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Gardner and Sutherland’s Chromosome Abnormalities and Genetic Counseling OXFORD MONOGRAPHS ON MEDICAL GENETICS General Editors: JUDITH G. HALL PETER S. HARPER LOUANNE HUDGKINS EVAN EICHLER CHARLES J EPSTEIN (DECEASED 2011) ARNO G MOTULSKY (RESIGNED 2011) R B McConnell: The genetics of gastrointestinal disorders A C Kopéc: The distribution of the blood groups in the United Kingdom E Slater and V A Cowie: The genetics of mental disorders C O Carter and T J Fairbank: The genetics of locomotor disorders A E Mourant, A C Kopéc, and K Domaniewska-Sobezak: The distribution of the human blood groups and other polymorphisms A E Mourant, A C Kopéc, and K Domaniewska-Sobezak: Blood groups and diseases A G Steinbert and C E Cook: The distribution of the human immunoglobulin allotypes D Tills, A C Kopéc, and R E Tills: The distribution of the human blood groups and other polymorphisms: Supplement I 10 D Z Loesch: Quantitative dermatoglyphics: Classification, genetics, and pathology 11 D J Bond and A C Chandley: Aneuploidy 12 P F Benson and A H Fensom: Genetic biochemical disorders 13 G R Sutherland and F Hecht: Fragile sites on human chromosomes 14 M d’A Crawfurd: The genetics of renal tract disorders 16 C R Scriver and B Child: Garrod’s inborn factors in disease 18 M Baraitser: The genetics of neurological disorders 19 R J Gorlin, M M Cohen, Jr., and L S Levin: Syndromes of the head and neck, third edition 21 D Warburton, J Byrne, and N Canki: Chromosome anomalies and prenatal development: An atlas 22 J J Nora, K Berg, and A H Nora: Cardiovascular disease: Genetics, epidemiology, and prevention 24 A E. H Emery: Duchenne muscular dystrophy, second edition 25 E G. D Tuddenham and D N Cooper: The molecular genetics of haemostasis and its inherited disorders 26 A Boué: Foetal medicine 27 R E Stevenson, J G Hall, and R M Goodman: Human malformations 28 R J Gorlin, H V Toriello, and M M Cohen, Jr.: Hereditary hearing loss and its syndromes 29 R J. M Gardner and G R Sutherland: Chromosomes abnormalities and genetic counseling, second edition 30 A S Teebi and T I Farag: Genetic disorders among Arab populations 31 M M Cohen, Jr.: The child with multiple birth defects 32 W W Weber: Pharmacogenetics 33 V P Sybert: Genetic skin disorders 34 M Baraitser: Genetics of neurological disorders, third edition 35 H Ostrer: Non-Mendelian genetics in humans 36 E Traboulsi: Genetic factors in human disease 37 G L Semenza: Transcription factors and human disease 38 L Pinsky, R P Erickson, and R N Schimke: Genetic disorders of human sexual development 39 R E Stevenson, C E Schwartz, and R J Schroer: X-linked mental retardation 40 M J Khoury, W Burke, and E Thomson: Genetics and public health in the 21st century 41 J Weil: Psychosocial genetic counseling 42 R J Gorlin, M M Cohen, Jr., and R C. M Hennekam: Syndromes of the head and neck, fourth edition 43 M M Cohen, Jr., G Neri, and R Weksberg: Overgrowth syndromes 44 R A King, J I Rotter, and A G Motulsky: Genetic basis of common diseases, second edition 45 G P Bates, P S Harper, and L Jones: Huntington’s disease, third edition 46 R J. M Gardner and G R Sutherland: Chromosome abnormalities and genetic counseling, third edition 47 I J Holt: Genetics of mitochondrial disease 48 F Flinter, E Maher, and A Saggar-Malik: Genetics of renal disease 49 C J Epstein, R P Erickson, and A Wynshaw-Boris: Inborn errors of development: The molecular basis of clinical disorders of morphogenesis 50 H V Toriello, W Reardon, and R J Gorlin: Hereditary hearing loss and its syndromes, second edition 51 P S Harper: Landmarks in medical genetics 52 R E Stevenson and J G Hall: Human malformations and related anomalies, second edition 53 D Kumar and S D Weatherall: Genomics and clinical medicine 54 C J Epstein, R P Erickson, and A Wynshaw-Boris: Inborn errors of development: The molecular basis of clinical disorders of morphogenesis, second edition 55 W Weber: Pharmacogenetics, second edition 56 P L Beales, I S Farooqi, and S O’Rahilly: The genetics of obesity syndromes 57 P S Harper: A short history of medical genetics 58 R C. M Hennekam, I D Krantz, and J E Allanson: Gorlin’s syndromes of the head and neck, fifth edition 59 D Kumar and P Elliot: Principles and practices of cardiovascular genetics 60 V P Sybert: Genetic skin disorders, second edition 61 R J. M Gardner, G R Sutherland, and L C Shaffer: Chromosome abnormalities and genetic counseling, fourth edition 62 D Kumar: Genomics and health in the developing world 63 G Bates, S Tabrizi, and L Jones: Huntington’s disease, fourth edition 64 B Lee and F Scaglia: Inborn errors of metabolism: From neonatal screening to metabolic pathways 65 D Kumar and C Eng: Genomic medicine, second edition 66 R Stevenson, J Hall, D Everman, and B Solomon: Human malformations and related anomalies, third edition 67 R Erickson and A Wynshaw-Boris: Epstein’s inborn errors of development: The molecular basis of clinical disorders of morphogenesis, third edition 68 C Hollak and R Lachmann: Inherited metabolic disease in adults: A clinical guide 69 V P Sybert: Genetic skin disorders, third edition 70 R J. M Gardner and D J Amor: Gardner and Sutherland’s chromosome abnormalities and genetic counseling, fifth edition GARDNER AND SUTHERLAND’S Chromosome Abnormalities and Genetic Counseling FIF TH E D ITION R J McKinlay GARDNER ADJUNCT PROFESSOR CLINICAL GENETICS GROUP UNIVERSITY OF OTAGO, DUNEDIN, NEW ZEALAND David J. AMOR LORENZO AND PAMELA GALLI CHAIR UNIVERSITY OF MELBOURNE VICTORIAN CLINICAL GENETICS SERVICES MURDOCH CHILDREN’S RESEARCH INSTITUTE ROYAL CHILDREN’S HOSPITAL, MELBOURNE, AUSTRALIA 1 Oxford University Press is a department of the University of Oxford It furthers the University’s objective of excellence in research, scholarship, and education by publishing worldwide Oxford is a registered trade mark of Oxford University Press in the UK and certain other countries Published in the United States of America by Oxford University Press 198 Madison Avenue, New York, NY 10016, United States of America © Oxford University Press 2018 All rights reserved No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, by license, or under terms agreed with the appropriate reproduction rights organization Inquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above You must not circulate this work in any other form and you must impose this same condition on any acquirer Library of Congress Cataloging-in-Publication Data Names: Gardner, R J M., author | Amor, David J., author Title: Gardner and Sutherland’s chromosome abnormalities and genetic counseling / R J McKinlay Gardner, David J Amor Other titles: Chromosome abnormalities and genetic counseling | Oxford monographs on medical genetics ; no 70 Description: Fifth edition | Oxford ; New York : Oxford University Press, [2018] | Series: Oxford monographs on medical genetics ; no 70 | Preceded by Chromosome abnormalities and genetic counseling / R.J McKinlay Gardner, Grant R Sutherland, Lisa G Shaffer c2012 | Includes bibliographical references and index Identifiers: LCCN 2017034126 | ISBN 9780199329007 (hardcover : alk paper) Subjects: | MESH: Chromosome Aberrations | Genetic Counseling Classification: LCC RB155.7 | NLM QS 677 | DDC 616/.042—dc23 LC record available at https://lccn.loc.gov/2017034126 This material is not intended to be, and should not be considered, a substitute for medical or other professional advice Treatment for the conditions