p?q?)x4,−6,i(6) (p10),+del(12)(?q11)x2,t(14;18)(q32;q21)x2, i(17)(q10),+der(18)

Lestou et al. (2003)

aRC3-1

MCL

44~46,X,del(X)(q13),der(1)t(1;2)(p36;p21), der(2)del(2)(p21)inv(2)(p21q13),r(3),inv(6) (p23q11),der(7)t(3;7)(p11;q36),i(17)(q10),−21

Pedrazzini et al. (2006)

aRC3-2

FL

46,XY,+X,−2,r(3)(q25q27),add(4)(q31), del(4)(p14),−6,del(8)(q13q22),add(9) (p13),add(9)(q34),+11,del(17)(p11),−18, +mar/46,XY,+X,−2, t(3;9)(p13;q34),−4,−6, del(8)(q13q22),+11,del(17)(p11),−18,+mar

Koduru et al. (1997)

aRC4

FL

52~55,X,+X,+add(X)(p22),+der(X)t(X;9) (p11;p11),+r(X)t(X;9)(p11;p11),add(1) (q44),+r(4),+5,del(6)(q13),add(8)(p23), +der(9)t(X;9) (p11;p11), der(14)t(1;14) (q12;p11),t(14;18)(q32;q21),−15,+16, del(17) (p11),+21,+mar

Lestou et al. (2003)

aRC6-1

BL

46,X,der(Y)t(Y;1)(q12;q11),r(6),t(8;14) (q24;q32)

Havelange et al. (2013)

aRC6-2

MCL

Struski et al. (2007) 39,X,−Y,der(1)t(1;18)(p?33;q1?2),r(6),−8,der(9)t(8;9)(q?22;p?21),t(11;14)(q13;q32),−13, der(13)t(13;16),der(14)t(14;21)(p?;?) t(14;22) (q?;?)t(16;22),der(15)t(3;15) (?;q?),−16,−18,−21,der(21)t(Y;21)(p?;q?),−22

aRC6-3

SMZL

Ott et al. (2000) 37~52,XX,+X,der(1)del(1)(p11)add(1)(q32), del(2)(q33),+3,t(3;7)(q27;q32),+4,+add(4)(p16) x2, r(6)(p25q27),der(7)t(3;7)(q27;q32),−8,+10, +del(10)(q22q24),+11,+12,+add(12) (p13),+13,−16,+18,+19,−20,−21,+mar

aRC6-4

MBL-NOS 43,XY,der(2)t(2;9)(p?16;q?),r(6),der(7)t(7;22) (?p11;?q11),der(8)t(8;12)(q?;?)t(8;12)(q?;?) t(8;12)(q?;?)t(8;12)(q?;?)t( 8;12)(q?;?),der(9) t(6;9)(?;q?)t(6;9)(?;q?)t(6;9)(?;q?),−10,−12, der(14)t(14;17)(?p11;?q11),−17,−22,+mar

Struski et al. (2007)

aRC7-1

HL

46,XY,r(?7)

Arthur and Bloomfield (1984)

aRC7-2

FL

46,XX,r(7)(p22q11)

Wang et al. (2015)

aRC7-3

HSTCL

47,XY,r(7),+8

Patkar et al. (2012)

aRC7-4

HSTCL

47,XY,r(7),+mar

Patkar et al. (2012)

aRC7-5

HSTCL

47,XY,r(7)(p22q36),+8

Shetty et al. (2006)

aRC7-6

LPL

46,XX,add(6)(q22),r(7)

Dascalescu et al. (1999)

aRC7-7

HSTCL

47,XX,r(7)(p13q36),+8

Jain et al. (2018)

aRC7-8

HSTCL

47,XX,r(7),+8,add(19)(p13)

Tamaska et al. (2006)

aRC7-9

DLBCL

46,XX,r(7)/44~45,XX,add(2)(p?),inc

Mohren et al. (2003)

aRC7-10

MCL

48,XX,r(7)(?p11q36),+12,del(14)(q22q32),+18

Oscier et al. (1996)

aRC7-11

SMZL

46,XY,der(7)t(7;7)(p22;q31)del(7)(q32q33),r(7) Viaggi et al. (2004) (p22q22)/45,idem,der(2)t(2;22)(p2?4;q11),add(8)(q24),−22 (continued)

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Y. S. Zou et al.

Table 31.8  (continued) Case

Disease

Karyotype

References

aRC7-12

AUL

44,XX,−5,r(7)(p22q11),dic(11:15) (p11;p11),add(12)(p13),add(16)(q13),dup(19) (q11q13.1),−21,−22,+mar1,+mar2/45,idem, +mar3/ 45,idem,+r(7)

Sato et al. (1995)

aRC7-13

DLBCL

76–80,XX,r(X)(p?q?),−1,del(2)(p?1q?1),del(3) Johnson et al. (2009) (p21),del(6)(q12),+7,+der(7)t(1;7) (?p13;?p22) t(7;16) (q?;?q11),+r(7)(p?q?), t(8;9)(q24; p13) x3,+10,+11,+der(12)t(2;12)(?;q24), t(14;18) (q32;q21)x2, −15,−16,−16,+der(18)t(14;18) (q32;q21),+20,+20,+21

aRC8-1

DLBCL

46,XY,r(8)

aRC8-2

MBL-NOS 46,XX,t(2;11)(p11;q13)/46,idem,del(1) (q22q42),r(8)

Wlodarska et al. (2004)

aRC8-3

T-PLL

46,t(X;14)(q28;q11),t(Y;14)(q12;q11),r(8) (q10q24),+mar

de Oliveira et al. (2009)

aRC10

MCL

47–48,XY,t(3;5)(p21;p15),t(7;18) (q21;q21),add(8)(p12),add(10)(p12),r(10) (p?p?),t(11;14)(q13;q32), der(15)t(7;15) (p11;p13),+19,?add(21)(p13),+1−3mar

Rouhigharabaei et al. (2013)

aRC11-2

ML

46,X,−5,−6,add(11)(q),r(11),+mar1,+mar2

GFCH (1984)

aRC11-1

DLBCL

48,XX,der(1)t(1;15)(p13;?),+7,t(8;14) (q24;q32),r(11),+13,−15,+18

Havelange et al. (2013)

aRC12-1

AL

49,XY,+6,t(6;8)(p21;q24),+r(12),+20/49, idem,inv(15)(q1?4q2?3),t(16;16)(q?;q?)/ 49,idem,t(3;5)(q?21;q?31)

Leroux et al. (2002)

aRC12-2

MBL-NOS 48,XY,der(3)t(3;8)(q22;q11),der(5)t(5;10) (q33;q22),+7,+del(7)(q21),der(9)t(4;9) (q11;p24),der(10)t(10;13)(q22;q21),r(12), del(19)(p13), del(22)(q11)

aRC12-3

DLBCL

Bosga-Bouwer et al. (2006) 45–49,X,−Y,del(2)(p13p11),+add(3)(q2?6), del(4)(q2?2),del(5)(q14q23),del(6)(p24),del(6) (q15q16),+add(7)(q31),del(9)(p22p21), add(10) (p1?2),dup(11)(q25q22),r(12)(p13q24),i(17) (p10),i(17)(q10), add(19)(p13),+20,+21

aRC12-4

NHL

Lambrechts et al. (1995) 45,X,−Y,del(2Xp12pp3),add(3)q26.1),del(4) (q22),del(5)(q15q31),del(6)(p24),del(6) (q21q23),add(7)(q22),del(9)(p23),add(10)(p12), der(11)(11pter→11q25::11q25→11q22::?), r(12),i(17)(p10),i(17)(q10),add(19)(p13)

aRC13-1

BL

46,Y,?dup(X)(q27q?),?dup(1)(q21q31),t(8;14) (q24;q32),r(13)

aRC13-2

MCL

47,XY,t(1;4)(q23;q33),+3,t(11;14) San Roman et al. (1982) (q13;q32),r(13)x2/47,idem,t(10;15)(p11.?;q11.?)

aRC13-3

BL

46,XY,dup(1)(q12q43),t(8;14) (q24;q32),r(13)/46,idem,t(2;16)(p11;q12)

Havelange et al. (2013)

aRC13-4

FL

49,XX,der(1)t(1;7)(p36;q22),t(2;6) (p25;q21),+9,r(13),t(14;18)(q32;q21),i(17) (q10),+19,+21

Dave et al. (1999)

Reeves and Pickup (1980)

Mark et al. (1978)

Havelange et al. (2013)

(continued)

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

449

Table 31.8  (continued) Case

Disease

Karyotype

References

aRC13-5

DCN

44,X,−Y,der(1)t(Y;1)(q12;q?21),der(3)t(1;3) (p11;q?),del(5)(q3?3q3?5),der(6)t(1;6)(q?;q22) t(1;8)(q?;q?), der(12)t(1;12)(?;p11),r(13)

Leroux et al. (2002)

aRC13-6

MCL

42~43,XY,−1,t(2;11)(p11~12;q13),der(10) t(1;10)(q3?1;p11~12),r(13)(p1?q3?),−17, der(20)t(1;20)(q?4;p1?3) t(1;17)(q?4;q?2) del(17)(q?),+1−2mar/43~44,idem,−17,+der(?) t(?;17)(?;?)

Woroniecka et al. (2007)

aRC(14;18) MBL-NOS 46,X,-X,dup(1)(q25q44),+5,r(14;18) (q32;q21),del(18)(q21)/46,X,−X,dup(1) (q25q44),+5,+r(14;18), del(16)(q22),del(18) (q21)

Juneja et al. (1997)

aRC15-1

DCN

42,X,−Y,der(2)t(2;6)(p11;q1?1)t(2;5)(p?;?),del(5)(p13),der(5)t(5;13)(q21;q?),der(6) t(2;6)(q?14;q1?1), t(6;18)(q2?2;q22),er(11), −13, der(13;21)(q10;q10),−14,der(14) t(Y;14)(q11;p11),r(15), der(19)t(3;19) (p21;q13),−21,i(22)(q10)

Leroux et al. (2002)

aRC15-2

AL

42,X,−Y,der(2)(6qter→6q1?1::2p10→2p?::5?), Leroux et al. (2002) del(5)(p13),der(5)(5pter→5q21::13q?), der(6)(6pter→6q1?1::2q?14→2qter),t(6;18) (q2?2; q22),der(11),−13,der(13)t(13;21) (q10;q10),−14,der(14)t(Y;14)(q11;p11),r(15), der(19)(19pter→19q13::3p21→3pter),−21,i(22) (q10)

aRC16-1

T-PLL

45,XY,inv(3)(p21q29),add(4)(q?),del(8)(q?), del(11)(q?),−12,inv(14)(q11q32),r(16)

Mecucci et al. (1988)

aRC16-2

T-PLL

45,XY,t(6;15)(q21;q22),i(8)(q10),del(11) (p11),inv(14)(q12q32),r(16),add(19) (p?),−20,t(21;21)(p11.?;q11.?),+mar

Taylor and Butterworth (1986)

aRC16-3

DLBCL

44,XX,t(3;14)(q27;q32),−6,der(9)t(9;15) (q34;q23),der(14)t(3;14)(q27;q32),t(6;14) (p11.?;p11.?),der(15) t(6;15)(q15;q23),r(16) (p13q24),−19/45,XX,idem,der(9)t(9;15) (q34;q23)

Keller et al. (2006)

aRC17-1

MCL

46,XY,del(2)(p22p12),t(5;9)(p15;q31~33),der(8) Nagel et al. (2010) t(3;8)(q13;p22~21),t(11;14)(q13;q32),r(17)

aRC17-2

FL

47,XY,trp(12)(q13q22),der(13)t(1;13) (q12;q34),t(14;18)(q32;q21),+r(17) [34]/46,XY[3]

aRC17-3

FL

Juneja et al. (1997) 51,XY,+X,+1,+3,+12,de1(3)(q21),de1(4) (q32),de1(7)(q22),dup(13)(q21q34),del(17) (p11),t( 1;2) (q11;p25),t(14;18)(q32;q21),+r(17) (p)

aRC17-4

T-PLL

Zver et al. (2004) 46,X,der(X)t(X;3)(q28;p25)t(X;16)(p14;q12), der(3)t(X;3)(q28;p25),der(6)t(X;6)(p14;q25),i(8) (q10),del(11)(q14q23),der(13)t(5;13)(q34;p11), der(13)t(13;14)(q22;q11),inv(14)(q11q32), der(16)t(X;16)(q28;q12),r(17)(p13q21),der(20) t(17;20)(q21;q13),add(22)(p?)

Fan and Rizkalla (2003)

(continued)

450

Y. S. Zou et al.

Table 31.8  (continued) Case

Disease

Karyotype

References

aRC17-5

T-PLL

44,X,add(X)(q25),del(3)(p11.?),der(4)t(3;4) (p12;p15),der(8)?t(X;8)(q25;p22), der(8)t(8;8)(p22;q23),−11,−13,inv(14) (q11q32),r(17)(p11.? q24)/44,XX,inv(1) (p12q25−31),−11,−13,inv(14)(q11q32), add(16)(q11.?),r(17)(p11.?q24)

Durig et al. (2007)

aRC18

DLBCL

46,X,−Y,t(3;3)(q11;q27),+6,add(6)(q21) x2,r(18)/46,idem,add(7)(p22),add(12)(p13)

Yoshioka et al. (2005)

aRC19-1

HCL

46,XY,r(19)

Haglund et al. (1994)

aRC19-2

DLBCL

50,XY,+Y,+5,der(6)t(5;6)(q14;q21),del(10) (p13),r(19),+21,der(22)(q13),+mar

Cabanillas et al. (1988)

aRC19-3

FL

45–50,XY,+X,−6,+7,+11,del(16)(p11.?),der(16) Bosga-Bouwer et al. (2003) del(16)(q2?q2?)dup(16)(p13p11.?),+ider(16) (q10)del(16)(q2?q2?),r(19)(p13q13),+mar

aRC20

DLBCL

44,X,−Y,del(4)(p12),t(11;14) (q23;q32),−17,r(20)(p13q13)/44,idem,t(1;9) (p36;q34)/45,idem,+21

Narayan et al. (2013) Gindin et al. (2015)

aRC22-1

T-PLL

45,X,dup(Y)(q11.?q12),t(2;9)(q35;q11),der(6) t(6;7)(q13;p13),der(7)t(6;7)(p21;p13),i(8) (q10),−11,add(13)(p?),inv(14)(q11q32),r(22)

Schlegelberger et al. (1990)

aRC22-2

SS

41–43,XY, ins(1;14;4)(p13?;q?;q?),del(2) (p21),der(4)t(4;14)(q?;q?),del(6)(q?),−1 0,−12,−14,−14,del(16)(p?),−17,der(18) t(17;18) (q11.?;p11.?),der(19)t(14;19) (q11?;p13),+r(22),+r(22)

Mohr et al. (1996)

aRC22-3

PTCL

45,X,dup(Y)(q12q11),t(2;9)(q35;q11),der(6) t(6;7)(q13;p13),der(7)t(6;7)(p21;p13),i(8) (q10),−11,add(13)(p?),inv(14)(q11.2q32.1), r(22)/46,XY,inv(14)(q11.2q32.3)

Schlegelberger et al. (1994)

aRC22-4

MCL

42,XY,add(4)(p16),t(8;12)(p10;p10), del(9)(q11q31),der(9),add(10) (p15),t(11;14)(q13;q32),−13, t(13;22) (p10;p10),−14,−17,add(19)(p13),−21,r(22), +2mar/42–43,idem,t(1;15)(p32;p11),add(11) (p15),der(16),+mar

Nowotny et al. (1996)

aRC(X)-1

MCL

46,X,r(X)(p22q28)

Goyns et al. (1993)

aRC(X)-2

T-PLL

42,X,r(X),t(6;14)(q16;q11.?),t(7;12)(p14;p12), Mecucci et al. (1988) del(10)(q21q24),−14,−15,del(16)(q?),−21,−21

aRC(X)-3

DLBCL

48,XX,r(X),t(1;6)(p22;q21),t(3;14)(q27;q32), der(5)t(5;22)(q11.2;q11.2),t(6;17)(q21;p13), der(8)t(X;8)(q22;p23)

Dave et al. (2002)

aRC(X)-4

MCL

45,der(X)t(X;3)(p22;q13),r(X)(p22q28), der(2)t(2;3)(q37;p23),der(8)t(8;10) (p22;q22),−10,t(11;14)(q13;q32),del(17) (p11.?),dmin

Goyns et al. (1993)

aRC(X)−5

BL

46,X,r(X),der(7)t(7;13)(q22;q12),+der(7)t(7;13) Havelange et al. (2013) (q22;q12),t(8;14)(q24;q32),der(13)t(13;18) (q13;?),del(15)(q?),−18 (continued)

31  Acquired Ring Chromosomes in Tumors of Hematopoietic and Lymphoid Tissues

451

Table 31.8  (continued) Case

Disease

Karyotype

References

aRC(X)-6

FL

46,XY,r(X)(q),der(3)del(3)(p23)add(3) (q27),add(4)(q3?3),add(5)(p15),add(6)(q11), der(12)t(9;12)(?; q11),t(14;18)(q32;q21),−15, del(15)(q11.?q15)x2,−17,−21,+2mar

Lestou et al. (2003)

aRC(X)-7

FL

52~55,X,+X,+add(X)(p22),+der(X)t(X;9) (p11.?;p11.?),r(X),t(X;9)(p11.?;p11.?),add(1) (q44),r(4),+5,del(6)(q13),add(8)(p23), +der(9)t(X;9)(p11.?;p11.?), der(14)t(1;14) (q12;p11.?),t(14;18)(q32,q21),−15,+16,del(17) (p11.?),+21,+mar

Lestou et al. (2003)

aRC(Y)-1

DLBCL

47,X,r(Y)(p11.?q12),+mar

Dave et al. (2004)

aRC(Y)-2

MBL-NOS 48,X,+X,r(Y),t(6;8;14)(p21;q24;q32),del(20) (q13)

Whang-Peng et al. (1995)

aRC(Y)-3

FL

48,X,r(Y),+2,+3,t(14;18)(q32;q21),der(22) t(1;22)(?;?)

