🧬 PART ONE BASIC CONCEPTS

Elements of Medical Cytogenetics  5 CHROMOSOMAL MORPHOLOGY Chromosomes have a linear appearance: two arms that are continuous at the centromere. Reflecting the French influence in the establishment of the cytogenetic nomenclature, the shorter arm is designated p (for petit), and the longer is q.2 In the early part of the cell cycle, each chromosome is present as a single structure, a chromatid, a single (but 2-stranded) DNA molecule. During the cell cycle, the chromosomes replicate, and two sister chromatids form. Now the chromosome exists as a double-chromatid entity. Each chromatid contains exactly the same genetic material. This replication is in preparation for cell division so that, after the chromosome has separated into its two component chromatids, each daughter cell receives the full amount of genetic material. It is during mitosis that the chromosomes contract and become readily distinguishable on light microscopy. Blood and buccal mucosal cells are the tissues from which DNA is extracted in routine chromosome analysis. From blood, the nucleated white cell is the tested component for microarray analysis, and in classical cytogenetic analysis, it is the lymphocyte. Buccal mucosal cells and white blood cells are obtained from a saliva sample. The chromosomal status of each small sample is taken as representative of the constitution of (essentially) every other cell of the body. In the case of invasive prenatal diagnosis, the cells from amniotic fluid or chorionic villi are the source material; these tissues are assumed (with certain caveats) to represent the fetal chromosomal constitution. Noninvasive prenatal testing exploits the presence of fetal blood cells and DNA in the maternal circulation. Preimplantation diagnosis analyzes a few cells taken from the trophoblast of an embryo at in vitro fertilization (IVF). The 46 chromosomes come in 23 matching pairs and constitute the genome. One of each pair came from the mother, and one from the father. For 22 of the chromosome pairs, each member (each homolog) has the same morphology in each sex: These are the autosomes. The sex chromosome (or gonosome) constitution differs: The female has a pair of X chromosomes, and the male has an X and a Y chromosome. The single set of 23 homologs—one of each autosome plus one sex chromosome—is the haploid set, and thus the haploid number (n) is 23. The haploid complement exists, as such, only in the gametocytes (ovum and sperm). All other cells in the body—the soma—have a double set: the diploid complement (2n) of 46. If there is a difference between a pair of homologs, in the sense of one being structurally rearranged, the person is described as a heterozygote. The chromosomes are classically distinguishable on the basis of their size, centromere position, and banding pattern. The centromere may be in the middle, off-center, or close to one end—metacentric, submetacentric, and acrocentric, respectively. The chromosomes are numbered 1 through 22, and X and Y, and are also assigned to groups A through G, according to their general size and the position of the centromere. The diagrammatic representation of the banding pattern is the ideogram (Appendix A). The numbering is based on size, largest to smallest (to split hairs, this order is not exact; for 2 Variously explained as being the next letter in the alphabet, a mistyping of g (for grand), for queue, or as the other letter in the formula p + q = 1.

6  BASIC CONCEPTS example, chromosomes 10 and 11 are shorter than chromosome 12, and chromosome 21 is smaller than 22). The classical format of a chromosome display, the karyotype, has the chromosomes lined up with p arms upward, in their matching pairs (Figure 1–3). Those coming from a DNA-based view may see the chromosome lying on its side, and microarray reports usually show a horizontal depiction of the chromosome arms, with the graph indicating duplications and deletions by a rise or a fall compared to baseline, respectively (although no one is proposing that short and long arms be renamed as left and right!). Karyotypes are described according to a shorthand notation, the International System of Human Cytogenetic Nomenclature (ISCN 2024); an outline is given in Appendix B. In this age of “molecular karyotyping,” chromosomes are more readily being thought of as lengths of DNA, rather than as segments of stainable chromatin. A laboratory report may be couched in terms of the actual DNA content of an imbalance (Chapter 2). It is almost as though we can “see” the genes, rather than just knowing intellectually that this is—of course—what a chromosome consists of. Table 1–1 and Figure 1–4 set out the amounts of DNA and the number of genes carried by each chromosome. CHROMOSOMAL STRUCTURE Chromatin exists in differently condensed forms: the less condensed euchromatin and the more condensed heterochromatin. Euchromatin contains the coding DNA—the genes—while heterochromatin comprises non-coding DNA. Chromosomes are capped Figure 1–3.  Chromosomes arranged as a formal karyotype, from a classical cytogenetic study based upon a mitotic cell. Elements of Medical Cytogenetics  7 at the terminal extremities of their long and short arms by telomeres, specialized DNA sequences comprising many repeats of the sequence TTAGGG, that can be thought of as “sealing” the chromatin and preventing its fusion with the chromatin of other chromosomes. The centromere3 is a specialized region of DNA that, at cell division, provides the Table 1–1.  The Lengths of the Chromosomes in Megabases, the Number of Genes per Chromosome, and the Density of Genes per Chromosome CHROMOSOME LENGTH IN Mb GENES GENES PER Mb 1 249.3 1959 7.86 2 243.2 1184 4.87 3 198.0 1029 5.20 4 191.2 721 3.77 5 180.9 835 4.62 6 171.1 1002 5.86 7 159.1 855 5.37 8 146.4 638 4.36 9 141.2 748 5.30 10 135.5 714 5.27 11 135.0 1236 9.16 12 133.9 987 7.37 13 115.2 305 2.65 14 107.3 577 5.37 15 102.5 547 5.33 16 90.4 783 8.67 17 81.2 1111 13.68 18 78.1 257 3.29 19 59.1 1332 22.53 20 63.0 518 8.22 21 48.1 213 4.43 22 51.3 418 8.15 X 155.3 806 5.19 Y 59.4 65 1.09 Notes: The relative sparsity of genes on the three classic “trisomic” chromosomes, 13, 18, and 21 (and as also evident in Figure 1–4) is to be noted. In contrast, chromosome 19, although one of the smallest, carries an extraordinary load of genes. This, presumably, is the basis of the extreme lethality of trisomy 19 (see Figure 20– 22). The lengths here (from hg19,a of the time) differ slightly from those listed in Table A–1, based upon hg38. a“hg”—human genome—refers to the UCSC (University of California Santa Cruz) Genome Browser assembly number. The similar GRCh (Genome Reference Consortium, human) database now uses the same numbering system, both currently 38; previously they had had different numbers, and hg37 matched CGRh19. These two databases are “reference genomes” that are periodically updated. Source: After S Scherer, Guide to the Human Genome, Cold Spring Harbor Laboratory Press, Woodbury, New York, 2010. 3 When considering the physical structure of rearranged chromosomes, it is useful to keep in mind the absolute requirement for a centromere to be present on every chromosome. There is also the need for each chromosome to have two telomeres, the single exception being when the rearranged chromosome forms a ring.

8  BASIC CONCEPTS site at which the spindle apparatus can be anchored and draw each separated chromatid4 to opposite poles of the dividing cell. Centromeric heterochromatin contains “satellite DNA,” so called because these DNA species have different buoyant densities and produce distinct humps on a density gradient distribution. (These are not to be confused with the satellites on acrocentric chromosomes.) A separate issue, of considerable academic interest (but which we shall take no further here), is the “packaging question”: how the centimeters of DNA are compacted into micron-length chromosomes, and which parts of the nucleus each chromosome occupies (Figure 1–5) (Sehgal et al. 2016; Sedat et al. 2022; Labade et al. 2024). Neither do we delve into the molecular anatomy of the chromosome, while it did not escape our notice5 that the DNA sequences within, now so well documented (Nurk et al. 2022), are, of course, their sine qua non. CHROMOSOME ABNORMALITY Chromosomes are distributed to each daughter cell during cell division in a very precise process—precise, but prone to error. From our perspective, the two cell divisions of meiosis, during which the gametes are formed, are of central importance. Much of the discipline of medical cytogenetics focuses on the consequences of disordered meiosis having produced a chromosomally abnormal gamete, causing a chromosomal abnormality in the conceptus. A chromosome abnormality that is present from conception and involves the entire body is a constitutional abnormality. If an additional cell line with a different chromosomal complement arises before the basis of the body structure is formed (that is, in embryonic or pre-embryonic life) and becomes an integral part of the organism, constitutional mosaicism results. In this book, we concern ourselves practically solely with constitutional abnormalities. Acquired chromosomal abnormality of course exists, and indeed it is a major initiating and sustaining cause in most cancers, a fact first proposed by Boveri in 1914 and voluminously attested in the work Figure 1–4.  A Display of the DNA Content, and the Gene Load, of Each Chromosome. Notes: Chromosomes are ordered by size (left) and gene content (right). The “viable” autosomes, 13, 18, and 21, are shown in blue, and the sex chromosomes in red and green. The low gene content of the viable autosomes is evident; the bar (right) is set at 327 genes. Source: From EM Torres, Consequences of gaining an extra chromosome, Chromosome Res 31:24, 2023. Courtesy EM Torres, and with the permission of Springer Nature. 4 Chromatids separate at mitosis and at meiosis II, and at “predivision” in meiosis I. 5 A nod to Watson and Crick. Elements of Medical Cytogenetics  9 of Mitelman et al. (2024); but this is more the field of study of the molecular pathologist than the genetic counselor. An incorrect amount of genetic material carried by the conceptus disturbs and distorts its normal growth pattern (from zygote → blastocyst → embryo → fetus). In trisomy, there is three of a particular chromosome, instead of the normal two. In monosomy, only one member of the pair is present. Two of each is the only combination that works properly! It is scarcely surprising that a process as exquisitely complex as the development of the human form should be vulnerable to a confused outflow of genetic instruction from a nucleus with a redundant or incomplete database. Trisomy and monosomy for a whole chromosome were the first cytogenetic mechanisms leading to an abnormal phenotype to be identified. More fully, we can list the following pathogenetic mechanisms that arise from chromosomal abnormalities: 1. A dosage effect, with a lack (deletion) or excess (duplication) of chromosomal material, whether for a whole chromosome or a part of a chromosome. This is by far the predominant category. 2. A direct damaging effect, with disruption of a gene at the breakpoint of a rearrangement. 3. A position effect, whereby a gene in a new chromosomal environment functions inappropriately. 4. An effect due to the incongruent parental origin of a chromosome or chromosomal segment (genomic imprinting). 5. Combinations of the above. We discuss these mechanisms in more detail in following chapters. Figure 1–5.  The Spatial Arrangement of Chromosomes within the Nucleus. Notes: The color coding indicates which part of the nucleus each chromosome occupies, in a human fibroblast. The figure at left is a composite from the analysis of all 23 chromosomes. At right, chromosome 3’s territory is outlined, which can be discerned in the green coloring in the composite image. This study is based on expansion in situ genome sequencing (ExIGS). Source: From AS Labade et al., Expansion in situ genome sequencing links nuclear abnormalities to hotspots of aberrant euchromatin repression, bioRχiv 2024.09.24.614614, 2024. Courtesy of JD Buenrostro and Colleagues, and with their permission.

10  BASIC CONCEPTS AUTOSOMAL IMBALANCE As noted earlier, imbalance may involve the gain or loss of a whole chromosome—full aneuploidy—or of part of a chromosome—partial aneuploidy. Full aneuploidies are trisomies and monosomies; partial aneuploidies are duplications (dup) and deletions (del). The abnormality may occur in the non-mosaic or mosaic state. Loss (that is, monosomy/deletion) of chromosomal material typically has a more devastating effect on growth of the conceptus than does an excess of material (that is, trisomy/duplication). Smaller dups and dels are referred to as copy number variants (CNVs), and Collins et al. (2022) document, at the level of individual dosage-sensitive genes within the CNV segments, which loci are critical to the generation of phenotypic abnormality; they speak of haploinsufficient (deletion intolerance) and triplosensitive (duplication intolerance) imbalance. Certain imbalances lead to certain abnormal phenotypes. The spectrum is listed in outline in Box 1–1. Most full autosomal trisomies and virtually all full autosomal monosomies set development of the conceptus so awry that, sooner or later, abortion occurs—the embryo “self-destructs” and is expelled from the uterus. This issue is further explored in Chapter 20. A few full trisomies are not necessarily lethal in utero, and many partial chromosomal aneuploidies are associated with survival through to the birth of an infant. Characteristically, “survivable imbalances” produce a phenotype of widespread dysmorphogenesis, and there may be malformation of internal organs and limbs. It is often in the facial appearance (facies) that the most recognizable physical abnormality is seen, with Down syndrome the classic example, although the physical phenotype in Box 1–1.  The Spectrum of Effects, in Broad Outline, Resulting from Constitutional Chromosomal Abnormality 1. Devastation of blastogenesis, with transient implantation or non-implantation of the conceptus. 2. Devastation of embryogenesis, with spontaneous abortion, usually in the first trimester. 3. Major disruption of normal intrauterine morphogenesis, with second or third trimester abortion/fetal death in utero, stillbirth, or early neonatal death. 4. Major disruption of normal intrauterine morphogenesis, but with some extrauterine survival. 5. Moderate distortion of normal intrauterine development, with substantial extrauterine survival and severe intellectual disability. 6. Mild distortion of normal intrauterine development, with substantial extrauterine survival and considerable intellectual compromise. 7. Minimal physical phenotypic effect, varying degrees of intellectual compromise; possible compromise of fertility. 8. No discernible physical phenotypic effect; cognitive function within the normal range, but less than expected from the family background.

Elements of Medical Cytogenetics  11 some cases of subtler deletion or duplication may be rather “bland.” The most complex organ of all, the brain, is the most vulnerable to a less-than-optimal genetic constitution, and some compromise of mental and intellectual functioning, usually to the extent of an obvious deficit, is nearly invariable, at least in imbalances of classical size. With several of the (much smaller) imbalances due to copy number variants, flawed neurodevelopmental progress or intellectual disability,6 with an outwardly normal physical phenotype, is well recognized as a chromosomal presentation. Thus, the central concern of most people seeking genetic counseling for a chromosomal condition is that of having a child who might have a physical, intellectual, or severe social handicap. Historically, the chromosomal basis of many syndromes was identified following analysis of groups of patients with similar phenotypes. This “phenotype-first” approach led to the identification of many of the well-known microdeletion syndromes (and of course such classic conditions as Down syndrome). With the advent of molecular karyotyping, new syndromes came to be identified based on their DNA aberration, a “genotype-first” approach. Representative examples of these newer syndromes are reviewed in Chapter 14. SEX CHROMOSOMAL ABNORMALITY Sex chromosome (gonosome) imbalance has a much less deleterious effect on the phenotype than does autosomal aneuploidy. The X chromosome is one of the larger and is gene-dense; the Y is small, comprising mostly heterochromatin, and carries very few genes (the key one being SRY, which determines maleness). In both male and female, one, and only one, completely functioning X chromosome is needed. X chromosomes in excess of one are inactivated, as the normal 46,XX female exemplifies; her second X does, however, maintain some segments genetically active. With X chromosome excess or deficiency, a partially successful buffering mechanism exists whereby the imbalance is counteracted, in an attempt to achieve the same effect as having a single active X. In such states as, for example, XXX, XXY, XXXX, XXYY, and XXXXX, excess X chromosomes are inactivated. In the 45,X state, the single X remaining is not subject to inactivation. If an abnormal X chromosome (e.g., an isochromosome, or a deleted X) is present, then, as a rule, cells containing this abnormal chromosome as the active X are selected against, perhaps due to preferential growth of those cells in which it is the normal X that is the active one. In X imbalance, the reproductive tract and brain are the organs predominantly affected. The effect may be minimal. As for Y chromosome excess, such as XYY, there is a limited phenotypic consequence—but again, the brain may be a vulnerable organ. 6 Words can be powerful, and choice of language can help, or hinder, a counseling consultation: Facts are to be conveyed clearly but also sensitively. The term “mental retardation,” once widespread in the genetics literature, has acquired a pejorative and somewhat harsh sense over the years, and we now prefer such expressions as “intellectual disability” or “cognitive impairment.” “Developmental delay” is a widely used term, and it can be perfectly appropriate in early childhood setting, but less so in dealing with a school-age child or adult; this distinction acknowledges that prediction of intellectual capacity is more precise in older children. As we write elsewhere, counselors will need to know to whom they speak, and what language is best to use.

12  BASIC CONCEPTS Functional Imbalance A correct amount of chromatin does not necessarily mean the phenotype will be normal. Inappropriate inactivation, or activation, of a segment of the genome can compromise the genetic message. Some segments of the genome require only monosomic expression, and the homologous segment on the other chromosome is inactivated. If this control fails, both segments can become activated or both inactivated, and the over- or under-expression of the contained loci can cause phenotypic abnormality. The classic example of this is genomic imprinting according to parent of origin, and we discuss this concept in Chapter 19. A rather specialized example arises with the X-autosome translocation. A segment of X chromosome can fail to be inactivated or, conversely, X-inactivation can spread into an autosomal segment, and in each case to deleterious effect (Chapter 6). THE FREQUENCY AND IMPACT OF CYTOGENETIC PATHOLOGY According to the window of observation (Figure 1–6), chromosomal disorders make a greater or lesser contribution to human mortality and morbidity. Looking at prenatal existence, the earliest window has been provided by the in vitro fertilization (IVF) clinic, from the procedure of preimplantation genetic testing (Chapter 23), at which cells taken from 5-day-old embryos are subjected to genetic analysis, and an extraordinary fraction are chromosomally abnormal. After implantation (about day 6), and through the first trimester of pregnancy (to week 13), chromosomal mortality is very high, and aneuploidy is the major single cause of spontaneous abortion (Chapter 20). Perinatal and early infant death have a significant chromosomal component, of which trisomies 18 and 21 (although the latter less so in more recent times) are major elements. In Table 1–2 we set out the birth incidences of the various categories of (classical) chromosomal abnormality; these data are from a Danish study, one of a number that have examined this question in the later decades of the 20th century with largely similar findings in each. Overall, around one in 135 liveborn babies have a classical chromosomal abnormality, and about 40% of these are phenotypically abnormal due to the chromosome defect. Figure 1–6.  The windows of observation open to chromosomal study. Note: Obviously, time on the x axis is depicted asymmetrically. FDIU, fetal death in utero. Elements of Medical Cytogenetics  13 (continued) Table 1–2.  Classical Chromosomal Rearrangements and Imbalances, Recorded in 34,910 Live Newborns in Århus, Denmark, Over a Total 13-Year Period, 1969–1974 and 1980–1988 NO. OF CASES PER 1,000a BIRTH FREQUENCY PER GROUP Sex Chromosomes Klinefelter Syndrome and Variants 47,XXY 20 1.12b 47,XXY/46,XY 7 0.39 46,XX ♂ 2 0.11 1 in 616 ♂ XYY 47,XYY 18 1.01 47,XYY/46,XY 2 0.11 1 in 894 ♂ XXX 47,XXX 17 1.00 1 in 1,002 ♀ Turner Syndrome and Variants 45,X 1 0.06 45,X/46,XX and 45,X/47,XXX 3 0.18 45,X/46,X,r(X) 1 0.06 45,X/46,X,i(Xq)/47,X,i(Xq),i(Xq) 1 0.06 Other Turner Variant 2 0.12 1 in 2,130 ♀ Other 45,X/46,XY 1 0.06 46,XX/47,XX,del(Yq) 1 0.06 46,XX/46,XY 1 0.06 Total 77 2.21 1 in 453 Autosomes Unbalanced Forms Trisomy 13 2 0.06 Trisomy 18 7 0.20 Trisomy 21 51 1.46 Trisomy 8 1 0.03 Supernumerary marker, ring 25 0.72 Deletions, duplications 6 0.17 1 in 379 Balanced Forms Robertsonian 13/14 translocation 34 0.97 Other Robertsonian 9 0.26 Reciprocal translocations 50 1.43 Inversions (other than of chromosome 2) 4 0.11 1 in 360 14  BASIC CONCEPTS The finer the cytogenetic focus, the greater the incidence, and it is now a task for the cytogenetic (or molecular genomic) epidemiologist of this century, in the microarray/molecular era, to derive new estimates of cytogenetic abnormalities in the different populations (Rosenfeld et al. 2013). While the incidence of classical data remains stable, the subtler del/dups have been increasingly observed in more recent years (Figure 1–7). A useful start has been made by Smajlagić et al. (2021) in their analysis of a population of newborns and their parents, in whom they tested for the presence of 13 of the recurrent deletions and duplications that have become well recognized as causative of known phenotypes/syndromes and are associated, in particular, with a neurodevelopmental component. They were able to make a distinction between those del/ dups inherited from a parent, and those arising de novo (Table 1–3). In this population, the notable figure of one in 200 children were born with a recurrent deletion or duplication; in other words, these abnormalities are not uncommon. And if we combine the overall estimates of Tables 1–2 (1 in 207) and 1–3 (1 in 200), and if we may extrapolate these Scandinavian data more generally, we approach a broad figure that about one in 100 individuals, at least in a newborn population, are chromosomally imbalanced and with an associated abnormal phenotype, whether of severe, moderate, mild, or barely discernible degree. Adolescence is a period during which many sex chromosome defects come to light, when pubertal change fails to occur, and in young adulthood when chromosomal causes of infertility are recognized. If the target population is fertile adults (ascertained incidentally), a much lower frequency of sex chromosome aneuploidy is observed (Samango-Sprouse et al. 2016). If we were to study a population of 70-year-olds, we could expect to see very few individuals with an unbalanced autosomal karyotype (but loss of the Y chromosome is a common observation in older men; p. 472). NO. OF CASES PER 1,000a BIRTH FREQUENCY PER GROUP Combined sex plus autosomal totals 266 7.62 1 in 131 Combined totals, excluding balanced autosomal forms 169 4.84 1 in 207 Notes: Not included in the 34,910 live newborns listing are four cases of induced abortion due to sex chromosome prenatal diagnosis, involving the karyotypes 47,XXY, 47,XYY, 47,XXX, and 45,X/46,X,del(Xq), and 15 cases of autosomal-diagnosis induced abortions, involving the karyotypes +21, +13, +18, and three different derivative chromosomes. Had these pregnancies proceeded to term, the frequencies in the relevant group category would have been marginally increased. These figures might continue to be broadly valid into this century, except that the category of deletions and duplications will substantially increase due to the more powerful detection now offered by molecular technology (Table 1–3). aPer 1,000 male, per 1,000 female, or per 1,000 both, as appropriate. The gender-specific denominators in this study were 17,872 males and 17,038 females. bAn increasing incidence of XXY in recent years has been suggested, and an Australian study, including data up to 2006, arrived at a figure of 1.91 per 1,000 (Herlihy and Halliday 2008; Morris et al. 2008; Herlihy et al. 2010). Source: From Nielsen and Wohlert (1991) Table 1–2.  Continued