described in this material is highly dependent on the individual circumstances And, while this material is designed to offer accurate information with respect to the subject matter covered and to be current as of the time it was written, research and knowledge about medical and health issues is constantly evolving and dose schedules for medications are being revised continually, with new side effects recognized and accounted for regularly Readers must therefore always check the product information and clinical procedures with the most up-to-date published product information and data sheets provided by the manufacturers and the most recent codes of conduct and safety regulation The publisher and the authors make no representations or warranties to readers, express or implied, as to the accuracy or completeness of this material Without limiting the foregoing, the publisher and the authors make no representations or warranties as to the accuracy or efficacy of the drug dosages mentioned in the material The authors and the publisher not accept, and expressly disclaim, any responsibility for any liability, loss or risk that may be claimed or incurred as a consequence of the use and/or application of any of the contents of this material 9 8 7 6 5 4 3 2 1 Printed by Edwards Brothers Malloy, United States of America This book is dedicated to Jocelyn, Geoffrey, and Craig, their parents, and all other families who seek our “chromosomal advice.” Jocelyn and Geoffrey (with lamb) have a partial trisomy for chromosome long arm, and Craig, the youngest, had a 46,XY result on amniocentesis Their father is a translocation carrier (see Fig. 5–1, lower) Craig, since married, came to the genetic clinic for confirmatory advice about his low genetic risk Heredity Inescapably, this is me—the diagnosis is cause for anger at those who brightly say we choose our destinies There is no store of courage, wit or will can save me from myself and I must face my children, feeling like that wicked fairy, uninvited at the christening, bestowing on my own, amidst murmurs of apprehension, a most unwanted gift—that of a blighted mind. No one could tell me of this curse when I was young and dreamt of children and the graces they would bear. Later, it seemed that a chill morning revealed deeper layers of truth For my romancing there is a price to pay— perhaps my children’s children will pass this tollgate after me My grandmothers gaze down from their frames on my wall, sadly wondering —Meg Campbell Dear DNA In real life you’re just a tangle of white filaments captured in a test-tube, and your first photo is not flattering: grey smudges like tractor tracks, or a rusty screw. Yet many say you are beautiful Online for a night with a hundred fantastic portraits and I’m head over heels In love with you, DNA, bewitched by your billions coiled in my cells, transcribing, replicating, mutating I see your never-ending dance A length of twisted ladder briefly unwinds, both strands duplicate, each copy drifts away on its secret mission to make a thought, feel sunshine, or digest this morning’s porridge Two winding parallel threads, a tiny tangle of gossamer designing my life DNA, you are astonishing and I am yours truly —Winifred Kavalieris Genes pass on our kind But our selves are transmitted In words left behind —J Patrick Gookin Curiosity is a virtue, perhaps an unsung and undervalued virtue, which should be the energizing fuel to the thinking geneticist —Willie Reardon Where is the wisdom we have lost in knowledge? Where is the knowledge we have lost in information? —T S. Eliot PREFACE TO THE FIFTH EDITION Chromosomal disorders have been, and will always be, with us; that is a given What is changing is our ability to recognize and detect them: detection both in terms of the subtlety of abnormalities and of the means we can use to find them Classical cytogenetics has now well and truly given way to “molecular karyotyping,” and this has been the extraordinary development of the early twenty- first century Readers will now be as accustomed to molecular nomenclature in defining a segment, such as chr5:1-18,500,000, as they had been to the classical description, 5p14.1→pter The very small deletions and duplications which molecular karyotyping can now reveal have become familiar to the clinicians and counselors who see patients and families in the clinic A large number of these are now on record, many attracting the nomenclature “copy number variant”: Some are very well understood, others becoming so, and yet quite a few—variants of uncertain significance, the acronym “VOUS” in daily parlance—whose roles in human pathology are imperfectly appreciated Many are not in the same mold as the deletions and duplications of classical cytogenetics, in which the single defect sufficed to cause a particular phenotype, and always did so: We now need to take account of the concept of incomplete penetrance, with some microdeletions or duplications not, of themselves, always leading to an abnormal phenotype Apparently clinically normal parents may carry the same alteration as their child with an abnormal phenotype Digenic, or “two-hit,” mechanisms may now require consideration These were not formerly notions much entering into the assessment of chromosomal disorders; discussion apropos in the clinic presents a new challenge The number of “new” del/ dup syndromes increases almost with each issue of the clinical genetic journals We include a mention of a considerable number of these here (Chapter 14), not intending to create an encyclopedic resource per se but believing that such a record may provide a useful first point of contact when these cases are encountered in the clinic Copy number variants • vii of uncertain significance, on the other hand, we mostly take only a broad rather than a detailed view (Chapter 17); the reader will need to consult other repositories for fuller information, as their interpretations evolve The new (or now, established) laboratory methodologies blur the boundaries between what might have been regarded as the classic chromosomal abnormalities and Mendelian conditions Some disorders recorded as being due not only to segmental deletion/duplication affecting a single locus but also to point mutation at that locus we continue to treat as “chromosomal”; and for most, their place in this book is secure But one major category, the fragile X syndromes, are now seen as essentially Mendelian disorders, their historic cytogenetic-based nomenclature notwithstanding, and they no longer claim their chapter Peripheral blood and skin have been the tissues in common usage for chromosome analysis, with an increasing role for cells got from the convenient and painless “spit sample.” Prenatal diagnosis has been based on amniocentesis and chorionic villus sampling, but latterly blastomeres from early embryos, and fetal DNA in the maternal circulation, have become targets for testing Now we can anticipate the potential for whole genome analysis to be applied to the prenatal diagnosis of the classic aneuploidies, from a simple maternal blood viii • P R E F A C E T O T H E F I F T H E D I T I O N sample, and this would widen such testing very considerably Questions such as these raise ethical issues, and a literature on “chromosomal ethics” is accumulating As we have previously written, however marvelous may be these new ways to test for chromosomes, the concerns of families remain essentially the same We may reproduce here the