Yunis (1982)

aRC(Y)-4

FL

47,X,r(Y)(p11.?q12),del(1)(p36),i(6) (p10),+7,add(8)(p21),t(14;18)(q32;q21)

Dave et al. (1999)

AL: Acute leukemia-CD4+ CD56+; AUL: Acute undifferentiated leukemia; BL: Burkitt lymphoma/leukemia; DCN: Dendritic cell neoplasm; DLBCL: Diffuse large B-cell lymphoma; FL: Follicular lymphoma; HCL: Hairy cell leukemia; HSTCL: Hepatosplenic T-cell lymphoma; HL: Hodgkin's lymphoma; LL: Lymphocytic lymphoma; LPL: Lymphoplasmacytic lymphoma; ML: Malignant lymphoma; MBL-NOS: Mature B-cell neoplasm, NOS; MCL: Mantle cell lymphoma; NMZL: Nodal marginal zone B-cell lymphoma; NHL: Non-Hodgkin’s lymphoma (the testis); PTCL: Peripheral T-cell lymphoma; PTCL: Peripheral T-zone lymphoma; SMZL: Splenic marginal zone B-cell lymphoma; SS: Sezary syndrome; T-PLL: T-prolymphocytic leukemia

9;22 with BCR::ABL1 gene fusion, have been reported in ALL. aRC like dmin and other chromosome derivatives like hsr or marker, are vehicles for oncogene amplification. Abnormal cancer genomes come with various losses, gains, amplifications, and complex structural rearrangements, which contribute to the pathogenesis of hematopoietic and lymphoid tumors. Accumulation of more aRCs with identified chromosome origins and genomic imbalances in various myeloid and lymphoid neoplasms in the future will further elucidate the clinical significance of aRCs in the formation and development of hematopoietic and lymphoid tumors.

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Acquired Ring Chromosomes in Solid Tumors

32

Jiadi Wen   and Mei Ling Chong

Abstract

Keywords

In human solid tumors, the occurrence of acquired ring chromosomes (aRCs) is a rare and sometimes secondary finding typically detected during chromosomal analysis. This chapter provides a summary of the prevalence of aRCs across various tumor sites in human solid tumors, as reported in the Mitelman database from 1971 to 2023. aRC aberrations exhibit specificity for certain tumor types, especially in bone and soft tissue tumors. Specific molecular consequences of aRCs, such as gene fusion, amplification, or deletion of oncogenes or tumor-suppressor genes, are reported for specific tumor types. However, some tumor types exhibit a high prevalence of aRCs but have not been thoroughly investigated due to the poor quality of banding in malignant cells from solid tumors. Therefore, further investigation into the formation and potential role of aRCs in tumor formation and progression could provide valuable insights into the diagnostic, prognostic, and treatment management of solid tumors.

Solid tumors · Bone · Soft tissue · Acquired ring chromosome (aRC) · Occurrence · Osteosarcoma · Liposarcoma · r(17;22) · r(12)

J. Wen (*) · M. L. Chong  Laboratory of Clinical Cytogenetics, Department of Genetics, Yale University School of Medicine, New Haven, CT, USA e-mail: [email protected] M. L. Chong e-mail: [email protected]

32.1 Introduction The presence of acquired ring chromosomes (aRCs) has been observed in a wide range of human tumors. The earliest documented reports of aRCs in solid tumors date back to 1959 (Makino et al. 1959). In 1983, Felix Mitelman compiled a comprehensive catalogue of all known chromosomal rearrangements, which was later made publicly accessible on the internet in 2000 under the name “Mitelman Database of Chromosome Aberrations in Cancer” (Mitelman 2023). This database provided free access to information on chromosomal aberrations in cancer. Gebhart (2008) conducted a summary of 760 cases of aRCs observed in human neoplasms as recorded in Mitelman database. In this work, Gebhart comprehensively reviewed aRCs in human neoplasms of various origins, including the molecular consequences of ring formation and their clinical significance based on information available in the literature up to 2007 (Gebhart 2008). This chapter provides a detailed illustration of aRCs in human solid tumors, based on a systematic assessment of 79,053 published tumor karyotypes up to early 2023.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_32

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32.2 Occurrence of aRCs in Solid Tumors Of the 79,053 published tumor karyotypes analyzed, 3% (2448/79053) showed the presence of aRCs. Specifically, solid tumors had a higher incidence of aRCs, accounting for 6% (949/16825) of all solid tumor cases, while hematologic tumors had a lower incidence at 2% (1499/62228). aRCs have been observed sporadically in many solid tumors, with varying frequencies among different tumor types. Mesenchymal tumors exhibit particularly high frequencies of aRCs and hold the most biological significance. Notably, parosteal osteosarcoma, dermatofibrosarcoma protuberans, well-differentiated liposarcoma, and dedifferentiated liposarcoma are among the bone and soft tissue tumors with the highest prevalence of aRCs, with frequencies of 90%, 64%, 63.4%, and 61.3%, respectively (Table 32.1). In these types of tumors, aRCs can be so common and characteristic that they may be considered cytogenetic signatures and can aid in differential diagnosis.

32.3 Formation of aRCs McClintock first showed that telomeric fusions cause broken chromosome ends, creating a series of breakage-fusion-bridge cycles (BFB) and chromosomal instability (McClintock 1941) (Fig. 32.1a). The phenomenon of telomeric fusion at the cytogenetic level is visualized in tumor cells when the telomeres of a single chromosome or two distinct chromosomes are associated or fused with minimal or no loss of material from either chromosome end. During tumor progression, aRCs frequently undergo breakage, which can result in resealing or conversion to rod-shaped giant marker chromosomes (Fig. 32.1b). The composition of the ring and giant marker chromosomes is often complex and may contain material from two or more chromosomes (Gisselsson et al. 2000; Heidenblad et al. 2006). Compared to constitutional RCs, aRCs occurring in these tumors are highly unstable. Due to their intrinsic mitotic instability, the size and composition of ring

J. Wen and M. L. Chong

and marker chromosomes can vary considerably among cells within the same tumor, and the number of aRCs per cell can also fluctuate. This variability creates a selective pressure for the preferential amplification of genes that promote tumor growth and a strong negative selection against sequences associated with proliferative disadvantage. However, passive bystander genes that neither promote nor inhibit tumor progression may be co-amplified solely due to their chromosomal proximity to driver oncogenes (Gisselsson et al. 1999).

32.4 Diagnostic Analysis of aRCs in Solid Tumors In clinical practice, cytogenetic techniques are frequently utilized to identify clonal numerical and structural rearrangements including aRCs in solid tumors. These techniques include conventional cytogenetic analysis, fluorescence in situ hybridization (FISH), chromosome microarray analysis (CMA) by array comparative genomic hybridization (aCGH) or single nucleotide polymorphism (SNP) array, reverse transcription PCR (RT-PCR), and sequencing. However, each approach has its own advantages and limitations, and they are frequently used in combination to identify the genetic makeup of the tumor. For instance, a thorough evaluation of aRCs should involve a combination of both cell-based G-banding and FISH analyses for detecting ring variants and dynamic mosaicism, and with DNA-based array and sequencing analyses for identifying genomic alterations. Thus far, cytogenetic changes are remained to be a crucial aspect of diagnosis, especially for bone and soft tissue tumors. During the 1970s, the development of chromosomal banding techniques by Caspersson and Zech enabled the identification of individual chromosomes through their unique banding patterns (Caspersson et al. 1970). This advancement allowed for a clearer understanding of chromosomal rearrangements in leukemias, such as the chromosomal rearrangement involving the ABL1 gene on chromosome 9, a tyrosine kinase, and the BCR gene on chromosome 22 (de Klein

32  Acquired Ring Chromosomes in Solid Tumors

477

Table 32.1  Occurrence of aRCs (%) in human solid tumors (1971–2023) Tumor type

Percentage  (RC/Total Cases)

Bone tumor Osteosarcoma, parosteal

90.0 (9/10)

Undifferentiated pleomorphic sarcoma

24.4 (30/123)

Osteosarcoma, nos

9.8 (16/164)

Osteogenic/bone tumor, special type

5.9 (1/17)

Ewing sarcoma

0.6 (3/476)

Bizarre parosteal osteochondromatous proliferation

16.7 (1/6)

Giant cell tumor of bone

12.5 (12/96)

Osteoblastoma

12.5 (1/8)

Synovial chondromatosis

11.1 (1/9)

Aneurysmal bone cyst

6.8 (3/44)

Chondroblastoma

6.3 (1/16)

Chondromyxoid fibroma Soft tissue tumor

3.8 (1/26)

Dermatofibrosarcoma protuberans/bednar tumor/giant cell fibroblastoma

64.0 (52/81)

Atypical lipomatous tumor/atypical lipoma/well-differentiated liposarcoma

63.4 (123/194)

Liposarcoma, dedifferentiated

61.3 (19/31)

Myxoinflammatory fibroblastic sarcoma

43.8 (7/16)

Low-grade fibromyxoid sarcoma

36 (9/25)

Liposarcoma, nos

33.3 (7/21)

Inflammatory myofibroblastic tumor/myofibroblastic sarcoma

25 (7/28)

Pleomorphic rhabdomyosarcoma

20 (2/10)

Spindle cell/sclerosing rhabdomyosarcoma

20 (1/5)

Leiomyosarcoma

10.6 (15/142)

Myogenic sarcoma, nos

9.1 (1/11)

Angiosarcoma

7.1 (1/14)

Undifferentiated round cell sarcoma

7.1 (1/14)

Chondrosarcoma, dedifferentiated

6.7 (2/30)

Liposarcoma, pleomorphic

6.3 (1/16)

Epithelioid sarcoma

5.3 (1/19)

Synovial sarcoma

5 (13/259)

Embryonal rhabdomyosarcoma

3.2 (3/94)

Liposarcoma, myxoid/round cell

3.2 (5/157)

Alveolar rhabdomyosarcoma

2.6 (3/115)

Fibroblastic/myofibroblastic tumor, nos

42.9 (3/7)

Soft tissue tumor, special type

19.4 (7/36)

Chondroid lipoma

16.7 (1/6)

Neurofibroma

16.7 (2/12)

Soft tissue tumor, nos

15.4 (2/13) (continued)

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J. Wen and M. L. Chong

Table 32.1  (continued) Tumor type

Percentage  (RC/Total Cases)

Myoepithelioma/myoepithelial carcinoma/mixed tumor

15.4 (4/26)

Epithelioid hemangioendothelioma

14.3 (1/7)

Angiomatoid fibrous histiocytoma

12.5 (1/8)

Benign fibrous histiocytoma

11.8 (2/17)

Malignant peripheral nerve sheath tumor/triton

11.7 (16/137)

Lipoblastoma

10.1 (7/69)

Localized giant cell tumor of tendon sheath

9.5 (2/21)

Leiomyoma

7.9 (42/529)

Myxofibrosarcoma

6.5 (6/92)

Hibernoma

5 (1/20)

Schwannoma

4.5 (4/88)

Fibroblastic/myofibroblastic tumor, special type

4.5 (1/22)

Adipocytic tumor, special type

4.2 (1/24)

Lipoma Tumors in other organs

3.2 (16/507)

Thymus

22.2 (4/18)

Gallbladder/biliary system

20.8 (5/24)

Pleural

15.3 (21/137)

Pancreas

15 (23/153)

Salivary gland

8.3 (43/518)

Peritoneum

7.7 (1/13)

Liver

6.3 (11/174)

Bladder

5.1 (10/197)

Breast

5 (57/1138)

Lung

4.3 (30/695)

Colorectal

4.2 (23/552)

Ovary

4.1 (27/661)

Vagina

3.9 (3/77)

Larynx

3.9 (5/129)

Skin

3.2 (11/339)

Oral cavity

2.9 (5/174)

Small intestine

2.9 (1/35)

Kidney

2.5 (50/2026)

Uterus, cervix

2.4 (2/84)

Tongue

2.2 (2/90)

Thyroid Central nervous system tumors

1.5 (5/342)

Astrocytoma, nos

6.7 (2/30)

Germ cell tumor, nos

5.3 (1/19) (continued)

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Table 32.1  (continued) Tumor type

Percentage  (RC/Total Cases)

Glioma, nos

4.4 (2/45)

Rhabdoid tumor

4.3 (2/47)

Meningioma

3.5 (33/954)

Ependymoma

2.4 (3/124)

Astrocytoma, grade I-II

2.2 (2/92)

Abbreviations: nos, not otherwise specified

Fig. 32.1  Formation of RCs. a Telomere-to-telomere fusion, or fusion of breakage and loss of distal ends of chromosome. b Rod-shaped giant marker chromosome contains amplified regions of various chromosomes. Reproduced from Garsed et al. (2009)

et al. 1982; Rowley 1973). The introduction of chromosomal banding techniques heralded a new era in cancer cytogenetics and played a significant role in advancing our understanding and becoming essential diagnostic and prognostic tools (Gebhart 2008). Until the 1990s, classical banding technology was the sole method used to detect and characterize RCs in clinical cytogenetics and cancer cytogenetics (Heim and Mitelman 1995). It is still the gold standard for detecting RCs. However, due to poor banding quality, as well as the small size or the intricate rearrangements of certain rings, it is difficult to precisely define the affected chromosomes or chromosomal regions. FISH techniques have significantly improved the identification of these segments and provided insights into the molecular changes

caused by ring formation. Various DNA probes utilized in FISH techniques have efficiently addressed this challenge (Gisselsson et  al. 1998). Chromosome-specific alphoid DNA repeat probes have been useful in detecting centromeric areas (Hopman et al. 1991; Koch et al. 1989), while breakpoint spanning cosmid and YAC probes have been employed to identify involved breakpoint regions and relevant genes (Kievits et al. 1990; Rowley et al. 1990). In addition, telomeric probes have been used to identify the lack of telomeres on the rings (Yan et al. 2003). Apart from these specific FISH probes, chromosome libraries such as spectral karyotyping (SKY) (Nishio et al. 2012) and reverse chromosome painting (Carter et al. 1992) allow the identification of the chromosome of origin and specific chromosomal

480

breakpoints in aRCs found in cancer cells. In addition, FISH can be used to identify specific translocations or amplification of certain genes in a case where a particular diagnosis is suspected with a relatively lower cost and a rapid turnaround time. The development of aCGH facilitates the detection of genomic losses, gains, and amplifications caused by ring formation in malignancies (Micci et al. 2002; Nishio et al. 2001; Szymanska et al. 1996). This technology contributes to tumor classification and diagnosis as well as aids in predicting the prognosis of some solid tumors. CMA can define somatic copy number abnormalities (CNAs) and discontinuous amplified CNAs as well as gene contents in the aRCs, revealing tumor-suppressor genes or oncogenes within the detected abnormalities (Chai et al. 2022; Storlazzi et al. 2010). More recently, the advent of massively parallel next-generation sequencing (NGS)-based assays have provided additional tools for assessing molecular alterations in neoplasms including solid tumors. Whole-genome sequencing (WGS) and transcriptome analysis by RNA sequencing have been reported in studies of both constitutional and somatic RCs (Alosi et al. 2015; Nord et al. 2014; Zhang et al. 2016). These new genetic approaches allow to characterize the breakpoints and fusion sequence at a base-pair level and to reveal differentially expressed genes in aRCs.