Elements of Medical Cytogenetics  15 While few classic cytogenetic defects come to attention later in adult life, many children with an aneuploidy can survive well into adulthood and some into old age, and some require lifelong care from their families or from the state. This latter group imposes a considerable emotional and financial burden. While some parents and caregivers declare the emotional return they experience from looking after these individuals, for others this responsibility is a source of continuing, unresolved, if attenuated, grief. As for morbidity, the brain, as mentioned above, is the most vulnerable organ, and chromosomal defects are the basis of a substantial fraction of all intellectual deficit. Many of these affected individuals will also have structural malformations that cause functional physical disability. Among an intellectually disabled population, Down syndrome is the predominant contributor in the fraction who have a classic chromosome abnormality (Phelan et al. 1996). Development of the heart is particularly susceptible to chromosomal imbalance, and in a population study from the US National Center on Birth Defects, 1 in 8 infants with a congenital heart defect had a chromosomal abnormality with, again. trisomy 21 the most common of these (53%), followed by trisomy 18 (13%), 22q11.2 deletion (12%), and trisomy 13 (6%) (Hartman et al. 2011). Figure 1–7.  The Increasing Detection of Deletions and Duplications. Notes: Shown are the numbers (y-axis) of terminations in central Denmark over the period 2008–2021 (x-axis), for the indications of an aneuploidy (upper line), and for a deletion or duplication (lower line). The baseline birth rate in this population is c. 14,000 per annum. Termination on the grounds of a fetal abnormality was available from 12 to 22 weeks gestation, but required approval from a local Regional Abortion Council. Source: From L Raaby et al., Has the introduction of increased genetic prenatal testing affected rates of termination of pregnancy due to fetal abnormality? Prenat Diagn 44:280–288, 2024. Courtesy L Raaby, and with the permission of John Wiley & Sons. Table 1–3.  The Population Prevalences of Some of the More Notable Deletions and Duplications, Derived from the Norwegian Mother, Father, and Child Cohort Study, 1999–2008 RECURRENT CNVs DE NOVO CNVs ALL CNVs Position (hg38) Del/Dup N Parental N Prevalence 1:147,107,276-147,924,476 del1q21.1 distal 2 pat ×2 6 4.9 1:147,107,276-147,924,476 dup1q21.1 distal 0 4 3.26 3:196,033,055-197,619,681 del3q29 1 pat 1 0.82 3:196,033,055-197,619,681 dup3q29 0 0 0 7:73,331,825-74,731,095 del7q11.23 (Williams-Beuren) 0 0 0 7:73,331,825-74,731,095 dup7q11.23 0 0 0 15:23,123,715-28,325,372 del15q11.2-13.1 (Prader-Willi/Angelman) 0 0 0 15:23,123,715-28,325,372 del15q11.2-13.1 2 mat ×2 3 2.450 15:30,783,588-32,154,652 del15q13.3 1 pat 5 4.08 15:30,783,588-32,154,652 dup15q13.3 1 pat 6 4.9 16:28,811,768-29,035,413 del16p11.2 distal 1 mat 3 2.45 16:28,811,768-29,035,413 dup16p11.2 distal 2 mat 8 6.53 16:29,639,423-30,183,727 del16p11.2 proximal 4 mat ×3, pat 6 4.9 16:29,639,423-30,183,727 dup16p11.2 proximal 0 5 4.08 17:1,344,540-2,685,615 del17p13.3 (Miller-Dieker) 0 0 0 17:1,344,540-2,685,615 dup17p13.3 0 0 0 17:16,908,429-20,313,519 del17p11.2 (Smith-Magenis) 0 0 0 17:16,908,429-20,313,519 dup17p11.2 (Potocki-Lupski) 0 0 0 17:36,460,758-37,856,053 del17q12 3 mat, pat ×2 3 2.45 17:36,460,758-37,856,053 dup17q12 0 2 1.63 17:45,628,753-46,087,790 del17q21.31 (Koolen-deVries) 0 0 0 17:45,628,753-46,087,790 dup17q21.31 0 0 0 22:19,037,347-21,114,846 del22q11.2 (DiGeorge) 1 mat 1 0.82 22:19,037,347-21,114,846 dup22q11.2 2 mat, pat 6 4.9 22:21,566,197-23,310,015 del22q11.2 distal 0 0 0 22:21,566,197-23,310,015 dup22q11.2 distal 0 0 0 Total 20 16/10,000 59 48/10,000 Notes: The extents of the del/dups are shown in molecular detail, at the level of the nucleotide. “hg38” refers to Human Genome build, version 38, also referred to as GRCh38 (Genome Reference Consortium human build 38). Prevalences are per 10,000. The study was based upon a material of 12,252 newborns and their parents. Source: From Smajlagić et al., Population prevalence and inheritance pattern of recurrent CNVs associated with neurodevelopmental disorders in 12,252 newborns and their parents, Eur J Hum Genet, 29:205–215, 2021.

Elements of Medical Cytogenetics  17 The Research Application of Cytogenetic Pathology The phenotypes that result from chromosome abnormalities can point the way to discovery of the causative genes. An early example of deletion mapping is the recognition that the gene for retinoblastoma was on chromosome 13, given the association of this cancer with the 13q– syndrome. Another cancer gene to be similarly mapped was APC (adenomatous polyposis coli), following the observation of polyposis of the colon in an individual with intellectual disability and del(5)(q22q23) (Hockey et al. 1989). The triple dose of chromosome 21 in Down syndrome (DS), and knowing that Alzheimer disease almost invariably affects DS persons aged 40 and older (Hithersay et al. 2019), was a signpost on the way to finding the β-amyloid precursor protein (APP) gene as one of the major Alzheimer loci; and the DS adult population may prove a valuable resource in the assessment of new dementia treatments (Rafii and Fortea 2023). A translocation with one breakpoint at 7q11.23 was found to disrupt the elastin gene in a family segregating supravalvular aortic stenosis. Further investigation of this locus in Williams syndrome proved this to be the site of deletion in this condition (Nickerson et al. 1995). The gene for CHARGE7 syndrome, CHD7, was discovered due to two patients with an 8q12 microdeletion (Vissers et al. 2004). David et al. (2023) studied two men presenting with infertility, of otherwise normal phenotype, in whom a balanced chromosome rearrangement had been identified: t(5;9)(q32;p21.1) and t(4;21) (p15.1;q22.3), respectively, leading them to propose YIPF5 near the 5q32 breakpoint, and SPATC1L within 21q23 as candidates contributory to the control of spermatogenesis. We have conducted reviews of chromosomal conditions in which epilepsy and kidney disease are features, with the aim of providing leads to epilepsy genes and renal genes (Singh et al. 2002a; Amor et al. 2003). The precision of microarray analysis, coupled with access to genome databases, now allows a much finer focus in the pursuit of causative genes. Ou et al. (2008) proposed, and Ballesta-Martínez et al. (2013) supported, that one of the genes SIX1, SIX6, or OTX2 might be the basis of one form of branchio-oto-renal syndrome, from their study of a child with a duplication of 14q22.3q23.3; SIX1 was eventually revealed as the culprit gene. We have shown WDR35 to be the gene for a short rib–polydactyly syndrome, having found a microdeletion on chromosome 2p24 by single nucleotide polymorphism (SNP) and copy number variant (CNV) analysis (Mill et al. 2011); and RAB39B was found to be the basis of a syndrome of early-onset Parkinson disease and intellectual disability, a 45 kb deletion at Xq28 leading us to this discovery (Wilson et al. 2014). It is a general principle that many important scientific discoveries are made serendipitously—or, as Louis Pasteur put it, “chance favors the prepared mind” (le hasard ne favorise que les esprits préparés). Voullaire et al. (1993) identified a small supernumerary marker chromosome (sSMC) in a child with a nonspecific picture of physical abnormality and intellectual deficit, which had no C-band positive centromere 7 CHARGE = coloboma, heart, choanal atresia, retardation, genital, ear. 18  BASIC CONCEPTS (only a constriction). Conventional wisdom has it (and indeed, as we have written above) that a chromosome cannot be stably transmitted at cell division if it has no centromere. These workers studied this sSMC and discovered that it did have a simple, but nevertheless functional, centromere. This observation led the way to the delineation of the “neocentromere” (p. 319).

2 CHROMOSOME ANALYSIS FOR THE FIRST HALF-CENTURY of clinical cytogenetics, analysis of chromosomes was an exercise in microscopy. This century, molecular methodologies are holding sway. But it behooves the counselor to have a good understanding of how things used to be, not least because one often needs to make reference to the historical literature. And it is, of course, an obligation to keep abreast of new developments. Modern cytogenomic (this word now entering the lexicon) reports are sophisticated documents, and those who read them, and who interpret them to patients and families, need to be well informed. CLASSICAL CYTOGENETIC ANALYSIS In classical methodology, chromosomes are analyzed under the light microscope, at a magnification of about 1000×. The chromosomes are stained to be visible, and a great many staining techniques were used to demonstrate different features of the chromosome. We list some of these, in particular those with a more immediate practical application to the clinical issues we discuss in this book, or which are of historical value when referring to the older literature. Plain staining (“solid staining”). Many histologic dyes, including Giemsa, orcein, and Leishman, stained chromosomes uniformly. Until the early 1970s, these were the only stains available. Giemsa or G-banding. This procedure required a trypsin (protein digestion) step, and is the main staining method in use in routine classical cytogenetics. It allows for precise identification of every chromosome, and for the detection and delineation of structural abnormalities. At the 400–550 band level, rearrangements down to about 5 megabases in length can be discerned, at least in regions where the banding pattern is distinctive. Its precision is increased by manipulations designed to arrest the chromosome in its more elongated state at early metaphase or prometaphase— high-resolution banding. Alternative methods to demonstrate essentially the same morphology are quinacrine, or Q-banding, and reverse or R-banding. In R-banded chromosomes, the pale staining regions seen in G-banding stain darkly, and vice versa. Constitutive or C-banding. This technique stains constitutive heterochromatin— mainly the centromeric heterochromatin, some of the material on the short arms of the acrocentric chromosomes, and the distal part of the long arm of the Y chromosome. Constitutive heterochromatin, by definition, has no direct phenotypic effect and, in general, is devoid of active genes. Replication Banding. This technique is used primarily to identify inactive X chromatin. A nucleotide analog (BrdU) is added either as a pulse at the beginning, or

20  BASIC CONCEPTS toward the end of the cell cycle, to allow the cytogenetic distinction between chromatin that replicates early from that which replicates late. It produces a banding pattern similar to that of R-banding. NOR (silver) Staining. This stain, of largely historic interest now, identified nucleolar organizing regions (NOR), which contain multiple copies of genes coding for rRNA, and which are sited on the satellite stalks of the acrocentric chromosomes. Distamycin A/DAPI Staining. This fluorescent stain identifies the heterochromatin of chromosomes 1, 9, 15, 16, and Y. A particular use was to distinguish the inverted duplication 15 chromosome from other small marker chromosomes. Fluorescence In Situ Hybridization (FISH) and variations thereupon. The major cytogenetic advance of the 1990s was the ability to identify specific chromosomes, and parts of chromosomes, by in situ hybridization with labeled probes. FISH has been widely used to detect submicroscopic deletions, and to characterize more obvious chromosome anomalies. A more focused use is in the assessment of the structural nature of imbalances revealed by microarray analysis (see below), with the probe from the genomic region targeted to the specific region identified by the array. Spectral Karyotyping (SKY). This is a variation upon the Multiplex-FISH theme. Judicious combinations of fluorophores allow every chromosome to appear of a different color (Liehr et al. 2004). The pictures resulting are certainly rather beautiful (Figures 1–1 and 2–2), and von Waldeyer-Hartz’s 1888 choice of the word “chromosome” becomes particularly apt. Targeted probes within segments of a chromosome enable some structural rearrangements to be very readily demonstrable (Figure 9–3). However, in fact it is more in the field of cancer cytogenetics that this methodology has been applied: a kaleidoscope of colors can rather clearly reveal the complexity of chromosomal changes accompanying tumor evolution. Comparative Genomic Hybridization (CGH). In CGH, differentially labeled, fluorophore-tagged DNA from the patient and a normal control (reference sample) is applied to a metaphase slide prepared from a “standard” normal person. Relative excesses and deficiencies of patient DNA bind competitively, with respect to the control, onto the reference chromosomes and yield different color intensities upon exciting the fluorophores. This procedure has been applied to archival pathology material. “High-resolution” CGH refers not to a more stretched chromosome preparation, but to a further level of sophistication of the computer software that is used to analyze the images, by adjusting for the idiosyncratic patterns that each homolog may have. Small imbalances may be identifiable by this approach, ~10 Mb or greater, and the nature of uncertain rearrangements clarified. Chromosomes examined by various techniques are illustrated in Figure 2–1. Full detail is to be found in Mark (2000), Miller and Therman (2001), Rooney (2023), Gersen and Keagle (2013), and Arsham et al. (2017), while Trask (2002) provides an historical span of the cytogeneticist’s skill. CHROMOSOMAL MICROARRAY ANALYSIS Since the 2010s, chromosomal microarray (CMA) has become the first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies (Manning and Hudgins 2010; Miller et al. 2010). There are basically two microarray techniques: The first uses a CGH approach, much like that described above for Chromosome Analysis  21 chromosomal CGH; but this has gradually been replaced by the second, which uses single nucleotide polymorphisms (SNP) to assess the number of alleles in a sample. Although microarrays can differ in their genomic composition and substrates used for the analysis, most comprise thousands of spots of reference DNA sequences, applied in a precisely gridded manner upon a slide (or “chip”) in which the locations can be Figure 2–1.  Chromosome pairs 1, 6, 15, 16, and Y and X stained by various techniques: plain stain (a), G-banding (b), replication banding (c), C-banding (d), Ag-NOR stain (e), and Q-banding (f). 22  BASIC CONCEPTS known by computer analysis. Some commercial microarrays combine CGH and SNP detection on the same array. Not that classical cytogenetics is likely to fade altogether from view: There are two crucial reasons for its continuing use in the laboratory. First, not all array results can give a definitive construction, and FISH is sometimes necessary to elucidate the cytogenetics. Second, the array cannot detect balanced rearrangements,1 and recognition of the carrier state will continue to need an old-fashioned chromosome test. And third, a rather subjective “reason” is that, by continuing to work with chromosomes, the molecular cytogeneticist/cytogenomicist will not lose the intuitive understanding of what chromosomes are really like, rather than seeing them merely as theoretical constructs or computer-screen displays. As mentioned above, the reporting of microarray results is a sophisticated exercise, and counselors need to be sophisticated readers of these reports. Many laboratories now use depictions from one of the genome browsers— with a classic chromosome ideogram laying on its side at the top—to illustrate the precise extent of the imbalance, and noting the genes contained within this segment. Comparative Genomic Hybridization The fundamental principle is essentially the same as in chromosomal CGH, noted above, but using the array, rather than the metaphase spread, as substrate. Patient and Figure 2–2.  Spectral karyotyping. Notes: Every chromosome is of a different color. Chromosome 4 stains light blue, and chromosome 8 is orange. In this example, a child with Wolf-Hirschhorn syndrome (p. 392) manifests a distal 4p deletion with a very small segment from chromosome 8, the orange tip only just discernible (arrowed), in its place, from the karyotype 46,XY,der(4)t(4,8). Source: From T Ried et al., Chromosome painting: a useful art, Hum Mol Genet 7:1619–1626, 1998, with the permission of Oxford University Press. 1 Albeit that whole genome sequencing is being used to address this shortcoming (Ordulu et al. 2016; Redin et al. 2017).

Chromosome Analysis  23 control DNA are labeled in two different fluorophores, usually one that appears red and one that appears green. These labeled DNAs are applied to the microarray, and hybridization takes place. Typically, if the number of copies between the control and the patient are the same, the spot looks yellow (produced from an overlapping of equal amounts of red and green). The fluorescent intensities of each dye are measured. If the patient has an excess at a locus (due to duplication or aneuploidy), the hybridization will more reflect the dye of the patient’s DNA. If the patient has a deficiency at a locus (loss due to deletion or unbalanced translocation), the hybridization will more reflect the dye of the control DNA (see the cover art of this book). Single Nucleotide Polymorphism (SNP) Array SNP arrays detect the number of alleles in a specimen (Figure 2–3) and provide two types of information. First, the intensity of the signal arising from each SNP can be measured to produce a log2 ratio: A relative increase in signal intensity corresponds to copy number gain, and a decrease in signal intensity corresponds to deletion. Second, SNP arrays produce genotyping information: Heterozygosity, with two distinct alleles, can be distinguished from homozygosity, and from the presence of three alleles. Apparent homozygosity may indicate a loss of DNA, such as a deletion, while three alleles may indicate a gain of DNA copy number, such as a duplication or trisomy. SNP-based microarrays have the added advantage of detecting uniparental disomy when the Figure 2–3.  SNP-based Microarray. Notes: Plot of chromosome 22 in a patient with a 22q11 deletion, using a SNP-based microarray that combines copy number (log2 ratio) and genotyping (B-allele frequency, blue dots) data. The copy number is reduced to a single copy in the region of the deletion, shown by the deviation of the log2 ratio (black dots) towards minus 1. The genotyping plot (blue dots) demonstrates an absence of A/B heterozygous probes in the deleted region, showing only hemizygosity for the A or B alleles, thus also allowing inference of a deletion (indicated by pink shaded region). Courtesy DI Francis.

24  BASIC CONCEPTS child’s results are compared to the parental genotypes. Isodisomy may be revealed, in the absence of parental samples, when the entire chromosome shows homozygosity, and chromosomal monosomy is an incompatible interpretation. POLYMERASE CHAIN REACTION-BASED APPLICATIONS A number of polymerase chain reaction (PCR) technologies can assess DNA copy number. These are targeted approaches to answer a specific question: How many copies of the target are present in the patient sample? Multiplex ligation-dependent probe amplification (MLPA) uses pairs of probes to detect specific sections of the genome, identifying exon-level copy number variation across one or many genes. Multiplexing of numerous exons allows detection in one reaction with high copy number accuracy. It can be combined with methylation-sensitive enzymes to detect the methylation status of a specific region. Quantitative real-time PCR (qPCR) combines amplification of a target DNA sequence with highly accurate quantification of the original template DNA target. Digital PCR (dPCR) partitions each PCR reaction strand to provide quantitation for a specific genomic location. Combined with oil droplet PCR (ddPCR) for partitioning of each PCR reaction, detection of variant fractions (in other words, mosaicism) as low as 1% is possible. Real-time Digital PCR (rdPCR) combines the above two methodologies, integrating the precision of dPCR with the real-time analysis capabilities of qPCR. DNA SEQUENCING Short-Read Sequencing (Next Generation Sequencing) DNA methodologies based on massively parallel genomic sequencing of short strands of DNA (reads of up to a few hundred base pairs long) allow the entire expressed genetic complement, the “exome,” and even the whole genome to be tractable to interrogation. These methodologies were originally called “Next Generation Sequencing” (NGS) to distinguish from the traditional Sanger sequencing, but are more accurately referred to as short-read sequencing (SRS). In the cytogenetic field, SRS is routinely applied as a highly accurate molecular counting tool, sequencing cell-free DNA circulating in the maternal plasma, and mapping each sequence read back to its chromosome of origin. A similar technique is applied to aneuploidy detection in preimplantation embryos. At the time of writing, chromosome microarray remains the gold standard methodology for molecular karyotyping, but SRS is a promising alternative. Low-coverage genome sequencing can detect, with 100% sensitivity, copy number variants diagnosed by microarray, and the technology offers the additional benefit of detecting balanced chromosome rearrangements (Dong et al. 2014, 2016). Given that genome sequencing at higher levels of coverage offers considerable diagnostic yield for the diagnosis of sequence-level mutations, it is expected that, in time, a single SRS-based test will generate both copy number and sequence data, and will Chromosome Analysis  25 become the first-line test for the investigation of children with developmental disabilities and for prenatal diagnosis. Long-Read Sequencing Long-read sequencing (LRS) analyzes DNA fragments of size tens to hundreds of kilobase pairs. Currently, the two main technologies are PacBio’s (Pacific Biosciences) single-molecule real-time (SMRT) sequencing, and ONT’s (Oxford Nanopore Technologies) sequencing. LRS can detect some structural variants that are missed by short-read sequencing, allowing these to be mapped with single base resolution, and is especially useful in resolving complex chromosome rearrangements. LRS can also be used to phase variants, generate haplotypes, and to interrogate epigenetic modifications. Optical Genome Mapping Optical Genome Mapping (OGM) can detect all classes of chromosome abnormalities encountered in the clinic, including aneuploidies, copy number variants from a few kilobases in size, and balanced structural rearrangements (Mantere et al. 2021; Xiao et al. 2024). Linearized strands of high molecular weight DNA are labeled with fluorescent markers at specific sites, evenly spaced throughout the genome. The labeled DNA strands are then loaded into nanochannels, in which they are imaged using a fluorescence microscope, so that the order of markers for each strand can be mapped to the human genome reference. Methylation Detection Several of the techniques listed above can be applied to the detection of DNA methylation, following modification of the DNA sequence to distinguish between the unmethylated and methylated regions. Methylation strand modification is commonly performed by bisulfite conversion of the CpG sequence, or by methylation-sensitive enzyme digestion. Direct detection of the methylated nucleotide can be achieved using nanopore-based LRS, whereby the methylated DNA strand passes through the nanopore at a slower rate than the unmethylated DNA strand. Cytogenetic (or Cytogenomic) Reports Chromosomal findings from molecular analyses are often presented in an intuitive pictorial form, such as, for example, the display in Figure 2–3. Although cytogenetics will continue to evolve, whatever techniques come to be used, the fundamental purpose of the cytogenetic report will of course remain the same. Descriptions about the technologies used will be important addenda to reports, because they may inform the clinician about the interpretation of the chromosome analysis and the possible need for further analysis. Reports may also include a listing of presumed significant genes in the region, a comment upon imprinting, and the likelihood of benign

26  BASIC CONCEPTS versus causative genomic changes. A pedantic but important point is that the genome “build” be noted. GENETIC COUNSELING CONSIDERATIONS Mostly, the abnormalities found by molecular technologies have clear clinical relevance for the patient and the family. However, higher resolution strategies may uncover DNA changes of unclear clinical significance (as we discuss at length in Chapter 18). Such findings may lead to testing of additional family members, parents, grandparents, and sometimes siblings, to understand the relationship, if any, between the DNA alteration and the clinical phenotype or medical condition of the patient. The possibility of findings of unclear clinical significance should be discussed when ordering the test, especially in the prenatal setting. Because these molecular-based tests have the ability to interrogate the entire genome, the pretest genetic counseling should include information about uncovering unwanted information, such as loci that could predispose to cancer, or to adult-onset disorders. The use of SNP arrays may uncover substantial stretches of homozygosity due to consanguineous or even incestuous relationships (Schaaf et al. 2011). These counseling caveats notwithstanding, the higher resolution potential of these new technologies will increase the detection rate of chromosome abnormalities, and will much improve our ability to make diagnoses, and to provide the answers that families seek. The counselor need not be expert in the nature of the technologies outlined above, but should have a broad understanding of the different applications and of what each can, or cannot, offer in the clinical setting.