final paragraph of the Preface of the first edition of this book, from 1989, as valid now as then: Families pursue genetic counseling in an effort to demystify the mysterious If they did not want to “hear it all,” they would not bother with genetic counseling Families want an honest evaluation of what is known and what is unknown, a clear explanation of all possibilities, both good and bad, and a sensitive exploration of all available information with which they can make knowledgeable decisions about future family planning Thus, Bloch et al (1979) succinctly convey the essence of why people go to the genetic counselor We hope this book will assist counselors in their task DunedinR.J.M.G MelbourneD.J.A February 2018 ACKNOWLEDGMENTS We thank John Barber, Rachel Beddow, Amber Boys, Cyril Chapman, Jane Halliday, Jan Hodgson, Caroline Lintott, Nicole Martin, Belinda McLaren, Fiona Norris, Mamoru Ozaki, Mark Pertile, Jenny Rhodes, Sharyn Stock-Myer, and Jane Watt for their critical advice We acknowledge Lisa Shaffer, who was a co-author of the previous edition, and much of whose work has flowed over into this edition We have made much use of the ideograms created by Nicole Chia The length of the Reference list, and the frequency with which we acknowledge, in legends to figures, the courtesy of colleagues whose work we use, speaks for the debt we owe to our colleagues in clinical cytogenetics worldwide Belatedly, R.J.M.G thanks Ngaire Adams and Dianne Grimaldi, whose need for chromosomal teaching at Dunedin Hospital in the 1980s provided the germination for writing this book We have appreciated the wise guidance, and the patience and forbearance of Oxford University Press, from Jeff House when this book made its first appearance, through to Chad Zimmerman and Chloe Layman in this fifth edition R.J.M.G thanks his wife Kelley for her patient help, once again, in document management; and the front cover art, and most of the new illustrations in this edition, have been drawn, or redrawn, by her • ix simple Mendelian, 50/50 (transmission naturally being gender-specific for X-linked variants) More so with CNVs, de novo generation is not uncommon The particular difficulty lies in the occasional penetrance of some (usually) nonpathogenic CNVs of genes coding for ribosomal RNA; because the nucleolus of the cell is formed by an aggregation of rRNA, this region is also called the nucleolar organizing region (NOR) Acrocentric short arm variation appears to be without any phenotypic effect CLASSICAL CYTOGENETICS B A N D I N G PATT E R N : E U C H R O M AT I N Microscopists from the era of classical cytogenetics became very familiar with the appearances of chromosomes and learned readily to distinguish normal structural variation The counselor of the twenty- first century may yet need to refer to historic literature and should have at least some familiarity with these classical concepts Homologs could differ in the respects discussed next Most of a chromosome consists of euchromatin, which contains the active genetic material, resident in greater amount in G-light bands (pale- staining on Giemsa banding) than in G- dark bands The light microscope cannot reliably enable detection of alterations of less than 3–5 Mb, and most deletions and duplications of more than this size can be presumed to have phenotypic consequences Exceptions to this rule include, first, euchromatic variants that involve common B A N D I N G PAT T E R N : copy-number variable regions that become visH E T E R O C H R O M AT I N ible when copy number is high enough, or when Heterochromatin is made up of highly repetitive the size of the copy-number variable tract is large DNA that has been distinguishable from euchro- enough Second, there are chromosomal segments matin for the larger part of a century (Heitz 1928).1 whose deletion or duplication has no phenotypic Heterochromatic variants are best seen on C- consequence banding, which specifically stains the extensive tracts of heterochromatin adjacent to the centroE U C H R O M AT I C VA R I A N TS meres of each chromosome (hence, the C), substantially comprising alpha-satellite DNA consisting of Euchromatic variants (EVs) due to copy-number hundreds of thousands of copies of a 171 base pair variable tracts (Table 17–1) can be considered, in repeat Certain chromosomes show quite marked a sense, as extreme forms of CNVs, either because differences in their C-band pattern, particularly chro- their copy number is at the high end or higher than mosomes 1, 9, 16, and the Y, and the large blocks of the normal range or because their size is greater than heterochromatin thus stained are labeled 1qh, 9qh, Mb (at which point they are excluded from the 16qh, and Yqh.2 They are of no phenotypic effect.3 Database of Genomic Variants; see below) Thus, EVs and the molecular CNVs (below) essentially form a continuum, with no fundamental genetic A C R O C E N T R I C S H O RT A R M S distinction For example, Tyson et al (2014) anaThe short arms of the acrocentric chromosomes lyzed the REXO1L1 gene and pseudogene clus(13, 14, 15, 21, and 22) can vary quite considerably ter which resides within a 12 kb tandem repeat in in their lengths Indeed, some p arms are apparently band 8q21.2, and of which the diploid copy number completely absent, and others are several times the ranges from approximately 100 to 200 This repeat typical length This reflects variation in the three may account for almost half of band 8q21.2 and, at components of the short arm: the centromeric the upper end of this range, additional G-light mateheterochromatin, the satellite stalk, and the satel- rial is discernible (Fig 17–1) Albeit that D’Apice lite material, identified as bands p11, p12, and p13, et al (2015) proposed that deletion of this segment respectively Band p12 contains multiple copies (but with several copies yet remaining) could be 1 The seminal contributions of Emil Heitz to the science of cytogenetics are reviewed in Passarge (1979) 2 Variation in the size of Yqh in an extended Canadian kindred could inferentially be traced back over three centuries, allowing Genest (1973, 1981) to claim that it was “the oldest known chromosome aberration.” 3 This has been the prevailing, if not universal view, for quite some time Reproduction may, however, be a vulnerable sphere; and Tempest and Simpson (2017) review the reported associations with infertility and unfavourable reproductive outcomes 370 • C h r omosom e Va r ian t s FIGURE 17–1 The likely benign euchromatic variant at 8q21.3, which reflects copy number variation of the REXO1L1 gene and pseudogene cluster This observation could be viewed, in a sense, as an intermediary between classical and molecular cytogenetic variation Source: From Tyson et al., Expansion of a 12-kb VNTR containing the REXO1L1 gene cluster underlies the microscopically visible euchromatic variant of 8q21.2, Eur J Hum Genet 22: 458–463 2014 Courtesy J. C K. Barber and C. Tyson, and with the permission of Nature Publishing Group Table 17–1. Euchromatic Variants due to Copy-Number Variable Tracts EUCHROMATIC VARIANT (EV) REPEAT/SEGMENT SIZE CONTROL COPY NUMBER EV COPY NUMBER dup 8p23.2 amp 8p23.1 amp 8q21.2 amp 9p12 del/dup 9p11.2p13.1 dup/trp/ins 9q12 del/dup/trp/amp 9q13q21.1 amp 15q11.2 2.5 Mb >260 kb 12 kb ~1 Mb ~5 Mb ~5 Mb ~5 Mb ~1 Mb 2-9 97–277 1–3 4 IGVH 1–3; NF1 1–4 amp 16p11.2 692–945 kb 3–8 8–12 265–270 7–12 3–5 5–6 3–8 IGVH 4–9; NF1 5–10 8–10 Abbreviations: amp, amplification; dup, duplication; EV, euchromatic variant; ins, insertion; IGVH, immunoglobulin variable heavy chain; NF1, neurofibromatosis 1; trp, triplication Source: From Tyson et al (2014) the basis of a new microdeletion syndrome, Barber et al (2016) argue that, more likely, it may typically be a benign EV The same interpretation of innocuousness likely applies to the other EVs listed in Table 17–1 Some of these EVs may have been confused, on classical karyotyping, with pathogenic imbalances On microarray analysis, however, the distinction should be clear; and in fact many microarrays exclude the repetitive regions that EVs involve I M B A L A N CES O F C H R O M OS O M A L SEG M E N TS W I T H N O A P PA R E N T P H E N OTY P I C E F F EC T In a review in 2005, Barber found only 23 examples of families with directly transmitted autosomal segmental imbalance in which two or more carriers were unaffected, and a few have since been published (Table 17–2) These cases were often ascertained for incidental reasons, such as prenatal diagnosis for maternal age The gene content is often lower than Table 17–2. Euchromatic Duplications and Deletions (and One Quadruplication) Detectable by Microscope Cytogenetics, and Without Phenotypic Effect, as Inferred from the Observation of Transmission from Phenotypically Normal Parent to Normal Child CHROMOSOME DEL 10 11 12 13 p12-p12 (x2) q13-q14.1 p25.3-pter (×2) q34.1-q34.3 p14.1-p14.3 (×2) q22.31-q23.1 p23.1/2-pter q24.13-q24.22 p21.2-p22.1 q11.2-q21.2 p12-p12 q14.3-q22.1 16 18 q14.3-q21.33 q21-q21 q21.1-q21.31 q21.1-q21.33 q13q22 (×4) p11.31-pter 22 q11.21-pter DUP QDP p21-p31 q31.1-q32 q28-q29 p16.1-p16.1 q12q13.1 p22.3-pter (×2) p22-p22 p23.1-p23.3 p12-p21.3 p11.1-q11.22 p13-p14 q21.31-q22 q13-q14.3 q14-q21 p11.2-pter q11.2-q12.2 Notes: The estimated sizes of the deletions and duplications range from 4.2 to 16.0 Mb (del) and from 3.4 to 31.3 Mb (dup) The numbers of studied families, where more than one, are shown in parentheses Source: From Barber (2005), and the Chromosome Anomaly Collection website at http://www ngrl.org.uk/wessex/collection (updated information is posted in the “What’s New” section) Additional material due to Chen et al (2011b), Coussement et al (2011), Kowalczyk et al (2013), and Liehr et al (2009b) 372 • C h r omosom e Va r ian t s the genome average, and the lack of phenotype is attributed to the absence of dosage-sensitive genes, or to dosage compensation by related genes Similar imbalances with no phenotypic consequence are recorded in more than one family for the gene- poor G-dark bands 2p12, 5p14, 13q21, and 16q21 Most of the families listed in Table 17–2 remain as isolated examples and may yet turn out to reflect segmental incomplete penetrance This may apply, for example, to the distal 3p cases, as other families with similar deletions are more often phenotypically affected This question of penetrance, in these few cases of cytogenetically visible imbalances, is somewhat of a harbinger of the immense challenge that came to be presented by the flood of CNVs of twenty-first century molecular analysis, as we discuss at length below Inversions. We mention normal variant inversions seen in certain chromosomes (1, 2, 3, 5, 9, 10, 16, and Y) in Chapter 9 Fragile Sites. Under certain stressed culturing conditions, some, indeed most, chromosomes show apparent rupture in one or, less commonly, both chromatids (Sutherland and Hecht 1985; Sutherland and Baker 2000; Arlt et al 2003; Sutherland 2003) This is almost always without phenotypic implication The spectacular exception is the fragile site FRAXA at Xq27, and indeed this laboratory observation lent its name to the well-known fragile X syndrome, originally referred to as a “marker” X (Lubs 1969) The fragile site observed by the microscopist reflected the trinucleotide expansion within the FMR1 gene Three other sites in the same region are FRAXB, FRAXD, and FRAXE, of which only the latter is pathogenic Otherwise, only two fragile sites may be of clinical import FRA11B, at 11q23.3, is possibly the basis of some (not all) Jacobsen syndrome 11q deletions (Michaelis et al 1998; and see p 288) A single case of a man with 46,XY,fra(16)(q22.1), the fragile site classed FRA16B/C, in whom 1% of sperm and two out of 10 PGD embryos showed chromosome 16 imbalance, is to be noted (Martorell et al 2014) We mention the fragile site FRA10A at 10q23, which may or may not be relevant at prenatal diagnosis, on p 495 COPY NUMBER VARIANTS The molecular lens, when it began to be applied from the late twentieth century, came up with a somewhat surprising observation: Short genomic segments could exist in deleted or duplicated state, invisible on routine classical cytogenetics, among individuals in the general, normal population They are certainly common, indeed universal: each of us has, on average, 1,000 CNVs of >450 bp, compared to a reference genome (Conrad et al 2010) The word “variant” can allow, as noted above, for agnosticism in terms of pathogenicity; adjectives and adjectival qualifiers can be added, accordingly as the interpretation unfolds, and a descriptive classification conferred (Figure 17–2): Pathogenic Likely pathogenic Uncertain significance (VOUS) Likely benign Benign FIGURE 17–2 CNV Gradations What is the actual basis of the variation? A short segment of chromatin—a “copy”—would normally exist on a chromosome in single state, and thus with one copy on each autosome (and one on each or one X chromosome, according to gender) Variation lies in the presence of these copies in absent or double (sometimes triple or quadruple or higher) states on a chromosome, and hence the expression copy number variant The copy size can vary from less than kb to approximately Mb.4 Some segments are in “gene deserts”; others contain known genes If there is an observation that no untoward effect exists in the presence of a single, triple, or higher multiple amount of these genes, this then allows the inference that these genes are not dosage-sensitive The difficulty lies in determining that a CNV is, indeed, a normal variant and of no phenotypic import (Hehir-Kwa et al 2013) The harmlessness 4 The lower limit of size may be taken as kb (a clinical viewpoint), or to as low as 50 bp (as seen by a molecular scientist) (Martin and Warburton 2015; Zarrei et al 2015) Elements below 50 kb are known as insertions or deletions, or “indels.” An upper limit of Mb is proposed, although many cases in the literature involving segments of up to a few megabases have been called CNVs The DGV database uses an arbitrary, somewhat higher cut-off of Mb; this could be seen as a pragmatic border between the euchromatic segmental variants described above and the CNVs as discussed here Normal Chromosomal Variation • 373 of many CNVs is attested by their segregation within a family, in which only the proband (through whom the CNV was ascertained) may have been of abnormal phenotype In these, the CNV can usually be taken as benign/ nonpathogenic, and its discovery merely coincidental The finding of a de novo change may more reasonably be considered as likely causative; but the gene content of this genomic segment should be considered in the context of the patient’s phenotype, and not losing sight of the fact that de novo CNVs are not uncommon in healthy individuals Interpretation may need to account for ethnicity: The frequencies of some CNVs vary depending on the background of the individuals tested Had it been possible