32.5 aRCs in Bone Tumors aRCs are a relatively uncommon finding in bone tumors, with a reported frequency ranging from 3.8% to 90%. However, they are more frequently observed in osteosarcomas, particularly in parosteal osteosarcoma (Table 32.1). Parosteal osteosarcoma is a well-differentiated malignancy that is rare but generally associated with a favorable prognosis, unlike conventional osteosarcomas which have a poor prognosis. Adequate initial resection of the tumor usually results in a cure for most patients

J. Wen and M. L. Chong

with parosteal osteosarcoma, and long-term survival rates range from 80% to 90% (Sandberg and Bridge 2003). The presence of aRCs containing amplified materials from 12q13 to q15 is characteristic for parosteal osteosarcomas. The aRC may present either as the sole anomaly or accompanied by only a few other abnormalities. In 1993, Mertens et al. identified aRCs in three cases of parosteal osteosarcoma without knowing the chromosome of origin (Mertens et al. 1993). Further investigation in several bone and soft tissue tumors by FISH and aCGH have revealed that the supernumerary ring of parosteal osteosarcoma contains materials from chromosome 12 (Gisselsson et al. 1998, 2002; Heidenblad et al. 2006; Szymanska et al. 1996; Zambrano et al. 2004). Figure 32.2 shows a representative result of FISH and aCGH from one of the parosteal osteosarcoma cases (Heidenblad et al. 2006). In this case, several amplified regions were detected in both 12p and 12q (Fig. 32.2a). FISH analyses were conducted using bacterial artificial chromosome (BAC) clones mapped to 12p11 and 12q15 and a whole-chromosome paint (wcp) Y probe, which confirmed the aCGH findings. The FISH analyses also revealed tumor heterogeneity with chromosome 12 sequences amplified in various combinations of rings and/or giant markers (Fig. 32.2b). Amplification of material from 12q leads to the amplification of multiple genes involved in tumorigenesis including MDM2, CDK4, and FRS2 (Heidenblad et al. 2006). The presence of aRCs is a useful prognostic indicator by distinguishing parosteal osteosarcoma from conventional osteosarcoma. Table 32.2 summarized the identified aRCs in bone tumors.

32.6 aRCs in Soft Tissue Tumors In certain subtypes of soft tissue tumors, there is a high prevalence of aRCs, particularly in fibroblastic and myofibroblastic tumors, adipocyte tumors, and smooth muscle tumors. The incidence and nature of aRCs aberrations were found to be highly nonrandom. For instance,

32  Acquired Ring Chromosomes in Solid Tumors

481

Fig. 32.2  Representative aCGH and FISH results of parosteal osteosarcoma. a aCGH data shows several amplifications along chromosome 12. b the FISH analysis

using BAC clones from two regions, 12p11.23 and 12q15, confirmed amplification in aRCs and heterogeneous staining regions. Reproduced from Heidenblad et al. (2006)

Table 32.2  Identified aRCs in bone tumors

r(17;22) was present in 48% (14/29) of dermatofibrosarcoma protuberans cases, chromosome 12 was involved in 91% (94/103) of adipocyte tumors, and r(1) was involved in 90% (27/30) of leiomyoma cases (Table 32.3). The occurrence of nonrandom aRCs events suggests the distinct ring aberrations may be driving factors in tumor initiation, formation, or progression.

aRCs

No of cases

Tumor type

r(1)

1

Osteosarcoma (1)

r(2)

1

Giant cell tumor of bone (1)

r(4)

1

Chondroblastoma (1)

r(6)

4

Chondrosarcoma, Nos (1) Synovial chondromatosis (1) Fibrosarcoma (1) Aneurysmal bone cyst (1)

r(7)

2

Giant cell tumor of bone (1) Osteoblastoma (1),

r(8),r(19)

1

Giant cell tumor of bone (1)

r(9) r(9),r(16)

2

Chondrosarcoma (1) Giant cell tumor of bone (1)

r(10)

2

Giant cell tumor of bone (1) Undifferentiated pleomorphic sarcoma (1)

r(11)

4

Giant cell tumor of bone (3) Osteogenic/bone tumor, special type (1)

r(12)

19

Osteosarcoma (17) Bizarre parosteal osteochondromatous proliferation (1) Giant cell tumor of bone (1)

r(15)

2

Chondromyxoid fibroma (1) Giant cell tumor of bone (1)

r(17) r(17),r(6)

2

Aneurysmal bone cyst (1) Giant cell tumor of bone (1)

r(19)

3

Undifferentiated pleomorphic sarcoma (1) Giant cell tumor of bone (1) Undifferentiated pleomorphic sarcoma (1)

r(1;7)

1

Undifferentiated pleomorphic sarcoma (1)

r(22) r(21),r(22)

5

Osteosarcoma (1); Ewing sarcoma (3) Undifferentiated pleomorphic sarcoma (1)

The number of cases for each tumor type is included in the bracket, indicated by (no of cases)

32.6.1 Dermatofibrosarcoma Protuberans Dermatofibrosarcoma protuberans (DFSP) is another important tumor for understanding the significance of aRCs. aRCs are present in a high percentage of DSFP cases, with 64% (52/81) of cases in the Mitelman database recording their presence (Table 32.1). DFSP is characterized by a gene fusion between the collagen 1A1 (COL1A1) gene and the platelet-derived growth factor beta (PDGFB) gene, resulting in the aberrant expression of the PDGFB protein and promoting tumor growth (O'Brien et al. 1998; Sandberg and Bridge 2003; Simon et al. 1997). This gene fusion is typically caused by an exchange of material between chromosome bands 17q21 (COL1A1) and 22q13 (PDGFB), which can result from a balanced or unbalanced translocation t(17;22), or from the formation of supernumerary RCs (Pedeutour et al.

482

J. Wen and M. L. Chong

Table 32.3  Identified aRCs in soft tissue tumors aRCs No of cases Fibroblastic and myofibroblastic tumors

Tumor type

r(1;?)

1

DFSP/Bednar tumor/giant cell fibroblastoma (1)

r(2) r(?;2)

2

Low-grade fibromyxoid sarcoma (1) Inflammatory myofibroblastic tumor/myofibroblastic sarcoma (1)

r(3) r(?;3)

3

Myxoinflammatory fibroblastic sarcoma (1); Myxofibrosarcoma (1) Myxoinflammatory fibroblastic sarcoma (1)

r(5)

1

DFSP/Bednar tumor/giant cell fibroblastoma (1)

r(7;16)

2

Low-grade fibromyxoid sarcoma (1) Inflammatory myofibroblastic tumor/myofibroblastic sarcoma (1)

r(8;17)

1

DFSP/Bednar tumor/giant cell fibroblastoma (1)

r(9), r(12;18)

1

Inflammatory myofibroblastic tumor/myofibroblastic sarcoma (1)

r(10)

1

Low-grade fibromyxoid sarcoma (1)

r(17)

12

DFSP/Bednar tumor/giant cell fibroblastoma (12)

r(17;22)/ r(4;17;22)/r(?;17;22)

14

DFSP/Bednar tumor/giant cell fibroblastoma (14)

r(20) Adipocyte tumors

1

Myxofibrosarcoma (1)

r(1)

3

Well-differentiated liposarcoma (2); lipoma (1)

r(?;1)

1

Well-differentiated liposarcoma (1)

r(12) r(?;12) r(?;1;12)

89

Well-differentiated liposarcoma (26); lipoma (4); liposarcoma (34) Well-differentiated liposarcoma (13); lipoma (1); liposarcoma, dedifferentiated (3) Well-differentiated liposarcoma (6); Liposarcoma, dedifferentiated (2)

r(11),r(12)

1

Liposarcoma, myxoid/round cell (1)

r(?;X;12)

2

Well-differentiated liposarcoma (1) Liposarcoma, NOS (1)

r(?;1;4;10;12;15) r(?;6;11;12;15)

2

Well-differentiated liposarcoma (1) Liposarcoma, NOS (1)

r(8) Smooth muscle tumors

5

Lipoblastoma (3); liposarcoma, myxoid/round cell (2)

r(1)/r(?;1)/ + r(7)/ + r(9;12)

27

Leiomyoma (27)

r(?5)

1

Leiomyoma (1)

r(8)

1

Leiomyosarcoma (1)

r(9)

2

Leiomyosarcoma (1); inflammatory myofibroblastic tumor (1)

r(13)

2

Leiomyosarcoma (1); leiomyoma (1)

r(19)

1

Leiomyosarcoma (1)

r(21)

1

Leiomyosarcoma (1)

r(X) 1 Leiomyoma (1) Abbreviations: DFSP, dermatofibrosarcoma protuberans; nos, not otherwise specified

1993, 1995). The majority of the identified rings include parts of chromosomes 17 and 22 resulting in a chromosomal gain of the bands 17q23q24 and 22q11q12 (Pedeutour et al. 1994, 1995).

Figure 32.3 displays the laboratory findings of a ring characteristic of DFSP, which were composed of chromosome 17 and chromosome 22, as confirmed by karyotyping and FISH analysis

32  Acquired Ring Chromosomes in Solid Tumors

483

Fig. 32.3  aRC with fusion of chromosome 17 and chromosome 22 in adult dermatofibrosarcoma protuberans. a representative R-banded karyotypes with aRC (arrow). b detection of chromosome 17 (whole-chromosome

painting probe to chromosome 17; green signal) and 22 (PDGFB probe; red signal) sequences on dermatofibrosarcoma protuberans rings. Reproduced from Sirvent et al. (2003)

(Sirvent et al. 2003; Pedeutour et al. 1994, 1995). In a 2020 study, Koster et al. (2020) analyzed 39 DFSP tumors and identified abnormal karyotypes in 32 patients. These included a balanced translocation t(17;22)(q21;q13) in one patient, one to two copies of an unbalanced der(22)t(17;22) in nine patients, and one to four aRCs in 25 patients. Metaphase FISH analysis was performed on nine tumors and showed a similar result (Koster et al. 2020). Clinicopathological association studies revealed that the balanced or unbalanced translocations have been observed mainly in pediatric or adolescent DFSP patients (Koster et al. 2020; Pedeutour et al. 1996), while the aRC r(17;22) occurs more frequently in older patients (Koster et al. 2020; Terrier-Lacombe et al. 2003). This suggests that these three chromosomal variants may arise through different mechanisms in different age groups.

Majority of the observed aRCs were constituted by chromosome 12 or segments of it, particularly in ALT/WDLPS. ALT/WDLPS is a locally aggressive mesenchymal neoplasm that represents the largest subgroup of adipocytic malignancies, accounting for approximately 40%–45% of all liposarcomas. Amplification of MDM2 and/or CDK4 is almost always present, suggesting that the amplification of these genes is essential for tumorigenesis in liposarcoma (Chai et al. 2022; Gebhart 2008; Hofvander et al. 2018; Italiano et al. 2009) (Fig. 32.4). Some reported rings were found to contain additional chromosomal material, aside from chromosome 12. Among these, chromosome 1 was the most frequently involved, accounting for approximately 30% of these rings (Gebhart 2008). Nilsson et al. investigated amplifications of 1q21-q23 and discovered three novel oncogenes (COAS1-3) within this amplicon. In the studied lipomatous tumors, the most common location of extra COAS signals was in supernumerary ring and giant marker chromosomes (Nilsson et al. 2004). The frequent co-amplification and colocalization of sequences from chromosomes 1 and 12 in these rings as observed raises the question of whether this is related to the mechanism of formation of supernumerary rings or to the pathogenetic process.

32.6.2 Lipoma, Liposarcoma, and Lipoblastoma aRCs are observed in over 60% of atypical lipomatous tumors (ALT) or well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPS). In contrast, they are only seen in about 3% of lipomas (Table 32.1).

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Fig. 32.4  Cytogenomic findings in the WDLPS or DDLPS cases with the giant ring (GR) or giant rod marker (GRM). a chromosome results showing GR or GRM in cases 1–4. b amplification levels of somatic copy number alterations and putative oncogenes in the

core segment of 12q14.1q15 in the three cases are given by number of copies and size of amplicons in Kb. Dash line “–” indicates normal two copies without amplification. Reproduced from Chai et al. (2022)

Lipoblastoma is a rare soft tissue tumor that occurs primarily in young children. Since the first report in 1986, 8q11q13 rearrangements involving PLAG1 have been documented as diagnostic cytogenetic features for lipoblastoma (Hibbard et al. 2000; Sandberg et al. 1986). aRCs have been seen in about 10% of lipoblastomas (Table 32.1). Interestingly, all of the aRC cases in lipoblastoma with available ring information contained aRC8 (Table 32.3). Several types of genetic aberrations have been described including translocation of 8q and splitting of the PLAG1 probe leading to “promoter swapping” and gains of chromosome 8 or PLAG1 foci (Wang et al. 2019). HAS2-PLAG1 fusion has been reported as the oncogenic consequence which leads to the upregulation of PLAG1 in a case of lipoblastoma with aRC8 (Hibbard et al. 2000). Several functional studies suggest that oncogenic capability of PLAG1 is exerted by activation of growth factors such as insulin-like growth factor 2 (IGF2) and its cognate receptor IGF1R (Van Dyck et al. 2007).

32.6.3 Leiomyoma Leiomyoma is a benign smooth muscle tumors of the uterus with approximately 40%–50% of the tumors harbor nonrandom and tumor-specific chromosomal abnormalities (Polito et al. 1999; Sandberg 2005). High mobility group (HMG) proteins have been shown to be pathogenetically involved in the uterine leiomyomas. Aberrations of either HMGIC or HMGIY have been identified as a primary tumor initiation event of leiomyomas with 12q14q15 or 6p21.3 abnormalities (Kazmierczak et al. 1996; Schoenmakers et al. 1995; Williams et al. 1997). aRCs presented in about 8% of leiomyoma cases (Table 32.1). Approximately 90% of leiomyoma contained r(1), suggesting that aRC1 is a nonrandom cytogenetic abnormality in leiomyoma. Further investigation on candidate gene HMG17 which also belongs to the HMG family on chromosome 1 identified this region was deleted in aRC1, suggesting that HMG17 is unlikely to contribute to leiomyoma similar to that observed

32  Acquired Ring Chromosomes in Solid Tumors

in other HMG proteins (Polito et al. 1999). aRC1 is often found in conjunction with other chromosomal alterations and is therefore considered a secondary abnormality (Sandberg 2005). As a result, the precise role of aRC1 in the development of leiomyoma remains unclear.

