Part 3 — Thompson & Thompson Genetics and Genomics in Medicine

Chapter 6: The Chromosomal and Genomic Basis of Disease

Ch6 · Pt1 chapter 6 The Chromosomal and Genomic Basis of Disease Disorders of the Autosomes and Sex Chromosomes Feyza Yilmaz ▶

Christine R. Beck Charles Lee In this chapter we present several of the most common and best understood chromosomal and genomic disorders encountered in clinical practice, building on the general pri...

Ch6 · Pt2 80 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE principles of gene dosage and the likely role of imbalance for individual genes that underlie specific developmental aspects of the phenotype apply to all aneuploid conditions; these are illustrated here in the context of Down syndrome, whereas the other conditions are summarized in Table 6.2. Down Syndrome Down syndrome is by far the most common and best known of the chromosome disorders and is the single most common genetic cause of moderate intellectual disability. The population incidence of Down syndrome (see Table 5.2) in live births is currently estimated to be approximately 1 in 700, reflecting the maternal age distribution for all births and the proportion of older mothers who make use of prenatal diagnosis and selective termination. At ~30 years of age, the risk begins to rise sharply, approaching 1 in 10 births in the oldest maternal age group (Fig. 6.1). Even though younger mothers have a much lower risk, their birth rate is much higher, and therefore more than half of the mothers of all newborns with Down syndrome are younger than 35 years. Down syndrome can usually be diagnosed at birth or shortly thereafter by its characteristic features, which vary among patients but nevertheless produce a distinctive phenotype (Fig. 6.2). Hypotonia may be the first abnormality noticed in the newborn. In addition to characteristic dysmorphic facial features (see Fig. 6.2), the patients are short in stature and have brachycephaly with a flat occiput. The neck is short, with loose skin on the nape. The hands are short and broad, often with a single transverse palmar crease and incurved fifth digits (termed fifth finger clinodactyly). A major cause for concern in Down syndrome is intellectual disability. Even though in early infancy the child may not seem delayed in development, the delay is usually obvious by the end of the first year. Although the extent of intellectual disability varies among individuals from moderate to mild, many children with Down syndrome develop into interactive and even self-reliant persons, and most attend local schools. There is a high degree of variability in the phenotype of Down syndrome individuals; specific abnormalities are detected in almost all patients, but others are seen only in a subset of cases. Congenital heart disease is present in about half of all liveborn infants with Down syndrome. Certain malformations, such as duodenal atresia and tracheoesophageal fistula, are much more common in Down syndrome than in other disorders. TABLE 6.1 Mechanisms of Chromosome Abnormalities and Genomic Imbalance Category Underlying Mechanism Consequences/ Examples Abnormal chromosome segregation Nondisjunction Aneuploidy (Down syndrome, Klinefelter syndrome) Uniparental disomy Recurrent chromosomal syndromes Recombination at segmental duplications Duplication/deletion syndromes Copy number variation Nonrecurrent chromosomal abnormalities Sporadic, variable breakpoints Deletion syndromes (Cri-du-chat syndrome, 1p36 deletion syndrome) De novo balanced translocations Gene disruption Unbalanced familial abnormalities Unbalanced segregation Offspring of balanced translocations Offspring of pericentric inversions Syndromes involving genomic imprinting Any event that reveals imprinted gene(s) Prader-Willi/ Angelman syndromes TABLE 6.2 Features of Autosomal Trisomies Compatible with Postnatal Survival Feature Trisomy 21 Trisomy 18 Trisomy 13 Incidence (live births) 1 in 700 1 in 6000–8000 1 in 5000–15,000 Clinical presentation Hypotonia, short stature, loose skin on nape, single palmar crease, clinodactyly Hypertonia, prenatal growth deficiency, characteristic fist clench, rocker-bottom feet Microcephaly, sloping forehead, characteristic fist clench, rockerbottom feet, polydactyly Dysmorphic facial features Flat occiput, epicanthal folds, upslanting palpebral fissures Receding jaw, low-set ears Ocular abnormalities, cleft lip and palate Intellectual disability Moderate to mild Severe Severe Other common features Congenital heart disease Severe heart malformations Severe CNS malformations Duodenal atresia Feeding difficulties Congenital heart defects Risk for leukemia Risk for premature dementia Life expectancy 60 yr Typically less than a few months; almost all <1 yr 50% die within first month, >90% within first year CNS, Central nervous system. ▶

80 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE principles of gene dosage and the likely role of imbalance for individual genes that underlie specific developmental aspects of the phenotype...