to make a clear distinction between all “CNVs” consistently harmless, and all those consistently pathogenic, the discussions in this chapter and in the chapters on autosomal and sex chromosomal microdeletions and microduplications (Chapters 14 and 15) could have been quite self-contained But that is not the case—at least as at the present writing The five-part classification is not necessarily as clear-cut as the grayscale above might imply With what confidence can a CNV be called indeed pathogenic, or benign? The bar is high: Only those “practically certain” to be so, can be called so How likely is “likely”? In the similar setting of Mendelian variants, an expert group5 views 90% as a suitable cut-off (Richards et al 2015) That leaves another 80% or so in the “variant of uncertain significance” (VOUS) territory Every counselor can expect to encounter, and to deal with, VOUSs The genic content of a CNV would seem, intuitively, to be a key—possibly the key—factor determining pathogenicity, or not This “common-sense” viewpoint is given formal support in Rice and McLysaght (2017), who determined that a pathogenic CNV is more likely to contain a gene or genes that are dosage-sensitive, that have a role in embryonic development, or that are evolutionarily conserved “Ohnologs” (footnote p 264) are especially represented in pathogenic CNVs Applying this understanding may, in due course, be helpful in allowing a more precise interpretation of which CNVs are of clinical significance We mentioned in Chapter 14 the concept of a deletion “unmasking heterozygosity” of a recessive allele coincidentally on the other chromosome The similar scenario may obtain with an otherwise benign deletion CNV, if a locus therein happens to code for a Mendelian recessive disease Thus, Liu, Li, et al (2016) diagnosed autosomal recessive spastic ataxia of Charlevoix and Saguenay (ARSACS) in a patient with a SACS mutation at 13q12.12 on one chromosome, and a 1.33 Mb CNV deletion encompassing the SACS locus on the other We have seen a very similar case, in a woman with ataxia and a Charcot-Marie-Tooth-like neuropathy inheriting a (normally nonpathogenic) paternal 0.2 Mb CNV deletion which removed SACS, and an accompanying maternal SACS mutation on the other homolog, and thus enabling a diagnosis of ARSACS DATA B A SE S The counselor dealing with a family in which a CNV has been shown, has formidable resources to which to appeal Collaborative efforts from around the world bring together data, and repositories are assembled to which enquiry may be made An important resource is DECIPHER, the Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources This database lists known or possibly pathogenic variants and also VOUSs The Internet link is http://decipher.sanger.ac.uk A panel displays CNVs that either match with or overlap with a segment of interest Duplications are shown in blue, and deletions are shown in red The distinction between pathogenic variants and VOUSs is indicated by the differing color intensity (the darker, the more likely to be pathogenic) The user will note that many cases show DDD as the data source: This is the database Deciphering Developmental Disorders, and it is accessible at http://w ww.ddduk.org A complementary resource is ClinGen, a “National Institutes of Health-funded resource dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for use in precision medicine and research.” Each listed variant has a thumbnail sketch of the 5 American College of Medical Genetics and Genomics and the Association for Molecular Pathology 374 • C h r omosom e Va r ian t s clinical history alongside; the link is http://www clinicalgenome.org.6 A more encyclopedic collection, including the smallest normal variants, is that due to DGVa—the Database of Genomic Variants (version a)—which is curated at The Center for Applied Genomics at the Hospital for Sick Children, Toronto, Ontario, Canada The Center records “genomic alterations that involve segments of DNA that are larger than 50 bp The content of the database is only representing structural variation identified in healthy control samples.” The data derive from upwards of 14,000 individuals, carrying more than 77,000 deletions and 660 duplications (MacDonald et al 2014; Zarrei et al 2015) The database is accessed directly at http://dgv.tcag.ca/dgv/app/home Continuing refining of the data leads to increasing accuracy and confidence, and a special track within the DGVa lists “gold standard structural variants” (GSSVs) The UCSC (University of California, Santa Cruz) genome browser site at http://genome ucsc.edu is another useful resource The site simultaneously displays information from DECIPHER, the Copy Number Variation Morbidity Map of Developmental Delay, OMIM, RefSeq genes, and GeneReviews The data from the laboratory need to be clearly conveyed to those for whom the report is intended, which will very often be the genetic counselor A pictorial display, with accompanying written detail, is a style of document with which the twenty- first century counselor is becoming well familiar, such as that produced by the Genoglyphix database (see Fig 17–6 below) Copy Number Variants and the Brain. The most complex organ, the brain, is the most susceptible to CNV imbalance, and typically presenting as cognitive/behavioral dysfunction; indeed, in most CNV imbalances, there is no observable physical phenotypic abnormality In a large study of children with intellectual deficiency/ developmental delay, an excess of those with a CNV compared to controls emerged significantly at a CNV size of 400 kb, and became more evident at 1.5 Mb (Cooper et al 2011) At least some of these CNVs, therefore, would have been pathogenic Again unsurprisingly, larger (>1.5 Mb) CNVs were overrepresented in de novo cases; presumably this reflects a reduced reproductive fitness of those with larger, and pathogenic, CNVs Similar conclusions are reached in Coe et al (2014) McCormack et al (2016) recorded frequencies of benign versus pathogenic CNVs in an abnormal population (Fig 17– 3) A subtler study was performed in Estonia (Männik et al 2015); in this study, the CNV status of a large population was shown to correlate with educational attainment (Table 17–3) These subjects had been selected due to attendance at a general medical practice, and could be considered as quite close to a true random population sampling Of the CNVs analyzed in these subjects, only smaller (0.25–1 Mb) duplications appeared to be consistently benign A detailed review of CNVs associated with a neurodevelopmental disorder is presented in Torres et al (2016): These authors list, in particular, CNVs at 1q21.1, 3q29, 15q11.2, 15q13.3, 16p11.2, 16p13.1, and 22q11 Autism spectrum disorder (ASD) is a clinical diagnosis for which molecular karyotyping is the first genetic investigation.7 The counselor may deal rather frequently with the challenge of interpreting a finding of a CNV, or of CNVs, and which may be de novo or inherited In a segment for which a causal link is well established, such as the del(16)(p11.2) (p. 296), the expression microdeletion/duplication may be more apposite, and counseling may be (relatively) straightforward For less well-understood segments, and especially when in combination, our understanding is a work in progress Certain regions, with spectacular contributions of chromosomes 15, 16, and 22, harbor ASD-related CNVs (Fig. 17–4) Developmental coordination disorder, also called dyspraxia or