32.6.4 Other Subtypes of Soft Tissue Tumors In 2014, Nord et al. conducted a study on the molecular characteristics of 47 high-grade soft tissue sarcomas that had not been well-characterized previously, including undifferentiated pleomorphic sarcoma, leiomyosarcoma, myxofibrosarcoma, malignant peripheral nerve sheath tumor, fibroblastic/myofibroblast sarcoma, atypical myofibroblast tumor, low-grade myofibroblastic sarcoma, giant cell tumor of soft tissue, spindle cell sarcoma of the heart, and pleomorphic liposarcoma (Nord et al. 2014). In this study, 30% (14/47) sarcomas with a distinct aRCs showed amplification of MDM2, and these tumors generally displayed relatively simple karyotypes, with multiple amplicons primarily clustering on 1q, 5q, 6q, and 12q. The remaining 70% (33/47) of the tumors were negative for MDM2 amplification and displayed complex karyotypes, but the number of amplicons was smaller than in MDM2positive cases, and few amplicons were recurrent. Recurrent losses were observed in regions on chromosome arms 8p, 9p, 10p, 13q, 16q, 17p, and 17q, and homozygous deletions affecting the genes CDKN2A, RB1, or TP53 were each found in these tumors. These finding suggests that the MDM2-positive and MDM2-negative aRCs likely have different roles in tumor development. The amplified or deleted genes in sarcomas with aRCs are involved in oncogenic pathways that are associated with cell proliferation or apoptosis.

32.7 aRCs in Epithelial Tumors While some aRCs have been well-characterized in bone and soft tissue tumors, they are typically considered a random occurrence in epithelial tumors

485

and tumors of the nervous system and adjacent tissues (Table 32.4). Although aRCs can be observed at a higher frequency in some tumor types, such as thymus, gallbladder, pleural, pancreas, and others, the reported case numbers are limited, and the specific chromosomal content remains undefined. Chromosomes 1 and 8 are more frequently observed in the identified rings (Table 32.4) than other chromosomes, with aRC8 showing a particularly high frequency in pleomorphic adenoma.

32.7.1 Pleomorphic Adenoma aRCs have been observed in approximately 8% of pleomorphic adenomas (PAs) in the salivary gland (Stenman et al. 2022). Among the identified aRCs, 16 cases of r(8) were detected in samples of PA (Table 32.4) (Mark et al. 1997; Persson et al. 2009). PA is the most common salivary gland tumor, and it has been extensively studied cytogenetically. Previous studies showed that the r(8)(p12q12.1) comprises a pericentromeric segment with recurrent breakpoints in FGFR1 at 8p12 and PLAG1 at 8q12.1, resulting in the amplification and overexpression of an FGFR1-PLAG1 gene fusion (Fig. 32.5) (Persson et al. 2008). Importantly, this fusion has been found to be 15-fold enriched in myoepithelial carcinoma-ex-PA compared to PA, suggesting that amplification and overexpression of the FGFR1-PLAG1 fusion may serve as a potential biomarker for malignant transformation of PA (Dalin et al. 2017). aRCs derived from other chromosomes have not resulted in gene fusions but have instead led to the loss of segments of chromosomes such as 8p, 5p, 5q, and/or 6q (Persson et al. 2008). It has been postulated that these regions may harbor genes with tumorsuppressor function, although this hypothesis requires further investigation and confirmation.

32.7.2 aRCs in Central Nervous System Tumors Similar to epithelial tumors, aRCs are rare in tumors of central nervous system (CNS), only

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Table 32.4  Identified aRCs in epithelial tumors and nervous system tumors aRCs

Epithelial

CNS

No of cases

Tumor type

No of cases

Tumor type

r(1), r(?1)

10

Adenocarcinoma (7); adenoma (2); benign epithelial tumor, special type (1)

4

Astrocytoma, grade I-II (1); meningioma (3)

r(1), r(5)

1

Adenoma (1)

0

NA

r(1), r(8;11)

1

Adenocarcinoma (1)

0

NA

r(2)

3

Adenoma (1); malignant epithelial tumor, special type (2)

0

NA

r(3)

7

Adenocarcinoma (5); Carcinoma, NOS (1); 0 malignant epithelial tumor, special type (1)

NA

r(3), r(17)

1

Malignant epithelial tumor, special type (1) 0

NA

r(4), r(?4)

4

Adenoma (3); adenocarcinoma (1)

0

NA

r(5)

7

Adenoma (7)

0

NA

r(6)

7

Adenocarcinoma (3); adenoma (2); benign epithelial tumor, special type (2)

1

Meningioma (1)

r(6;11)

0

NA

1

Rhabdoid tumor(1)

r(7)

3

Adenocarcinoma (1); adenoma (1); benign epithelial tumor, special type (1)

1

Meningioma (1)

r(8), r(?8)

20

Adenocarcinoma (3); adenoma (16); benign 1 epithelial tumor, special type (1)

Meningioma (1)

r(8;11)

1

Adenocarcinoma (1)

0

NA

r(9)

4

Adenoma (2); benign epithelial tumor, special type (1); Hyperplasia (1)

0

NA

r(11), r(?11)

3

Adenocarcinoma (2); benign epithelial tumor, special type (1)

2

Meningioma (2)

r(12)

2

Adenocarcinoma (1); adenoma (1)

2

Astrocytoma, NOS (1); meningioma (1)

r(14)

1

Basal cell carcinoma (1)

1

Ependymoma (1)

r(15), r(?15)

2

Adenocarcinoma (2)

0

NA

r(16)

1

Adenocarcinoma (1)

1

Meningioma (1)

r(17)

4

Adenocarcinoma (3); benign epithelial tumor, special type (1)

1

Meningioma (1)

r(17;17)

1

Adenocarcinoma (1)

0

NA

r(17;18)

1

Mucoepidermoid carcinoma (1)

0

NA

r(18)

2

Adenocarcinoma (1); adenoma (1)

0

NA

r(19)

1

Adenocarcinoma (1)

2

Meningioma (2)

r(19;?)

1

Adenocarcinoma (1)

0

NA

r(21)

0

NA

1

Meningioma (1)

r(22)

0

NA

5

Astrocytoma, grade I-II (1); Ependymoma (1); meningioma (2); rhabdoid tumor(1)

r(X)

2

Adenocarcinoma (1); adenoma (1)

1

Meningioma (1)

The number of cases for each tumor type is included in the bracket, indicated by (no of cases). NA: not available Abbreviations: NA, not available; nos, not otherwise specified

32  Acquired Ring Chromosomes in Solid Tumors

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Fig. 32.5  aRC8 in pleomorphic adenomas. a schematic illustration of the formation of the r(8), resulted in fusion of FGFR1-PLAG1 (Stenman et al. 2022). b profiles of overlayed chromosome 8 from 4 cases with r(8) showed

copy number gain of 8p12–8q12.1 and loss of 8pter-p12. The transcriptional direction of the FGFR1 and PLAG1 genes is indicated by arrows. Reproduced from Persson et al. (2008)

observed in about 2%-7% of tumors (Table 32.1). Our understanding of the significance of aRCs in tumors of CNS tissues is even more limited due to the small number of reported cases, and the fact that most of the observed rings cannot be definitively assigned to specific chromosomes. Among the reported 25 rings in CNS, aRC22 showed a higher frequency that has been reported in six cases, including meningioma, astrocytoma, ependymoma, rhabdoid tumor, and neuroglial neoplasm (Table 32.4). However, none of these cases have undergone molecular analysis, and the genes affected by the ring rearrangements remain unknown.

Amplification of chromosomal segments due to ring formation. (3) Loss of acentric chromosomal segments that are created by the ring rearrangement, leading to the loss of genes located on them. The role of aRCs in neoplasias with highly complex karyotypes remains unclear. It is uncertain whether the presence of aRCs or the overall complexity of the karyotype affects the prognosis in these cases. Further molecular analysis of aRCs could help shed light on their role in complex karyotypes. A more extensive and intensified examination of aRCs in human neoplasias, particularly those with distinct patterns of aRCs, is needed to gain deeper insights into the mechanisms underlying ring formation and the actual role of rings in malignancy.

32.8 Future Diagnostics and Clinical Implication of aRCs in Neoplasia Previous studies have shown that the formation of an aRC can result in genetic consequences that are associated with, or contribute to, the development of cancer or tumor progression. In sarcoma, three main types of aRCs have been characterized, including: (1) Fusion of genes or genetic regions that result in the coding of new proteins or the contribution of new protein functions. (2)

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32  Acquired Ring Chromosomes in Solid Tumors McClintock B (1941) The stability of broken ends of chromosomes in Zea mays. Genetics 26(2):234–282. https://doi.org/10.1093/genetics/26.2.234 Mertens F, Mandahl N, Orndal C, Baldetorp B, Bauer HC, Rydholm A, Wiebe T, Willen H, Akerman M, Heim S, Mitelman F (1993) Cytogenetic findings in 33 osteosarcomas. Int J Cancer 55(1):44–50. https:// doi.org/10.1002/ijc.2910550109 Micci F, Teixeira MR, Bjerkehagen B, Heim S (2002) Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding, CGH, M-FISH, and chromosome- and locus-specific FISH. Cytogenet Genome Res 97(1–2):13–19. https://doi. org/10.1159/000064038 Mitelman F (2023) Mitelman database of chromosome aberrations and gene fusions in cancer. https://mitelmandatabase.isb-cgc.org/ Nilsson M, Meza-Zepeda LA, Mertens F, Forus A, Myklebost O, Mandahl N (2004) Amplification of chromosome 1 sequences in lipomatous tumors and other sarcomas. Int J Cancer 109(3):363–369. https:// doi.org/10.1002/ijc.11716 Nishio J, Aoki M, Nabeshima K, Iwasaki H, Naito M (2012) Characterization of giant marker and ring chromosomes in a pleomorphic leiomyosarcoma of soft tissue by spectral karyotyping. Oncol Rep 28(2):533–538. https://doi.org/10.3892/or.2012.1835 Nishio J, Iwasaki H, Ishiguro M, Ohjimi Y, Yo S, Isayama T, Naito M, Kikuchi M (2001) Supernumerary ring chromosome in a Bednar tumor (pigmented dermatofibrosarcoma protuberans) is composed of interspersed sequences from chromosomes 17 and 22: A fluorescence in situ hybridization and comparative genomic hybridization analysis. Genes Chromosomes Cancer 30(3):305–309.  https://doi. org/10.1002/1098-2264(2000)9999:99993.0.CO;2-R Nord KH, Macchia G, Tayebwa J, Nilsson J, Vult von Steyern F, Brosjo O, Mandahl N, Mertens F (2014) Integrative genome and transcriptome analyses reveal two distinct types of ring chromosome in soft tissue sarcomas. Hum Mol Genet 23(4):878–888. https:// doi.org/10.1093/hmg/ddt479 O’Brien KP, Seroussi E, Dal Cin P, Sciot R, Mandahl N, Fletcher JA, Turc-Carel C, Dumanski JP (1998) Various regions within the alpha-helical domain of the COL1A1 gene are fused to the second exon of the PDGFB gene in dermatofibrosarcomas and giant-cell fibroblastomas. Genes Chromosomes Cancer 23(2):187–193.  https://doi.org/10.1002/ (SICI)1098-2264(199810)23:23.0.CO;2-L Pedeutour F, Coindre JM, Nicolo G, Bouchot C, Ayraud N, Carel CT (1993) Ring chromosomes in dermatofibrosarcoma protuberans contain chromosome 17 sequences: Fluorescence in situ hybridization. Cancer Genet Cytogenet 67(2):149. https:// doi.org/10.1016/0165-4608(93)90171-h Pedeutour F, Coindre JM, Sozzi G, Nicolo G, Leroux A, Toma S, Miozzo M, Bouchot C, Hecht F, Ayraud

489 N, Turc-Carel C (1994) Supernumerary ring chromosomes containing chromosome 17 sequences. a specific feature of dermatofibrosarcoma protuberans? Cancer Genet Cytogenet 76(1):1–9. https://doi. org/10.1016/0165-4608(94)90060-4 Pedeutour F, Simon MP, Minoletti F, Barcelo G, TerrierLacombe MJ, Combemale P, Sozzi G, Ayraud N, Turc-Carel C (1996) Translocation, t(17;22) (q22;q13), in dermatofibrosarcoma protuberans: A new tumor-associated chromosome rearrangement. Cytogenet Cell Genet 72(2–3):171–174. https://doi. org/10.1159/000134178 Pedeutour F, Simon MP, Minoletti F, Sozzi G, Pierotti MA, Hecht F, Turc-Carel C (1995) Ring 22 chromosomes in dermatofibrosarcoma protuberans are lowlevel amplifiers of chromosome 17 and 22 sequences. Cancer Res 55(11):2400–2403 Persson F, Andren Y, Winnes M, Wedell B, Nordkvist A, Gudnadottir G, Dahlenfors R, Sjogren H, Mark J, Stenman G (2009) High-resolution genomic profiling of adenomas and carcinomas of the salivary glands reveals amplification, rearrangement, and fusion of HMGA2. Genes Chromosomes Cancer 48(1):69–82. https://doi.org/10.1002/gcc.20619 Persson F, Winnes M, Andren Y, Wedell B, Dahlenfors R, Asp J, Mark J, Enlund F, Stenman G (2008) Highresolution array CGH analysis of salivary gland tumors reveals fusion and amplification of the FGFR1 and PLAG1 genes in ring chromosomes. Oncogene 27(21):3072–3080. https://doi.org/10.1038/ sj.onc.1210961 Polito P, Dal Cin P, Kazmierczak B, Rogalla P, Bullerdiek J, Van den Berghe H (1999) Deletion of HMG17 in uterine leiomyomas with ring chromosome 1. Cancer Genet Cytogenet 108(2):107–109. https://doi.org/10.1016/s0165-4608(98)00128-9 Rowley JD (1973) Letter: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 243(5405):290–293. https://doi. org/10.1038/243290a0 Rowley JD, Diaz MO, Espinosa R, Patel YD, van Melle E, Ziemin S, Taillon-Miller P, Lichter P, Evans GA, Kersey JH et al (1990) Mapping chromosome band 11q23 in human acute leukemia with biotinylated probes: Identification of 11q23 translocation breakpoints with a yeast artificial chromosome. Proc Natl Acad Sci U S A 87(23):9358–9362. https://doi. org/10.1073/pnas.87.23.9358 Sandberg AA (2005) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: Leiomyoma. Cancer Genet Cytogenet 158(1):1–26. https://doi.org/10.1016/j.cancergencyto.2004.08.025 Sandberg AA, Bridge JA (2003) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors. Dermatofibrosarcoma protuberans and giant cell fibroblastoma. Cancer Genet Cytogenet 140(1):1–12. https://doi.org/10.1016/ s0165-4608(02)00848-8

490 Sandberg AA, Gibas Z, Saren E, Li FP, Limon J, Tebbi CK (1986) Chromosome abnormalities in two benign adipose tumors. Cancer Genet Cytogenet 22(1):55– 61. https://doi.org/10.1016/0165-4608(86)90137-8 Schoenmakers EF, Wanschura S, Mols R, Bullerdiek J, Van den Berghe H, Van de Ven WJ (1995) Recurrent rearrangements in the high mobility group protein gene, HMGI-C, in benign mesenchymal tumours. Nat Genet 10(4):436–444. https://doi.org/10.1038/ ng0895-436 Simon MP, Pedeutour F, Sirvent N, Grosgeorge J, Minoletti F, Coindre JM, Terrier-Lacombe MJ, Mandahl N, Craver RD, Blin N, Sozzi G, Turc-Carel C, O’Brien KP, Kedra D, Fransson I, Guilbaud C, Dumanski JP (1997) Deregulation of the plateletderived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma. Nat Genet 15(1):95–98. https://doi.org/10.1038/ng0197-95 Sirvent N, Maire G, Pedeutour F (2003) Genetics of dermatofibrosarcoma protuberans family of tumors: From ring chromosomes to tyrosine kinase inhibitor treatment. Genes Chromosomes Cancer 37(1):1–19. https://doi.org/10.1002/gcc.10202 Stenman G, Fehr A, Skalova A, Vander Poorten V, Hellquist H, Mikkelsen LH, Saba NF, GuntinasLichius O, Chiesa-Estomba CM, Andersson MK, Ferlito A (2022) Chromosome translocations, gene fusions, and their molecular consequences in pleomorphic salivary gland adenomas. Biomedicines 10(8). https://doi.org/10.3390/biomedicines10081970 Storlazzi CT, Lonoce A, Guastadisegni MC, Trombetta D, D’Addabbo P, Daniele G, L’Abbate A, Macchia G, Surace C, Kok K, Ullmann R, Purgato S, Palumbo O, Carella M, Ambros PF, Rocchi M (2010) Gene amplification as double minutes or homogeneously staining regions in solid tumors: Origin and structure. Genome Res 20(9):1198–1206. https://doi. org/10.1101/gr.106252.110 Szymanska J, Mandahl N, Mertens F, Tarkkanen M, Karaharju E, Knuutila S (1996) Ring chromosomes in parosteal osteosarcoma contain sequences from 12q13-15: A combined cytogenetic and comparative genomic hybridization study. Genes Chromosomes