28  BASIC CONCEPTS Chapter 14) or to a deletion of a gene (e.g., Pitt-Hopkins syndrome, Chapter 14), and thus diagnosable on cytogenomic technology. The analysis of copy number variants (CNVs)—short segments of genomic material in excess or deficiency—is generally regarded as a chromosomal exercise. We will largely confine ourselves (Chapter 18) to those CNVs which are known to be pathogenic; but there is also blurring, here, in terms of CNVs, whose harmlessness (or not) is uncertain. We focus first upon the classic chromosomal disorders. Most of these arise at meiosis. A gamete from a 46,N person may acquire an extra (but normal) chromosome, and this would lead to a full aneuploidy in a conceptus of theirs: a trisomy. Or, partial aneuploidy may be due to meiotic malsegregation having taken place in gametogenesis of a 46,rea (rea = rearrangement) parent carrying a balanced rearrangement. De novo (“of new”; that is, with normal parental karyotypes) partial aneuploidies may have been generated at a meiotic division, or at a premeiotic germ cell mitosis; where there is normal/aneuploid mosaicism, a postmeiotic event in the embryo is implicated. While it may not be possible to presume with reasonable confidence where the original error lay, a theoretical consideration of the point at which a chromosomal defect arose—before, during, or after meiosis—can underpin a useful understanding. It thus behooves us to appreciate the broad processes of meiotic and mitotic cell divisions. MEIOSIS Meiosis in Chromosomally Normal Persons The purpose of meiosis is to achieve the reduction from the diploid state of the primary gametocyte (2n = 46) to the haploid complement of the normal gamete (n = 23); and to ensure genetic variation in the gametes. The latter requirement is met by enabling the independent assortment of homologs (the physical basis of Mendel’s second law)2 and by providing a setting for recombination between homologs. While we do not dwell on recombination per se, this is, to the classical geneticist, a raison d’être of the chromosome: “From the long perspective of evolution, a chromosome is a bird of passage, a temporary association of particular alleles” (Lewin 1994). The mature gamete is produced after the two meiotic cell divisions: meiosis I and meiosis II, the cells going through stages of oögonium/spermatogonium → primary gametocyte → secondary gametocyte → mature gamete (or polar body). As per the classical description (Figure 3–1), the chromosomes of the (diploid) primary gametocyte entering meiosis I do not divide at the centromere, and they remain, following cell division into secondary gametocytes, as double-chromatid chromosomes. With only one of each homolog, the cell is in the haploid state. In meiosis II, the chromosomes of the secondary gametocytes separate into their component single chromatids, and this cell division gives rise to the (haploid) gametes. In the male, the cells produced from one primary gametocyte are the four spermatids, which mature into spermatozoa. In the female, the cells are the mature ovum and its polar bodies. (In fact, 2 The law of independent assortment: During gamete formation, the segregation of the alleles of one allelic pair is independent of the segregation of the alleles of another allelic pair. The exception: If two loci are close together—“linked”—on the same chromosome. Origins and Consequences of Chromosome Pathology  29 Figure 3–1.  Meiosis I. Chromosomal behavior during meiosis I, according to the classical model. Circles represent germ cells: at (a) oögonia and spermatogonia (gonocytes); at (b–d) primary oöcytes and spermatocytes (gametocytes); and at (e) secondary oöcytes and spermatocytes. One crossover has occurred between the long arms of one chromatid of each homolog. Following meiosis I, the chromosome number has halved (reduction division). In oögenesis, one of the two cells at (d) would be the first polar body, and would typically not enter meiosis II. 30  BASIC CONCEPTS it is not until sperm penetration that meiosis II in the ovum is completed.) Figure 3-2 shows the appearances of the chromosomes at these different stages of meiosis. Each gamete thus contains a haploid set of chromosomes. The diploid complement is restored at conception, with the union of two haploid gametes. The moment of conception, as the embryologist sees it, is not at sperm penetration but only when the two pronuclei have fused to form a single nucleus (“syngamy”). Note that spermatogenesis divides the cytoplasm evenly, so that after meiosis II there are four gametes of equal size. The sperm head that penetrates the ovum comprises almost entirely nuclear material; the tail is cast off. In oöcytes, cytoplasmic division is markedly uneven, producing a secondary oöcyte and first polar body after meiosis I, and the mature ovum and second polar body at meiosis II.3 The ovum and the polar bodies each have a haploid chromosome set, but the ovum retains almost all of the cytoplasm.4 Another major sex difference concerns the timing of gamete maturation. In the female, meiosis is partway through, in the late prophase of meiosis I, by the eighth month of intrauterine life (the actual process of recombination taking place during weeks 16–19 of fetal life). At birth, a female baby has around a third of a million oöcytes (Figure 20–30). Most of this pool is gradually lost, but those eggs destined to mature stay in a “frame-freeze” until they enter ovulation some one to five decades thereafter,5 and meiosis recommences. Testicular stem cells (spermatogonia), on the other hand, Figure 3–2.  The Chromosomes in Human Gametes. Notes: The karyotype from a 23,X egg (left), showing the haploid set of double-chromatid chromosomes following the completion of the reduction division of meiosis I (cf. Figure 3–1d). The chromosome spread (center) from a 46.XY spermatocyte, the homologs having come together as bivalents in mid-meiosis I (cf. Figure 3–1b,c). The autosomal bivalents have their homologous segments aligned, whereas the X-Y bivalent aligns only at the tips of Xp and Yp. The single-chromatid chromosomes of a 23,Y sperm (right) at meiosis II (cf. Figure 3–1e). Sources: From O Samura et al., Sperm and oocyte chromosomal abnormalities, Biomolecules 13:1010, 2023; and L Uroz and C Templado, Meiotic non-disjunction mechanisms in human fertile males, Hum Reprod 27:1518–1524, 2012. Courtesy O Samura, and with the permission of MDPI; and courtesy L Uroz and C Templado, and with the permission of the European Society of Human Reproduction and Embryology and Oxford University Press. 3 The first polar body does not normally undergo (a biologically pointless) meiosis II. 4 Cytoplasm contains the mitochondria, and transmission of mitochondrial DNA is maternal. The mitochondrial genome has been described, somewhat whimsically, as chromosome 25, or the M chromosome. In not otherwise referring to this “chromosome” we are not seeking to deny its importance or interest! 5 As Eichenlaub-Ritter (2012) points out, oöcytes are one of the longest-lived cells in the body.

Origins and Consequences of Chromosome Pathology  31 do not begin to enter meiosis until the onset of puberty. Thereafter, millions of mature sperm are continuously produced. Meiosis in Detail. We now examine more closely the details of meiosis, according to the classical model. During the final mitotic division in the primary gametocyte, the homologous pairs of chromosomes have (as with any mitosis) replicated their DNA to change from the single-chromatid to the double-chromatid stage. They now enter into the meiotic cell cycle (Figure 3–1a). As meiosis I proceeds to prophase, chromosomes conduct a “homology search” and come together and pair, with matching loci alongside each other (Figure 3–1b). This process—synapsis—continues with a more intimate pairing of the homologs, starting at the tips of the chromosomes and proceeding centrally, and the synaptonemal complex is formed (Barlow and Hultén, 1996). The paired chromosomes themselves are called bivalents.6 Synapsis sets the stage for an exchange of matching chromosome segments; this is the process of recombination, or crossing-over (Figure 3–1c). Next, desynapsis occurs (the diplotene stage), with dissociation of the synaptonemal complex and the formation of chiasmata. Now, the two homologous chromosomes disjoin and go to opposite poles of the cell. This is the anaphase stage; the orderly movement of chromosomes during this sequence is facilitated if synapsis, recombination, and chiasmata formation have proceeded normally. Finally, the cell divides into the two daughter cells (Figure 3–1d). How the chromosomes are distributed—which chromosome goes to which pole—is called segregation. Normally, each daughter cell gets one of each of the pair of chromosomes, and this is referred to as one-to-one (1:1) segregation. Uniquely in the meiosis I cell division daughter cells are produced with double-chromatid chromosomes. These gametocytes then enter meiosis II (with the exception of the first polar body). In this cycle, the chromosomes do not replicate because they are already in the double-chromatid state. The chromosomes separate at the centromere, and the resulting single-chromatid chromosomes disjoin, one going to each pole, resembling a mitotic division (Figure 3–1e). The foregoing, classical construction held sway since practically the beginning of human cytogenetics, and it remains very useful conceptually; but its primacy has since been challenged. One alternative description puts the events of meiosis I and II in the reverse order: That is, the chromosomes separate into chromatids at meiosis I and then segregate into daughter cells at meiosis II (Figure 3–3). This has been called reverse segregation (Ottolini et al. 2015; Webster and Schuh 2017). The other, very important, retelling of the events of meiosis is premature separation of sister chromatids (PSSC). This is, in fact, the predominant mechanism in the female, and is important in the male (Ottolini et al. 2015; Sakakibara et al. 2015). It refers to the “precocious” separation of chromatids during meiosis I, as initially proposed by Angell (1997), and involves three sequential events (Figure 3–4). First, the (double-chromatid) homologs fail to pair during meiosis I; or, if they do pair, they separate again before meiosis I is complete. In other words, instead of the two (double-chromatid) chromosomes existing as a conjoined bivalent, they exist as two separate univalents. Second, these univalents are prone to “predivide”—that is, the separation of the two chromatids that should (on the classical plan) happen 6 Since, at the level of the chromatid, the bivalent pair contains four elements, the word tetrad can also be used in this setting; in this sense, the cell at this stage of the cycle has 23 × 4 = 92 chromatids. 32  BASIC CONCEPTS Figure 3–3.  Reverse Segregation. Chromosomal behavior during meiosis, specifically ovarian meiosis, according to the model of “reverse segregation.” Circles represent germ cells: at (a) oögonia; at (b–d) primary oöcyte; at (d) first polar body (PB1); and at (e) secondary oöcyte and the second polar body (PB2). One crossover has occurred between the long arms of one chromatid of each homolog. The single-chromatid chromosomes separate at (d); this is the step that defines “reverse segregation.” Meiosis II follows at (e). Note that the pairs of cells after meiosis II (ovum + second polar body) have homologs of opposite parental origin (non-sister chromatids). In the classical model (Figure 3–1), the homologs in these pairs would always be of the same parental origin (sister chromatids). It was this distinction which, along with other evidence, pointed Ottolini et al. (2015) toward proposing this new model. Origins and Consequences of Chromosome Pathology  33 at meiosis II, instead takes place while they are still in the first meiotic cycle. This could happen to both univalents or just the one, and these would then exist as single-chromatid chromosomes. The oöcyte in Figure 3–6 (lower) may be an example of asymmetric segregation due to this process, having received a double-chromatid and a single-chromatid chromosome 21. Third, at anaphase of meiosis I, these double- or single-chromatid chromosomes segregate independently to the oöcyte and polar body, or mature spermatocytes. Chromosomal pathology arises when these processes of disjunction and segregation go wrong—malsegregation and nondisjunction. Figure 3–4.  Premature Separation of Sister Chromatids. Nondisjunction following “predivision” of one homolog into its component chromatids in meiosis I (Angell’s hypothesis). The asterisked gamete reflects the complement of the oöcyte in Figure 3–6 (lower). In öogenesis, one of the two cells following meiosis I would be the first polar body, which might or might not proceed to meiosis II.

34  BASIC CONCEPTS Malsegregation and Nondisjunction in Meiosis Malsegregation (or missegregation) is remarkably frequent at meiosis, and in consequence many human conceptions are trisomic or monosomic. Malsegregation is a “catch-all” term; in principle, nondisjunction specifically refers to the failure of homologous chromosomes to segregate symmetrically at cell division, although in practice it is often considered (and we sometimes do) as “the inclusion of both daughter chromosomes in the same nucleus, by whatever mechanism” (Miller and Therman 2001). The process of malsegregation is described according to two models: the classical description of nondisjunction (Figure 3–5), and a modern description based upon the PSSC model as described above. While the classical model has long been seen as the typical process, in fact, as Gabriel et al. (2011) write, “it appears to be a relatively minor player” and “the received wisdom that non-disjunction [sensu stricto] is the primary mechanism leading to human aneuploidy should be reconsidered.” Nevertheless, and taking a conservative viewpoint,7 we first set out in detail the model that has appeared in textbooks for generations, but then pay due attention to the more recent knowledge. The classical description of the mechanism of meiotic nondisjunction is as follows: In a chromosomally normal person, if the pair of homologs comprising a bivalent at meiosis I fail to separate (fail to disjoin8), one daughter cell will have two of the chromosomes, and the other will have none. This is 2:0 segregation (Figures 3–5 and 3–6, upper). In other words, one gametocyte is disomic for that homolog, and the other is nullisomic. Or, nondisjunction may occur in meiosis II, meiosis I having proceeded normally. In meiosis II, it is the chromatids that fail to separate (Figure 3–5b). Following these nondisjunctional errors, the conceptus, at fertilization, ends up trisomic or monosomic, assuming the other gamete to be normal (Figure 3–7a, b). Trisomy or monosomy in the offspring of normal parents is called primary trisomy or primary monosomy. Concerning meiotic malsegregation following the alternative premature separation of sister chromatids scenario Figure 3–4 sets out the picture. As the oögonium and spermatogonium enter meiosis, one double-chromatid homolog “pre-divides” and now consists of a pair of single-chromatid chromosomes. If these chromosomes were then to segregate symmetrically, at meiosis I, the primary gametocytes resulting would have a balanced constitution; but if it is asymmetric—as in the example in Figure 3–4—one gametocyte has an extra chromatid, while the other has only one. As meiosis II proceeds, giving rise to the secondary gametocytes, it transpires that two are normal haploid, one is diploid, and one “nulliploid.” The oöcyte in Figure 3–6 (lower) may be an example of asymmetric segregation due to this process, having received a double-chromatid and a single-chromatid chromosome 21.9 Polar body and embryo studies in the IVF laboratory suggest that, at least at an older maternal age, half of all aneuploidy in the oöcyte results from meiosis I,10 about a third from meiosis II and a small fraction from meiosis I nondisjunction (Verdyck et al. 2023). 7 “Be not the first by whom the new are tried, Nor yet the last to lay the old aside.” Alexander Pope, An Essay on Criticism, 1711. 8 Note that disjunction is a normal process, and nondisjunction is not; there is no such word as dysjunction. 9 Certain terminologies and nomenclature may be mentioned here. A gamete with an extra chromosome is hyperhaploid, with a karyotype written as, for example, 24,X,+21. A gamete missing a chromosome is hypohaploid (e.g., 22,Y,–21). If, at meiosis I, the extra chromosome is present only as a single chromatid (e.g., the asterisked oöcyte in Figure 3–4), the abbreviation cht (for chromatid) is used: thus, 24,X,+21cht. The ISCN (2024) provides nomenclature for meiotic cells, and an extra 21 at meiosis I, present as a univalent, would be denoted as MI,24,+I(21). 10 This category may also have included examples of reverse segregation. Origins and Consequences of Chromosome Pathology  35 Figure 3–5.  The classical view of the mechanics of nondisjunction. The asterisked gamete reflects the complement of the oöcyte in Fig. 3–6 (upper), with respect to one of the G-group chromosomes. In oögenesis, one of the two cells following meiosis I would be the first polar body, which typically does not proceed to meiosis II. A process somewhat intermediate between these two major mechanisms is achiasmate nondisjunction, in which the homologs had never joined and then segregate together to the same daughter cell. The end result is the same as if classical nondisjunction had occurred, but without any recombination (Uroz and Templado 2012). 36  BASIC CONCEPTS Figure 3–8 summarizes these several scenarios, with particular reference to the more vulnerable gamete, which is to say, the egg. Frequencies of Meiotic Malsegregation The very considerable majority of human aneuploidy due to meiotic malsegregation, takes place during oögenesis. Remarkably high fractions of mature oöcytes are Figure 3–6.  Oöcyte chromosomes at metaphase of meiosis II, showing nondisjunction of a G-group chromosome having occurred at the preceding first meiotic division. Upper, oöcyte with classical nondisjunctional disomy, showing an additional G-group double-chromatid chromosome. Possibly the arrowed pair are chromosome 21s, and the karyotype 24,X,+21. Lower, oöcyte with “predivisional” disomy, showing an additional G-group single chromatid. The arrowed pair may be chromosome 21s, and the karyotype 24,X,+21cht. Source: From Kamiguchi et al. Chromosomal analysis of unfertilized human oocytes prepared by a gradual fixation-air drying method, Hum Genet 90:533–541, 1993. Courtesy Y Kamiguchi.

Origins and Consequences of Chromosome Pathology  37 Figure 3–7.  Aneuploid gametes producing an aneuploid conceptus (a and b), and aneuploid gametes producing uniparental disomy (c). aneuploid, at least as measured in a population of women presenting to an in vitro fertilization (IVF) clinic (Figure 3–9). In women aged 28–37 years, nearly half of eggs are disomic or nullisomic, whereas in 38- to 47-year-olds the figure rises to three-quarters (Fragouli et al. 2013). In a different population—women from teenage to mid-30s presenting for cryopreservation of eggs due to a diagnosis of cancer, and otherwise perhaps more representative, of the general population—Nikiforov et al. (2020) determined a rather smaller fraction, one-third of oöcytes at the second meiotic division being aneuploid. If we look at the immediate downstream consequence of these abnormalities, which is to say, the picture in preimplantation embryos at day 3 to 4, again we see a very striking maternal age effect, aneuploidy rates ranging from about 10% to 70% from ages 20s through early 40s (Figure 3–10). By day 5—that is, at the appearance of the blastocyst—aneuploidy figures have reduced somewhat, likely reflecting lethality due to the most severe aneuploidies, and range from about a quarter (maternal age under 35) to a half (over age 35) from IVF data (Kubicek et al. 2019). The maternal basis of aneuploidy is again very apparent, with only minor contributions from paternal meiotic error or postmeiotic mitotic error (Figure 3–11). Certain aneuploidies show predilections for one or other meiotic stage. For example, essentially all trisomy 16 may be due to a maternal meiosis I error, whereas most trisomy 18 reflects meiotic II malsegregation. In those aneuploidies which are capable of carrying on through to birth, it is the classic trisomies of chromosomes 21, 18, 13, and of the sex chromosomes, which have a strong advanced maternal age association, but not triploidy and monosomy X (Elmerdahl Frederiksen et al. 2023). 38  BASIC CONCEPTS Figure 3–9.  Aneuploidies in Oöcytes of Women of Ages 36–40. Note: Disomies per chromosome are shown above the 0% baseline, nullisomies below These findings are based on inferences from the analysis of the first and second polar bodies in 676 oöcytes, from women undergoing PGT-A. The mirror-like appearance, above and below the line, reflects the agency of meiotic error, with essentially equal likelihoods of the missegregating chromosome going to the egg (for disomy), or to the polar body (for nullisomy); the similar observation of equal numbers of disomies and nullisomies in seen in Oberle et al. (2024). Light green = chromatid gain, darker green = 2-chromatid chromosome gain; Light red = chromatid loss, darker red = 2-chromatid chromosome loss. Source: Adapted from P Verdyck et al., Aneuploidy in oocytes from women of advanced maternal age: analysis of the causal meiotic errors and impact on embryo development, Hum Reprod 38:2526–2535, 2023. Courtesy P Verdyck, and with the permission of Oxford University Press. Figure 3–8.  Different Ways in which Meiotic Error may Happen in the Egg. Notes: Some errors at meiosis I can be corrected at meiosis II, resulting in the egg being euploid. In the case of reverse segregation, this correction may be mediated by chromatin threads. Source: From L Wartosch et al., Origins and mechanisms leading to aneuploidy in human eggs, Prenat Diagn 41:620–630, 2021. Courtesy ER Hoffmann, and with the permission of John Wiley and Sons. Origins and Consequences of Chromosome Pathology  39 Spermatogenesis makes very little contribution to meiosis-based aneuploidy (Dviri et al. 2021; Ivanova and Semenova 2023); a reason may be the existence of a postmeiotic checkpoint excluding most aneuploid spermatozoa from full maturation (Uroz and Templado 2012). Figure 3–12 sets out the range of abnormality in a very large sample; the comparison with oöcytes (Figure 3–9), in terms of overall fractions, comparing the scales of the y axes, is notable. Given the frequency with which nondisjunction happens, it is not at all surprising that instances of multiple aneuploidy are known, the observed numbers during stages of pregnancy reducing as nonviability takes its toll. In spontaneous abortions toward the end of the first trimester, double autosomal combinations, from simultaneous nondisjunctions, may be seen in the analysis of products of conception Figure 3–10.  Aneuploidy Rates in Day 3 to 4 Embryos. Note: These data were derived from material at day 3 to 4 (the morula stage), through different maternal ages, in patients from an IVF clinic. Observe the near straight-line upward slope of the smoothed data (in red) of the graph, attesting to the strong correlation of aneuploidy of the embryo cf. maternal age. Source: From J Li et al., Chromosome aneuploidy analysis in embryos derived from in vivo and in vitro matured human oocytes, J Transl Med 19:416, 2021. Courtesy Y Xu, and with the permission of Springer Nature. Figure 3–11.  Aneuploidies in Day 5 to 6 Blastocysts. Note: This is a display of the relative frequencies of the different origins, in day 5-6 blastocysts, of uniform (non-mosaic) aneuploidies of the different chromosomes. Maternal meiotic error is very much the predominant, with only very small contributions due to paternal meiotic error, or from mitotic error in the conceptus. These maternal meiotic data closely resemble the similar data in Figure 20–12. The data are from an analysis of SNP genotyping from 2,277 trophectoderm biopsies, of which 29% were aneuploid, from women of a wide maternal age range. Source: From B Rana et al., Identifying parental and cell-division origins of aneuploidy in the human blastocyst, Am J Hum Genet 110:565–574, 2023. Courtesy NR Treff, and with the permission of Cell Press.