the “clumsy child syndrome,” is not uncommonly diagnosed in school-age children, and quite often in coexistence with attention deficit disorder (Gibbs et al 2007) In a relatively small Canadian cohort of such children, the burden of CNV deletions or duplications in the 0.5–1.0 Mb range was significantly increased, and CNVs more often spanned brain- expressed genes, compared with a control population (Mosca et al. 2016) 6 This database also provides a list of loci according to their dosage sensitivity, at https://www.ncbi.nlm.nih.gov/projects/dbvar/ clingen (Hunter et al. 2016) 7 It is necessary to distinguish “idiopathic autism” from neurogenetic syndromes which may include, in some, autistic-like features (e.g., Rett syndrome, fragile X syndrome, tuberous sclerosis) Harris (2016) offers a useful commentary; he refers to CNVs in idiopathic ASD as “common variation, individually of small effect, [which] may have substantial impact en masse.” Normal Chromosomal Variation • 375 250 200 150 Total CNVs Pathogenic CNVs 100 50 10 11 12 13 14 15 16 17 18 19 20 21 20 X Y FIGURE 17–3 Frequencies of pathogenic CNVs, compared to total CNV frequencies, in an abnormal population These data derive from a series of 5,369 postnatal (single or multiple congenital abnormalities, neurodevelopmental delay with or without neuropsychiatric disorders) and prenatal (two or more abnormalities detected on ultrasound) samples Source: From McCormack et al., Microarray testing in clinical diagnosis: An analysis of 5,300 New Zealand patients, Mol Cytogenet 9: 29, 2016 Courtesy D. R Love and A. M George, and with the permission of BioMed Central, per the Creative Commons Attribution License Table 17–3. Educational Attainment in Three Estonian Cohorts, with Respect to Copy Number Variant Carriage GROUP TOTALS EDUCATIONAL NOT REACHING SECONDARY EDUCATION ATTAINMENT* NO Estonian population DECIPHER-listed CNV carriers Deletion carrier by CNV size >1 Mb 500 kb–1 Mb 250–500 kb Duplication carrier by CNV size >1 Mb 500 kb–1 Mb 250–500 kb 7,877 56 4.08 3.64 2,000 28 % 25 50 37 47 164 3.51 3.93** 3.84 17 16 50 46 34.0 30.5 115 149 319 3.69 4.10 4.14 45 43 78 39.1 28.9 24.5 Notes: In the general population, the average attainment score is 4.08 In those with DECIPHER-listed CNVs, it is less, at 3.64 The averages in those with other deletion CNVs is also less, ranging from 3.51 to 3.93 Likewise, the score is less in the larger duplication category (>1 Mb), but in those with smaller (0.25–1 Mb) duplications, it is essentially the same as that for the general population These average figures match those of the fractions of those not reaching secondary education *The mean educational attainment score is derived from these levels, based on the Estonian education curriculum: less than primary, 1; primary, 2; basic, 3; secondary, 4; professional or college, 5; university or academic, 6; and scientific degree, 7 **This fraction, although slightly less than that of the general population, does not reach statistical significance Source: From Männik et al (2015) FIGURE 17–4 Chromosomal locations of the top-ranked 11 autism spectrum susceptibility CNV loci Copy number gains (duplications) are open bars; copy number losses and gains (both duplications and deletions) are filled bars The length and width of bars are proportional to the CNVs’ genomic size and burden score, respectively; note the disproportionate roles of chromosomes 15 and 22 in particular, and also of chromosomes and 16 Source: From Menashe et al., Prioritization of copy number variation loci associated with autism from AutDB—An integrative multi-study genetic database, PLoS One 8; 8: e66707, 2013 Courtesy I. Menashe, and with the permission of the Public Library of Science, according to the Creative Commons Attribution License The X chromosome is rich in CNVs Isrie et al (2012) studied a cohort of 2,222 males with intellectual disability and found 3% to have an X-borne CNV Some could quite confidently be termed as pathogenic; in others, the interpretation was unclear These authors developed a decision tree, whereby a CNV could be “called.” Those interpreted as pathogenic ranged in size8 from 0.5 kb to 4.4 Mb; those regarded as VOUSs were of a rather similar range, kb to 4.3 Mb, but the distribution skewed toward smaller sizes An inference is, therefore, that many of the smaller ones would have been nonpathogenic CNVs Family studies can cast light, as we exemplify in the family with a trp(X)(q27.1) mentioned below CNVs comprising a duplication of a specific segment within Xp22.33 which includes, but may extend beyond, the SHOX locus convey a low-penetrance risk for autism (3.6%) or other neurodevelopmental disorder (8.6%) (Tropeano et al. 2016) P E N T R A N CE A N D E X P R ESS I V I TY The concepts of variable penetrance and expressivity,9 more traditionally invoked in Mendelian genetics, impose a real concern with respect to the CNV (Grayton et al 2012) A CNV may be, in one genomic environment (e.g., in a parent), of no clinical effect, but it may be pathogenic in a child, if a different CNV—a “second hit”—comes from the other parent Subtle examples come from studies in autism (Coe et al 2014) Or, a microduplication or microdeletion of recognized incomplete penetrance may become penetrant in the company of a CNV (Fig 17–5) The concept of “digenic inheritance” may, in some, understate the genetic complexity: Oligogenic, or even polygenic, mechanisms may be the basis of some CNV combinations determining a boundary beyond which phenotypic abnormality appears Or, to use the common terminology, a two-hit or more-hit scenario may apply The other issue to add into this mix is the matter of defining a boundary of abnormality—which can be a 8 One outlier of size 11 Mb, a triplication at Xq27 encompassing 48 loci, including FMR1, and visible on karyotyping, might be seen as a microtriplication rather than a CNV 9 Penetrance refers to the proportion of individuals with an imbalance that shows any trait resulting from that imbalance, whereas expressivity refers to the variability in phenotype of those who carry the imbalanced region Normal Chromosomal Variation • 377 FIGURE 17–5 A display of microduplications and microdeletions (a), alongside concomitant second-hit CNVs (b) that may influence phenotype, typically for the worse Microduplications and microdeletions are ranked, from top down, according to the frequency with which second-hit CNVs are observed Those at the top of the list can sometimes be (apparently) nonpenetrant, and thus the second-hit CNV may be necessary to lead to overt pathogenicity (in the top four, asterisked in b, the enrichment of CNVs is statistically significant) Those further down the list have second-hit CNVs at no greater frequency than in the control population, and are “stand-alone” pathogenic The fractions of microduplications and microdeletions due to parental or de novo origin are indicated in panel a, according to the shading of the bars Compare Figure 14–70, which shows second-hit CNVs in the dup(22)(q11.12) syndrome Source: From Girirajan et al., Phenotypic heterogeneity of genomic disorders and rare copy-number variants, N Engl J Med 367: 1321–1331, 2012 Courtesy S. Girirajan, and with the permission of the Massachusetts Medical Society subtle question in the case of intellectual and behavioral traits The Estonian study noted above leads to an inference that earlier assumptions, that some heterozygotes for “syndromic CNVs” could