J. Wen and M. L. Chong Cancer 16(1):31–34. https://doi.org/10.1002/ (SICI)1098-2264(199605)16:13.0.CO;2-4 Terrier-Lacombe MJ, Guillou L, Maire G, Terrier P, Vince DR, de Saint Aubain Somerhausen N, Collin F, Pedeutour F, Coindre JM (2003) Dermatofibrosarcoma protuberans, giant cell fibroblastoma, and hybrid lesions in children: Clinicopathologic comparative analysis of 28 cases with molecular data—a study from the French Federation of Cancer Centers Sarcoma Group. Am J Surg Pathol 27(1):27–39. https://doi. org/10.1097/00000478-200301000-00004 Van Dyck F, Declercq J, Braem CV, Van de Ven WJ (2007) PLAG1, the prototype of the PLAG gene family: Versatility in tumour development (review). Int J Oncol 30(4):765–774. https://doi.org/10.3892/ ijo.30.4.765 Wang G, Guzman MA, Batanian JR (2019) Three novel aberrations involving PLAG1 leading to lipoblastoma in three different patients: High amplification, partial deletion, and a unique complex rearrangement. Cytogenet Genome Res 159(2):81–87. https://doi. org/10.1159/000503158 Williams AJ, Powell WL, Collins T, Morton CC (1997) HMGI(Y) expression in human uterine leiomyomata. Involvement of another high-mobility group architectural factor in a benign neoplasm. Am J Pathol 150(3):911–918 Yan J, Whittom R, Delage R, Drouin R (2003) A unique clone involving multiple structural chromosome rearrangements in a myelodysplastic syndrome case. Cancer Genet Cytogenet 140(2):138–144. https://doi. org/10.1016/s0165-4608(02)00682-9 Zambrano E, Nose V, Perez-Atayde AR, Gebhardt M, Hresko MT, Kleinman P, Richkind KE, Kozakewich HP (2004) Distinct chromosomal rearrangements in subungual (Dupuytren) exostosis and bizarre parosteal osteochondromatous proliferation (Nora lesion). Am J Surg Pathol 28(8):1033–1039. https:// doi.org/10.1097/01.pas.0000126642.61690.d6 Zhang R, Chen X, Li P, Lu X, Liu Y, Li Y, Zhang L, Xu M, Cram DS (2016) Molecular characterization of a novel ring 6 chromosome using next generation sequencing. Mol Cytogenet 9(1):33. https://doi. org/10.1186/s13039-016-0245-9

Part IV

Ring Chromosome Research

Molecular Mechanisms of Ring Chromosome Formation and Instability

33

Stanislav A. Vasilyev   and Igor N. Lebedev  

Abstract

Keywords

Ring chromosome (RC) formation is a result of “rescue” chromosomes with DNA doublestrand breaks. The mechanisms of formation of RCs are not limited to incorrect repair of DNA double-strand breaks (DSBs), but also include recombination errors that lead to the occurrence of such breaks. The main mechanisms of instability of RCs in cells are also associated with meiotic and mitotic recombination. The chapter discusses the main causes and mechanisms of the occurrence and instability of RCs, as well as possible factors affecting the degree of such instability of RCs in vitro and in vivo.

Ring chromosome (RC) · DNA repair · Double-strand breaks (DSBs) · Dynamic mosaicism · Breakage-fusion-bridge (BFB) cycle · Sister chromatid exchange (SCE)

S. A. Vasilyev (*)  Laboratory of Genomic Tools, Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation e-mail: [email protected] I. N. Lebedev  Laboratory of Ontogenetics, Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russian Federation e-mail: [email protected]

33.1 Introduction With few exceptions, prokaryotes have genomes consisting of circular DNA molecules, whereas eukaryotic genomes are represented by linear chromosomes. There are no eukaryotic species found in nature that have one or more ring chromosomes (RCs) permanently maintained in the nuclear genome. Such cases occur either sporadically or are the result of directed chromosomal engineering (Shao et al. 2019; Murata et al. 2013). At the same time, eukaryotes constantly maintain the genomes of mitochondria and plastids of bacterial origin in the circular state. In addition, it has recently been shown that both somatic cells and germ line cells contain a large number of extra chromosomal DNA fragments existing in a circular form (Moller et al. 2018). RCs are usually found in various plant species (Yu 2018), fungi (Klar et al. 1983; Naito et al. 1998) and animals, including humans, rarely being inherited (Li et al. 2022). As a

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_33

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rule, the presence of RCs is associated with phenotypic abnormalities ranging from mild defects to embryonic death (Pristyazhnyuk and Menzorov 2018). In humans, only a few cases of stable transmission of RCs between generations have been described (Jenderny et al. 1993; Unterberger et al. 2019) (see also chapters for specific RCs of this book). RCs are more stable in organisms capable of asexual reproduction. Thus, in yeast, the production of several strains with individual RCs (Naito et al. 1998; Nakamura et al. 1998; McEachern et al. 2000) and even the creation of a synthetic line in which all 16 chromosomes were combined into one RC have been described (Shao et al. 2019). In somatic cells, RCs are more common, especially in cells with an increased mutation rate. Such an increased mutation rate may be associated with chromosomal instability. This is confirmed by the frequent occurrence of RCs in tumor cells of various localization (Gebhart 2008). In addition, RCs together with dicentric chromosomes are specific markers of exposure to ionizing radiation on cells (Jiang et al. 2000; Sun et al. 2019). However, even when RCs occur in somatic cells, they are characterized by increased instability and some of them are lost during cell division (Nikitina et al. 2021).

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33.2 Mechanisms of Ring Chromosome Formation

In the first mechanism, no genes are lost, and therefore, no haploinsufficiency is arisen (Fig. 33.1a). In the second scenario, again no genes are lost, but the absence of telomeres, at least for some chromosomes, might have an epigenetic influence upon nearby genes expression. At last, the third mechanism will lead to deletion of genes and if these genes are dosagesensitive, then clinically significant effect might be expected. From the molecular cytogenetics point of view, all these mechanisms are closely related to the repair of DNA double-strand breaks (DSBs). This is supported by the frequent occurrence of RCs when cells are exposed to various mutagens, e.g., ionizing radiation (Jiang et al. 2000; Sun et al. 2019; Melnikov et al. 2012), and the formation of RCs after mutations in DNA DSB repair genes (Naito et al. 1998). A RC is the result of incorrect repair of two DNA DSBs occurring on the same chromosome (Fig. 33.1b). When such DNA DSBs are located on different arms of the chromosome, a RC containing a centromere arises, and the result of connecting DNA DSBs in the same arm is an acentric RC. Distal fragments of chromosomes are lost in the form of acentric fragments. The molecular mechanisms of repair of DNA DSB leading to the formation of rings remain unclear; however, first of all they are most likely related to the non-homologous end joining (NHEJ) in the G1-phase of the cell cycle.

From the classical cytogenetics point of view, there are three cytological mechanisms of RC formation (Gardner and Amor 2018):

33.2.1 Telomeric or Subtelomeric Fusion

1. Fusion of telomeres without loss of other chromosomal material. These RCs are truly balanced and named as “complete rings.” 2. Deletion of subtelomeric region on p and/ or q arms with fusion of the exposed ends. This mechanism led to loss of subtelomeric repeats and known as “telomere healing.” 3. Deletion of some amounts of euchromatin at p and/or q arms with fusion of the exposed ends.

Telomere erosion can also be associated with the formation of RCs, since the ends of chromosomes with shortened telomeres begin to be perceived by the cell as unrepaired DNA DSB. The fusion of the ends of different chromosomes leads to the formation of dicentric chromosomes, and the fusion of the ends of the same chromosome leads to the formation of complete RCs (Fig. 33.1a). At the same time, there is no direct loss of chromosomal material. The role of

33  Molecular Mechanisms of Ring Chromosome Formation and Instability

Fig. 33.1  Mechanisms of ring chromosome formation. a telomeric or subtelomeric fusion. b DNA double-strand breaks and repairing. c inverted duplications associated with terminal deletions (inv dup del rearrangements) by (1) U-type exchange mechanism, (2) low-copy repeat

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dependent mechanism, (3) paracentric inversion dependent mechanism, and (4) fold-back mechanism. DSB, double-strand breaks; c-NHEJ, classical non-homologous end joining; a-EJ, alternative end joining, IR, inverted repeats

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telomeres in maintaining the stability of chromosome ends is confirmed by an increase in the frequency of occurrence of RCs with mutations in telomere maintenance genes and telomere erosion (Gisselsson et al. 2001; Artandi et al. 2000).

33.2.2 DNA DSBs and Repairing A combined mechanism of the occurrence of RCs is also possible as a result of the fusion of a telomeric region with the opposite end of the same chromosome, formed as a result of a DNA DSB inside the arm of the chromosome (Fig. 33.1b). This mechanism is most frequently seen and could explain rings with distal deletions on one or both chromosome arms. Recently, it has been shown that when two DNA DSBs on both arms of a chromosome 9 are repaired using an alternative end joining pathway, a more complex rearrangement is formed in a RC9 with distal deletions and interstitial duplication (Chai et al. 2020) (see also Chap. 13).

33.2.3 Inverted Duplication Deletion Rearrangement An alternative mechanism for the formation of RCs is associated with inverted (inv) duplication (dup) deletion (del) rearrangements (Seghezzi et al. 1999; Knijnenburg et  al. 2007; Rossi et al. 2008; Nikitina et al. 2021). Four molecular mechanisms have been proposed to explain the origin of inv dup del structure (Burssed et al. 2022) (Fig. 33.1c). All of them are related to appearance of a dicentric chromosome with subsequent broken and formation of deleted and duplicated regions. The first mechanism is a U-type exchange between sister chromatids (Fig.  33.1c-1). It starts from a DSB in the telomeric region of sister chromatids that lead to a terminal deletion. Next, the fusion of the broken ends will produce a U-type structure and a dicentric chromosome which undergoes a second double strand break

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forming inv dup del chromosome without spacer between the duplicated regions. The second mechanism is related to exchange between inverted low-copy repeats in subtelomeric region of sister chromatids which leads to the formation of dicentric chromosome (Fig. 33.1c-2). During cytokinesis, a chromatid break is occurred in the region between two centromeres, leading to the duplication with a normal copy number spacer between the duplicated regions. Then, the chromosome is closed into the RC as a result of fusion of distal end with telomere of the opposite arm of the same chromosome (Nikitina et al. 2021). This leads to the RC, which contains terminal deletion and inverted duplication of adjacent region with disomic spacer. The third mechanism requires the presence of a paracentric inversion in one of the parents (Fig. 33.1c-3). During meiosis, the inversion loop will formed with normal homologue. A cross over within inversion loop leads to the appearance of dicentric chromosome. Like in previous mechanism, a chromatid break will result an inv dup del chromosome with a normal copy number spacer between the duplicated regions. The second and third mechanisms are responsible for the most recurrent inv dup del rearrangement—the inv dup del at 8p. Non-allelic homologue recombination (NAHR) between the two olfactory receptor gene clusters in 8p23.1 (REPP and REPD) leads to the origin of inv dup del chromosome with a 7–8 Mb terminal deletion and inverted duplication with a 4–5 Mb spacer. A polymorphic 8p23.1 paracentric inversion present up to 26% of a population of European descent (Giglio et al. 2001) and usually present in the mother, promotes the risk of inv dup del of 8p and related RC8 occurrence. Therefore, a prenatal diagnosis should always be considered for carriers of an 8p23.1 inversion. The fourth, a fold-back mechanism was introduced by Hermetz with colleagues in 2014 (Fig. 33.1c-4) (Hermetz et al. 2014). In this mechanism, a DSB creates a terminal deletion. Since the region is no longer protected by telomere, the DNA from the free end can suffer

33  Molecular Mechanisms of Ring Chromosome Formation and Instability

from 5’ to 3’ resection. As a result, a reparative DNA synthesis is initiated from the 3’-end in a region of microhomology, DNA synthesis restore the resected gap forming monocentric chromosome. Following DNA replication, the dicentric chromosome is appeared with a normal copy number spacer between duplicated copies of the fold-back loop region. The size of the spacer depends on the amount of resected DNA and the distance between regions of microhomology. It is important to note, that inv dup del rearrangement leads to appearance of a partial trisomy in the karyotype due to the presence of duplicated region. This feature can explain the known from the clinical practice fact that some carriers of RC may demonstrate a phenotype specific not only to deleted subtelomeric and adjusted euchromatin regions but for interstitial partial trisomy also. From this point of view, precise determination of RC structure by current molecular cytogenetic and cytogenomic approaches, like chromosome microarray analysis (CMA), next-generation sequencing (NGS), and Hi-C technologies, is strongly required to predict the course of disease.

33.2.4 Other Mechanisms The basic principle of all mechanisms of RC formation apparently remains the same: the fusion of two terminal ends of the same chromosome. Sometimes, one of the two DNA DSBs necessary for this is formed in a more complex way as a result of abnormal recombination and further rupture of the bridge. The formation of the second DNA DSB can be either spontaneous or occur due to telomere erosion. Alternatively, multiple DNA DSBs occur simultaneously as a result of chromothripsis due to degradation of lagged chromosome in micronucleus (Fig. 33.2). At the same time, the repair of shattered chromosomes takes place with the loss of some fragments and amplification of several other fragments of chromosomes. Stabilization of the resulting rearranged chromosome is possible due to its closure into a RC.

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An example of such a mechanism is the occurrence of RCs containing interstitial amplification of individual regions of chromosome 21 associated with acute lymphoblastic leukemia (Li et al. 2014). The risk of such chromosomes occurrence is increased in carriers of inherited Robertsonian translocations between chromosomes 15 and 21 (Harrison and Schwab 2016). It is assumed that in this case, a chromosome with a Robertsonian translocation is sometimes lost during mitotic division due to the presence of two centromeres, included into the micronucleus and undergoes chromothripsis (Fig. 33.2). Another result of chromothripsis may be the so-called double minute chromosomes containing individual chromosome regions closed in small RCs and subjected to multiple amplification (Holland and Cleveland 2012) (Fig. 33.2) Such chromosomes are often found in tumor cells and often contain amplified oncogenes (e.g., myc) (see also Chaps. 31 and 32 on acquired RCs in leukemia and solid tumors).