40  BASIC CONCEPTS (Micale et al. 2010). As for livebirth, the reader with a sense of history will want to review the 48,XXY,+21 case described in Ford et al. (1959); a very few other cases have followed, the most common combination being trisomy 21 along with an additional sex chromosome (Li et al. 2004; Tennakoon et al. 2008). Sequential nondisjunctions at both meiotic divisions could lead to tetrasomy, and this is the basis of some X-chromosomal polysomy (Hassold et al. 1990; Deng et al. 1991). Complete nondisjunction is an expression that could be applied in the case of triploidy—a 69 chromosome count—when this is due to the retention of the polar body within the ovum (Martin et al. 1991). Simultaneous parental nondisjunctions, both gametes being disomic, is rare but not unknown, and is another route to double aneuploidy, and for example Robinson et al. (2001) describe 48,+14[pat],+21[mat] in a spontaneous abortion. If one gamete is disomic and the other nullisomic, for the same chromosome, this means that one parent has contributed both members of the homologous pair, and the other none (Figure 3– 7c). This is uniparental disomy due to gametic complementation, an event of extreme rarity. Simultaneous errors of nondisjunction and other rearrangement would typically be quite coincidental, such as a child having both XXY Klinefelter syndrome (maternal nondisjunction) and del(15)(q11.2q13) Prader-Willi syndrome (paternal deletion) (Nowaczyk et al. 2004). Causes of Meiotic Malsegregation As discussed above, most aneuploidy due to malsegregation arises in oögenesis. “Quality checking,” which is stringently applied in the male, is poorly effective in the female, and so the maturing of an aneuploid oöcyte is not prevented; and, as Hunt and Hassold (2002) comment, “Nature seems to have erred in putting less protective investment into the more scarce gamete.” A particular vulnerability of maternal meiosis likely lies in the degradation, over time, of factors that underpin the adhesion of the homologous chromatids of the Figure 3–12.  Aneuploidies in Sperm. Notes: Disomies per chromosome are shown above the 0‰ baseline, nullisomies below. In most sperm, there is a single abnormality; some may have more than one. Overall, around 0.3% of sperm in this study were nullisomic, and 0.2% disomic; this excess of nullisomy over disomy is also seen in other such studies (García-Mengual et al. 2019). Additionally, 0.4% of sperm were diploid. 99% were normal haploid. These data are derived from ten normozoöspermic men, from whom near a quarter million sperm were analyzed. Note the orders of magnitude difference in the y axis measurements of eggs (Figure 3–9 above) cf. sperm: whole numbers % vs. fractions ‰. Source: Drawn from data in Table 3 in S Zhu et al., Comprehensive chromosome FISH assessment of sperm aneuploidy in normozoospermic males, J Assist Reprod Genet 39:1887–1900, 2022. Origins and Consequences of Chromosome Pathology  41 bivalent. This failure of snug apposition leads the chromosomes to adopt unstable positions when meiosis resumes; or, homologs may become separate from each other, and this then sets the scene for pre-division or for achiasmate nondisjunction (Duncan et al. 2012; Eichenlaub-Ritter 2012). This cohesion, or its lack, is the explanation most often raised (Lagirand-Cantaloube et al. 2017). Other possibilities include a role for the spindle apparatus, a component of the cellular machinery which draws chromosomes to their positions in dividing cells, and its compromised function could cause aneuploidy (Mihajlović et al. 2023). While these meiosis-control factors may be the proximate cause of failed disjunction, what background attributes might lead to a loss in its integrity? Of course, older childbearing age is an obvious answer. A very telling insight comes from the work of Battaglia et al. (1996). These investigators sampled oöcytes at meiosis II metaphase from younger (20–25 years) and older (40–45 years) volunteers who were having normal menstrual cycles. They did not look at individual chromosomes but, rather, at the disposition of the spindle and the metaphase chromosomes as a whole. They made the most striking findings according to the ages of the women: A symmetrical and neatly arrayed complex was seen in the younger women, while in the older women the spindle was askew, and the chromosomes a-jumble, as shown in Figure 3–13. It is not difficult to accept that this structural disorganization would undermine the capacity of the chromosomes of the oöcyte then to undergo regular segregation (Wasielak-Politowska and Kordowitzki 2022). Not that the young are immune. Fragouli et al. (2006), in a paper dedicated to the memory of the 18-year-old patient whom they had studied, analyzed oöcytes harvested ahead of her chemotherapy for a marrow malignancy which, had she lived, might have enabled fertility. Of 11 oöcytes and 7 first polar bodies able to be analyzed, one egg had a single-chromatid X and could have gone on to a monosomy X conception, while another egg was inferred (via its polar body) to have an additional X and 21 chromatid, and the conception could have been 48,XXX,+21. The introductory sentence of this paper is worth quoting: “Humans as a species are not as fertile as other mammals”; and, as already noted, it is in meiosis of the oöcyte that much of this (relative) weakness resides. An alternative to the foregoing meiosis-focused scenarios is to suppose that, at least in some cases, the error in the gamete had arisen at a premeiotic stage, and that the parent is actually a gonadal mosaic for the aneuploidy (see below, PreMeiotic Mitotic Error in the Gamete, and Gonadal Mosaicism). Meiosis in Chromosomally Abnormal Persons The classic major category is the phenotypically normal person heterozygous for a balanced structural rearrangement (translocation, inversion, and insertion being the main forms), and meiosis can present considerable complication. A class of increasing importance is the individual who may carry a molecular-defined microdeletion or microduplication, and whose own phenotype may be normal or only mildly or subtly abnormal; and meiosis here is straightforward (although the interpretation of risk is often not, as we discuss below). Rarely, we see persons who are themselves chromosomally unbalanced with either a full or a partial aneuploidy, and who are clearly phenotypically abnormal, presenting with questions about their reproductive potential. We

42  BASIC CONCEPTS Figure 3–13.  Oöcytes in Younger and Older Women. Notes: The disposition of the chromosomes at meiosis II in the oöcytes from younger and older women is shown, illustrating what may be the physical basis of the maternal age effect. Meiosis II oöcytes (a) from younger and older women, illustrating what may be the physical basis of the maternal age effect. The microtubules of the spindle stain green, and the chromosomes stain orange. The tracing (b) identifies these components, and the smooth or wavy lines suggest, respectively, an intact or a degenerating spindle apparatus (the ages of the women indicated). The chromosomes are well organized at the metaphase plate at the equator of the cells in the younger women (the 22-year-old’s oöcyte, on the upper left, is viewed on a tilt). In contrast, the 40-year-old’s oöcyte shows the chromosomes in disarray. The 42-year-old woman’s oöcyte has one chromosome, at the top, dislocated from the metaphase plate, and the disposition of the other chromosomes at the equator is not as regular as in the younger women. Source: From Battaglia et al., Influence of maternal age on meiotic spindle assembly in oöcytes from naturally cycling women, Hum Reprod 11: 2217–2222, 1996. Courtesy DE Battaglia, and with the permission of the European Society of Human Reproduction and Embryology and Oxford University Press.

Origins and Consequences of Chromosome Pathology  43 will deal in detail with each situation in separate chapters, but we consider the broad principles here. BALANCED CARRIERS OF CLASSIC STRUCTURAL REARRANGEMENTS In heterozygotes for some balanced rearrangements involving only small segments, the chromosomes may “ignore” the nonhomologous material they contain, and pair (this is heterosynapsis) and segregate, much as would happen at a normal meiosis. In other balanced rearrangements, the inherent tendency to pairing dictates that homologous segments of rearranged chromosomes will align, as well as they are able, in order to achieve full pairing (homosynapsis). This may require the chromosome to be something of a contortionist, forming complex configurations such as multivalents and reversed loops. According to either scenario, the stage is set for the possibility of unbalanced segregation. The gametes produced—and therefore the conceptuses that arise—are frequently imbalanced. In this context, a segmental aneuploidy is usually involved—that is, a part of a chromosome is present in the trisomic or monosomic state; or, quite frequently, a combination of trisomy for one segment and monosomy for another. Partial trisomy and partial monosomy are also referred to as duplication and deletion, respectively. In some rearrangements, recombination presents a further hazard. Inversions and insertions may produce a new recombinant (rec) chromosome that has a different genetic composition from that of the original rearrangement. A conceptus forming from a gamete containing it would inevitably be genetically unbalanced. CARRIERS OF COPY NUMBER VARIANTS These imbalances, typically detectable only on molecular karyotyping, are small relative to chromosome length, mostly of kilobase size. CNVs are not known to interfere with normal cell division; thus, meiosis is typically symmetric, 1:1—an even probability of transmitting the abnormal chromosome (although the resultant phenotype is far less predictable). These microdeletions and microduplications are to be distinguished, in practice, from the partial aneuploidies (deletions and duplications) of classical cytogenetics noted above. FULL AND PARTIALLY ANEUPLOID INDIVIDUALS In the individual who him- or herself has a full aneuploidy and in whom gametogenesis is able to proceed, in theory a trivalent may form, or a bivalent and an “independent” univalent. Either could lead, effectively, to a 2:1 segregation. This appears actually to be the case in trisomy 21, whereas in sex chromosomal states (XXX, XXY, and XYY) the “third” chromosome is, as it were, disposed of, and most gametes are normal. In the person with a classic partial aneuploidy due to an unbalanced rearranged chromosome, whether 46,(abn) or 47,+(abn), the abnormal chromosome may have an even (or near-even) chance to be transmitted in the gamete. 44  BASIC CONCEPTS Nondisjunction in Mitosis and the Generation of Mosaicism The purpose of a mitotic cell division is faithfully to pass on an intact and complete and balanced copy of the parental cellular genome to the progeny cells. An error in this process can lead to an aneuploid cell. If the error occurs in a mitosis during gametogenesis, mosaicism of the gonad is the consequence, and this is a premeiotic mitotic error. An error in early embryogenesis, perhaps as early as the very first mitosis after conception, can lead to a constitutional mosaicism of the embryo, typically comprising a mix of normal and aneuploid cells. This is a postmeiotic mitotic error. An early error typically leads to a widespread mosaicism, affecting many and possibly all tissues of the embryo. An error in later embryogenesis typically affects a lesser fraction of the soma, and may be restricted to certain tissues, and the expression “low-grade mosaicism” is used. If the mosaicism affects a cell line destined to give rise to both some somatic tissue and gonadal tissue, this is somatic-gonadal mosaicism. The mitotic cycle consists of the following sequence: gap-1 period (G1) → synthesis period (S) → gap-2 period (G2) → mitosis (cell division). The G1 → S → G2 components together comprise the interphase period of the cell cycle. During the S period, the chromosomes replicate their DNA, thus converting from the single-chromatid to the double-chromatid state. Genetically active segments of chromosomes replicate earlier during the S period, while inactive segments, which include almost the entire inactivated X chromosome in the female, are late-replicating. The cell division period is further subdivided into prometaphase → metaphase → anaphase → telophase. The chromosomes condense to enter prometaphase, and condensation continues into metaphase. Metaphase chromosomes align on the equatorial plate (Figure 3–14), and the spindle apparatus becomes attached to the centromere of each chromosome, consisting of its two kinetochores. Pulled at the kinetochores (centromeres), the chromatids of each chromosome then separate (disjoin) and are drawn in opposite directions (anaphase) and arrive at the opposite poles of the cell (telophase). Then the chromosomes decondense, the nuclear membrane reconstitutes, the cytoplasm constricts and divides, and two daughter cells now exist. PreMeiotic Mitotic Error in the Gamete, and Gonadal Mosaicism A mitotic error occurring in a primary gametocyte, oögonium or spermatogonium, prior to its entering into meiosis, can produce an aneuploid cell, which could then lead to a lineage containing this abnormality and comprising a “wedge” or an “island” within the gonad—in other words, gonadal mosaicism with a mix of normal and aneuploid gametocytes. As the abnormal gametocytes enter meiosis, the mature gametes then resulting from them would11 be aneuploid. Note that the conceptus resulting from such an aneuploid gamete would be uniformly (that is, non-mosaic) aneuploid. We now consider the case in more detail. Cells destined to give rise to gametocytes originate from the yolk sac in early embryogenesis and migrate to the gonadal ridge on the dorsal wall of the abdominal cavity, where, along with the supporting cells, they come to comprise the tissue of the gonad (De Felici 2013). In doing so, gametocytes 11 Barring a “correction” during meiosis (Maiato and Silva 2023).

Origins and Consequences of Chromosome Pathology  45 must replicate over and over. Looking at the male case, going through about 30 cycles of division produces 230 (about 1,000,000,000) progeny spermatogonia, and the potential for error exists at each cell division contributing to this population. These errors could be nondisjunctions, or the production of structural rearrangements. Consider this startling statistic: The total length of the seminiferous tubule in a man is about half a kilometer, a third of a mile (Johnson et al. 1998). If a mutation were to occur in a spermatogonium in, for example, the 20th cycle of division, its progeny would then go through 10 more cycles and comprise 210 (about 1,000) cells. This would be only a millionth (1,000/1,000,000,000) of the ½ km of tubule—a mere ½ mm. So a man mosaic in such a way would have a risk of only one in a million to father a conception with this particular abnormality, given the improbability of the fertilizing sperm coming from this imperfect ½ mm. From similar reasoning, a defect arising at the tenth cycle could affect half a meter of tubule and carry a risk of 1 in 1,000. Oögonia need go through a lesser number of mitotic cycles (about 22), but the same principles broadly apply. If the error took place in embryonic existence before gonadal precursors had formed, and may thus have involved a cell giving rise to both gonadal and somatic tissue, this is Figure 3–14.  Mitosis. A culinary whimsy. The cake is a cell about to undergo a mitotic division. The peanut pairs are three double-chromatid chromosomes, submetacentric and metacentric; the reader will need to imagine the other twenty. The other nuts are the spindle mechanism and kinetochores. Courtesy Dr Nooshin Sheidaei. 46  BASIC CONCEPTS somatic-gonadal mosaicism. If a significant fraction of the soma carries the aneuploid cell line, an abnormal phenotype may result; but if not, the fact of a somatic component may not become known until a gonadal event—that is to say, a child born with the (non-mosaic) aneuploidy—has come to notice (e.g., Figure 3–23). Given the huge number of premeiotic mitoses, and, as noted above, opportunity thus for mitotic error, gonadal mosaicism might, in theory, be expected to be common. This indeed seems to be so, and direct analysis of gametes gives similar results for male and female, with premeiotic aneuploidy12 (disomy or nullisomy) rates around 10% (Ghevaria et al. 2022; Zhu et al. 2022b).13 Findings from family searches for gonadal mosaicism have had mixed results. Nimmakayalu et al. (2013) report a case from molecular karyotyping: two siblings with macrocephaly and intellectual disability with a 399 kb 19p13.13 microdeletion, this the only case of gonadal mosaicism being recognized in a cohort of 1,800 patients studied. Campbell et al. (2014) undertook a systematic analysis of parents from a prospectively collected cohort of 100 children with a microdeletion, and they showed four parents to be mosaic for their child’s deletion (the mosaicism level, on blood, was from <1% to 9%). Four out of 100 is a quite surprising number, but perhaps more widely indicative. (These latter cases represent somatic-gonadal mosaicism, and thus could equally have had mention in the following section.) PostMeiotic Mitotic Error and Constitutional Mosaicism A mitotic error can cause phenotypic abnormality by generating, in an initially normal conceptus, an abnormal cell line at some point during early embryogenesis. If we focus on the end result, the feature distinguishing post-conception mitotic from meiotic (or premeiotic mitotic) errors is that the post-conception error typically produces a mosaic conceptus, whereas meiotic/premeiotic errors lead to non-mosaic abnormality. We define constitutional chromosomal mosaicism as the coexistence, within the one conceptus, of two (or, rarely, more) distinct cell lines which are otherwise genetically identical except for the chromosomal difference between them, these cell lines having been established by the time that embryonic development is complete (the point at which the embryo becomes a fetus, around eight weeks post-fertilization). Thus, the different cell lines are fixed in the individual and are a part of his or her chromosomal constitution. The earlier in embryogenesis that a mitotic error occurs, the greater the likelihood for a substantial fraction of the soma to be aneuploid, leading to increasing departure from normality of the phenotype. But it is probable that many mitotically arising abnormalities lead to cell death, leaving no trace, as we go on to discuss. Mosaicism may involve any type of chromosomal abnormality. In a large study in Pham et al. (2014), these authors identified 57 cases of somatic mosaicism among just over 10,000 patients, who had presented with a wide range of phenotypic abnormality (and so, naturally, a selected population). The abnormalities included classic 12 Note that detection of premeiotic aneuploidy requires analysis of immature oöcytes, or alternatively, the analysis of both the metaphase II oöcyte and its corresponding first polar body. 13 Hultén et al. (2013) controversially suggest mitotic errors during ovariogenesis are inevitable and that “most women may be trisomy 21 ovarian mosaics.”

Origins and Consequences of Chromosome Pathology  47 autosomal and sex chromosomal aneuploidy, isochromosomes, ring chromosomes, marker chromosomes, single del/dups, multiple del/dups, and unbalanced translocations. The level of mosaicism in these cases ranged from low (5%) to high (80%). Mosaicism may affect a cell destined to give rise to both soma and gonad: this is somatic-gonadal mosaicism (quite likely the case in some of the patients in Pham et al. above). If the proportion of abnormal cells in the mosaic parent is higher or differently distributed, that parent may manifest some signs of the partial aneuploid state. The father reported in Kennedy et al. (2001) had a dup(8)(p23.1p23.1) in mosaic state, in the ratio normal:duplication of 17:8 on blood sampling, and he himself had a heart defect, as did his non-mosaic dup(8) daughter. Her defect was, however, rather more severe than his. Notably, the daughter was described as “achieving top grades in school,” a very unusual phenotypic commentary in a child with a non-mosaic classical chromosome duplication. A mother and daughter in Freitas et al. (2012) carried a 6.2 Mb deletion at 2q36.1q36.3, mosaic (~15% on blood) in the mother, non-mosaic in the daughter. The intellectually disabled daughter presented an obvious facial dysmorphism, but in the mentally normal mother this was very mild, and only appreciated in retrospect. While “clinical chromosomology” is widely seen as a discipline of application particularly to a pediatric or obstetric population, older age groups are beginning to get a look-in.14 The quite common finding of acquired loss of an X or a Y chromosome in an occasional cell in an older female or male population (and more notably in centenarians) may reflect “normal” age-related anaphase lag (Russell et al. 2007). Mosaicism involving an X or an autosome is linked to advanced maternal age, and inferentially to an increased risk for infertility (Kouvidi et al. 2021). The potential significance of the observation of mosaic loss of a Y chromosome (mLOY) as a marker of biological aging, and an associated risk for a number of common diseases of aging (Sano et al. 2023; Kuznetsova et al. 2023), is discussed in Chapter 15 (p. 472). The suggestion that Alzheimer disease might have a basis in mosaic trisomy 21 or X aneuploidy of some brain tissue is intriguing, but leaves open the question of the time in life at which the putative aneuploid cell line may have become incorporated (Graham et al. 2019; García-González et al. 2023). The bowel, an organ constantly replenishing its epithelium, accumulates microdeletions and microduplications with age, which are not necessarily harmful (Hsieh et al. 2013). Finally, we make mention, but no more than that, of the role of shortening of the telomeres (the chromosomal “caps”) in aging, and potentially in the diseases of aging (Chakravarti et al. 2021); but this is not what we usually think of as being a part of the repository of classic chromosomal abnormality. Vulnerability to Error in the First Few Mitoses in a Euploid Zygote or Early Embryo The first few mitotic divisions from the normal (46,N) one-cell zygote, the fertilized egg, are particularly vulnerable to error, and especially the very first, the number-one 14 Actually, the classic example of disease due to acquired somatic chromosomal mosaicism is, of course, cancer, usually an age-related condition; and a textbook of much larger size than this one would be needed to review the role of tissue-confined aneuploidy apropos (Oromendia and Amon 2014); we make no further mention here. 48  BASIC CONCEPTS mitosis (Currie et al. 2022). Thus it is that the embryo is at risk of constitutional mosaicism. The coexistence of a normal and an aneuploid cell line is called diploid-aneuploid mosaicism. Insight into this vulnerability has come from experience in the IVF laboratory (Chapter 23), and retrospective inferences can be drawn from study of mosaic individuals. In an initially normal zygote, a mitotic nondisjunction at the very first cell division would generate mosaicism for a trisomic and a concomitant monosomic line; in the next few mitoses, the monosomic cell line would likely fail, leaving an embryo with a non-mosaic trisomy. A mitotic error occurring in one of the two cells at the next, the second mitosis, would produce mosaicism with, as well as a trisomic and a monosomic cell line, a normal cell line (Figure 3–15a). In mosaicism due to autosomal nondisjunction, growth of a monosomic cell line is severely compromised, and it will likely die out at a very early stage, leaving just the trisomic cell line (mitosis no. 1 error) or normal and trisomic cell lines (mitosis no. 2 et seq. error) to continue growing.15 The picture may be different with the X chromosome (see below). Surprisingly large fractions, a quarter to a third, of cleavage-stage embryos (day 3) subjected to preimplantation genetic testing (PGT) are chromosomally mosaic, typically with complete aneuploidies (at least in a population of usually older women presenting at an IVF clinic). Mosaicisms are frequently observed at day 4 (the morula); this stage is seen as a watershed, and very many aneuploid conceptuses arrest development here (Mertzanidou et al. 2013). At the blastocyst stage, the study of entire embryos allows the analysis of more than 50 single cells per embryo,16 and at this timeframe even higher levels of mosaicism are revealed: around 80% of embryos contain at least some cells with numerical and/or structural abnormalities, with the majority being euploid-aneuploid mosaic (Chavli et al. 2024). Survival or demise of these very early mosaic diploid-aneuploid conceptuses will depend upon the particular aneuploid chromosome involved, and the extent within the cell mass of the normal cell line. Particularly where a “large”17 aneuploid chromosome is involved, and if it comprises a substantial fraction of the soma, a morula or blastocyst will early succumb and fail to implant or, if proceeding to an embryonic stage, will subsequently miscarry. But “small” chromosome mosaicisms may survive, and mosaic Down syndrome with the karyotype 46,N/47,+21 is the classic example. 15 A very rare example of autosomal monosomy/disomy/trisomy mosaicism was identified in the abnormal baby reported in Stefanou et al. (2006), mentioned below. Only one cell in 200 on blood showed 47,XY,+20, and disomy was demonstrated on buccal mucosal FISH and skin fibroblast analysis, but 39/50 cells from urinary sediment were monosomic. 16 In these experiments, embryos are destroyed, as distinct from embryo biopsy in PGT, where only 5–10 cells are removed. 17 A “large” chromosome refers to size but also to content, in terms of the number, and roles, of the genes therein (Figure 1–4). In this context, chromosome 19, while of short length, is particularly gene-dense and so could be considered functionally large. Chromosome 21 is “small,” both in terms of length and in being gene-sparse.