Ch6 · Pt3 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 81 Only ~20% to 25% of trisomy 21 conceptuses survive to birth (see Table 5.2). Among Down syndrome conceptuses, those least likely to survive are those with congenital heart disease; approximately one-fourth of the liveborn infants with heart defects die before their first birthday. There is a 15-fold increase in the risk for leukemia among individuals with Down syndrome who survive the neonatal period. Premature dementia, associated with the neuropathologic findings characteristic of Alzheimer disease (cortical atrophy, ventricular dilatation, and neurofibrillary tangles), affects nearly all individuals with Down syndrome several decades earlier than the typical age at onset of Alzheimer disease in the general population. As a general principle it is important to think of this constellation of clinical findings, their variation, and likely outcomes in terms of gene imbalance—the relative overabundance of specific gene products; their impact on various critical pathways in particular tissues and cell types, both early in development and throughout life; and the particular alleles present in an individual’s genome, both for genes on the trisomic chromosome and for the many other genes inherited from the parents. The Chromosomes in Down Syndrome The clinical diagnosis of Down syndrome usually presents no particular difficulty. Nevertheless, karyotyping is necessary for confirmation and to provide a basis for genetic counseling. Although the specific abnormal karyotype responsible for Down syndrome usually has little effect on the phenotype of the patient, it is essential for determining the recurrence risk. 1988 20 22 24 26 28 30 32 34 36 Cases Controls Population 1990 1992 1994 1996 Birthyear Mean Maternal Age 1998 2000 2002 2004 2006 Figure 6.1 Maternal age dependence on the incidence of trisomy 21. Comparison of mean maternal ages at the time of birth. Case (top): maternal age at birth of infant with trisomy 21; control: maternal age at birth of infant without trisomy 21; population: maternal ages at birth of infants in the population from cases and controls. (Data from Allen EG, Freeman SB, Druschel C, et al: Maternal age and risk for trisomy 21 assessed by the origin of chromosome nondisjunction: a report from the Atlanta and National Down Syndrome Projects, Hum Genet 125:41–52, 2009; Bull MJ: Down syndrome, NEJM 382:2344–2352, 2020.) Figure 6.2 Phenotype of Down syndrome. Kayla is representing the National Down Syndrome Society (ndss.org). Her hand of greeting shows characteristic short fingers; other typical features include her flattened nasal bridge, small low-set ears, and eyes displaying epicanthal folds and upslanting palpebral fissures. (Photograph by Rick Guidotti, Positive Exposure, www.positiveexposure.org.) ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 81 Only ~20% to 25% of trisomy 21 conceptuses survive to birth (see Table 5.2). Among Down syndrome conceptuses, those least likely to survive...

Ch6 · Pt4 82 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Trisomy 21. In at least 95% of all patients, the Down syndrome karyotype has 47 chromosomes, with an extra copy of chromosome 21 (see Fig. 5.8). This trisomy results from meiotic nondisjunction of the chromosome 21 pair. As noted earlier, the risk for having a child with trisomy 21 increases with maternal age, especially after the age of 30 years (see Fig. 6.1). The meiotic error responsible for the trisomy usually occurs during maternal meiosis (~90% of cases), predominantly in meiosis I, but ~10% of cases occur in paternal meiosis, often in meiosis II. Typical trisomy 21 is a sporadic event, and thus recurrences are infrequent, as will be further discussed later in this chapter. Approximately 2% of Down syndrome patients are mosaic for two cell populations – one with a normal karyotype and one with a trisomy 21 karyotype. The phenotype may be milder than that of typical trisomy 21, but there is wide variability in phenotypes among mosaic patients, presumably reflecting the variable proportion of trisomy 21 cells in the embryo during early development. Robertsonian Translocation. Approximately 4% of Down syndrome patients have 46 chromosomes, one of which is a Robertsonian translocation between chromosome 21q and the long arm of one of the other acrocentric chromosomes (usually chromosome 14 or 22) (see Fig. 5.10). The translocation chromosome replaces one of the normal acrocentric chromosomes, and the karyotype of a Down syndrome patient with a Robertsonian translocation between chromosomes 14 and 21 is therefore 46,XX or XY,rob(14;21)(q 10;q 10),+21 (see Table 5.1 for nomenclature). Despite having 46 chromosomes, patients with a Robertsonian translocation involving chromosome 21 are trisomic for genes on the entirety of 21q. A carrier of a Robertsonian translocation, involving, for example, chromosomes 14 and 21, has only 45 chromosomes; one chromosome 14 and one chromosome 21 are missing and are replaced by the translocation chromosome. The gametes that can be formed by such a carrier are shown in Fig. 6.3, and such carriers are at risk for having a child with translocation Down syndrome. Unlike standard trisomy 21, translocation Down syndrome shows no relation to maternal age but has a relatively high recurrence risk in families when a parent, especially a mother, is a carrier of the translocation. For this reason, karyotyping of the parents and possibly other relatives is essential before accurate genetic counseling can be provided. A 21q21q translocation chromosome is seen in a few percent of Down syndrome patients and is thought to originate as an isochromosome. It is particularly important to evaluate if a parent is a carrier because all gametes of a carrier of such a chromosome must either contain the 21q21q chromosome, with its double dose of chromosome 21 genetic material, or lack it and have no chromosome 21 representative at all. The potential progeny therefore inevitably have either Down syndrome or monosomy 21, which is not viable. Mosaic carriers are at an increased risk (100%) for recurrence, and thus prenatal diagnosis should be considered in any subsequent pregnancy. A C B Normal Balanced Unbalanced 21 14 Figure 6.3 Chromosomes of gametes that theoretically can be produced by a carrier of a Robertsonian translocation, rob(14;21). (A) Normal and balanced complements. (B) Unbalanced, with one product containing both the translocation chromosome and the normal chromosome 21, and the reciprocal product containing chromosome 14. (C) Unbalanced, one product with both the translocation chromosome and chromosome 14, and the reciprocal product with chromosome 21 only. Theoretically, there are six possible types of gametes, but three of them appear unable to lead to viable offspring. Only the three shaded gametes (left) can lead to viable offspring. Theoretically, the three types of gametes will be produced in equal numbers, and thus, the theoretical risk for a child with Down syndrome should be 1 in 3. However, extensive population studies have shown that unbalanced chromosome complements appear in only ~10% to 15% of the progeny of carrier mothers and in only a few percent of the progeny of carrier fathers who have translocations involving chromosome 21. ▶