33.3 Mechanisms of Ring Chromosome Instability The reason for the ban on the existence of RCs in eukaryotes, apparently, is meiosis. This is supported by a disruption of meiosis in all cases of artificial production of RCs (Naito et al. 1998; Nakamura et al. 1998; McEachern et al. 2000; Shao et al. 2019). Incompatibility of RCs and meiosis may be the result of several mechanisms. First, the crossover between sister chromatids of RCs in meiosis leads to the formation of interlocked rings and dicentric RCs, which then break during cytokinesis at the end of meiosis I (Endow et al. 1984; Sutou 1997). Second, the formation of RCs leads to the disappearance of telomeres, which play important roles in meiosis (Ishikawa and Naito 1999). The attachment of telomeres to the nuclear lamina in meiosis is necessary for the convergence of the telomeres of all chromosomes in one region of the lamina and the formation of the so-called “bouquet stage” (reviewed in Wang et al. 2022). Such a convergence of chromosomes is necessary for

Fig. 33.2  Other mechanisms for ring chromosome formation through chromothripsis. DSB, double-strand breaks; BFB, breakage-fusion-bridge; iAMP21, intrachromosomal amplification of chromosome 21

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the formation of bivalents and the normal crossover (Niwa et al. 2000). The absence of telomeres in the RC can lead to its mis-attachment to the nuclear lamina and the absence of crossover (Ishikawa and Naito 1999). In this case, the abnormal crossover is closely related to the subsequent chromosome non-disjunction and the occurrence of aneuploidy in meiosis. In addition, the attachment of chromosome telomeres to the nuclear lamina itself is necessary for the normal process of chromosome segregation (Moiseeva et al. 2017). Thus, RCs are incompatible with meiosis due to interference with the processes of crossover and chromosome segregation. The formation of a RC is a way to stabilize a chromosome with chromosomal breaks. However, the further passage of RCs through mitosis faces difficulties, as with meiosis. In mitosis, telomeres do not play a critical role in the chromosome segregation. However, as with crossover in meiosis, mitotic recombination takes place in mitotic cells as a result of Holliday junction resolution after repair of DNA DSBs by homologous recombination mechanism. Therefore, similar to meiosis, there is instability of RCs in somatic cells associated with the occurrence of sister chromatid exchanges (SCEs). An odd number of SCEs leads to the formation of dicentric rings, and an even number of SCEs leads to inter-locked rings (Brewen and Peacock 1969; McClintock 1938; Gatti et al. 1979) (Fig. 33.3). Both types of derived rings form anaphase bridges when passing through mitosis and break during cytokinesis, leading to the formation of DNA DSBs at new sites (Endow et al. 1984; Sutou 1997). This leads to a new round of RC formation, which in the next cell cycle may break again. Thus, the instability of RCs is a dynamic process developing by the mechanism of breakage-fusion-bridge (Fig. 33.3). An alternative to the rupture of the RC in mitosis is anaphase lagging, chromosomal non-disjunction or the formation of micronuclei. As a result, so-called “dynamic mosaicism” occurs in cells of carriers of RCs with the formation of RC variants, linear derivatives, and aneuploidy.

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The instability of RCs is well known in radiation mutagenesis, where the death of most cells in the first two divisions after irradiation is associated with mitotic death due to instability of dicentrics and RCs. In the first division, about 55% of dicentrics and RCs are lost after irradiation (Bauchinger et al. 1986). At the same time, in the second division after irradiation, the frequency of RCs doubles in relation to dicentrics, which indicates greater stability of RCs (Pala et al. 2001; Krishnaja and Sharma 2004). A separate analysis for RCs showed that 19% is lost in the first division and 61% in the second division after exposure to ionizing radiation in vitro (Kaddour et al. 2017). All these indicate a high degree of instability of the RCs formed as a result of exposure to mutagens. Despite the chromosomal instability of RCs in mitosis, dynamic mosaicism is not characteristic of all carriers of RCs, and in its presence, most of the patients’ cells most often continue to contain RCs. This indicates a relatively low probability of RC instability. In addition, yeast strains with a RC show only a slight lag in proliferation relative to strains with all linear chromosomes (Klar et al. 1983; Naito et al. 1998; Shao et al. 2019). On the other hand, a much greater degree of instability of RCs is observed in the case of induced pluripotent stem cells, in which their loss or reduction of RCs in size is often recorded (Nikitina et al. 2021). Apparently, the stability of RCs can be influenced by factors specific to different chromosomes and cell types.

33.4 Factors Affecting Ring Chromosome Instability A key factor in the instability of RCs in mitotic cells is the probability of mitotic recombination. Therefore, factors affecting the intensity of such instability can be all factors affecting the frequency of occurrence of SCEs on an individual RC. These are the size of the RC, the presence of somatic recombination hotspots, cell type, number of divisions, and exposure to mutagens.

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Fig. 33.3  Mechanisms of ring chromosome instability. BFB cycle, breakage-fusion-bridge cycle. Adapted from Nikitina et al. (2021)

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The probability of occurrence of SCEs per unit chromosome length is approximately constant in different regions of the genome (Claussin et al. 2017), which leads to an increased probability of occurrence of SCEs in larger chromosomes. At the same time, regions with an increased frequency of occurrence of SCEs, often unrelated to DNA DSBs, are distinguished in the genome (Heijink et al. 2022). Such hot spots of somatic recombination include nucleolus organizer regions (NORs) located in humans on the short arms of acrocentric chromosomes 13, 14, 15, 21, and 22. The frequency of SCEs is increased in the NOR regions in different species (Claussin et al. 2017; Satoh and Obara 1995). In addition, there is an increased frequency of occurrence of SCEs near telomeres (so-called T-SCE) (Nieves et al. 2023; Wang et al. 2005). Finally, stalled replication forks and common fragile sites that are difficult to replicate can make a certain contribution to the formation of SCE and, consequently, to the instability of RCs (Kumar et al. 2019; Hamadeh et al. 2022; Heijink et al. 2022; Lukusa et al. 1991). At the same time, the occurrence of SCE is suppressed in common fragile sites located near centromeres (Waisertreiger et al. 2020). However, testing the hypothesis of increased instability of RCs depending on the presence of NOR or common fragile sites on the chromosome requires further research. As far as the authors are aware, there are currently no systematic reviews that allow comparing the frequency of mitotic recombination events in different types of mammalian and human cells. Nevertheless, it is known that in pluripotent cells (both in embryonic stem cells and in induced pluripotent stem cells), the homologous recombination pathway leading to the formation of SCEs is predominant pathway used to repair DNA DSBs. This appears to be the reason for the increased frequency of SCEs found in mouse embryonic stem cells (Claussin et al. 2017; Falconer et al. 2012). The phenomenon of karyotype correction due to the loss of RCs and uniparental disomy (UPD) in induced pluripotent human stem cells (Nikitina et al. 2021; Bershteyn et al. 2014) may also be

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associated with the effect of this factor. Given the probabilistic nature of the occurrence of SCEs, the stability of RCs should also depend on the number of cell divisions necessary for the formation and maintenance of a tissue or organ. One of the most important factors affecting the occurrence of chromatid exchanges and indirectly on the stability of RCs is the effect of mutagens. SCEs are a consequence of the repair of DNA DSBs using the homologous recombination pathway. This indicates a possible influence on the frequency of SCEs of mutagens inducing DNA DSBs or single-strand DNA damage leading to stalled replication forks. Indeed, such an effect is shown for exposure to ionizing radiation (Kanda et al. 2004). However, the most pronounced increase in SCEs is observed after exposure to replication inhibitors, including topoisomerase and PARP inhibitors (Heijink et al. 2022; Ribas et al. 1996). Different levels of DNA damage leading to replication disruption may be another factor affecting the instability of RCs in ontogenesis.

33.5 Conclusions The appearance of RCs is primarily a way of “rescue” of chromosomes that have one or two DNA DSBs, which allows the cell to pass the cell cycle checkpoints. Recent data show that in addition to the repair of DNA DSBs, recombination also plays an important role in the formation of rings. The “rescue” of the damaged chromosome due to its closure into the RC is not complete, as it is associated with increased instability of the RC in cell divisions. The results available to date show that the ban on the ring shape of DNA in eukaryotic cells is primarily associated with passage through meiosis, where the ring shape of the chromosome leads to chromosomal instability during meiotic recombination, and the absence of telomeres leads to abnormal attachment of chromosomes to the nuclear lamina, errors in chromosome pairing and chromosome segregation. During mitotic divisions, RCs can be maintained in cells for a long time, but are characterized by

502

increased instability, the sources of which are the breakage-fusion-bridge cycle, the occurrence of DNA DSBs and replication errors. The resulting “dynamic mosaicism” with the presence of secondary RC variants, linear derivative and marker chromosomes, and aneuploidy in the cells significantly complicates the characterization of the karyotype of patients. Conventional cytogenetic analysis keeps the brand of “a gold standard” for visualization of RCs and related mosaic patterns at cellular level in the genomic era. At the same time, current genomic technologies, like CMA, long-read NGS, optical genome mapping (OGM), and methods of 3D-genomics, provide new opportunities for better understanding of RC origin, structure, and behavior. Additional fundamental studies of the instability of various RCs, depending on various factors, are needed. Given the different probability of instability of RCs in different tissues, it is desirable to analyze the degree of mosaicism in several tissues in patients with RCs, to assume the presence of marker chromosomes of different sizes and aneuploidy in cells. All these intentions are significant for better understanding the nature of RC and improving the medical care and genetic counseling of patients with RC.

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504 origin are common in healthy human somatic tissue. Nat Commun 9(1):1069. https://doi.org/10.1038/ s41467-018-03369-8 Murata M, Shibata F, Hironaka A, Kashihara K, Fujimoto S, Yokota E, Nagaki K (2013) Generation of an artificial ring chromosome in Arabidopsis by Cre/LoxP-mediated recombination. Plant J 74(3):363–371. https://doi.org/10.1111/tpj.12128 Naito T, Matsuura A, Ishikawa F (1998) Circular chromosome formation in a fission yeast mutant defective in two ATM homologues. Nat Genet 20(2):203–206. https://doi.org/10.1038/2517 Nakamura TM, Cooper JP, Cech TR (1998) Two modes of survival of fission yeast without telomerase. Science 282(5388):493–496. https://doi.org/10.1126/ science.282.5388.493 Nieves M, Puntieri F, Bailey SM, Mudry MD, Maranon DG (2023) Exploring the relationship between spontaneous sister chromatid exchange and genome instability in two cryptic species of non-human primates. Animals 13(3):510. https://doi.org/10.3390/ ani13030510 Nikitina TV, Kashevarova AA, Gridina MM, Lopatkina ME, Khabarova AA, Yakovleva YS, Menzorov AG, Minina YA, Pristyazhnyuk IE, Vasilyev SA, Fedotov DA, Serov OL, Lebedev IN (2021) Complex biology of constitutional ring chromosomes structure and (in)stability revealed by somatic cell reprogramming. Sci Rep 11(1):4325. https://doi.org/10.1038/ s41598-021-83399-3 Niwa O, Shimanuki M, Miki F (2000) Telomere-led bouquet formation facilitates homologous chromosome pairing and restricts ectopic interaction in fission yeast meiosis. EMBO J 19(14):3831–3840. https:// doi.org/10.1093/emboj/19.14.3831 Pala FS, Moquet JE, Edwards AA, Lloyd DC (2001) In vitro transmission of chromosomal aberrations through mitosis in human lymphocytes. Mutat Res 474(1–2):139–146. https://doi.org/10.1016/ s0027-5107(00)00172-x Pristyazhnyuk IE, Menzorov AG (2018) Ring chromosomes: From formation to clinical potential. Protoplasma 255(2):439–449. https://doi. org/10.1007/s00709-017-1165-1 Ribas G, Xamena N, Creus A, Marcos R (1996) Sisterchromatid exchanges (SCE) induction by inhibitors of DNA topoisomerases in cultured human lymphocytes. Mutat Res 368(3–4):205–211. https://doi. org/10.1016/s0165-1218(96)90062-2 Rossi E, Riegel M, Messa J, Gimelli S, Maraschio P, Ciccone R, Stroppi M, Riva P, Perrotta CS, Mattina T, Memo L, Baumer A, Kucinskas V, Castellan C, Schinzel A, Zuffardi O (2008) Duplications in addition to terminal deletions are present in a proportion

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iPSC Models of Ring Chromosomes, Genome Editing, and Chromosome Therapy

34

Tatiana V. Nikitina and Igor N. Lebedev

Abstract

Induced pluripotent stem cells (iPSCs) are beneficial for studying of chromosomal diseases, including ring chromosomes (RCs), because iPSCs can be differentiated to various cell types, some of which are difficult to access in vivo. Patient-derived iPSCs are an infinite source of cells that can be genetically edited, and used for disease modeling, pathogenesis studies, as well as for gene therapy. Here, we review the studies of patientderived iPSCs carrying a RC and consider mitotic instability of RC in pluripotent state. We also discuss normalization of the iPSCs karyotype through a loss of the RC due to its mitotic instability and a compensatory rescue by replicating the normal homolog, which has been proposed as an approach for karyotype correction named “chromosome therapy.” Some issues of difference in RC mitotic stability between somatic and pluripotent cells are considered, as well as possible reasons for diversity of RC stability between

T. V. Nikitina (*) · I. N. Lebedev  Laboratory of Ontogenetics, Research Institute of Medical Genetics, Tomsk National Research Medical Center of the Russian Academy of Sciences, Tomsk, Russia e-mail: [email protected] I. N. Lebedev e-mail: [email protected]

long-term cultures of isogenic lines. We also review the stability of small supernumerary marker or ring chromosomes (sSMC/sSRCs) and the appearance and stabilization of RC in pluripotent cells. Overall, patient-derived iPSCs seems to be a promising tool for studies of RCs in human cells.

Keywords

Induced pluripotent stem cells (iPSC) · Mitotic instability of RC · Uniparental disomy · Chromosome therapy

34.1 Introduction Studies of human chromosomal abnormalities, including ring chromosomes (RCs), are among others complicated by the involvement of many cell types and tissue types, some of which being difficult to access. It is also hard to apply animal disease models because chromosomal structure and content are variable among different species. The ability to produce induced pluripotent stem cells (iPSCs) from somatic cells (Takahashi et al. 2007) offers new opportunities for studies of human diseases, as previously inaccessible cell types, such as neurons, can now be generated through iPSCs from individuals with chromosomal variants. These patient-derived iPSCs are highly beneficial for studying chromosomal

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 P. Li and T. Liehr (eds.), Human Ring Chromosomes, https://doi.org/10.1007/978-3-031-47530-6_34

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diseases and have been widely utilized for recapitulating pathogenesis in vitro, thereby contributing to disease modeling and allowing the study of the effect of human genetic variation on developmental processes (see Fig. 34.1).

T. V. Nikitina and I. N. Lebedev

First attempts to generate iPSCs with RCs were considered not entirely successful, because the RCs in iPSCs were found to be unstable

(Bershteyn et al. 2014). When primary fibroblasts from a Miller–Dieker syndrome patient with RC17 were reprogrammed into iPSCs, the RC17 was lost and the normal homologous of chromosome 17 was duplicated in four out of six clones, thereby establishing uniparental disomy (UPD) of chromosome 17. Remarkable, no RC17 was found in metaphases of iPSCs, suggesting that such cells may be terminal and non-dividing in the pluripotent state. Similar results were obtained in this study for most of iPSC clones with an RC13 (Bershteyn et al. 2014).

Fig. 34.1  Application of human iPSCs for chromosomal disease modeling. Illustration of human iPSCs modeling for RC: reprogramming of somatic cells from patient with RC to iPSC with RC; spontaneous karyotype correction during iPSC propagation with formation of isogenic lines of iPSC with isoUPD of indexed

chromosome; directed differentiation of iPSC to target cell types (i.e., neurons) or organoids; comparative analysis of data from isogenic cell lines; in future using of this data for improved diagnosis, development of treatment or using of cells with normalized karyotype for therapy

34.2 iPSC Cellular Modeling for RCs

34  iPSC Models of Ring Chromosomes, Genome Editing, and Chromosome Therapy

This cell-autonomous correction was proposed further as the possible way for the correction of the large-scale chromosomal aberrations. While the derivation of genome-engineered iPSC cell lines with single nucleotide and gene variants is now routine, the correction of large chromosomal rearrangements is a challenging problem. Normalization of the karyotype through RC mitotic instability has been proposed as an approach for karyotype correction in cells not only with RCs, but also with other large-scale rearrangements like deletions, duplications, and inversions (Kim et al. 2014; Plona et al. 2016). The circularization of damaged chromosomes could result in its elimination during cell line propagation, with following monosomy correction via duplication of the remaining intact linear homologue to form isodisomy.

34.3 Cell-Autonomous Correction and Chromosome Therapy The method of probable karyotype correction using RC loss and compensation was called “chromosome therapy” (Kim et al. 2014; Plona et al. 2016). Site-specific Cre-lox recombination technology has been proposed to force a linear chromosome to close into a RC. However, experimental results using this method have not yet been published. Another way to generate RCs is using a CRISPR/Cas9 system, followed by end-to-end DNA ligation (Møller et al. 2018). Extra chromosomal ring DNA molecules from a few hundred base pairs in length to a 47.4 megabase-sized RC18 were obtained using this method in human fibroblasts and 293T embryonic kidney cells (Møller et al. 2018). Because these experiments were carried out on cell types other than iPSCs, the question remains about its feasibility for the pluripotent cells. The method should be adopted for pluripotent cells since mechanisms of maintaining genetic stability in iPSCs have some peculiarities from somatic cells (Chen et al. 2021). Except for RCs, some studies demonstrated spontaneous karyotype normalization in iPSC lines with trisomies through “trisomy-biased

507

chromosome loss” mechanisms without any genetic manipulation or chemical treatment (Hirota et al. 2017; Inoue et al. 2019; Akutsu et al. 2022). Karyotype correction was also observed in cell lines with sex chromosome monosomy: four fibroblast lines from chorionic villi with 45,X karyotype reprogrammed to iPSC; one line restored the normal 46,XX karyotype through UPD of whole X-chromosome (Luo et al. 2015). It indicates that reduplication of single homologue in iPSC can occur in another genetic context than RC loss.