Origins and Consequences of Chromosome Pathology  49 In one particular well-known survivable autosomal aneuploidy, trisomy 8 mosaicism, somatic nondisjunction accounts for practically all cases (Karadima et al. 1998). Insight into the timing of the abnormality can be gained from inference in the study of mosaic individuals, and X chromosome mosaicism has been particularly informative. An aberrant mitosis involving the X chromosome, in an initially 46,XX zygote, may generate X and XXX cell lines, both of which would be survivable. If this happens at the first mitosis, X/XXX mosaicism would result. If at any later mitosis, a normal cell line would exist, and the mosaic state would be X/XX/XXX (Essouabni et al. 2023) (Figure 3–15b). The same can happen in a 46,XY zygote, with an X/XYY or an X/XY/XYY mosaicism resulting (the gender in the embryo being determined according to the sex chromosome composition of gonadal tissue). Similarly, Jacobs et al. (1997), in a study of Turner syndrome, observed that patients with Xq isochromosome mosaicism hardly ever have a Figure 3–15.  The Generation of Mosaicism. (a) Postzygotic nondisjunction in an initially normal conceptus. In this example, one cell line (monosomic 21) is subsequently lost, with the final karyotype 46,N/47,+21. (b) Postzygotic nondisjunction in an initially normal 46,XX conceptus, resulting in 45, X/46,XX/47,XXX mosaicism. (c) Postzygotic anaphase lag in an initially abnormal 47,+21 conceptus; this leads to a “corrected,” or “rescued,” normal cell line. 50  BASIC CONCEPTS 46,XX cell line: Most are 45,X/46,X,i(Xq). This is what would be expected if the error happened at the very first mitosis of the initially 46,XX zygote. If it happened at the next two or three divisions, a 46,XX cell line would also have been present, and thus three cell lines, 45,X/46,XX/46,X,i(Xq). The presence of three cell lines in Stefanou et al. (2006), who describe an abnormal infant with diploid/aneuploid trisomy 20 mosaicism on blood, but with a monosomic 20 cell line identified in urinary epithelial cells, similarly allows the inference of an initiating error at, or a little later than, the second mitosis. Actually, about 5% of standard apparently non-mosaic 47,+21 is due to a postmeiotic mitotic defect from a 46,N zygote (Antonarakis et al. 1993), with the “third” chromosome 21 equally likely to be maternal or paternal. In 3% of apparently non-mosaic 47,XXY and 9% of 47,XXX, the error was post-zygotic, presumably prior to the formation of the inner cell mass18 (MacDonald et al. 1994). As noted above, the nature of the mosaicism can indicate the likely time of its generation. More than one mitotic error can happen, separate in time and place; for example, DeBrasi et al. (1995) identified concomitant 45,X, 46,X,+8, and 47,XX,+8 in a woman with clinical features of both trisomy 8 and Turner syndrome, in whom the molecular study supported the hypothesis of an originally 46,XX conception. Mosaicism Arising from Error at Later Mitoses in a Euploid Embryo The categories of mosaicism described above are associated with phenotypes of varying degrees of severity, and with the mitotic event having caused the mosaicism of early occurrence during embryogenesis. But if the aberrant mitosis takes place later in embryogenesis, perhaps some thousands or millions of cell divisions along the way to the establishment of the fetal, and thus, eventually, post-natal anatomy, the effect upon the phenotype may be minimal, indiscernible, or without any effect at all.19 Perhaps, as we now go on to discuss, every apparently 46,XX or 46,XY person is, somewhere in their anatomy, a mosaic. Consider the following. A classic chromosome test on any normal person—a routine analysis from a sample of peripheral blood, or from a “spit” sample—would probably get a normal result (46,N). We would conclude from an analysis of a dozen or so cells from one particular tissue that the rest of the soma is also 46,N. In most of the person’s tissue, this will be truly the case. But the body comprises a vast number of cells—ten trillion (1013) or so—which required a vast number of mitoses for their generation. The dozen cells checked in the laboratory are only a ten-billionth of a percent of all the person’s cells, and we routinely (and, for practical purposes, not unreasonably) regard this minute fraction as a valid representative of the remaining 99.9999999999%. Notwithstanding, we can surely suppose that one or more errors will have happened during one or some of the many mitoses, and these will have produced a chromosomally abnormal cell line, and the person is really a chromosomal mosaic. It seems plausible to 18 The “inner cell mass” is seen as a clump of cells on part of the inner wall of the blastocyst, and these cells are destined to form the embryo (Figure 23–6). The remaining cells, comprising the shell of the blastocyst, are trophoblasts, and will form the placenta. 19 But not overlooking, as noted above, that some chromosomal mosaicisms of restricted extent, or of late acquisition, can lead to certain adult-onset diseases.

Origins and Consequences of Chromosome Pathology  51 imagine that unrecognized islets of mosaicism, involving a tiny number of cells—only a few thousand, perhaps—could well be a frequent state. Almost certainly, somewhere in their soma, everyone may be such a mosaic; but this fascinating academic matter is not a question of much practical relevance in the genetic counseling clinic. Classical cytogenetics can (if the tested tissue is representative) show mosaicism unequivocally, with the recognition of two different karyotypes, but this is dependent on there being enough cells in the less frequent line for the observation to be made. In molecular karyotyping, detection is a subtler exercise, and it is based on an appreciation of a quantitative shift in the log2 graph, or, if single nucleotide polymorphism array is the methodology, the genotyping pattern may be revealing; but in principle, mosaicism that might otherwise have slipped through on classical analysis can be picked up (Repnikova et al. 2012). The practicalities of this question are discussed below, Tissue Sampling in the Detection of Mosaicism. Mosaicism Arising from Error in an Aneuploid Zygote or Embryo Nondisjunction can occur in a post-zygotic mitosis in a conceptus that is initially trisomic for an autosome (for example, 47,+21). Thus, one copy of the homolog in question is lost. The same result may be due to the mechanism of anaphase lag.20 This converts the trisomy in this cell to 46,N and is sometimes referred to as “correction” or “rescue.” Its descendant cells are 46,N, and the karyotype of the conceptus is, for example, 47,+21/46,N (Figure 3–15c). Most mosaic trisomy/disomy 13, 18, 21, and X, arises in this way: for example, 47,XXY → 46,XY/47,XXY (Robinson et al. 1995). A conceptus with what might be called interchange tertiary trisomy—that is, a 47-chromosome count, with the two translocation chromosomes and an additional copy of one of the derivative chromosomes—might generate a cell line with the balanced state, if one of the derivatives is lost post-zygotically. Thus, a zygote with, for example, a 47,t(1;2),+der(1) karyotype might acquire a cell line with 46,t(1;2). If this cell line included blood-forming tissue, but if much of the soma otherwise consisted of cells with the unbalanced state, a phenotypically abnormal child could have, on blood sampling, a balanced translocation karyotype. Such a case is presented in Dufke et al. (2001) concerning an abnormal child who, on blood, had a familial balanced karyotype 46,t(17;22), but who on skin biopsy karyotyped 47,der(22)t(17;22), and was thus shown to be duplicated for segments of 17 and 22. These authors speculate that this scenario might be a rare contributor to the apparent slight excess of abnormal children among the balanced carrier offspring of translocation carrier parents (p. 123). Gonadal mosaicism can arise due to this mechanism, and again the classic example comes from mosaic trisomy 21, in which a 47,+21 embryo at a very early post-zygotic 20 In this latter case, the chromosome fails to connect to the spindle apparatus, or is tardily drawn to its pole, and fails to be included in the reforming nuclear membrane. On its own in the cytoplasm, it will form a micronucleus and soon be lost. 52  BASIC CONCEPTS stage discards the extra chromosome in one cell, thus giving a 46,N lineage. This 46,N line then contributes to most tissues, and an apparently normal physical phenotype results; but the gonad is not so fortunate, as it were, and may receive a larger fraction of 47,+21 cells (Kovaleva 2010). A clinical observation supporting this conclusion is that the low-level mosaic mothers (who had presented due to their having had a non-mosaic Down syndrome child) are of a typical maternal age range, whereas their mothers— the grandmothers of the Down syndrome children—were of older maternal age at the time their daughters had been born. This is consistent with their daughters having been conceived as (maternal-age-influenced) trisomy 21, but subsequently, at least in their soma, “corrected.” Post-zygotic “Correction” of Aneuploidy and Uniparental Disomy. If the conversion of trisomy to disomy occurs prior to the formation of the inner cell mass, and if the 46,N line then gives rise to the inner cell mass, the embryo will be non-mosaic 46,N. According to which one of the three chromosomes was lost, normal biparental disomy in the embryo could be restored, or uniparental disomy (UPD) could result (Figure 3–16). This is much the usual mechanism of formation of UPD. It is at prenatal diagnosis, typically, that the fact of this rescue mechanism comes to be discovered, with trisomy seen at chorionic villus sampling (CVS) and disomy at a subsequent amniocentesis (Sirchia et al. 1998). Chromosome 15 is of particular concern, and Purvis-Smith et al. (1992) and Cassidy et al. (1992) provide historic illustrations in pregnancies showing 47,+15 at CVS, with conversion to 46,N at amniocentesis— but the infants had UPD(15)mat, and so they were born with Prader-Willi syndrome. A contemporary example is the diagnosis of UPD(15)mat following the detection of trisomy 15 at noninvasive prenatal testing (NIPT) (Hong et al. 2023). An inference of “rescue” may be made in the case of UPD discovered because of isozygosity for a recessive gene, and an example of this is deafness due to the connexin-26 gene. Yan et al. (2007) report a child presenting with deafness due to homozygosity for the common 35delG mutation, for which his father, but not his mother, was a carrier. As it transpired, the child had UPD(13)pat, with isodisomic and heterodisomic segments of chromosome 13. The segment in 13q12.1, which contains the connexin-26 locus, was one of the regions of isodisomy. Quite possibly, this had been a trisomic 13 conception but rescued due to discarding one of the chromosomes, which happened to be the maternal chromosome 13. Had it not been for the coincidence of the father’s heterozygosity for the 35delG mutation, the rescue would have been entirely successful. Post-zygotic correction can also happen in the other direction, as it were: to convert a monosomic zygote into a disomic one. It is very rarely recognized (Schinzel et al. 1993). Quan et al. (1997) report a girl, 46,XX, with Duchenne muscular dystrophy due to a homozygous deletion of exon 50 of the dystrophin gene. She had homozygosity of the X chromosome for all of the tested marker loci, apparently a complete maternal uniparental isodisomy X. Even a meiosis II nondisjunction would likely have had some heterozygosity, due to recombination at meiosis I; and so Quan and colleagues propose a mitotic mechanism. A 45,X0 conception, from a “22,0” sperm + 23,X egg at syngamy, underwent duplication, or possibly nondisjunction, of the single X chromosome. Unfortunately, this X chromosome carried a de novo Duchenne mutation.

Origins and Consequences of Chromosome Pathology  53 Mosaicism Due to Somatic Recombination in Homologs Genetic exchange can take place as a normal event during a mitotic cycle, involving either the pair of homologous chromosomes or the sister chromatids of one chromosome. The cytogenetic demonstration of sister chromatid exchange (SCE) is rather dramatic (Figure 16–3). Should the SCE be unequal, tandem duplication and deletion lines may be generated. If the deletion line is lost, a normal/duplication mosaicism results (Rauen Figure 3–16.  Uniparental disomy from “correction” of a trisomic conceptus by loss of a homolog. Nondisjunction* at meiosis I, followed by postzygotic loss** of one homolog, causes uniparental heterodisomy. (If, for example, this were chromosome 15, and the meiotic nondisjunction occurred in the mother, the child would have Prader-Willi syndrome.) Nondisjunction at meiosis II would cause uniparental isodisomy. 54  BASIC CONCEPTS et al. 2001). According to the somatic extent of the abnormal cell line, the phenotype may or may not be affected. CHIMERISM Chimerism, which is to be distinguished from mosaicism, is the coexistence of more than one cell line in an individual due to the union of two originally separate (“sibling”) conceptions (Chen et al. 2013f; Madan 2020).21 It could be imagined that dizygotic twin blastocysts happen to make contact and then fuse, and this may be the more typical scenario. Since four gametes will have contributed to the person, we may hear the expression “tetragametic” chimerism. Alternatively, but rarely, there might have been two sperm fertilizing an ovum and a polar body. A 46,XX//46,XX or 46,XY//46,XY chimera would most probably present as a normal female or male, whereas 46,XX//46,XY could manifest an abnormality of sexual differentiation (Wimmer et al. 2022). The discovery of chimerism can cast a most remarkable light in certain cases in which parenthood is being tested. A mother apparently “could not have been” the mother of two of her three sons, when she and the family underwent HLA (immune histocompatibility) testing ahead of a planned kidney transplant. But it transpired that she was a tetragametic 46,XX//46,XX chimera. Her ovaries presumably comprised tissue from both fused conceptuses, but blood-forming tissue came from only one. Thus, she could have children who had neither of her blood-test HLA haplotypes (Yu et al. 2002). Similarly, a father who “failed” a paternity test from a son conceived at IVF— could this have been a laboratory mix-up?—turned out himself to be a tetragametic 46,XY//46,XY chimera. The child came from sperm due to tissue deriving from the father’s absorbed fraternal co-twin, and his genetic profile was that of a nephew of his father (Baird et al. 2015). The more usual form is “confined” chimerism, in which only one tissue—that is, blood—possesses the two cell lines. This is due to twin-to-twin (or feto-fetal) transfusion, when dizygous twins have intimately opposed placentae, allowing vascular connections (“anastomoses”) to form between them, with marrow colonization by the other twin’s hematogenous cells. Sudik et al. (2001), for example, describe a woman typing XY in almost all (99%) of peripheral lymphocytes, but she was 46,XX on three other tissues, including ovarian; she had had a twin brother who had died as a neonate. Somewhat stretching the analogy, Bianchi (2000) makes the intriguing suggestion that, due to the retention and persistence of fetal blood cells following delivery, every mother is, in a sense, a hematologic (micro)chimera. TWINNING Dizygous twinning is more frequent in mothers in their late 30s, and so it is not remarkable that occasionally twins are born, one with normal chromosomes and the other with a maternal-age-related aneuploidy. Monozygous twinning could happen in an abnormal conception just as in a normal one, and the occasional instance of twins concordant for an abnormal karyotype is to be expected (Schlessel et al. 1990). Rather more remarkable is the case of monozygous (MZ) twins discordant for karyotype—clearly, 21 For the record, the chimera of classical mythology was “in the forepart a lion, in the hinder a serpent, and in the midst a goat.” Note the // descriptive format.

Origins and Consequences of Chromosome Pathology  55 the adjective “identical” is inappropriate here! Rogers et al. (1982) studied monochorionic twin brothers, one 46,XY and the other 47,XY,+21 with Down syndrome, in whom genetic analysis supported a diagnosis of monozygosity; similarly, Chanes et al. (2021) describe MZ twins, one normal, the other with ring(13) mosaicism. In this type of twinning, the assumption is that either a mitotic nondisjunction or a mitotic rearrangement occurred in one of the two monozygous embryos from an initially normal conception; or, vice versa, an initially 47,XY,+21 conceptus underwent splitting, with loss of a chromosome 21 then occurring in one of the newly created embryos. A number of similar cases are on record,22 including monozygous twins of opposite gender (Lewi et al. 2006; Stemkens et al. 2007; Zech et al. 2008). Perhaps the most extraordinary circumstance of chromosomal discordance in monozygous twins concerns the acardiac (that is, lacking a heart) fetus. Trisomy 2 is one of the aneuploidies observed (Mihci et al. 2009). An initially normal conceptus might generate a trisomy 2 cell line that then separates and produces the co-twin, or an initially trisomic conceptus gives rise to a “corrected” lineage. It is only the presence of the normal twin that allows the acardiac co-twin to survive, at least temporarily, with placental vascular connections providing blood circulation (twin reverse arterial perfusion, TRAP) from normal to abnormal twin. We have seen such a case due to trisomy 3, with the affected acephalic, acardiac fetus surviving through to the second trimester, of barely recognizable human form (and see Figure 20–24). STRUCTURAL REARRANGEMENT The following classical structural rearrangements may be listed: translocations, insertions, inversions, isochromosomes, duplications, deletions, rings, and complex rearrangements. These may be very obvious on classical karyotyping or, for smaller deletions or duplications (3–5 Mb), may have required high-resolution banding for identification. With molecular karyotyping, imbalances of submicroscopic size, typically measured in kilobases, are detectable: these are referred to as “microdeletion” and “microduplication,” or alternatively, as “copy number variants” (CNVs). In general parlance, the terms microdeletions and microduplications are often used in a setting of assumed pathogenicity, whereas CNV may need a qualifying adjective of benign, pathogenic, or of uncertain status. All arose de novo at one point—whether with the index case in whom the abnormality was discovered, or in a parent or more distant ancestor, with a balanced or unbalanced form transmitted thereafter in the family. Jacobs (1981) derived the following mutation rates for the generation of de novo classical rearrangements: 1.6 × 10–4 per gamete for the balanced reciprocal translocation, and 2.9 × 10–4 per gamete for unbalanced rearrangements.23 Concerning CNVs, the mutation rate for larger (>500 kb) microdeletions/duplications is 6.5 × 10–3 per gamete (Itsara et al. 2010). In other words, out of 100,000 gametes, on average 16 will have a balanced and 29 an unbalanced de novo classical rearrangement, while 650 will have a de novo larger CNV. 22 It is plausible that in some instances, the presence of different cell lineages contributes to the monozygotic twinning process. 23 These conclusions were derived from data of several studies of pregnancies that were able to “survive long enough to give rise to a recognized pregnancy”: in other words, from clinical miscarriage and newborn data. Jacobs acknowledges that these figures will surely be underestimates. 56  BASIC CONCEPTS Mechanisms of Formation of Structural Rearrangement The human karyotype is hostage to the fine detail of its structure. Molecular analysis of structural chromosome rearrangements and their breakpoints has shed light on the underlying generative mechanisms. In particular, it is clear that different mutational mechanisms apply for recurrent versus non-recurrent rearrangements. Recurrent rearrangements share essentially the same size and genomic content in unrelated individuals, the breakpoints being fixed by the presence of highly homologous long flanking repeats. In contrast, non-recurrent rearrangements have a size and genomic content that is unique to the individual. Carvalho and Lupski (2016) and Burssed et al. (2022) provide comprehensive reviews. Recurrent Rearrangements and Nonallelic Homologous Recombination The common basis for recurrent del/dup rearrangements lies in the existence of long tracts of DNA sequences, generally of some thousands of base pairs, known as segmental duplications or low copy repeats (LCRs). These LCRs are seen throughout the genome (comprising 6.6% of the genome; Nurk et al. 2022), and are sufficiently similar (“paralogous,” rather than exactly homologous) that they enable the erroneous coming-together of different chromosome regions. The two sequences involved in a particular exchange have a length of near-perfect homology, and this is the site of the actual strand exchange. In other words, “ectopic synapsis” sets the scene for a subsequent “ectopic homologous recombination.” This is referred to as nonallelic homologous recombination (NAHR; Figure 3–17), and meiosis is the usual setting. Non-Recurrent Rearrangements and Nonhomologous End Joining Non-recurrent structural rearrangements are characterized by the seemingly random locations of breakpoints, leading to individual del/dups of unique size and genomic content. The absence of any long-tract sequence homology surrounding these rearrangements is indicative of nonhomologous end joining (NHEJ). During mitotic cell division (and including in the premeiotic gametocyte), different chromosomal segments may happen to be in close proximity due to their “geographical space” within the nucleus. Then, in the simple case, if breaks occur during replication, instead of the correct ends being brought back together, the broken ends of different segments may inappropriately be ligated. More complex mechanisms underlying this breakage and rejoining include fork-stalling and template-switching, and microhomology-mediated break-induced replication (Burssed et al. 2022). Setting in Which De Novo Rearrangement Occurs While rearrangement could, in principle, occur during either meiosis or mitosis, and in the gonad of either sex, in fact, different chromosomal forms differ in this respect. Most

Origins and Consequences of Chromosome Pathology  57 Robertsonian translocations arise in oögenesis, at a maternal meiosis (Bandyopadhyay et al. 2002). Microdeletions/duplications can occur in both gonadal types and mostly at a meiosis, at least inasmuch as the common cases of 7q11, 15q11q13, and 22q11 may be considered to be representative (Thomas et al. 2010). On the other hand, spermatogenesis is the setting for almost all de novo non-Robertsonian reciprocal rearrangements, and both meiosis and premeiotic mitoses may be the site (Höckner et al. 2012). Balanced, Apparently Balanced, and Functionally Unbalanced Rearrangements Structural rearrangements can be balanced, with the correct amount of genetic material in a cell, or unbalanced, with a deletion and/or duplication of genetic material. Arguing somewhat circularly, in the phenotypically normal person it is inferred that although such an individual’s genetic material is in a different chromosomal arrangement, it is present in the correct (balanced) amount, and functioning properly. It is irrelevant to the person’s health, other than his or her reproductive health. It may be helpful in explaining this to think of the person’s genome as a recipe book—a series of instructions for everything that is genetically determined. If an error occurs in the pagination (a translocation) and, for example, pages 17–24 are inserted between pages 36 and 37, the recipes are all Figure 3–17.  Non-Allelic Homologous Recombination. Notes: NAHR is the basis of many deletions and duplication. In this construction, the grey arrows represent DNA segments of 90%–98% similarity, and orange arrows of segments of >98% similarity. Between the two >98% sequences is a segment of unique DNA (black line, asterisked). An inappropriate lining-up of a pair of very similar sequences is followed by crossing-over, at X above. A new hybrid “orange segment” is created from part of one and a part of the other (the two parts either side of the dotted lines), but this will have practically the same DNA sequence as it did before. In consequence of this recombination, chromosomes are generated, one with a deletion of the segment of unique DNA, and the other with a duplication. Two classic examples both reside in chromosome 17: Smith-Magenis syndrome and Potocki-Lupski syndrome are due to deletion and duplication, respectively, for the segment 17p11.2p11.2. A little further up the 17 short arm, a 1.7 Mb segment within 17p12, including the PMP22 gene, is deleted in hereditary pressure-sensitive neuropathy, and duplicated in Charcot-Marie-Tooth neuropathy. Source: Drawn after CMB Carvalho and JR Lupski, Mechanisms underlying structural variant formation in genomic disorders, Nat Rev Genet 17:224–238, 2016. Courtesy JR Lupski, and with the permission of Springer Nature. 58  BASIC CONCEPTS still there; they are still perfectly capable of being read. If a sequence of pages is inserted upside down (an inversion), one need only turn the book around to read them. If a phenotypically abnormal person has a rearrangement that is balanced on classical laboratory study, one can only speak, at the cytogenetic level, of the rearrangement being “apparently balanced.” In the case of an associated cognitive impairment, one can suggest (and more so in a de novo case), but not necessarily state with certainty, that the observed phenotype may be due to the identified karyotype. Modern methodologies can clarify such cases as being either indeed truly balanced, or with very subtle imbalances. A rearrangement that is balanced at the genomic level may yet lead to a phenotypic consequence, due to gene disruption or due to “position effect.” As an example of gene disruption, consider the well-known PMP22 gene at 17p11.2, the basis of Charcot-Marie-Tooth and pressure-sensitive neuropathy (Chapter 14). Nadal et al. (2000) studied a mother and son, both of whom presented with pressure-sensitive neuropathy. The classical cytogenetic study showed them to be heterozygotes for the apparently balanced translocation t(16;17)(q12;p11.2). Applying the technology of the day, the chromosome 17 breakpoint was shown to have been sited actually within the PMP22 gene. This disruption would have led to a functional haploinsufficiency, which is known to be the basis of pressure-sensitive neuropathy. Another such example is described in Dupont et al. (2013), who identified a COL2A1 disruption in a family with Stickler syndrome, the affected persons having inherited a rcp(12;15) (q13;q22.2); COL21A is located at 12q13.11. Redin et al. (2017) and Lowther et al. (2022), applying molecular methodology, address the question in the study of large cohorts of translocation carriers. Comparing those with a normal (n = 304) and those with an abnormal (a developmental disorder) phenotype (n = 406) in Lowther et al., those with an abnormal phenotype had a sevenfold increased likelihood, compared to the normal group, to have the breakpoint disrupt a known developmental gene. As one such example, the TCF4 gene (Chapter 14; Pitt-Hopkins syndrome) at 18q21 was disrupted in some of the phenotypically abnormal cases but none of the normals. Concerning position effect, one of the earliest examples is of the SOX9 gene at 17q25.1: Chromosomal rearrangement in the vicinity can result in loss of long-range regulation and hence non-expression of the (intact) gene, with sex reversal, campomelic dysplasia, and Pierre Robin syndrome possible phenotypic consequences (Gordon et al. 2014) (Figure 3–18). Sophisticated methodologies can reveal in closer detail the Figure 3–18.  Position Effect in an Apparently Balanced Translocation. Notes: An apparently balanced translocation causing the syndrome of campomelic dysplasia (which includes skeletal, genital, and brain defects). One breakpoint is at 17q25.1, on or close to the SOX9 locus (shown as dot on the cartoon karyotype), where the basis of the syndrome lies. One possibility is that the gene is disrupted. Or, an influence of adjacent chromosome 5 chromatin (“position effect”) leads to inactivation of the SOX9 gene on the der(17), the functional SOX9 haploinsufficiency then being responsible for the phenotype. Source: From R Savarirayan and A Bankier, Acampomelic campomelic dysplasia with de novo 5q;17q reciprocal translocation and severe phenotype, J Med Genet, 35:597–599, 1998.