82 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Trisomy 21. In at least 95% of all patients, the Down syndrome karyotype has 47 chromosomes, with an extra copy of chromosome 21 (see Fig. 5....

Ch6 · Pt5 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 83 Partial Trisomy 21. Very rarely, Down syndrome is diagnosed in a patient in whom only a part of the long arm of chromosome 21 is present in triplicate. These patients are of particular significance because they can show what region of chromosome 21 is likely to be responsible for specific components of the Down syndrome phenotype and what regions can be triplicated without causing that aspect of the phenotype. The most notable success has been the identification of a less than 2-Mb region that is critical for the heart defects seen in ~40% of Down syndrome patients. Sorting out the specific genes crucial to the expression of the Down syndrome phenotype from those that merely happen to be syntenic with them on chromosome 21 is critical for determining the pathogenesis of the various clinical findings. UNIPARENTAL DISOMY Chromosome nondisjunction most commonly results in trisomy or monosomy for the particular chromosome involved in the segregation error. However, less commonly, it can also lead to a disomic state in which both copies of a chromosome derive from the same parent, rather than one copy being inherited from the mother and the other from the father. This situation, called uniparental disomy, is defined as the presence of a disomic cell line containing two chromosomes, or portions thereof, that are inherited from only one parent (see Table 6.1). If the two chromosomes are derived from identical sister chromatids, the situation is described as isodisomy; if both homologs from one parent are present, the situation is heterodisomy (Fig. 6.4). The most common explanation for uniparental disomy is trisomy rescue due to chromosome nondisjunction in cells of a trisomic conceptus to restore a disomic state. The cause of the originating trisomy is typical meiotic nondisjunction in one of the parental germlines; the rescue results from a second nondisjunction event, this one occurring mitotically at an early postzygotic stage, thus rescuing a fetus that otherwise would most likely be aborted spontaneously (the most common fate for any trisomic fetus; see Table 5.2). Depending on the stage and parent of the original nondisjunction event (i.e., maternal or paternal meiosis I or II), the location of meiotic recombination events, and which chromosome is subsequently lost in the postzygotic mitotic nondisjunction event, the resulting fetus or liveborn can have complete or partial isodisomy or heterodisomy for the relevant chromosome. Although it is not known how common uniparental disomy is overall, it has been documented for most chromosomes in the karyotype by demonstrating uniparental inheritance of polymorphisms in a family. Clinical abnormalities, however, have been demonstrated for only some of these, typically in cases when an imprinted region is present in two copies from one parent (see the section on genomic imprinting later in this chapter) or when a typically recessive condition (which would ordinarily imply that both parents are obligate carriers; see Chapter 7) is observed in a patient who has only one documented carrier parent. It is important to stress that, although such conditions frequently come to clinical attention because of variants in individual genes or in imprinted regions, the underlying mechanism in cases of uniparental disomy is abnormal chromosome segregation. Other Disorders Due to Uniparental Disomy Although it is unclear how common uniparental disomy is, it may provide an explanation for a disease when an imprinted region (see the section on genomic imprinting later in this chapter) is present in two copies from one parent. Thus physicians and genetic counselors must keep imprinting in mind as a possible cause of genetic disorders. For example, a few patients with cystic fibrosis and short stature have been described with two identical copies of most or the entirety of their maternal chromosome 7. In these cases, the mother happened to be a carrier for cystic fibrosis (Case 12), and because the child received two maternal copies of the mutant cystic fibrosis gene and no paternal copy of the normal allele at this locus, the child developed the disease. The growth failure was unexplained but might be related to loss of unidentified paternally imprinted genes on chromosome 7. STRUCTURAL VARIANTS AND CLINICAL IMPACT Structural variants (SVs) are typically defined as changes in DNA that are 50 bp or greater in size. SVs have different types, such as deletions, duplications, insertions, inversions, and translocations, and can potentially impact molecular and cellular processes, regulatory functions, 3D structure, and transcriptional machinery (see Chapter 4). SVs also include insertions and deletions of mobile elements. Some examples of mobile elements include long interspersed element 1 (LINE-1), Alu, short interspersed element (SINE), variable-number tandem repeat (VNTR), and SINE-R/VNTR/Alu (SVA) (see Chapter 2). Mobile element insertions into the DNA of gametes or the early embryo can disrupt genes or regulatory elements leading to disease (Table 6.3) (see Chapter 4). The Impact of Genetic Diversity The 1000 Genomes Project (1000GP) was initiated to identify genetic variation in the human genome across diverse populations, and it has been instrumental in generating the largest catalog of genomic variants. The 1000GP structural variation analysis group, known as Human Genome Structural Variation Consortium (HGSVC), aims to identify a high-quality map of SVs ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 83 Partial Trisomy 21. Very rarely, Down syndrome is diagnosed in a patient in whom only a part of the long arm of chromosome 21 is present in...