34.4 Stability of RC in Long-Term Cell Culture In any way, the question remains how inevitable is the loss of the constitutional RC in iPSCs? Several studies showed that stable iPSC lines with RCs can be generated, at least for some chromosomes and for some time or passages (Nikitina et al. 2018a, 2018b; Gridina et al. 2020; Khabarova et al. 2020). There are not enough results to make strong conclusions, but current studies in this area suggest the smaller the RC size, the more stable it is in iPSCs. As shown in Table 34.1, RC21 and RC22 were found most stable in iPSCs, and RC17 and RC8 appear to be less able to maintenance in the pluripotent state. Chromosome size correlates with the number of sister chromatid exchanges (SCE), which appears to affect RC stability in mitosis. In addition, the specific gene content influences the stability of the RC. RC8 and RC17, both include genes that contribute to accelerated iPSCs proliferation, so trisomies of these chromosomes are recurrent in iPSC lines of different origins (International Stem Cell Initiative et al. 2011; Taapken et al. 2011; Na et al. 2014; Assou et al. 2020). The fact that RC17 was not found in iPSC metaphases (Bershteyn et al. 2014) may be associated with the localization of the antiapoptotic BIRC5 gene, which provides a proliferative advantage (Blum and Benvenisty 2009), such that RC instability or monosomy 17 may strongly compromise the ability of pluripotent cells to divide.

58

80

90

46,XY,r(13)/46,XY, -13, +mar/45,XY,-13

46,XY,r(13)/ 45,XY,-13

46,XY,r(13)/ 45,XY,-13

13

46,XY,r(18) (p11.1q23)

46,XX,r(21) (p11.2q22.3)

18

21

dup22q13.32, del22q13.32-q13.33

two deletions in 21q22.3

del18p11.32-p11.21, del18q23

del17p13.3- p13.2

del14q32.33

del13q34-q21.31

del13q34-q31.3

del13q34

Stable

Stable

Stable/fragmentation

Not found, loss during reprogramming

Loss during culture

Loss during reprogramming/culture

Loss during reprogramming/culture

Loss, fragmentation

17/33c

0/2

0/ND

0/3

4/6

1/3

2/4

4/5

0/4

6/6

Nikitina et al. (2021)

Bershteyn et al. (2014)

Cherry (2014)

Bershteyn et al. (2014)

Bershteyn et al. (2014)

Nikitina et al. (2021)

Nikitina et al. (2021)

Refs.

–

Nikitina et al. (2021)

Ring chro- Schuy et al. (2022) mosome stable through multiple passages of NES

–

–

Able to form NPC and neural rosettes

–

–

–

–

Rate of cor- Neuronal rection via derivatives isoUPDb

Abbreviations: a main cell subpopulations karyotypes; b number of lines with spontaneous karyotype correction via isoUPD of the total number of lines studied; NES—neuroepithelial stem cells; NPC—neural progenitor cells; ND—no data; c does not include the number of r(21)-iPSC lines analyzed, since it is not given

Total

ND

100

95

46,XX,r(22)/45,XX,-22 73

46,XY,r(17)/ 45,XY,-17

17

22

46,XY,r(14)/ 45,XY,-14

14

86

67

46,XY,r(8)/ 45,XY,-8

8

RC “behavior” in iPSC

invdup8p23.1-p11.22, del8p23.3-p23.1 Loss during culture

Percentage of RC structure cells with RC

Karyotypea

Ring chr Fibroblasts

Table 34.1  iPSC lines from patients with RC and the number of lines with spontaneous karyotype correction resulting to isoUPD

508 T. V. Nikitina and I. N. Lebedev

34  iPSC Models of Ring Chromosomes, Genome Editing, and Chromosome Therapy

Mitotic stability of RCs in cultured cells differs markedly between somatic cells and iPSCs. Slow growth of fibroblasts from patients with a RC was discovered over fifty years ago (Bobrow et a1. 1971). Cell viability and cloning efficiency of fibroblasts from patients with RCs were decreased compared to control cells with normal karyotype; problems in the passage of RCs through mitosis were suggested as reason therefore (Kosztolányi 1987). There is some diversity in mitotic “behavior” of RC between iPSCs lines and their initial fibroblasts. One can hypothesize that this discrepancy is caused by the predominance of the homologous recombination pathway in double-strand break (DSB) repair in iPSCs (Adams et al. 2010; Tichy et al. 2010). Due to homologous recombination, the Holliday junctions between chromatids are formed, resulting in increased SCE formation in the pluripotent stem cell cycle. Replication stress during the reprogramming process and cell propagation increased frequency of DSB (Bai et al. 2015; Ruiz et al. 2011; Ji et al. 2012). As a result, the frequencies of dicentric and interlocked rings increase, followed by anaphase lagging, non-disjunction, or fragmentation (Nikitina et al. 2021). Surprisingly, marked variability in the mitotic stability of RCs between isogenic, i.e., derived from one source, iPSC lines was found (Nikitina et al. 2021). This instability manifests both as the loss of the entire RC and in the form of structural variability of the RC. The latter pointed to multiple rearrangements, which the corresponding RC underwent. For example, two out of four cell lines with RC13 in the study of Nikitina et al. (2021) had relatively stable ring structure with terminal deletions similar to that of the initial RC13 of patient, but the other two “unstable” lines had significant losses of RC13 content along with higher rate of cells with monosomy 13. The structural variability of RCs in iPSCs may be a result of ring fragmentation or chromothripsis, when the lagging chromosome is encapsulated within a micronucleus and then reincorporated into the nucleus of one of the daughter cells with loss of some DNA material. Overall, the reasons for striking difference

509

of RC stability between long-term cultures of isogenic lines remain unclear yet. As for RC8, loss of RC and forming of isoUPD8 happened in all studied iPSC lines by a similar pattern (Fig. 34.2).

34.5 iPSC as a Cellular Model iPSCs are similar to embryonic stem cells (ESCs) and simulate early stages of embryo development, so study of chromosomal instability in iPSCs can help elucidate the origins of RC mosaicism, since each iPSC clone is a descendant of one cell. Even so, one needs to keep in mind that the results obtained on cell cultures cannot be directly extrapolated to a whole organism. The number of cells in culture is limited and cells passed through the bottleneck in culture establishing that increases both the selective pressure of culture conditions and the likelihood of the mutation event detecting, especially if new genetic variant provides a proliferative advantage. Nevertheless, in vivo examples of RC rescue via monosomy appearance followed by chromosome duplication, resulting “compensatory” UPD, were reported in patients with RC8 (Gradek et  al. 2006) and RC21 (Petersen et al. 1992) by mechanisms similar to reported iPSC events (Bershteyn et al. 2014; Nikitina et al. 2021). As spontaneous correction of the RC would produce “pseudonormal” karyotype with isodisomy, one should beware of the possible negative UPD consequences, since UPD itself can be the cause of the pathology either due to manifestation of recessive mutations or through the abnormal expression of the imprinted genes (Kotzot 2008; Liehr 2022). If there is an imprinted gene on a RC, then ring loss with appearance of monosomy in the part of cells may be an etiological mechanism of mosaic imprinting disorders, presumably with less severe phenotype, as it was demonstrated for patient with Birk–Barel syndrome and RC8 (Kashevarova et al. 2020). Because iPSC-based disease modeling use cells differentiated in specific direction, taken into account the frequent impairments

Fig. 34.2  Spontaneous karyotype correction in iPSCs from patient with RC due to the loss of the RC8 and the formation of uniparental isodisomy of the intact homologue. Top row: outline of events that result in iPSC-mediated spontaneous normalization of karyotype with RC. Middle row: fragments of GTG analysis with a pair of chromosome 8 shows the presence of RC8 in iPSCs of early passages and loss of RC8 during iPSC culture; cells with normal karyotype appear and begin to prevail on later passages. Bottom row: microarray analysis of chromosome 8 shows RC8 in iPSCs of early passages, then mitotic instability of RC8 in pluripotent cells result in multiple rearrangements followed by loss of RC8 during iPSC culture; cells with normal karyotype appear and begin to prevail on later passages. STR arrays on later passages shows the presence of only one homologous chromosome that prove isoUPD(8)

510 T. V. Nikitina and I. N. Lebedev

34  iPSC Models of Ring Chromosomes, Genome Editing, and Chromosome Therapy

511

Table 34.2  ESC and iPSC lines with spontaneous occurrence of the RC Refs.

Cell type

Initial karyotype

Karyotype detected

The number of lines with RC occurrence

Karamysheva et al. (2013)

ESC

46,XX

46,XX,r(18)(p11.31q21.2)

1

Vaz et al. (2021)

iPSC

46,XY

36~45,XY,~6dmin,+r1,+r2[6]/46,XY[14]

1

Chang et al. (2015)

iPSC

46,XY

46,XY,r(22)(p13q13.3)/45,XY,-22

2/4

46,XY,r(22)(p12q13)/46,XY

of intellectual development, epilepsy, etc., in patients with RCs, differentiation in the neuronal direction is of the most interest. Currently only two studies of differentiation of iPSCs with RC to a neural lineage are known, indicating the possibility of obtaining neural progenitor cells (Cherry 2014) and neuroepithelial stem cells (Schuy et al. 2022) (Table 34.1). RC21 was found stable through passages of neuroepithelial stem cells. Surprisingly, RCs can also appear and then remain stable in pluripotent cells (Table 34.2). Generally, the karyotypes of most iPSC lines are quite stable; however, in 5–15% of the lines, large chromosomal abnormalities emerge that occur either during reprogramming or during long-term culture (International Stem Cell Initiative et al. 2011, Taapken et al. 2011; Halliwell et al. 2020; Assou et al. 2020). An example of the RC emergence in ESC is the subline hESM01-18, where cells with karyotype 46,XX,r(18) were retained as a modal class for many passages, indicating that this rearrangement has some selective advantage (Karamysheva et al. 2013). Cases of the appearance of RCs in iPSCs are rare. iPSC from a patient with long QT syndrome type 2 exhibited RCs in 6.7% of cells. As authors suggest, ring formation is caused by shortening of telomeres associated with oxidative stress in cells of such patient (Vaz et al. 2021). In iPSCs lines obtained from fibroblasts of healthy man, two lines had a normal karyotype 46,XY, and another two lines had RC22 in mosaic state with different types of mosaicism: mos 46,XY,r(22) (p13q13.3)/45,XY,-22 and mos 46,XY,r(22)

(p12q13)/46,XY (Chang et  al. 2015) (Table 34.2). Detection of RCs in iPSCs with presumably normal karyotype may be a consequence of low-level mosaicism preexisted in the initial tissues, which was not detected by banding cytogenetic methods. However, the appearance of RC22 in two iPSCs lines from one person may also be a consequence of the structural peculiarities of the chromosome, predisposing to a ring formation. Interestingly, the size of the RC22 in two lines was different, which confirms the latter assumption. Such cases may be of interest for studying the mechanisms of ring formation. Small supernumerary marker chromosomes (sSMC) also can be ring-shaped (sSRCs), and the creation of iPSC models will help to predict the stability of such extra chromosomes and their effect on the carrier phenotype. It has been shown that sSRCs and centric minute–shaped sSMCs are similarly mitotic unstable, while inverted duplication–shaped sSMC are much more stable (Hussein et al. 2014). In iPSCs generated from the carriers of sSMC, some clones were found without sSMC and other clones with varying rates of the marker chromosome, typically below the rate detected in the source fibroblasts (Tcw et al. 2017; Gridina et al. 2022). Interestingly, neural differentiation was not affected by the frequency of sSMC mosaicism and was similar for different lines, suggesting that the level of mosaicism in neural progenitor cells did not reflect the source iPSCs (Tcw et al. 2017). In contrast to the correction of constitutional RCs, karyotype rescue in sSMC-positive cells with RC lead to the formation of mosaic karyotypes with an actually normal cell clone (Lebedev et al. 2021).

512

34.6 Future Directions Cell reprogramming technologies for RC studies assumes several frontier directions. One of them is investigation of mechanisms that control the “behavior” of RCs during embryo development and cell/tissue differentiation. The potential role of cell competition in determining the composition of cell populations may be investigated in iPSCs to model cell–cell interactions during different stages of development and disease (Price and Barbaric 2022). Such studies may shed light also on the phenomenon of aneuploidy rescue and karyotype self-correction in mosaic preimplantation embryos (Lebedev and Zhigalina 2021). Lineage-specific behavior of aneuploid cells in early human embryogenesis was recently showed using BMP4-stimulated human ESCs in self-organized structures, named “gastruloids” (Yang et al. 2021). In the context of the study of RCs, the ideal control for similar experiments could be isogenic iPSC lines with RC and with corrected disomic karyotype. Karyotype correction using RC loss and compensation, named as “chromosome therapy” (Kim et al. 2014; Plona et al. 2016), seems to be a promising direction for repair of large-scale chromosomal abnormalities in human cells. Correction in iPSC is possible both as cellautonomous event (Bershteyn et al. 2014; Luo et al. 2015; Hirota et al. 2017; Nikitina et al. 2021), and as genome editing (Adikusuma et al. 2017; Zuccaro et al. 2020). Further development of methods for closing a linear chromosome into a ring in different cell types is needed along with studies of ring loss and duplication of remaining homologue. Differentiated derivatives (e.g., neuronal) will provide researchers with an appropriate tool to investigate the etiology of developmental delay, epilepsy, seizure, and other pathogenic features on the cellular level (Vadodaria et al. 2020). In addition, the study of the RC emergence and stability in reprogrammed cells may be of importance for oncogenetic researches, because RCs may arise as acquired genetic abnormalities in cells from solid tumors or leukemia.

T. V. Nikitina and I. N. Lebedev

Importantly, there are some shared characteristics between iPSCs and tumor cells, such as activation of similar signaling pathways, high level of telomerase activity, loss of contact inhibition, capacity for unlimited self-renewal and proliferation (Blum and Benvenisty 2009; Hatina et al. 2022). In summary, cell reprogramming and genome editing technologies drive genetic studies of human RCs to a new level both in the study of the influence of RCs to the cell proliferation and function, and in the perspective of large-scale chromosomal abnormalities correction. Acknowledgements  The study was supported by the Russian Science Foundation, grants 16-15-10231 (for the study of ring chromosome instability in iPSCs) and 21-65-00017, https://rscf.ru/en/project/21-65-00017/ (for the study of ring sSMC(10) and literature review).

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Genetic Mosaic Analysis in Model Organisms

35

Hui Zong  

Abstract

Keywords

The definition of genetic mosaicism is the presence of cells with various genotypes within the same animal, which could be caused by somatic transposition, mitotic recombination, or genomic aberrations, including the formation and irregular segregation of ring chromosomes. Because naturally occurring mutant cells in genetic mosaic animals cannot be effectively studied due to the difficulty in tracking them down, a genetically engineered mosaic mouse model termed mosaic analysis with double markers (MADM) was established to enable one to perform phenotypic analysis of mutant cells in vivo by unequivocally labeling them with green fluorescent protein (GFP) and red fluorescent protein (RFP). With its singlecell resolution, MADM is highly suitable for studying developmental biology, neuroscience, and regenerative biology problems caused by stochastically distributed mutant cells in mice.