be unaffected, may be incorrect, albeit that the degree of affection is quite mild (Lupski 2015; Männik et al. 2015) IN PRACTICE The following is a very common situation the counselor faces: An imbalance is detected on molecular karyotyping, and the segment concerned contains CNVs and possibly known genes An example is a 268 kb triplication at Xq27.1, trp chrX:138,414,910-138,683,873,10 that we have seen in a child with epilepsy and intellectual deficiency The extent of the segment is displayed in Figure 17– 6, according to the Genoglyphix database Two known genes are included, Factor IX (F9) and MCF2; the latter is incompletely present and thus unlikely to be of pathogenic significance 10 These coordinates according to hg19, as this is the build Genoglyphix, ClinGen, and DECIHPER were using at this writing 378 • C h r omosom e Va r ian t s FIGURE 17–6 An example of a CNV display using the Genoglyphix database, based on the trpX:139,332,751-139,601,714 bp described in the text (here seen according to the hg19 numbering, chrX:138,414,910-138,683,873) The Factor IX gene (F9) is completely contained within the segment; the MCF2 gene is partially included (Case of J. Watt.) If this sequence is interrogated in DECIPHER, a list is displayed of several segments of larger and smaller size, which overlap with the sequence of interest The closest in this example is a case of dup X:138,556,249-138,764,448, and this segment can be called up and displayed in the row “This patient: copy number variants” (Fig 17–7) This case is annotated (click on the “Affected patient” bar) as “Paternally inherited, constitutive in father.” No comment is made under Pathogenicity, and the curators have left this interpretation open (perhaps awaiting further cases; and this one of ours has since been submitted) But the assessment is not inconsistent with the CNV being, at least in terms of brain function, benign A summary of the genes resident within a region and a commentary on haplo/triplo-sufficiency status where applicable, and with links to synoptic data about each locus, are accessible through ClinGen (https://www.clinicalgenome.org) The display according to the trp(X) under discussion is shown in Figure 17–8 (on hg19) By going to the DGVa link11 mentioned above and entering the coordinates of the trp(X) (q27.1), the CNVs contained therein are displayed 11 Or, if a segment is identified in the University of California Santa Cruz (UCSC) browser, and the track “DGV Struct Var” under the Variation category is chosen, the region will be displayed, and segments of deletion (red) and duplication (blue) within the vicinity indicated Clicking on to a CNV within the segment of interest will link to the DGV database Normal Chromosomal Variation • 379 (a) (b) FIGURE 17–7 (a) An example of an interrogation using the DECIPHER database, based on the trpX:139,332,751-139,601,714 bp described in the text (here seen according to the hg19 numbering, chrX:138,414,910-138,683,873) Entering these coordinates, and then scrolling through a list of cases that DECIPHER presents with some degree of overlap, the closest variant is dup X:138,556,249-138,764,448 Choosing this case, it is then shown as the prominent bar “Affected patient” in the track “This Patient: Copy Number Variants” (upper) (b) Clicking on this bar (lower) gives a dialog box with detailed information, although in this case a call was unable to be made on pathogenicity In the track below, other annotated CNVs from the DECIPHER database are depicted (Fig 17–9) The largest is a 15,273 bp deletion, and smaller dels and dups are listed But these, by definition in DGVa, are normal variants and can therefore be dismissed as pheno- contributory Only two genes are noted, and one of these, MCF2, is disrupted by the distal breakpoint and thus, as mentioned above, unlikely of concern (and no Mendelian disease is due to this gene) The remaining gene, coding for clotting Factor IX, could in principle be associated with a disorder of coagulation The important next step is a family study, if feasible In the example just given, it transpired that the brother and mother both had the same trp(X) and were both normal intellectually But interestingly, they had both suffered thrombotic episodes, with elevated levels of Factor IX The conclusion to be drawn is that the neurological compromise12 in 12 Brain imaging was normal, and there was no evidence that cerebral vascular thromboses could have been the basis of her abnormality 380 • C h r omosom e Va r ian t s FIGURE 17–8 The display according to the ClinGen database, of the trpX:139,332,751-139,601,714 (on hg19) described in the text The links at the right (ICSA ID) take the reader to synoptic data about each locus FIGURE 17–9 An example of an interrogation using the Database of Genomic Variants (DGVa) based on the trpX:139,332,751-139,601,714 bp described in the text The website is accessed at http://dgv.tcag.ca/ gb2/gbrowse/dgv2_hg38 Nucleotide numbers are entered according to the appropriate “build” chosen (here, hg38) Duplicated CNV segments (blue on screen; here, dark gray) and deleted CNV segments (red; here, light gray) are presented These are mostly labeled nsv and esv (sv, structural variant, archived and accessioned by dbVAR and DGVa, respectively) The largest duplication is nsv524240 (upper center), whereas esv2658705 (upper left) is the largest deletion Clicking on each entry links to detail about the variant, including the lengths (here, 46,585 and 15,273 bp, respectively) The default display also shows actual genes (here, F9 and MCF2) within the chosen segment, by exons (blocks) and introns (wavy lines) the presenting child is likely coincidental A genetic diagnosis yet awaits, if indeed there is one (The reader may well have similar stories to tell.) A Rare Complexity The Multiple De Novo CNV Phenotype Whereas de novo CNVs are generated at a mutation rate considerably higher than that seen in Mendelian genetics, the number of independent de novo CNVs observed in this rare “CNV mutator” phenotype is on an altogether different scale and reflects a different mechanism (Liu et al 2017) The original cases, through whom the syndrome had been delineated, were defined by the possession of four or more independent de novo CNVs of >100 kb, and they had been ascertained at a frequency of in 12,000 among children with “various developmental disorders” referred for clinical microarray testing These CNVs are typically duplications, and they may number in the low single digits to just double digits; an example is shown in Table 17–4 Further investigation of the de novo CNVs in these individuals suggests that they arise in the perizygotic time interval, due to a transient fault in the DNA replicative repair process According to one proposed construction, a de novo mutation arising in the gamete leads to the production of a mutant mRNA that compromises the repair of DNA replicative error, Table 17–4. Nine De Novo Copy Number Variants Observed in a Child with the Multiple De Novo Copy Number Variant Phenotype SITE SIZE NATURE PARENTAL ORIGIN 1p34p35 3p14p21 8q24 10q24q25 16p11 16q23 16q24 19q13 Xp11 1.7 Mb 4.2 Mb 4.5 Mb 4.7 Mb 322 kb 4.2 Mb 312 kb 4.3 Mb 214 kb Dup Dup Dup Dup IDD IDD Dup Dup Dup Maternal Maternal Maternal Maternal Paternal Maternal Maternal Maternal Maternal Dup, duplication; IDD, insertional double duplication Source: From Liu et al (2017) 382 • C h r omosom e Va r ian t s thereby leading to the “CNV