Origins and Consequences of Chromosome Pathology  59 pathogenetic mechanism in some such cases, in which a region of influence—a “topologically associated domain” (TAD)—is implicated. Wallis et al. (2021) studied a family with a history of lymphedema and distichiasis (LDS), segregating a rcp(16;22) (q24;q13.1), and showed that the (normal) LDS locus at 16q24.1, FOXC2, this locus lying within 16q24, had been separated from its normal control due to the translocation breakpoint being close by, 120 kb upstream. This compromise of genetic control led to the appearance of the syndrome. There may be no loss or gain of DNA in such cases; in other words, the rearrangement is genomically balanced, but functionally unbalanced. MICRODELETIONS, MICRODUPLICATIONS, AND COPY NUMBER VARIANTS OF INCOMPLETE PENETRANCE AND VARIABLE EXPRESSIVITY While the classic chromosome disorders are characterized by complete penetrance,24 the picture with the molecular-defined microdeletions/duplications is different. Some carriers may display no clinical abnormality, and yet a child of theirs may have presented with obvious symptomatology. If the “normal” parent is studied more closely, microsigns of the phenotype associated with that imbalance may be discerned; but they remain within the range of what is considered normal in the general population, and they can function as independent, productive adults. These “normal” parents are considered to be nonpenetrant with respect to the imbalance in question. It is more particularly with autism (Chapter 25) and non-dysmorphic cognitive impairment that these matters apply. The basis of this variation may lie in the coexistence of different CNVs or SNP variants, most likely on another chromosome, and inherited quite coincidentally. Each imbalance might not suffice to cause a phenotype on its own, but the two together may add up to abnormality. This is the “two-hit” hypothesis (Girirajan et al. 2010; Atli et al. 2022). Most often, one hit (the first hit) will be the more important, and the second hit could be due to any one of a number of different “lesser” imbalances, which typically would warrant being called no more than a “VUS” (variant of uncertain significance).25 It might be more accurate to speak of two or more hits multiplying together, rather than adding up, to produce a phenotype of combined effect. Multiple hits contribute to a “liability threshold” beyond which a phenotype emerges (Pizzo et al. 2019; Jensen et al. 2021; Smolen et al. 2023). This concept is illustrated in study of the well-known 16p12.1 deletion (Figure 3–19). These further hits comprise CNVs, rare sequence variants of variable impact, or the combined effect of multiple common variants (in other words, polygenic inheritance). Rosenfeld et al. (2013) reviewed a number of the more common microdeletions/ duplications (“first-hit” imbalances), establishing penetrance estimates ranging from 10% to 62% (the 62% referring to the 16p11.2 microdeletion just mentioned). Their 24 Penetrance is a quantitative descriptor and refers to the percentage fraction of a particular genetic cohort who show phenotypic abnormality. In such a population, in which the fraction is less than 100%, we may speak of incomplete penetrance; in a single individual showing no abnormality, this is nonpenetrance. Expressivity is qualitative, and reflects the range of clinical manifestation in those in whom a condition has been penetrant. It may sometimes become a matter of semantics whether a subtly abnormal person is considered to represent nonpenetrance, or penetrance with very mild expressivity. 25 A rare and different form of two-hit mechanism concerns the “unmasking” of a recessive allele on a normal homolog, due to a deletion of that segment on the other chromosome (Poot 2012; Paciorkowski et al. 2013).

60  BASIC CONCEPTS work was since followed up by others (Coe et al. 2014; Kendall et al. 2019; Redaelli et al. 2019), and culminating in the review of Goh et al. (2025) as listed in Appendix C. EPIGENETICS AND GENOMIC IMPRINTING A formal definition of an epigenetic effect includes these points: The DNA sequence of a particular gene remains unaltered, but the capacity of this gene to be expressed is altered. The expression “genomic imprinting” is applied in the setting of epigenetic effects that are imposed during germline transmission. Some parts of some chromosomes are subject to genomic imprinting as a normal occurrence, and this imprinting is parent-specific; that is, genes in the chromosomal segment are expressed, or not expressed, according to whether the chromosome had been transmitted in the sperm or in the ovum (“parent-of-origin effect”). An imprinted segment takes up an “epigenetic mark,” and the gene or genes in this segment are not expressed, leaving it to the corresponding locus or loci on the homologous chromosome from the other parent to be the only source of expression. When the phenomenon was first appreciated in humans, it was naturally suspected that many forms of congenital abnormality might be due to aberrant imprinting. As it has transpired, however, the practical application of genomic imprinting appears to be confined to a rather small number of cytogenetic conditions (Chapter 19). Nevertheless, the theoretical interest is considerable. Most of the autosomal genome is not subject to imprinting, and it is functionally disomic. That is, with each locus having a pair of alleles, each of the pair is functionally active, contributing more or less equally to the genetic output from that locus.26 This is biallelic gene expression. A minority of the genome is subject to imprinting and requires only one of the pair of alleles to be active, while the other one becomes inactivated 26 But exceptions exist, and approximately 5% of autosomal genes are randomly expressed from only one or other parental allele (Gimelbrant et al. 2007). Figure 3–19.  The 16p12.1 Deletion in a Proband, and in the Carrier and Non-Carrier Parent. Notes: The thresholds are shown for developmental and psychiatric manifestation, according to an individual’s genetic load: the presence of the 16p deletion; the carriage of second-hit variants; and the load otherwise of variants relating to an autism polygenic risk score. The deletion is carried by the proband and the carrier parent. Some carrier parents may manifest a relatively mild psychiatric phenotype. The non-carrier parent may have a number of different rare variants (blue), which of themselves, in this setting, are harmless, along with a variable polygenic load. But the combination, then, in the child, of the 16p deletion, along with a number of rare second-hit variants (yellow), combined with a polygenic load (polygenic risk score, blue), combine to cross a threshold sufficient to convey a developmental phenotype.

Origins and Consequences of Chromosome Pathology  61 (“silent”); in other words, the locus is functionally monosomic, with a genetic output from only one allele. This is monoallelic expression. If the allele of maternal origin is inactivated, only the allele of paternal origin is functionally active, and vice versa. Following conception, the imprint remains through cycles of post-conceptional somatic mitoses: The chromosome “remembers” the sex of the parent who contributed it (put differently, it retains its epigenetic mark). The imprinting pattern may be specific to a certain tissue or to a certain developmental stage (Ideraabdullah et al. 2008). Thus, in some tissues a gene may express monoallelically, whereas in other tissues biallelic expression is retained; or a gene may express monoallelically in a specific tissue at one stage in embryogenesis, and biallelically thereafter. X chromosome inactivation is a special case. Parent-of-origin imprinting is a normal mechanism of gene regulation. It is mediated through a process taking place during gametogenesis, of which the physical basis includes methylation of cytosine bases within the gene(s) or in controlling sequences upstream of it. This process is reversible, and in the “life” of an autosomal allele or chromosomal segment, as it passes from individual to individual down the generations and across the centuries, imprinting—the epigenetic mark—will be acquired, maintained, lost (“erased”), reacquired (“reset”), and lost again, according to the sexes of the individuals through whom it is transmitted. Throughout, it retains the same DNA sequence. Mechanisms Whereby Functional Genetic Abnormality Can Arise In the context of imprinting, we may consider three categories of functional genetic defect. These are as follows: uniparental disomy with overexpression or non-expression of genes in certain chromosomal segments; deletion with non-expression; and relaxation of imprinting with overexpression. Uniparental disomy will lead to either biallelic expression, or to no expression, at the locus or loci within the imprintable segment. If a deletion removes a chromosomal segment that would otherwise have been “silenced,” all that is lost is a nonfunctioning genetic segment, and there is no untoward consequence. On the other hand, if the deletion removes the segment on the active chromosome, the corresponding part of the other homolog is inactive, and so neither chromosome will be genetically functioning in this segment; in a sense, the silent allele is unmasked. Relaxation of imprinting allows a segment that should be non-expressed to lose its imprint. The locus or loci contained therein will be operating biallelically, which will be, theoretically, at double normal capacity. These mechanisms are dealt with in detail in Chapter 19. CONSEQUENCES OF GENETIC ABNORMALITY Structural Imbalance Our physical anatomy is due to our chromosomes (Gardner 2016; Anastasiadou et al. 2024). Chromosome imbalances are harmful because of the fundamental reason that many genes are dosage-sensitive. And if megabase amounts of chromatin are involved, it is highly likely dosage-sensitive genes will be included in the imbalanced segment(s). In duplications, there is 150% of the normal amount of this chromosomal segment, 62  BASIC CONCEPTS and in the deletion there is 50% of the normal amount. The imbalance involves a whole chromosome (full aneuploidy) or a part of a chromosome. From classical cytogenetics, the latter state is described as partial or segmental aneuploidy, a deletion or a duplication. In molecular karyotyping, in which the focus is on much smaller segments, the terms used are microdeletion and microduplication, or CNV deletion or duplication. An incorrect amount of dosage-sensitive genetic material in every cell of the conceptus distorts its development to a greater or lesser extent. As discussed above, large losses or gains almost invariably set early anatomical development so awry that natural abortion occurs. Lesser imbalances may be compatible with continued intrauterine survival, but with the eventual production of a phenotypically abnormal child. Very minor partial aneuploidies may cause defects that are not readily detectable in early infancy; and some chromosomal “defects” may be without phenotypic effect. However, as a first principle, anything but 100% of the normal amount of genetic material (in classical, megabase cytogenetic terms) produces a less than 100% normal phenotype. Cognitive impairment is the almost universal consequence of classical autosomal imbalance, while much intellectual disability is due to a chromosome abnormality. On the other hand, imbalances detectable only by molecular karyotyping (CNV del/ dups) may be so small that no dosage-sensitive material is affected, and the phenotype is unaffected; or, the imbalance may only lead to phenotypic abnormality when it exists in the company of another micro-imbalance elsewhere in the genome (“second-hit” effect, as discussed above). The distinction between very small classical and larger molecular imbalances is not as clear-cut as the foregoing might suggest, and indeed some cases could carry either description; but it is useful nevertheless to consider these as two categories. It is generally too simplistic to think of deletions and duplications leading to opposite qualities of phenotype (Neri and Romana Di Raimo 2010). But in some instances the concept of “type and countertype,” originally proposed by Lejeune (1966), may be invoked. Deletion of 7p15 may cause the cranial bones to fuse prematurely (craniosynostosis) due to abnormal behavior of osteoblasts at their periphery, whereas duplication leads to underdevelopment of the skull, with a large and confluent fontanelle (Stankiewicz et al. 2001) (Figure 14–31). Deletion of 15q26.1qter (which removes the growth factor locus IGFR1) is associated with intrauterine growth retardation, whereas dup(15)(q26.1qter) may cause a syndrome of post-natal overgrowth (Faivre et al. 2002; Nagai et al. 2002). Similarly, carriers of reciprocal CNVs at 16p11.2 exhibit mirror phenotypes of obesity/macrocephaly (deletion) and underweight/microcephaly (duplication) (Loviglio et al. 2017). Assessment of Imbalance With respect to classical degrees of cytogenetic imbalance, the blunt quantitative tool of haploid autosomal length (HAL; see Appendix A) measurement can be applied, although this may now be of somewhat historic interest in the molecular era. The largest chromosome, no. 1, comprises 8.66% of the HAL, whereas chromosome 21, the smallest, is 1.77% (Table A–1). As a very general rule, if the imbalance consists of less than 1% of HAL (corresponding to ~30 Mb), the conceptus is often viable in utero, and live birth frequently results. If the excess is greater than 2%, in utero lethality, with spontaneous

Origins and Consequences of Chromosome Pathology  63 abortion, is likely. Imbalance involving autosomal deficiency (partial monosomy) is generally much less survivable than is duplication (partial trisomy). A qualitative assessment is more to the point: chromosomal segments vary considerably in respect of their genetic content. Insight is gained on the empiric observations of phenotypes. Some large segments (e.g., 9p, all of 21) appear to have a substantial pre- and post-natal survivability in the trisomic state, whereas a lesser number of segments (e.g., distal 4p) are often viable when monosomic. Chromosome 13 provides the most impressive examples of viability for a large autosomal imbalance. Trisomy for the whole of chromosome 13—fully 3.7% of the HAL—frequently goes through to live birth, and in the 13q– deletion syndrome, monosomy occurs for up to 2.5% of HAL. This reflects the low gene density on this chromosome, only 6.5 genes per Mb (Table 1–1). The same principle applies to chromosomes 18 and 21.27 On the other hand, chromosome 19, although small, carries a high concentration of genes, indeed the highest per length (Figure 1–4), and segmental imbalances are thus rarely viable. Occasionally, imbalance detectable classically is so “small” that the effect on the child’s physical phenotype is only very minor, and intellectual function can remain within the normal range, albeit toward the lower end of that range. Indeed, there are some segments of Mb size which, when duplicated or deleted, appear to cause no abnormality at all (Stumm et al. 2002; Barber 2005; Atli et al. 2022). The concept of heritable “euchromatic deletions and duplications without phenotypic effect” is discussed in Chapter 17. Molecular karyotyping has enabled a finer view of the genome, and microdeletions and microduplications of kilobase size are routinely detectable. These imbalances may impose a phenotype in which cognitive and behavioral abnormality is the predominant observation. Dysmorphism may be evident, but can be of quite minor degree. This may reflect that these small imbalances affect only a few or even a single gene, or regulatory factor. Since the organ commanding the largest component of a person’s genome is the brain, it is plausible to suppose that “brain genes” will be the most likely type of gene to reside within the microdeletions/duplications concerned. Differing lengths of deleted or duplicated segments enable a dissection of the specific segmental contributions to components of an abnormal phenotype. A broad-brush “malformation map” can be produced from documenting the association of certain congenital defects or known syndromes with particular segmental aneusomies (Brewer et al. 1998, 1999; Carey and Viskochil 2007) (Figure 3–20). Specific malformations can be interrogated: van Karnebeek and Hennekam (1999) document imbalances associated with congenital heart disease, as do Thorsson et al. (2015); Tyshchenko et al. (2009) have assembled a (very preliminary) brain list; Marcelis et al. (2011) record chromosomal segments associated with anorectal malformations; and we have undertaken phenotype mapping studies with respect to epilepsy (Singh et al. 2002a) and to kidney defects (Amor et al. 2003). Catelani et al. (2009) have searched for molecular imbalances in children with syndromic deafness. The chromosome regions thus illuminated may serve as candidate regions for the discovery of culprit genes. Note that a one-to-one connection between a deleted/duplicated segment and a specific trait cannot necessarily be drawn; and, for example, we have proposed that the particular nervous system malformation of periventricular 27 Chromosome 21 has a similar density, at 6.7 genes/Mb, whereas chromosome 18 is the least dense, at 4.3 genes/Mb (Nusbaum et al. 2005). Figure 3–20.  A duplication-malformation correlation map. Some chromosomal regions, in the duplicated state, are particularly associated with certain types of malformation. Presumably, these regions harbor genes that have roles in the formation of these particular organs. Other regions (including all of chromosome 19) are unrepresented, and some of these may contain “triplo-lethal genes.” ACC, agenesis of the corpus callosum; ASD, atrial septal defect; AVSD, atrioventricular septal defect; PDA, patent ductus arteriosus; VSD, ventricular septal defect. A similar map has been drawn for deletions (Brewer et al. 1998). In a somewhat similar vein, autism-susceptible copy number variant loci have been mapped (Figure 25–2). Source: From Brewer et al. (1999), A chromosomal duplication map of malformations: Regions of suspected haplo- and triplo-lethality—and tolerance of segmental aneuploidy—in humans, Am J Hum Genet 64: 1702– 1708. Courtesy C Brewer and DR FitzPatrick, and with the permission of the University of Chicago Press.

Origins and Consequences of Chromosome Pathology  65 nodular heterotopia might be an epiphenomenon accompanying a number of microdeletion syndromes, rather than the direct consequence of specific segmental imbalances (van Kogelenberg et al. 2010). Looking at segments as a whole is, as mentioned, to take a broad-brush approach. But bear in mind that an aneuploid segment of interest is of course a length of DNA, typically containing a number of protein-coding genes, possibly as few as one, or indeed perhaps none; in the latter case, non-coding DNA may contain regulatory elements that influence activity of genes elsewhere. In those segments in which a single major locus is involved, such as the PMP22 gene of Charcot-Marie-Tooth disease, a (relatively) simple one-to-one genotype-phenotype relationship may apply, and the other loci resident within the segment, not being dosage-sensitive, are non-contributory. Many imbalances, however, involve a number of pheno-contributory loci; or in other words, a number of loci within the segment may be dosage-sensitive. Many authors have collected particular cases from their own experience and from the literature. As the data from cohorts of cases are brought together, we may be able to tease out the individual genes or regulatory elements responsible for the different components of an abnormal phenotype. As an example of a multigene pathogenesis, Engels et al. (2012) studied five patients with molecular-defined deletions at 14q32.3, refining the phenotype map, and they hone down to a region containing just seven known genes that may be assumed, in total, to produce the phenotype as defined by them. A number of individual gene-level resources are now available to assess the pathogenicity of a given deletion or duplication based on its genetic content, including gene constraint metrics and dosage-sensitivity scores (Chapter 18). The concept of genes acting “in total,” or perhaps better said “in concert,” is addressed by Carvalho et al. (2014) in their review of the 17p13 deletion (Chapter 14). They studied the small number of genes within this segment and judged the relative roles of these genes in determining one particular aspect of the phenotype of this syndrome (microcephaly); they propose there to be functional interconnections (that is, epistasis) between some of these genes (Figure 3–21). The end result of this interaction may be a “second-level” or higher-level effect upon the phenotype; in other words, the whole may be different from the sum of the individual parts (and see also Figure 3–19). Surely, similar scenarios apply rather widely in the generality of the chromosomal syndromes. For the most part, the clinical states due to chromosomal imbalance are fixed and static. Structural defects such as a cardiac septal defect, or facial dysmorphism, are not progressive (although they may be evolving) conditions: They were established during embryogenesis and fetal development, and in essence, and unless surgically repaired, will stay that way. They may, of course, set the stage for consequential progressive change, such as a urinary tract defect that has back-pressure effects upon a kidney, affecting renal function; but this is a secondary factor. The brain, the most vulnerable organ, is similarly fixed in terms of its underlying anatomy, and chromosome disorders would not, as a general rule, be described as neurodegenerative. The most notable exception to that rule is the long-recognized dementia that typically commences around age 40 years in Down syndrome, and which reflects the effects of a triple dose of the amyloid precursor protein gene on chromosome 21, with a gradual accumulation in the brain of the abnormal protein. It is an obvious point, but worth restating: The defect in these aneuploid states involves too much or too little of what is normal chromosome material. The “third”

66  BASIC CONCEPTS chromosome in standard trisomy 21 is a perfectly normal chromosome 21, with a perfectly normal complement of chromosome 21 genes. How, therefore, could it be that an additional amount of normal genetic message leads to an abnormal interpretation of that message? How is the “dosage effect” mediated? This is one of the great remaining unanswered questions of biology (Liu et al. 2023), which we touch upon (no more than that) in Chapter 13. THE SEX CHROMOSOMES Sex chromosome imbalances need to be considered separately. Any X chromosomes in excess of one are, almost entirely, genetically inactivated. Thus, indicating the inactivated X in lowercase, normal females are 46,Xx; normal males are 46,XY; Turner females are 45,X; Klinefelter males are 47,XxY; and other X aneuploidies are 47,Xxx, 48,Xxxx, 48,XxYY, 49,XxxxY, and 49,Xxxxx. As for the Y chromosome, its active genetic material is confined to only a small segment, these genes being mostly related to sex determination and testicular function. Thus, despite the presence of one or more whole X or Y chromosomes in excess in the 47-, 48-, and 49-chromosome states, in utero survival remains possible. Indeed, for 47,XXX, 47,XXY, and 47,XYY, survival from conception is apparently uncompromised. Gonadal development in X aneuploid males is particularly affected, and intellectual function is jeopardized to a mild or moderate or severe extent in the n ≥ 47 states in both sexes. The cognitive compromise may reflect, inter alia, an influence upon normal Figure 3–21.  Interaction Between Genes Influencing the Phenotype: The Interactome. The17p13 deletion syndrome: effect of individual loci upon head size, and interaction of genes within this region. Above, gene map with nine loci within the deletion segment depicted. Cross-hatched locus, severe impact upon phenotype (microcephaly); dotted locus, moderate effect upon phenotype; open locus, no apparent effect. Below, proposed interaction between some of these loci, as inferred from zebrafish study. Source: From Carvalho et al., Dosage changes of a segment at 17p13.1 lead to intellectual disability and microcephaly as a result of complex genetic interaction of multiple genes, Am J Hum Genet 95:565–578, 2014. Courtesy CMB Carvalho and JR Lupski, and with the permission of Elsevier.