Ch6 · Pt6 84 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE and develop new methods to take advantage of both traditional and new genome analysis techniques. Two recent publications from HGSVC not only identify novel variants, including single-nucleotide variants (SNVs), insertions, and deletions, and SVs but also indicated the importance of adopting new technologies, such as longread sequencing, Strand-Seq, and optical mapping to reveal previously uncharacterized regions of the genome and detect novel variants. The vast majority of genomic TABLE 6.3 Examples of Mobile Element Insertions and Clinical Phenotypes Mobile Element Disease/Disorder LINE-1 Hemophilia A, Duchenne muscular dystrophy, β-thalassemia trait, hemophilia B, cancer, neurofibromatosis Alu Geographic atrophy, familial hypercholesterolemia SVA Fukuyama-type congenital muscular dystrophy Figure 6.4 Uniparental disomy (UPD), isodisomy, and heterodisomy examples. (A) The formation of UPD. Blue color represents paternal and maroon color represents chromosomes of maternal origin. The nondisjunction could arise in either the maternal or paternal germline. (B) Heterodisomy and isodisomy examples represented. Three offsprings with the following chromosomes are observed: normal biparental inheritance of the example chromosome, maternal heterodisomy with one of each of the mother’s chromosomes, and two potential types of uniparental isodisomy with two copies of either of the mother’s chromosomes (denoted as a and b). (Modified from Preece MA, Moore GE: Genomic imprinting, uniparental disomy and foetal growth, Trends Endocrinol Metabol 11:270–275, 2000.) ▶

84 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE and develop new methods to take advantage of both traditional and new genome analysis techniques. Two recent publications from HGSVC not only...