mutator” phenotype In the male, the homolog harboring the mutation in a meiotic spermatocyte is preferentially segregated into a daughter nontransmitted sperm, leaving the actual fertilizing sperm to have the normal homolog, but yet retaining some of the abnormal mRNA The chromosomes of this sperm are vulnerable to this mRNA, but the effect is short-lived, and by the time the zygote comes into existence, no mRNA is left; thus, the de novo CNVs are all of paternal origin In the female, albeit that the homolog with the mutation is, in similar fashion, directed out of harm’s way into the polar body, mRNA is nevertheless retained in the cytoplasm of the oöcyte, and its influence carries over into the zygote and the first one or two mitoses Thus, the de novo CNVs are of both maternal and paternal origin (e.g., the case in Table 17–4) Thereafter, these CNVs are transmitted stably in the soma GENETIC COUNSELING Classic Cytogenetic Variant A person carrying a classical chromosome variant has, practically by definition, no increased risk for having abnormal offspring, pregnancy loss, or any other reproductive problem Some view it as at best pointless and at worst counterproductive even to mention to the individual that a variant chromosome has been found; others feel obliged to pass on the observation If it is discussed, it must be made clear that it is a normal finding—perhaps interesting but of no practical importance For the heterochromatic size variants (C-band and NOR) and euchromatic variants, the point can simply be made that some chromosomes come in short, medium, and long forms, and where a chromosome happens to fit in this continuum is without significance For segmental imbalances ascertained in apparently unaffected individuals, careful clinical assessment should be made, if practicable, of carriers from the same family; incomplete penetrance and variable expressivity should be borne in mind in assessing innocuousness, or not, of the variant Fragile sites are, almost always, normal findings The primacy, in the twenty-first century, of molecular karyotyping in fact means that discovery of variants such as these will be rather infrequent events There is considerable potential for iatrogenic anxiety, whereas in reality the biology of the supposed anomaly has no pathogenic implication The counselor may thoroughly understand the presumed harmlessness of a variant chromosome, but the person in whose family it has been discovered may react “nonscientifically.” To put a stark setting, the worst possible response might be for a couple to choose to terminate a pregnancy because of an overinterpreted variant chromosome, as has actually happened with the 16p11.2 euchromatic variant (López Pajares et al 2006) Primum non nocere: First no harm Copy Number Variants The distinction between harmful and harmless variants is a much subtler exercise in the case of CNVs As we outlined above, interrogating databases such as DECIPHER and DGVa may be a first court of appeal If a parent, or other family member, has the same variant, and is of normal phenotype, the CNV may be adjudged a “likely benign” variant; or, other data, and especially pedigree data, in the public domain, may be sufficiently powerful to indicate indeed a nonpathogenic CNV A qualitative assessment of the genic content, as mentioned above, and as understanding progresses, may well be valuable A de novo CNV may need to be considered as “likely pathogenic” unless there is solid evidence otherwise; a data resource is the website http://denovo- db.gs.washington.edu (Turner et al 2017) A detailed format for the practical assessment of a CNV is outlined in Di Gregorio et al (2017), who assessed a little over 1,000 individuals with developmental delay/intellectual disability (Fig 17–10) These variants were classified into CNVs of size greater than Mb (which we might equally call microdeletion/ duplications); del/ dups associated with known syndromes; CNVs spanning known Mendelian disease genes; likely pathogenic CNVs, and noting the genes contained within them; and VOUSs/likely benign These authors referred to the databases of a number of publicly-available sources, including material from large autism repositories, in order to judge the possible pathogenicity of abnormal copy number of the loci contained within CNVs In some, a diagnosis was clear enough at the outset, with a number of known syndromes seen In others, it required a detailed weighing of the nature of the loci, and appealing to information from the several sources The reader wishing further demonstration of the rationale in CNV assessment is referred to this paper FIGURE 17–10 A schema for the analysis of copy number variants The data from a series of 1,015 cases of developmental delay/intellectual disability were assessed, and in 10%, a pathogenic CNV was identified The criteria by which the CNVs were judged are set out in fine detail in the Tables S1-S7 in the original paper Sources referred to: DGV, DECIPHER as noted above; OMIM, Online Mendelian Inheritance in Man; HGMD, Human Gene Mutation Database; SFARI, Simons Foundation Autism Research Initiative; NDAR, National Database for Autism Research; NDD, neurodevelopmental disorders; ExAC, Exome Aggregation Consortium; GO, Gene Ontology Consortium Source: From Di Gregorio et al., Copy number variants analysis in a cohort of isolated and syndromic developmental delay/intellectual disability reveals novel genomic disorders, position effects and candidate disease genes, Clin Genet 92: 415–422, 2017 Courtesy A Brusco and G.B Ferrero, and with the permission of John Wiley & Sons Normal Chromosomal Variation • 383 The question of nonpenetrance, or at least reduced expressivity, of a CNV is a challenging one Attempting to dissect out “micro-phenotypes” in a parent may prove rather fraught Adding to this is the problem of “second hit” CNVs, and the degree to which they may modify or exacerbate a phenotype In the meantime, in advising about the risk to a future child, the counselor will need to consult current sources and to seek expert advice A problem of long familiarity in genetic counseling, that of dealing with uncertainty, certainly applies here (Wilkins et al 2016) Conveying the information about a CNV to 384 • C h r omosom e Va r ian t s counselees is an exercise to which genetic counselors are becoming more accustomed, which is not to say that they find it straightforward Finally, a question of well-considered clinical judgment, and of which the answer might differ between families: Having discovered a CNV that would qualify as a VOUS, would it, or might it not, be helpful to pursue a family study? Concerning the CNV-mutator phenotype, if the theory of a fresh mutation at a meiotic stage (see above) is correct, then occurrence would be sporadic, and no increased risk would apply to a subsequent pregnancy ... 1, 000a Sex Chromosomes Klinefelter Syndrome and Variants 47,XXY 47,XXY/46,XY 46,XX 20 1. 12b 0.39 0 .11 XYY 47,XYY 47,XYY/46,XY 18 1. 01 0 .11 XXX 47,XXX 17 1. 00 1 0.06 0 .18 0.06 0.06 0 .12 Other... abnormalities and genetic counseling, fifth edition GARDNER AND SUTHERLAND’S Chromosome Abnormalities and Genetic Counseling FIF TH E D ITION R J McKinlay GARDNER ADJUNCT PROFESSOR CLINICAL GENETICS... Translocations 11 3 Robertsonian Translocations 14 2 Insertions 15 8 Inversions 17 7 10 Complex Chromosomal Rearrangements 2 01 11 Autosomal Ring Chromosomes 210 12 Centromere Fissions, Complementary Isochromosomes,