Origins and Consequences of Chromosome Pathology  67 cortical asymmetries in the brain (Lin et al. 2015). The severe language disorder in one of the polysomic states, 49,XXXXY, may be due at least in part to poor development of the white matter tract from the language area (Broca’s area) of the frontal cortex to the premotor cortex (Dhakar et al. 2016). Monosomy X, in contrast, has a high in utero lethality, although the small fraction surviving to term as females with Turner syndrome show, in contrast, a remarkably mild phenotype. PHENOCOPIES Similar phenotypes may flow from different genotypes. Syndromes resembling Silver-Russell syndrome, Prader-Willi syndrome, and Angelman syndrome, but due to other chromosomal imbalances, are described, and some examples are noted in Chapter 14 and Chapter 15. “Pseudotrisomy 13” might more usefully be known as holoprosencephaly and polydactyly syndrome (Bous et al. 2012). The expression “DiGeorge syndrome” refers to an ensemble of signs that characterize the 22q11 deletion. Somewhat similar clinical pictures can be seen in deletions of 10p13 and of 4q34.2. THE MOSAIC STATE Whether constitutional mosaicism matters depends upon which tissue, and how much of that tissue, is abnormal. If a majority of the soma is chromosomally abnormal, then naturally the phenotype is likely to be abnormal. If only a tiny fraction of some tissue were involved, in which the aneuploidy would have essentially no effect—if, for example, some of the bony tissue of the distal phalanx of the left little toe were trisomic 21, and the rest of the person 46,N—it would never be known. Indeed, as mentioned above, possibly everyone has mosaicism, essentially harmlessly, in certain tissues or organs. More to the point, a very minor degree of mosaicism could yet convey pathogenicity if a crucial tissue carried the imbalance. An abnormal chromosome confined to tissues of, say, a localized area or cell type in one part of the brain, could theoretically cause neurological dysfunction (Rohrback et al. 2018; Graham et al. 2024).28 Mosaicism for a chromosome abnormality is detectable, using a sophisticated molecular approach, in normal adults, and a quarter of those analyzed in one study carried a “clonally-expanded mosaic chromosome alteration (mCA)” in at least one tissue (Gao et al. 2023). The older the person, the more such mCAs. These mCAs may have been expansions from a constitutional origin, or—more likely—may have arisen in post-natal life. The particular relevance of this state may be to do with cancer. Abnormality involving a gonad or part of a gonad (gonadal mosaicism) could lead to a child being conceived with that aneuploidy, as discussed above. Mosaicism confined to extraembryonic tissue may be without phenotypic effect, although it can certainly 28 Mosaic aneuploidy of the brain arising in prenatal or post-natal life may be a basis of neurological disease, having somewhat of a parallel with the evolution of some cancers (Rosenkrantz and Carbone 2017); but here we are considering constitutional mosaicism generated in early embryonic life and established ab initio over the period of intrauterine brain development. 68  BASIC CONCEPTS cause anxiety if it produces an abnormal test result at prenatal diagnosis: This is confined placental mosaicism (CPM). CPM may exist unbeknownst in pregnancies producing normal infants, as Lestou et al. (2000) showed in a study of 100 placentas, with five revealing CPM for trisomies 2, 4, 12, 13, and 18. Mosaicism may frequently be observed at the IVF laboratory at blastocyst biopsy: a state of affairs that becomes very relevant in preimplantation genetic testing (Chapter 23). Mosaicism for a Full Aneuploidy As a general principle, an individual with an aneuploid line in only some tissues is likely to have a less severe but qualitatively similar phenotype to someone with the non-mosaic aneuploidy. The ascertainment of these individuals is biased: those with a more obvious phenotypic defect are, naturally, more likely to be detected. Mosaic Down syndrome—47,+21/46,N—can be less obvious than standard trisomy 21, and with a lesser compromise of intellectual function (Papavassiliou et al. 2015). The existence of 46,N cells in some of the brain tissue presumably has a moderating effect. Some aneuploidies can only, or almost only, exist in the mosaic state, the non-mosaic form being lethal in utero. Examples of this are mosaic trisomies 8, 9, and 16. If the distribution of the aneuploid cell line is asymmetric, body shape may be asymmetric, generally with the hypoplasia present in regions of aneuploidy. De Ravel et al. (2001) described hemifacial microsomia (one side of the face being underdeveloped) and other body asymmetry in two children with autosomal mosaicism, one for trisomy 9, and the other trisomy 22. The child with 47,XY,+22/46,XY had 9/10 cells + 22 on skin fibroblasts from the arm on the right (underdeveloped) side, compared with 5/11 on the left arm (the child’s blood karyotype was 46,XY). Molecular analysis supported there having been a post-zygotic anaphase lag that had produced the 46,XY line from an initially 47,XY,+22 conception. Niessen et al. (2005) studied in some detail a girl with three shades of skin pigmentation—hypopigmented, normally pigmented, and hyperpigmented (“cutis tricolor”)—following the lines of Blaschko (see next section). She karyotyped 45,X on blood, and 47,XX,+7 on skin biopsied from the darker skin. A surprising case is that of Greally et al. (1996): a child with mosaic trisomy 16, a cardiac malformation, and otherwise (barring a unilateral simian crease) not dysmorphic, and her neurodevelopmental progress was quite normal. One might suppose (but could not prove) that the trisomic cell line was confined in distribution and excluded the brain. Mosaicism excluding the bone marrow will give a normal blood karyotype, while mosaicism confined to marrow would be seen on routine peripheral blood analysis but not on other samplings; mosaic trisomy 8 may provide examples in both directions. Examples of presumed very low-level trisomy mosaicism have come to light through prenatal diagnosis, such as trisomy 13 mosaicism in an apparently normal child with one cell out of 400 on cord blood (Delatycki et al. 1998; Figure 22–6). In sex chromosome mosaicism, fertility can exist when otherwise infertility is the rule—for example, in “formes frustes” of Turner syndrome with 45,X/46,XX and of Klinefelter syndrome with 47,XXY/46,XY.

Origins and Consequences of Chromosome Pathology  69 Mosaicism for a Structural Rearrangement Mosaicism for a classical structural rearrangement—a translocation, an inversion, a deletion, or a duplication—is rarely recognized. Kovaleva and Cotter (2016) reviewed 104 cases from the literature, either balanced or unbalanced. In the experience of one Australian laboratory, two cases of unbalanced translocation mosaicism were seen among 75,000 karyotypes from 1989 to 2013. One was a normal woman presenting with recurrent miscarriage, with 46,XX,der(6)t(6;8)(q27;q22.1)/46,XX; and the other was a globally delayed infant with 46,XY,der(22),t(14;22)(q32.1;q13.3)/46,XY (Dalzell et al. 2013). Molecular karyotyping increased the yield: in Francis et al. (2023), mosaic deletions and duplications were detected in 0.7% of clinical samples. With an unbalanced karyotype, the broad (indeed, obvious) rule applies, that the mosaic form is likely to be less severe than the non-mosaic form. Pigmentary skin anomaly is a notable and clinically useful phenotypic trait that can characterize this type of unbalanced mosaicism, the important categories being hypomelanosis of Ito (Figure 3–22), linear and whorled nevoid hypermelanosis, and “phylloid” (leaf-like) pigmentary disturbance (Vreeburg and van Steensel 2012). Figure 3–22.  Hypomelanosis of Ito in a child with mosaicism 46,XX,dup(3)(q26.3qter)/ 46,XX. 70  BASIC CONCEPTS The distribution of the abnormal cells in hypomelanosis of Ito, and thus of dyspigmentation, follows the lines of Blaschko, and Magenis et al. (1999) use the expression “Blaschkolinear malformation complex.” Asymmetry is a further clinical pointer (Woods et al. 1994). An interesting category of mosaicism for a structural rearrangement is that in which two lines of opposite imbalance coexist, with or without a normal cell line as well. Here, the error must have happened at a very early stage, and quite possibly, in those cases lacking a normal cell line, at the very first mitosis of the zygote. Such a case is described in Morales et al. (2007a), who analyzed a boy with the karyotype at birth of 46,XY,del(7q)/46,XY,dup(7q), although by age 12–14 months, the deletion cell line had disappeared, at least from blood and exfoliated urinary epithelial cells. Presumably, the karyotype at conception was 46,XY, but then the two chromosome 7 homologs underwent an unequal exchange of q21.1q31.3 material, generating, in the two daughter cells from the first mitosis, the deletion and duplication lineages. If, for example, the error had occurred one division later, at one of the two second mitotic divisions, a normal cell line might have been retained and have contributed to the inner cell mass. Tissue Sampling in the Detection of Mosaicism Clearly, detecting—or failing to detect—a mosaic state will depend upon which tissue is studied and how many cells are counted/analyzed. Blood, buccal cells, amniocytes, chorionic villi, trophectoderm, skin fibroblasts, and urinary sediment cells have been the usually tested tissues. A normal blood result in a child with an indicative phenotype, or following an abnormal prenatal test, might then warrant testing other tissues. Blood is a specialized tissue, and an abnormal cell line, otherwise widespread through the soma, can potentially be excluded from blood marrow following untold numbers of mitoses and if normal cells exert a superior survivability. Presumably this was the case, for example, in the child described in De Ravel et al. 2001 above, in whom skin fibroblasts, but not blood, retained the aneuploidy. Skin fibroblast analysis has been a mainstay of mosaicism detection since the earliest days of cytogenetics (and actually predated the use of blood). Another tissue of historic provenance is the buccal mucosa, the inner lining of the cheek obtained from a “cheek swab,” and this was the basis of a simple analysis of X chromosome aneuploidy via the demonstration of a compacted inactivated X (the Barr body)—a methodology little used these days. But this tissue has come to the forefront in the current century with the development of technologies to extract DNA from buccal mucosal cells that are contained in saliva, the “spit sample.” The buccal mucosal cells present in a saliva sample are representative of ectodermal tissue: the tissue from which, among others, the nervous system derives. There is an obvious practical attraction in the ability to avoid even the (for most people) minor pain of a venipuncture, especially with children; no more is involved than simply spitting into a tube. The DNA can then be subjected to a methodology such as single nucleotide polymorphism (SNP) chromosome microarray (CMA). The suitability of this approach was demonstrated in a landmark study in Francis et al. (2023), who compared the diagnostic utility of saliva-based against blood-based

Origins and Consequences of Chromosome Pathology  71 CMA. In the analysis of 370 patients in a pediatric setting, including 224 presenting with syndromic or non-syndromic intellectual disability, in whom both types of testing were applied, the saliva-based test picked up 20 cases of mosaicism, a CNV or a trisomy, which were missed on the blood sampling. Notably, these mosaic cases were confined to the group of patients presenting with a syndromic intellectual disability. (For non-mosaic results, the blood and saliva samples were completely concordant.) These observations lead to the conclusion that saliva-based chromosome analysis should be a first-tier test, at least in the case of syndromic intellectual disability: that is to say, intellectual disability in the context of such other observations as organ malformation, facial and other dysmorphism, or growth disturbance. Gonadal (and Somatic-Gonadal) Mosaicism Gonadal mosaicism is suspected upon the observation of a chromosomally normal couple (at least on blood testing) having had two or more children with the same abnormal karyotype. Molecular analysis can allow an inference of who is the carrier parent, such as Tosca et al. (2010) show in the family study of two children with a dup(4)(q22.2q32.3), in which the microsatellite pattern indicated a maternal origin. In Kuroda et al. (2014), even though chromosome studies on the mother were normal, a maternal origin could be assumed in two siblings with Angelman syndrome due to a chromosome 15 inversion, which had deleted (among other genes) the UBE3A gene (Chapter 19). Direct proof is provided by analysis of gametes. For example, in a case that had come to notice through an IVF clinic, Somprasit et al. (2004) report a couple having had a 21q duplication in two embryos subjected to PGT, and then showed the same duplication in 6.6% of 1,002 of the father’s sperm. The abnormality was not present in his blood. Similarly, Alkaya et al. (2020) describe a father of three children with cri du chat syndrome (Chapter 14) due to an unbalanced translocation t(5;19)(p13.3;q13.4); the translocation was present in his sperm, but in neither blood nor skin biopsy. Somatic-gonadal mosaicism can be inferred on the observation of one parent of an aneuploid child carrying, in mosaic state, the same aneuploidy. Sachs et al. (1990) studied a mother who had had one Down syndrome child and three other trisomic 21 pregnancies, and her blood karyotype was 47,+21[3%]/46,N[97%]. Ovarian biopsies showed almost half the cells in each ovary to be 47,XX,+21. We have seen a similar case, a woman who presented having had two trisomic 21 pregnancies. On blood with a 60-cell count, she and her partner were non-mosaic 46,N. She recounted a story that her own mother had had an amniocentesis when pregnant with her, which had shown a single + 21 cell. Given this information, “spit sample” microarray was done, and she proved to have ~5% of cells trisomic, inferentially revealing her state of somatic-gonadal mosaicism. Figure 3–23 shows an example of somatic-gonadal mosaicism for a structural rearrangement, del(1). The index case was identified with a small intrachromosomal del(1) at routine prenatal diagnosis. The father was mosaic for this deletion in 20% of lymphocytes. Of his two other children, one had normal chromosomes, and the other had the same deletion. The father is phenotypically normal, and the older child with the deletion has an IQ in the low normal range. A similar circumstance is recorded in Fan et al. (2001): A university-educated man working as a financial planner, having the blood karyotype 46,XY,dup(8)(p21.3p23.1)[6]‌/46,XY[24], fathered two daughters 72  BASIC CONCEPTS with 46,XX,dup(8)(p21.3p23.1). These girls had poor language development, clumsy motor abilities, and minor facial dysmorphism. Pitt-Hopkins syndrome (Chapter 14) is due to an 18q microdeletion, and normal parents are on record as having him- or herself a low-level mosaicism demonstrable on blood (Figure 14–73) (Doudney et al. 2013; Kousoulidou et al. 2013) or on blood, urinary, and salivary (but not hair) cells (Steinbusch et al. 2013). Mosaicism at Prenatal Diagnosis About 1%–2% of placentas can have a different chromosomal constitution from that of the embryo, with usually the embryo being normal and the placenta trisomic. This is “confined placental mosaicism.” Thus, in 1%–2% of chorionic villus sampling (which can be considered a placental biopsy) or in noninvasive prenatal testing (which can be considered, in a sense, as a “wet” chorionic villus sampling), there will be a potentially misleading result. Fortunately, these uncommon instances can, as a rule, be recognized as such, although often not without causing some anxiety at the time. In a few confined placental aneuploidies, the function of the placenta may be compromised, and fetal well-being may be affected. Infrequently, true mosaicism is recognized at amniocentesis. Occasional cells with a chromosomal abnormality, if they are solitary or involving a single clone, are generally regarded as having arisen in vitro (“artifactual mosaicism”). At least most of the time, Figure 3–23.  Somatic-Gonadal Mosaicism. A family exemplifying somatic-gonadal mosaicism. (a) Pedigree. The father had the mosaic karyotype 46,XY,del(1)(q25q31.2)[16]/46,XY[4]‌ on lymphocyte study. Two children have the del(1)(q25q31.2) in nonmosaic state. The family was ascertained following routine prenatal diagnosis. The older sibling’s development was judged, at age 5 years, to be in the low average range; height, weight, and head circumference were in the range 20–25th centiles. The father worked as an electrician. (b) Partial karyotype showing the father’s two cell lines: two normal no. 1 chromosomes, and one normal and one deleted chromosome 1. The segment 1q25q31.2 is shown cross-hatched. (Courtesy G Dawson.)

Origins and Consequences of Chromosome Pathology  73 this is probably the correct interpretation (but see the case in Somatic-gonadal mosaicism above). We consider mosaicism at prenatal diagnosis in detail in Chapter 22. FUNCTIONAL IMBALANCE The idea that abnormality could be due to unequal parental contributions of an overall correct amount of chromosome material seemed most remarkable in 1980 when Engel first made the suggestion and coined the expression “uniparental disomy.” It came to be accepted fact. The two disorders that, par excellence, exemplify the concept of qualitative imbalance are Prader-Willi syndrome (PWS) and Angelman syndrome (AS). The concept of genomic imprinting, discussed above, is central to an understanding of the etiology. Each syndrome is due to the non-expression of different (but neighboring) segments within the proximal long arm of chromosome 15. A “PWS critical region” is normally expressed from only one chromosome, in this case the paternally originating chromosome. The maternal-originating region is normally inactive, and alleles in this region are not transcribed. Thus, there is a “functional monoallelism.” If the paternal PWS region is absent, the maternal one cannot “fill the gap,” and the consequential functional nullisomy is the root cause of PWS. An “AS critical region” exists, lying just a little distal from the PW region. Likewise, it needs only monoallelic expression for normal phenotypic function. In this case, it is the maternal region that is active, and the paternal region, having been imprinted, is inactive. If the maternal region is absent there can be no genetic activity, and this causes the AS phenotype. Absence of the paternal PWS region or maternal AS region can flow from two major mechanisms. First, in UPD, the chromosome 15 from one parent is lacking, and the “correcting” presence of two copies from the other parent cannot restore a proper balance. This can be heterodisomy (the two homologs being different) or isodisomy (they are identical). Second, there can be a deletion within proximal 15q that removes a segment of chromatin containing the PWS and AS regions. These issues are dealt with in some detail in Chapter 19. Uniparental disomy for the entire chromosome set—“uniparental diploidy”—has a devastating effect on development. If a conceptus has lost its maternal complement, and the paternal complement is doubled, embryonic development arrests, leaving only grossly abnormal chorionic villi comprising the pregnancy. This is a hydatidiform mole (Chapter 20). If a 46,XX ovum at meiosis I attempts a parthenogenetic development, a grossly disorganized mass of embryonic tissue results: an ovarian teratoma. If a triple set of chromosomes (triploidy) is present at conception, there is either a diploid maternal set plus a haploid paternal set or vice versa. These different parental origins determine quite different but very abnormal fetal and placental phenotypes (Chapter 13). As the imprinting story has evolved, it has emerged that most of the genome appears not to be subject to imprinting.29 For most chromosomes and with both homologs equally genetically active, regardless of the parent of origin, UPD will have no untoward effect. Only if the UPD-contributing parent should happen to be heterozygous for a recessive gene, and if this is the isodisomy category of UPD, will the child be affected, 29 Several small segments across the karyotype show an imprinting effect, but a clinical implication of this remains uncertain (Joshi et al. 2016; and see Figure 19–2).

74  BASIC CONCEPTS displaying the Mendelian condition concerned due to homozygosity (“isohomozygosity”) for that recessive gene. Rare instances of this scenario are known. Similar considerations may apply in the trisomies. Naturally, one parent must have contributed more than one homolog. Considering the example of Down syndrome, does the parent from whom the disomic gamete came contribute two different chromosome 21s? In other words, does the child inherit a chromosome 21 from three of the grandparents—“heterotrisomy”? Or does the parent contribute two identical (isodisomic) chromosome 21s? Whether phenotypic differences may flow from these different possibilities is quite uncertain, although Baptista et al. (2000) suggest that heterotrisomy 21 may, of itself, convey a greater risk for a specific heart malformation, ventricular septal defect, speculatively due to a damaging interaction of three subtly different protein products from a 21q “heart locus.” Segmental Uniparental Disomy A mitotic mechanism that can lead to functional imbalance, if the segments exchanged are in a region subject to imprinting, is somatic recombination. The first shown example of this causing a dysmorphic syndrome is the segmental paternal uniparental disomy for 11p that underlies some Beckwith-Wiedemann syndrome, 11p being a segment that is normally maternally imprinted. In the partial UPD(pat) cell line, this segment will now be expressing biallelically at distal 11p, instead of the normal monoallelic expression. The asymmetry of body growth that may be observed in this syndrome reflects the body distribution of two cell lineages: the normal biparental disomic line, and the functionally imbalanced UPD(pat) line. SPORADIC AND RECURRENT ABNORMALITIES Chromosomally normal parents can produce abnormal gametes by nondisjunction, rearrangement, or one of the other mutational mechanisms we have discussed above. The combination of factors that causes these defects in an individual case is unknown. No convincing case has ever been made for an important agency of diet, illness, chemical exposure, or “lifestyle factors” in maternal chromosome 21 meiotic nondisjunction (Chapter 26), nor is there much support from epidemiological studies (Chapter 13). Noting the similarity of Down syndrome prevalence rates worldwide, Carothers et al. (1999) comment that “the totality of published data could well be consistent with no real variation at all, and [this] might explain why a search for environmental factors associated with Down syndrome has been so unproductive.” The maternal age effect is of course important, indeed central, and any search for causes of chromosomal aneuploidy must take this into account. A plausible view is that there is a natural degeneration of the oöcyte, as we discussed above, and with reference to Figure 3–13. Simply put, eggs get older, and they show their age. Chromosomes are plastic, dynamic entities, and cell division is a complex mechanical process; and these qualities alone may suffice to endow the vulnerability that causes human aneuploidy and rearrangement. Given the assumption that all persons with intact gametogenesis are capable of producing an abnormal gamete, one view is Origins and Consequences of Chromosome Pathology  75 that it may simply be so, that a certain background abnormality rate is intrinsic to the human species and at least in the majority of cases, it is a chance matter whether this or that couple will have the misfortune to conceive the abnormality which, inevitably, someone has to bear. Parental Predisposition to Nondisjunction or Deletion/Duplication? An alternative view is that some 46,XX and 46,XY people are more prone than others to produce chromosomally unbalanced gametes. An intrinsic fault, or at least a vulnerability, in the mechanism of chromosome distribution at cell division could be the basis of the rare examples of recurring defects. The synaptonemal complex gene SYPC3, and the mismatch repair genes, with particular reference to MLH1 (otherwise familiar to the counselor in Lynch syndrome) and MLH3, and the related meiosis genes MSH4 and MSH5, would all be plausible candidates in which subtle variation might affect integrity (Singh et al. 2021). Given the complexity of the apparatus and process of meiotic cell division, it is logical that error-causing mutants in the controlling genes (whether or not this might include any of the aforementioned) would exist. Whether there might be milder alleles at postulated cell-division or recombination loci, which could more widely be the cause of occasional nondisjunction or del/dup, remains a matter for speculation. A genome-wide association study for genes associated with maternal nondisjunction of chromosome 21 identified some loci of interest, but none were conclusive (Chernus et al. 2019). Nevertheless, a geneticist could scarcely ignore that there might exist subtle genetic variation potentially setting the stage for chromosomal aberration. A Note on the Diagrams. Following the progress of rearranged chromosomes during meiosis is not easy, so we have taken some liberties in simplifying the diagrams. Most of these diagrams depict the synapsing chromosomes at meiosis with just one chromatid; of course, the chromosome has actually replicated at this point and exists as a double-chromatid entity (Figure 3–24). Figure 3–24.  Chromosomes at synapsis exist as double-chromatid structures (e.g., the reciprocal translocation quadrivalent at right). But, for simplicity, we generally represent them with just the one chromatid (left).