Mouse genetic mosaic system · Mosaic analysis with double markers (MADM) · Single cell knockout · Twin spot analysis · Lineage tracing

H. Zong (*)  Department of Microbiology, Immunology, and Cancer Biology, University of Virginia Health System, Charlottesville, VA 22908, USA e-mail: [email protected]

35.1 Brief History of Genetic Mosaic Analysis To understand genetic regulations in normal development and human diseases, gene knockout technology was developed in mice (Thomas and Capecchi 1987; Doetschman et al. 1987; Mansour et al. 1988), the most used mammalian model organism, and then applied to many biological questions. Owing to its fundamental impact on scientific research, three scientists who pioneered the gene knockout technology were awarded the Nobel Prize in 2007 (Abbott 2007), 20 years after their initial successes. While powerful, it was found that whole-body knockout often led to embryonic lethality due to the requirement for the gene-of-interest at early developmental stages, precluding the analysis of its functions in somatic tissues. To overcome this hurdle, a genetic mosaic analysis technology, known as conditional knockout (CKO), was developed in mice using the Cre/loxP sitedirected recombination system (Gu et al. 1994; Lewandoski 2001). To target a specific gene,

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two loxP sites are inserted in introns flanking critical exon(s), which would still allow normal gene expression in the absence of Cre expression. To achieve gene knockout in a tissue of interest, Cre transgene can be engineered to have restricted expression in target tissue(s) at a desired time. Upon Cre expression, the exon(s) between two loxP sites would be excised, rendering the loss of expression for the gene of interest specifically in target tissue(s) but not other places in the body. By knocking out a gene of interest in a defined cell population at a desirable time, genetic mosaic analysis with Cre/ loxP-based CKO has been widely used to study many biological processes of somatic tissues in mice. While CKO models were used broadly, a few problems arose. First, due to the varied level of Cre expression, gene KO in the target cell population could be incomplete, meaning that some cells would be homozygous null, others heterozygous, while the remaining wildtype (Terry et al. 2020). The intractability of the genotype variations leads to variegated phenotypes that can be difficult to interpret. Second, even when the CKO system works well by inactivating the gene of interest in an entire cell population, it could lead to profound tissue disorganization, especially when cell polarity or adhesion genes are disrupted (Li et al. 2003), precluding precise analysis of the cell-level phenotypes and compounding one’s understanding of genetic regulation of cell autonomous versus non-cell autonomous functions. Finally, significant cell loss due to gene knockout in many cells in a given tissue could lead to compensatory outgrowth that could mask important phenotypes (Wojcinski et al. 2019). Considering the inherent complexity of in vivo studies, these technical issues of CKO greatly hamper our understanding of genetic control of developmental process and disease onset/progression. Therefore, a refined genetic mosaic system is needed to overcome these problems. Ideally, the system needs to be able to knockout genes in rare cells in a target tissue to preserve tissue integrity. Furthermore, mutant cells need to be unequivocally identifiable

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among normal sibling cells and neighboring environmental cells, revealing both cell autonomous gene functions in the mutant cells and non-cell autonomous effects toward neighboring normal cells.

35.2 Engineering Genetic Mosaic in Model Animals The pioneering work on genetic mosaic model using site-directed recombinase was initially done in Drosophila. With the Flp/FRT system, genetic mosaics were created through Flpdependent mitotic recombination, generating colorless mutant cells among colored wildtype cells (Xu and Rubin 1993). While highly successful, identifying a few colorless cells among the sea of colored ones is difficult. Therefore, a Gal4/Gal80 trans-activator/suppressor pair was introduced into the Flp/FRT system to mark mutant cells with GFP among colorless wildtype cells, resulting in a system termed mosaic analysis with a repressible cell marker (MARCM) (Lee and Luo 1999). To establish an equivalent system in mice terms mosaic analysis with double markers (MADM), a pair of reciprocal GFP-RFP chimeric coding sequences separated by a loxP-containing intron was targeted into equivalent locus of homologous chromosomes. From a colorless, heterozygous mother cell, Cremediated mitotic recombination followed by X segregation results in a GFP+ mutant cell and its RFP+ sibling wildtype cell (Fig. 35.1) (Zong et al. 2005). The type of cell labeling can be controlled by cell-specific Cre drivers, and the timing can be controlled by either Tet-controlled Cre expression or tamoxifen-inducible CreER translocation into the nucleus. It should be noted that, even with the high-expressing Cre lines, the labeling in MADM is always sparse, owing to the much lower efficiency of inter-chromosomal recombination than that of intra-chromosomal recombination in CKO models. Definitive correlation between color and genotype allows us to investigate the aberrant behaviors of mutant cells with a few unique advantages: (1) with RFP+ sibling wildtype

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Fig. 35.1  Schematic of the MADM system. a from a colorless, heterozygous mother cell, G2 recombination followed by X segregation generates a GFP-labeled homozygous mutant cell and a RFP-labeled wildtype sibling cell; while G2 recombination followed by Z

segregation generates a colorless and a yellow heterozygous cell. b recombination during G1 or post-mitotic G0 phase results in a yellow heterozygous cell. Reproduced from Zong et al. (2005)

cells as a internal control for GFP+ mutant cells, MADM enables precise assessment of cellular growth defects based on the ratio of greento-red cells (Muzumdar et al. 2007; Liu et al. 2011; Terry et al. 2020); (2) the co-existence of mutant and wildtype cells in the MADM mice

enables the study of cell competition, a critical biological process during development and adult homeostasis, which is generally masked when conventional mouse models are used (Morata and Ripoll 1975; Merino et al. 2016); (3) the immediate and permanent labeling of mutant

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cells with GFP allows one to trace the lineage of mutant cells to discover trans-differentiation in a unequivocal fashion (Yao et al. 2020). By design, MADM can be used to generate mutant cells null of any gene (or genes) located between the MADM-insertion site and the telomere, even a relatively big chromosomal deletion (Fig. 35.1). With the availability of Crispr-based gene mutations, MADM can be used to study many kinds of genetic aberrations in somatic tissues without the need of making floxed alleles of these genes. To enable the analysis of most mouse genes with this technology, through a heroic effort, MADM cassettes have been inserted into all 19 autosomes near the centromere of each chromosome (Contreras et al. 2021). With the availability of this genome-wide collection of MADM mice, 96% of the entire mouse genome now can be subjected to genetic mosaic analysis.

35.3 Gene Functions in Growth Regulation By generating pairs of GFP+ mutant and RFP+ wildtype sibling cells, MADM provides a powerful tool to precisely analyze genetic control of cellular growth. In its first application, MADM was used to study the role of a cell cycle regulator, p27kip1, in regulating the proliferation of cerebellar granule neuron precursors (GNPs), which go through a burst of exponential expansion after birth and promptly exit cell cycle by the weaning age in mice (Muzumdar et al. 2007). While GNPs merely increased by 70% in conventional p27kip1 KO model (Miyazawa et al. 2000), the ratio between GFP+ p27kip1-null and RFP+ wildtype GNPs reached 6, nearly one order of magnitude beyond the whole mouse KO. Two reasons can explain the power of MADM to reveal “subtle” phenotypes: (1) biologically, by generating sporadic mutant cells, MADM enables cell autonomous functional analysis without the compound effect of tissue-level global regulation, such as negative feedback for organ size control that would stunt the expanding potentials of p27kip-null GNPs when every cell is mutant;

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(2) technically, RFP+ wildtype cells provide a perfect internal control that enable the precise calculation of the extent of mutant cell expansion despite the high variability of clonal expansion of individual GNPs (Espinosa and Luo 2008). Mechanically, as a cell cycle regulator, p27kip1 could reduce proliferation by either lengthening G1 or G2 phases or ensuring prompt cell cycle exit. While this problem is extremely difficult to study in vivo, MADM-based spatial analysis and mathematic modeling pointed to the latter as the mechanism (Muzumdar et al. 2007), demonstrating the power of MADM for revealing mechanistic cell biology insights in a physiologically intact setting. Later MADM was applied again to study the genetic control of GNP proliferation by a positive regulator, IGF1R (Terry et al. 2020). It is nearly impossible to study the loss of a positive regulator in GNP proliferation due to two confounding problems: (1) technically, Cre efficiency is not high enough to achieve completely knockout in a timely fashion, leading to significantly masked phenotypes; (2) as an exponentially expanding population, remaining IGF1R-wildtype GNPs can go through compensatory cell divisions to mask the phenotypes of mutant cells even further. MADM circumvents both problems readily because (1) MADM ensures the correlation between labelings and genotypes: GFP+ cells are always mutant cells, and RFP+ cells are always wildtype siblings; (2) the rarity of mutant cells avoids eliciting compensatory growth, which would not be GFP+ even if it occurs. Using MADM, this study firmly established the critical role of IGF1R in GNP proliferation and revealed the potential mechanism through the down-regulation of p27kip1, thereby delaying cell cycle exit. Since the proliferation of GNPs is best known to be regulated by Shh signaling, the finding of IGF1R-p27kip1 axis points to an intricate regulatory network that provides a check-andbalance mechanism to ensure robust regulation of GNP number at the population level despite stochasticity at the clonal level. In addition to studying cell proliferation in normal development, MADM is a powerful tool in analyzing aberrant proliferation that leads to

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tumor formation. Malignant glioma is the most common type of cancers in the central nervous system (Louis et al. 2007; Louis 2006). The unstoppable progression of low-grade tumors and inevitable relapse despite visually complete resection of cancerous tissue make it one of the deadliest cancer types (Louis et al. 2007; Louis 2006). We reasoned that intrinsic regenerative potentials of glioma cell-of-origin could contribute to its malignant nature. However, the cellof-origin of glioma is difficult to identify with human tumor tissues and conventional mouse models because malignant glioma cells have greatly altered morphologies and gene expression profiles (Visvader 2011; Zong et al. 2015). Using MADM, the Zong lab established a glioma model by mutating Trp53 and NF1 (Liu et al. 2011), two key TSGs involved in human gliomas based on TCGA studies (Parsons et al. 2008; Cancer Genome Atlas Research Network 2008). Prior to our study, two progenitor cell types have been proposed to be the cell-of-origin for glioma: While neural stem cell (NSC) is a usual suspect, more restricted progenitors are also potential culprits. We used MADM to resolve this issue based on the following predictions: If NSC is the

cell-of-origin, then green mutant NSCs would outnumber red ones and subsequently lead to increased G/R ratio in all cell lineages; if a progenitor cell type is the cell-of-origin, then the G/R ratio would remain close to 1 in all lineages except for that particular lineage. Data showed that the latter is true, unequivocally pinpointing oligodendrocyte precursor cell (OPC) as the cell-of-origin (Fig. 35.2a) (Liu et al. 2011). Importantly, the massively expanded precancerous field could have been easily missed in a conventional mouse model because the overall OPC density barely changed in mutant brains (top row of Fig. 35.2b). In the MADM model, the premalignant expansion of mutant cells is readily detectable by the GFP-labeling of mutant cells (bottom row of Fig. 35.2b).

Fig. 35.2  MADM reveals OPC as the cell-of-origin for glioma, and the massive expansion of precancerous field a dramatic pre-malignant increase of G/R ratio in OPC but not other lineages suggests that OPC is the cell-of-origin for

glioma b over-expansion of mutant OPCs is barely detectable based on the staining of OPC-specific marker PDGFRα (top row), but is readily revealed by the GFP-labeling of mutant OPCs in the MADM model (bottom row)

35.4 Genes Related to Brain Functions During the development of neocortex, layering of neurons occurs in an “inside-out” fashion, during which deep layer neurons are born first while later-born neurons migrate past them to

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settle in their destined upper laminae (Angevine and Sidman 1961; Rakic 1974). Defects in neuronal migration lead to brain malformation known as lissencephaly (smooth brain), manifesting as the absence of folds in the cerebral cortex, severely disrupted neuronal lamination, and microcephaly (small head). Genetic analysis showed that mutation in the LIS1 gene is a key reason for these defects (Reiner et al. 1993). Patients with a ring chromosome 17 involving a deletion of the LIS1 (PAFAH1B1) gene showed phenotype of Miller-Dieker lissencephaly syndrome (see Chapter 21). Further studies showed that LIS1 and NDEL1 form a protein complex (Niethammer et al. 2000) and that the inactivation of either Lis1 or Ndel1 genes in mice resulted in severe defects in neuronal migration (Sasaki et al. 2005; Shu et al. 2004). Because these defects could be either cell autonomous or non-cell autonomous when these genes are inactivated in all neurons, MADM was used to create Lis1or Ndel1-mutant neurons in a mosaic pattern to deepen our understanding (Hippenmeyer et al. 2010). Immediately, it was found that MADMgenerated LIS1- and NDEL1-mutant phenotypes varied significantly: 1) while Lis1 loss led to significant proliferation defects, Ndel1 loss had no impact on cell divisions at all; 2) while neuronal migration was delayed upon Lis1 loss, they caught up later in development, in stark contrast to permanent migratory defects of Ndel1-mutant cortical neurons. As importantly, while Ndel1-KO in all neurons led to complete loss of neuronal migration, MADMbased analysis, at both population and clonal levels, revealed that Ndel1-mutant neurons can migrate through the ventricular zone (VZ) and intermediate zone (IZ), but are unable to enter the developing cortical plate (CP). Live imaging of organotypic embryonic cortical slice culture further confirmed the normal migration of Ndel1-mutant neurons through IZ and their stop at IZ-CP border. These experiments clearly illustrated the power of MADM in distinguishing cell autonomous from non-cell autonomous gene functions with excellent spatial resolution during brain development.

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In addition to neuronal migration, MADM is also a lineage-tracing tool because the labeling of GFP and RFP is permanent. Comparing to conventional Cre reporter-based lineage-tracing system, MADM is particularly powerful in ruling out alternative interpretations when one discovers trans-differentiation of a cell lineage. In one study, when we used MADM to model medulloblastoma (Yao et al. 2020), a brain tumor originating from cerebellar granule neuron precursors (GNPs) that are known to be unipotent, i.e., only giving rise to granule neurons but never other brain cell types including astrocytes (Zhang and Goldman 1996). Using a GNP-specific Math1-Cre (Matei et al. 2005), we generated a MADM-based medulloblastoma model. Surprisingly, all astrocytes in the tumor mass were GFP+, rather than unlabeled as one would predict based on the unipotency of GNPs (Yao et al. 2020). Knowing that the claim of trans-differentiation is often hampered by technical issues, we examined many alternative interpretations. First, although Math1-Cre faithfully labels GNPs during normal development, could it mis-express in astrocytes within the tumor mass due to aberrant promoter regulation? While this possibility is nearly impossible to be ruled out by conventional lineage-tracing tools, MADM-based analysis refuted it unequivocally because mis-expressing Cre in astrocytes would lead to yellow (both GFP+ and RFP+) astrocytes while all of them are singly GFP+ in MADM medulloblastoma model. Second, the possibility of cell fusion was refuted because these GFP+ astrocytes were never bi-nucleated (Yao et al. 2020). Encouraged by these findings, we collaborated with pathologists to carefully analyze patient samples, using a dual staining scheme: FISH pinpoints tumor and tumor-derived cells based on a stereotypic chromosomal loss; GFAP staining reveals all astrocytes in the tumor mass. Results showed that, while other cells in the tumor mass have normal karyotype, GFAP+ astrocytes shared the same chromosomal loss as tumor cells, revealing their lineage relationship (Yao et al. 2020). It should be noted that the presence of astrocytes in medulloblastoma and their correlation with worse prognosis have

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been known for more than a century, no one ever suspected their origin from trans-differentiated tumor cells until this MADM-based study (Yao et al. 2020).

35.5 Summary While cells in the culture dish mostly behave in an autonomous fashion, cells in living animals not only behave individually but also collectively to ensure tissue integrity. Due to this reason, genetic mosaics often lead to disorganized and malfunction tissues due to sibling cell rivalry, community responses, and compensatory effect, and other intricate cell–cell interactions. Therefore, to understand diseases caused by genetic mosaicism, it would be critical to pinpoint the defective cell type(s) and how their impact on neighboring normal cells leads to these diseases. By generating unequivocally labeled mutant cells along with their wildtype sibling cells labeled with another color, MADM enables phenotypic analysis in vivo at the single-cell resolution. The broad application of MADM in genetic research should lead to fundamentally groundbreaking discoveries on both normal development and aberrations, including but not limited to ring chromosome-related diseases.

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