4 DERIVING AND USING A RISK FIGURE RISK IS A CENTRAL CONCEPT in genetic counseling. By risk, we mean the probability that a particular event will happen. Probability is conventionally measured with a number ranging from 0 to 1. A probability (p) of zero means never, and a probability of 1 means always. For two or more mutually exclusive possible outcomes, the individual probabilities sum to 1.0 (or 100%). Thus, someone who is a heterozygote for a particular chromosomal rearrangement might, in any given pregnancy, have a probability of 0.10 (10%) of having an abnormal child and a probability of 0.90 (90%) of having a normal child. We may speak in terms of risks of recurrence or of occurrence: the probability that an event will happen again, or that it will happen for the first time. Risk can also be presented as odds: the ratio of two mutually exclusive probabilities. The odds for the hypothetical heterozygote just mentioned would be 9:1 in favor of a normal child. The word risk has two important meanings in the English language. First, there is the scientific sense of probability that we already discussed. Second, as most people use the word, it conveys a sense of exposure to danger. Our hypothetical heterozygote runs the risk that an unfortunate outcome may occur (an abnormal child, or an abnormal result at prenatal diagnosis). In the genetic counseling clinic, these meanings of risk coalesce in some ways, to which the counselor needs to be sensitive.1 We might instead use such everyday words as chance or likelihood, which have no negative connotation, to refer to the fortunate outcome of normality. The words fortunate and unfortunate are also chosen deliberately: The wanted or the unwanted event will occur entirely by chance, analogous to tossing a coin, throwing a dice, or being dealt a card. DIFFERENT TYPES OF RISK FIGURE Geneticists arrive at risk figures in a number of ways (Clarke 2019), two of which, empiric and Mendelian, have particular application to cytogenetics. 1. Empiric risks. In the great majority of chromosomal situations, no clear theory exists from which a risk figure can be derived, and one must observe what has happened previously (as far as one can judge) in the same situation in other families and make an extrapolation to the family in question. Empiric risks thus appeal to experience, and they only estimate the intrinsic, true probability. The data may be available in the literature record or in specific databases; or the counselor may need to derive a “private estimate” from an analysis of the client’s family. The 1 There has been a move in the UK to replace the word risk with the word chance when used in the context of prenatal screening, prompted by opinions from some parents of children with Down syndrome. Wald et al. (2022) disagree, even seeing Orwellian overtones, arguing that “risk” and “chance” are not perfect synonyms, and the subtle distinction in meaning is important in the public understanding of prenatal screening. 78  BASIC CONCEPTS risk estimate has a greater or lesser degree of precision depending on how much data has been accumulated upon which the estimate is based. 2. Mendelian risks. If a clear model of inheritance is known, risk figures derived by reference to that theory may be used. In practice, only Mendel’s law of segregation is applied in this context. When a pair of homologous chromosomes segregates at meiosis, which chromosome enters the gamete that will produce the conceptus is typically a random matter. Each has an equal chance: a probability of 0.5. Thus, a parent who carries a microdeletion 16p11.2 has a 50/50 likelihood to transmit this chromosome to a child, a 1:1 segregation. This is assumed to be a true risk, not an estimate: It is 0.5 exactly. Consider, for example, the common situation of a young couple having had a child with Down syndrome. Nothing is known about nondisjunction that could provide a theoretical model on which to base a recurrence risk figure. We therefore use empiric data—that is, information obtained from surveying large numbers of other such families. It may be observed, for example, that in these families about one pregnancy in 100, subsequent to the index case of Down syndrome, produced another child with Down syndrome. Formally expressed, this is a segregation analysis. From this rate of 1/100 we can derive a risk figure of 1%, which we then have as the basis for advising patients. (Actually, it is not quite as straightforward as this in Down syndrome; see Chapter 13.) If a theoretical construct can be applied, this may allow a more precise calibration of the empiric figure. The del 17q21.31 of Koolen-de Vries syndrome (Chapter 14), which has a population frequency of 1/16,000, offers an example. This particular deletion may have, as a necessary but not sufficient basis for its generation, a 17q inversion encompassing the length of the deleted segment (chr17:45.6-46.1 Mb). The risk is related to the inversion status of the parents, the dimorphism referred to as H1 (normal 17q21.31 sequence, N) and H2 (inverted 17q21.31 sequence, V). Koolen et al. (2012) apply some fundamental genetic concepts in order to tailor the risk figure according to the possible parental inversion genotypes, NN, NV, and VV, and thus the six possible mating combinations: NN × NN, NN × NV, NV × NV, NN × VV, NV × VV, and VV × VV. The adjusted risk figures of 0.03% (for VV × VV parents) and 0.008% (NN × NV parents), versus the population figure of 0.006%, barely dent the >99.9% chance of non-recurrence, but the principle behind the exercise is to be acknowledged. A somewhat similar approach may apply to the inverted duplication of 8p (inv dup 8p), as discussed on Chapter 14. An inversion polymorphism at chr8:7.6-12.3 Mb, which has a high (26%) frequency in the general population, predisposes to a misalignment during meiosis (Giglio et al. 2001). Indeed, generation of the rearrangement may only be possible in the setting of this parental inversion. However, the absolute risk among this quarter of the (at least European) population must remain extremely low, given the rarity with which the inv dup 8p is seen, and the absence of any report of recurrence. The risk to the non-carrier may be a true 0.0%. Likewise, for the circumstance of the parent heterozygous for a chromosomal rearrangement, the counselor can consult data that have been accumulated by workers in the field, foremost among whom, in respect of reciprocal translocations, are Stengel-Rutkowski et al. (1988), Cohen et al. (1992, 1994), and Midro et al. (2000). Since almost

Deriving and Using a Risk Figure  79 all reciprocal translocations are unique to one family, it is not necessarily simple to estimate a figure for a family with a “new” translocation, but an attempt can be made (see Chapter 5). On the other hand, for the Robertsonian translocations, each type of which can generally be regarded as the same between families, extrapolation of risk figures from historical data to a current family is usually valid. Risk may apply more generally in the sense of a successful or unsuccessful outcome. Consider the case of couples having had a previous aneuploid pregnancy and now seeking blastocyst testing to be sure of avoiding a recurrence: What are their odds of having a euploid blastocyst, suitable for transfer? In an observational study, L Zhang et al. (2023b) saw a 30% rate of an aneuploid blastocyst in this group, substantially above the 21% rate of those presenting with no previous aneuploidy history. These authors caution that these rates were derived from a population of women who were able to produce at least four blastocysts, and who had good ovarian reserve; hence they should be applied as risk estimates only to those in this same category. Hook and Cross (1982) note the importance of distinguishing between the rate (which may be thought of as “past tense”) and the risk (which is “future tense”). They emphasize that although geneticists routinely extrapolate from rates in one population at one point in time, and may use these figures as risk estimates in another population and certainly at a later point in time, they should be on their guard for any evidence that a condition varies with time, geography,2 or ethnicity. But actually, there is little indication that any important variation exists. Chromosomal biology appears to be rather consistent throughout the human race and across the centuries. Doing a Segregation Analysis Segregation analysis is essentially a simple exercise. A farmer who surveys a flock of newborn lambs and notes that 3 are black and 97 are white has done a segregation analysis. In human cytogenetic segregation analysis, the exercise involves looking at a (preferably large) number of offspring of a particular category of parent: parents who carry some particular chromosome rearrangement, or those who have had a child with a chromosomal abnormality while they themselves are karyotypically normal. The proportion of these parents’ children who are abnormal is noted (say, 3 out of 100), and this datum serves as the point estimate of the recurrence risk (thus, 3%). Although segregation analysis is simple in principle, there are potential pitfalls in its application, the most important of which is ascertainment bias. We will deal with this problem only briefly. It is important that the counselor know of ascertainment bias, and recognize whether it has been accounted for in the published works consulted. But it is not necessary to understand the complex and sophisticated mechanics of segregation analysis in detail. The reader wishing fuller instruction is referred to Murphy and Chase (1975), Emery (1986), and Stene and Stengel-Rutkowski (1988). The classic example of ascertainment bias is that of the analysis of the sex ratio in sibships of military recruits in World War I. Adding up the numbers of brothers and sisters, there was a marked excess of males. But of course (in 1914–1918) the recruit himself had to be male. Once he was excluded from the total in each sibship, the overall sex ratio was normal, namely 2 A curious difference in the types of chromosome abnormality from prenatal testing in different regions of China (J Zhang et al. 2023a) remains unexplained.

80  BASIC CONCEPTS 1.0. Likewise, in a cytogenetic segregation analysis, the individual whose abnormality brought the family to attention—the proband—is excluded from the calculation. That person had to be abnormal. Furthermore, for very many classical chromosomal scenarios, that individual’s carrier parent, grandparent, and so on in a direct vertical line, had to be phenotypically normal to have been a parent. These individuals must also be excluded from an analysis of their own sibship, if that generation is available for study. Other sibships may be included in full. These manipulations—dropping the proband and the heterozygous direct-line antecedents—are the major steps to be taken to avoid the distorting effects of ascertainment bias. Another potential methodological confounder for the aficionado is ascertainment probability. For example, families with more affected members may be more likely to come to medical attention, which would unduly weight the data. There are means to overcome this problem. Family/population studies on the microdeletions, microduplications, and copy number variants (CNVs) of 21st-century chromosomology present a more difficult problem. Non-penetrance and variable expressivity, and phenotypes confined to intellectual/behavioral traits and in some of mild degree, complicate the picture. Where is the threshold to be taken as affected/unaffected? The pioneers in this field are Vassos et al. (2010) and Rosenfeld et al. (2013), who compared prevalences of CNVs in affected cohorts versus a presumed normal population. The work of Vassos et al. was focused specifically on schizophrenia. Goh et al. (2025) undertook a formal review of several published studies, calculating penetrance values for many CNVs, and these data are listed in Appendix C. We discuss these conditions in Chapter 14 and Chapter 18. Essential to a good analysis is good data, or at least as good as possible. Some retrospective information may be uncertain. In a family translocation study, did a phenotypically abnormal great uncle who died as a child in 1930 have the “family aneuploidy”? (Old photos may be very helpful in this respect.) Some family skeletons may remain in cupboards unopened to the interviewer. Particularly in the follow-up of prenatal diagnosis results, it is important to know the endpoint of data collection of the child and how the data were collected: at birth or until school age, by formal examination or by anecdotal report. The investigative zeal, clinical judgment, and personal qualities of the researcher are crucial in getting the right information, and getting it all. THE DERIVATION OF A “PRIVATE” RECURRENCE RISK FIGURE We will demonstrate some of the previously noted principles in estimating a private recurrence risk figure for the hypothetical family depicted in Figure 4–1. Six sibships are available for analysis: one in generation II, two in generation III, and three in generation IV. We determine the segregation ratio in each. It is conventional to form a table with a row for each sibship, noting the numbers of phenotypically normal (carrier, non-carrier, unkaryotyped) and phenotypically abnormal offspring (Table 4–1). The figures in parentheses give raw totals in these sibships, but then the proband (IV:4) and his heterozygous antecedents (II:1 and III:1) are excluded from their sibships. Note that I:1’s heterozygosity must be inferred from his wife’s and children’s karyotypes. (It is a subtle question whether his offspring should properly be included in the analysis, which we will not pursue here.) We see that the offspring of heterozygous parents total 14, the proband and the heterozygous antecedents having been excluded. The proportion of abnormal children is 3/14 (0.21). This, then, is a point estimate of the risk for recurrence in a future pregnancy of a heterozygote. The reader should know intuitively Deriving and Using a Risk Figure  81 that an estimate based on just 14 children is not going to be very precise (but not to be discarded). And what of children who had died before the family cytogenetic study was done? Let us suppose this was the case with III:4 and 5. If there was good historic evidence for their having been chromosomally abnormal, a better estimate would be 5/14 (0.36). VanDerwerken (2015) takes the sophistication of a “private” risk assessment to a further level, in applying the principles of Bayesian analysis. He proposes that a prior probability due to relevant literature data is usefully to be taken into account, and that this can fine-tune the accuracy of advice given. The mathematically sophisticated reader is referred to his paper. Genetic Heterogeneity and the Use of Empiric Risk Data It is not necessarily valid to extrapolate from one family’s experience to a prediction for another. Different factors may cause an abnormality in different families. As an obvious example, it would be misleading to “lump” all Down syndrome families to determine Table 4–1.  Calculating a Recurrence Risk Due to a Familial Translocation PARENT OF SIBSHIP SIBSHIPS AFFECTED CARRIER NON-CARRIER UNKARYOTYPED TOTAL I:1 0 1 (2) 2 0 3 II:1 1 1 (2) 0 2 4 II:2 0 1 0 1 2 III:1 2 (3) 0 1 0 3 III:2 0 1 0 0 1 III:7 0 0 1 0 1 Total 3 4 4 3 14 Notes: These data come from the family shown in Figure 4–1. The numbers in brackets refer to the raw totals, before the removal of the proband and the heterozygous antecedents. Figure 4–1.  A Family Study. Notes: Hypothetical pedigree in which a chromosomal rearrangement is segregating. Filled symbol, abnormal individual with unbalanced karyotype; half-filled symbol, balanced carrier; N in symbol = 46,N. The proband is, as is conventional, indicated by an arrow.

82  BASIC CONCEPTS a recurrence risk figure. We need to “split” into the different karyotypic classes of standard trisomy, familial translocations, and de novo translocations. The standard trisomic category requires further splitting in terms of maternal age. In a unique case, a woman had three trisomy 21 conceptions and displayed a tendency to produce multiple cells with differing (“variegated”) aneuploidies in at least skin, blood, and gonad (Fitzgerald et al. 1986). She required unique advice. And in reciprocal translocation families, uniqueness is the rule! It is generally reasonable (and often all that is feasible or possible) to apply a risk figure derived from the study of families with a similar, albeit not exactly identical, chromosomal arrangement. But occasionally a family is large enough for a “private” estimate of the recurrence risk to be made from the family itself. This estimate, if it is precise enough (see the later discussion of confidence limits and standard error), is the most valid to offer that family. Pregnancy Outcomes to which the Risk Figures Refer With particular reference to the situation of a parent heterozygous for a chromosomal rearrangement, risk figures are generally presented in terms of “the risk that a liveborn child would have a chromosome imbalance related to the parental translocation.” The numerator is the number of aneuploid babies, and the denominator is the number of all babies. Thus, considering the example of the common t(11;22)(q23;q11) translocation (Chapter 5), Stengel-Rutkowski et al. (1988) accumulated data on a total of 318 births (the denominator) to carrier parents, of whom, after ascertainment correction, nine (the numerator) had the 47,+der(22) aneuploidy, and 9/318 gives the risk expressed as a percentage, 2.8%. Separating out mothers and fathers, the respective risk figures are 3.7% (9/241) and <0.7% (0/77). For those choosing prenatal diagnosis, the risk figure of interest relates to the timing of the procedure, generally chorionic villus sampling (usually done at 10–12 weeks) and amniocentesis (15–17 weeks). In other words, they want to know how likely it is they will have to face the actuality of a decision about termination. The risk here is likely to be higher (7% in the case of the 11;22 translocation), given that some of the abnormal pregnancies would have spontaneously aborted some time after that period of gestation. Table 4–2 sets out these and other possible ways of considering risk. Table 4–2.  Different Ways of Looking at the Quantum of Reproductive Risk Due to a Parent Being a Carrier of a Chromosomal Rearrangement NUMERATOR DENOMINATOR Abnormal liveborn baby All liveborns Abnormal liveborn baby All recognized pregnancies Abnormal amniocentesis result (early second trimester) All pregnancies at ~16 weeks 8–14 week miscarriage All recognized pregnancies Abnormal embryo on biopsy All embryos from one in vitro fertilization procedure Deriving and Using a Risk Figure  83 Association: Coincidental or Causal? The counselor not infrequently encounters the problem of a chromosomal “abnormality” discovered in a phenotypically abnormal individual but in whose family, others—who are quite normal—are then shown to have, apparently, exactly the same rearrangement. Does a genetic risk apply, then, to children of the carrier, to whom the same rearranged chromosome may be transmitted? From classical cytogenetics, the familial paracentric inversion is a good example. In a review of 69 probands, Price et al. (1987) list the phenotypic abnormalities that led to these individuals coming to a chromosome study. There was a collection of various clinical indications, with no consistent pattern (other than that intellectual disability was frequent), and several ascertained quite by chance at prenatal diagnosis. By definition, one parent carries the same inversion; and if the net is widened, often other relatives do so as well (Groupe de Cytogénéticiens Français 1986a). In this context, and provided of course that the carrier relatives are phenotypically normal, one would reach the conclusion that the chromosome rearrangement was balanced, with no functional compromise of the genome, and that it was coincidence that led to its discovery (Romain et al. 1983). But when some very unusual clinical picture is associated with a paracentric inversion that is rare or previously undescribed (as many inversions are), some writers are skeptical of coincidence and propose a causal link (Fryns et al. 1994; Urioste et al. 1994). Similarly, Wenger et al. (1995), noting the coincidence of children with an apparently balanced familial translocation and being phenotypically abnormal, wrote that “the chance that two rare events in the same individual are unrelated seems unlikely to us.” Here, there is a risk of deception due to “Kouska’s fallacy”—Kouska was a fictional 19th-century philosopher who concluded that the combination of unlikely events that led to his parents meeting was too implausible to believe, and that therefore he himself could not exist (Lubinsky 1986). As does Lubinsky, we must insist on the point: The proband had to be phenotypically abnormal, and the coexistence of a subsequently discovered different abnormal event (the karyotype) need not be seen as necessarily remarkable. (Having made that point, we cannot, nevertheless, discount the alternative interpretation that these authors may indeed have concluded correctly.) A similar question arises when two rare karyotypes are seen in the same family, or when one individual has more than one aneuploidy. A double aneuploidy such as Klinefelter plus Down syndrome, 48,XXY,+21, could be interpreted as two separately arising nondisjunctions but with each occurring on the basis of the same underlying predisposing factor (such as maternal age). The two conditions occur together more often than the product of the frequency of each singly, which would be consistent with that interpretation. Alternatively, if the XXY component could be shown to reflect a paternal meiotic error, while the trisomy 21 was of maternal origin, then the association could be seen as coincidental. Two different types of abnormality, such as Klinefelter plus Prader-Willi syndrome (a handful of cases of which have been published; see Nowaczyk et al. 2004), might also be judged to reflect two unrelated abnormal events, at least for the deletional form of Prader-Willi syndrome, given that the mechanisms leading to nondisjunction and to deletion are quite different. The prior probability of two abnormal karyotypes coinciding might be a very small figure (1/2000 × 1/15,000 = 1/30,000,000 in the foregoing example); but recalling that the range of abnormal karyotypes is very wide, it should not necessarily be seen as reflecting some extraordinary predisposition when two

84  BASIC CONCEPTS abnormalities are diagnosed in the one individual or family. Coincidences do happen, and interesting coincidences are publishable (Schneider et al. 2004). In the molecular era, the matter of CNVs brings the question of causality into a sharp focus, although some of the answers may be less than sharp. A small molecular duplication, for example, which might at first sight appear to be a plausible candidate as explanation for a child’s abnormal phenotype, may be judged less likely as culpable if the same observation is made on the DNA sample from a parent. And yet, in the complexity that CNVs present, there may yet remain a possibility that such a duplication could contribute to abnormality when existing on a different genetic background. In other words, and as discussed above, a particular CNV may be nonpenetrant in a parent but penetrant in the child—a concept that hitherto has had little relevance in clinical cytogenetics. We can expect that CNV associations and their causing, or not, of abnormality will continue to be an active area of study (and see Chapter 18, Chapter 25, and Appendix C). PRESENTATION OF A RISK FIGURE A risk figure is a probability statement, and it should be presented as such to the counselee in everyday language—for example, “There is a 50/50 chance for such and such an event,” and “The risk for such and such to happen is around one chance in 10.” The raw probability figure may not of itself be sufficient, and it is a test of the counselor’s skill to interpret figures so as to provide empathic guidance rather than presumptuous direction. Loaded interpretative comments such as “The risk is quite high that . . .” or “There is only a small chance that . . .” should be used with great care. The perception of a risk figure as high or low may vary greatly according to an individual’s personality and life experiences, and the way he or she uses the language of numbers; the very act of discussing the risk may help the client see it in a less threatening light (Kessler and Levine 1987). Some counselors use diagrams with cartoons showing a crowd of 100 people, with the risk fraction shown in a different color. Dealing with risk advice in a pregnancy, in particular, can be anxiety-inducing. Nagle et al. (2009) examined the views of 294 Australian mothers in the postpartum period and recorded preferences for how these women felt, in retrospect, that a risk of having a child with Down syndrome might best have been conveyed. The choices were as follows, with the fractions of the women choosing each category shown: 1. As a number in percentage, such as “1%” or “0.05%” 13% 2. In words such as “no increased risk” or “increased risk” 13% 3. As numbers such as “1 in 10” or “1 in 1000” 37% 4. In words such as “high risk” or “low risk” 19% 5. Other (please specify) 0% A combination of the above 18% None of these stood out as an obvious best to help the counselor decide on the most appropriate approach. People are different! And people can see the same risk from different positions. For example, older women having an increased age-related risk (say, 1 in 100) for a child with Down syndrome may decide against an amniocentesis if a screening test gives a risk (say, 1 in 200) that

Deriving and Using a Risk Figure  85 is above the cut-off for access to amniocentesis (1 in 250) but lower than their “starting figure”; whereas a younger woman with an age-related risk of, for example, 1 in 500 is likely to opt for amniocentesis if she were to have the same 1 in 200 result from the screening test (Beekhuis et al. 1994). Responses to risk figures might not always be what we, as scientifically trained professionals, would necessarily consider objective. This is in the nature of the human condition! Urquhart (2016), a folklorist, gives her own perspective on the counseling she received during the course of prenatal diagnosis (in this case not for a chromosome condition, but rather for a Mendelian disorder, albinism). She had “always had an insatiable urge to know the future. Coupled with a keen interest in the supernatural—as a folklore scholar and as a layperson—this has led me to forms of soothsaying like tarot cards and runes but also to the people who trade in clairvoyance.” When she was about to hear the results of her amniocentesis test, she writes, “First, she [the counselor] tells me the odds. But the numbers never meant anything to me. I put as much faith in those predictions as I might in a palm reading. This child will either have albinism, or he will not.” In the event—to her initial consternation, but then fierce acceptance—he did not. PRECISION OF THE RISK FIGURE As noted above, theoretical risk figures are true, and empiric risk figures are estimates; the former are exact, and the latter are not. For an empiric figure we have a point estimate (e.g., 10%) and a likely range (e.g., 5%–15%) of where the risk actually is. The more data that have been gathered, the more accurate the estimate and the narrower the likely range—and the more confidently, therefore, can the counselor present the figure. The likely range can be measured in different ways. The standard error, which formally measures the precision of the estimate, can be used to give a sense of the region within which the true risk can realistically be considered to lie. The 95% confidence limits define the broad range that very probably (p = 0.95) encompasses the true risk.