Ch6 · Pt7 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 85 variant data derive from individuals of European des cent residing in Western countries, which might cause incorrect clinical interpretation of genomic variants. Strikingly, a recent study showed that the African pangenome, built using sequence data from 910 individuals of African descent, contained ~10% more DNA not present in the human reference genome assembly, GRCh 38, suggesting that the current reference genome may not fully represent genomic variation in diverse human populations. This is further evidence of the need for de novo assemblies of a large number of genomes from underrepresented populations, in order to comprehensively assess the variation in the human genome. A study by Kessler and colleagues suggested that the lack of individuals of African ancestry in variant databases may have resulted in the mischaracterization of variants in the Clin Var and the Human Gene Mutation Databases highlighting the fact that additional studies are required to get a better understanding of the human genome diversity and its clinical impact. Several recent large-scale sequencing studies in underrepresented populations, including people of African, Asian, Latinx, and Native American ancestry, have been uploaded to publicly available resources, such as the Genome Aggregation Database (gnom AD). Segmental Duplications, Copy Number Variants, and Nonallelic Homologous Recombination Approximately 5% of the human genome consists of low copy repeats called segmental duplications (SDs) that are 1000 bp or greater in size, with paralogous copies (duplicated copies descended from the same origin) sharing 90% or more sequence identity. Paralogous copies of SDs either can be in tandem, exist on the same chromosome at some distance (i.e., intrachromosomal SDs), or they can be found on different chromosomes (i.e., interchromosomal SDs). Different SDs can be clustered together into complex regions called SD blocks (Fig. 6.5). High sequence identity between paralogous copies of SDs makes them an excellent substrate for nonallelic homologous recombination (NAHR) (Fig. 6.6A). NAHR refers to aberrant recombination resulting from the misalignment of two highly similar paralogous copies of SDs, which further leads to SVs, including deletions, duplications, translocations, and inversions. NAHR is a mechanism that has been shown to cause several genomic disorders, such as Williams-Beuren syndrome (WBS) on chromosome 7q11.23, 15q13.3 microdeletion/microduplication syndrome, 16p11.2 microdeletion/microduplication syndrome, and 22q11.2 deletion syndrome (DS) and cateye syndrome (CES) (22q11.2 duplication syndrome) on chromosome 22q11.2. The Impact of Inversions in Genomic Disorders It is estimated that there are more than 3400 inversions described among humans (based on Database of Genomic Variants 2020-02-05 GRCh 38 variant list). An average human genome carries as many as 156 inversions. Inversions can predispose a person to the formation of a new SV that may lead to a genomic disorder. For instance, a study has shown that a ~1.2-Mb inversion in the 7q11.23 region is found in 25% of the parent of origin chromosomes of probands with WBS. In other cases, inversions do not seem to have an increased propensity to form a new SV that causes a genomic disorder. For example, a recent study showed that among the Figure 6.5 Features of segmental duplications (SDs) in the 7q11.2 region of the human genome are represented. Black rectangles, SDA and SDB, represent two SD blocks. Yellow, black, and gray colored rectangles within SD blocks represent individual SDs, and signs embedded to each SD show orientation (>: direct, <: inverted). Text next to each SD shows the genomic position of the other highly similar copy, known as paralogous copy. SDs can be intrachromosomal (A, red rectangles), paralogous copy is present on the same chromosome (chr 7), or interchromosomal (B, blue rectangles), paralogous copy is present on a different chromosome (chr 10). (Image is obtained from University of California Santa Cruz Genome Browser.) ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 85 variant data derive from individuals of European des cent residing in Western countries, which might cause incorrect clinical interpretatio...

Ch6 · Pt8 86 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE 22 parents of probands with the 3q29 deletion syndrome, six carried the ~289-kb inversion within SDA and SDB, and three of the affected probands inherited the inversion on the intact chromosome. However, none of the parent of origin chromosomes carried the larger inversion (~2 Mb) within SDA and SDC, which might caused a pathogenic deletion in probands. Deletion and Duplication Syndromes Genomic disorders result from gain or loss of hundreds of kilobases of DNA. There are at least two mechanisms whereby SVs can be formed that lead to genomic disorders. NAHR leads to recurrent SVs, whereas nonhomologous end joining (NHEJ) and other nonhomologous recombination repair mechanisms (see Fig. 6.6B and C) lead to nonrecurrent SVs. Recurrent Structural variants NAHR is the key mechanism causing recurrent SVs. These rearrangements usually have the same size in unrelated individuals because the breakpoints are localized to interspersed, highly similar paralogous copies of SDs. Extensive analysis of over 30,000 patients worldwide has now implicated this general sequence-dependent mechanism in 50 to 100 syndromes involving contiguous gene rearrangements, which collectively are sometimes referred to as genomic disorders. Here we focus A C B Figure 6.6 Model of rearrangements underlying genomic disorders. (A) Nonallelic homologous recombination (NAHR), unequal crossing over between misaligned sister chromatids or homologous chromosomes containing highly homologous copies of segmental duplications can lead to deletion or duplication. (B) Nonhomologous end joining (NHEJ), double-strand breaks (DSBs) occur and NHEJ polymerase, nuclease, and ligase complexes initiate SV formation. Red dashed boxes represent microhomology between the two DNA ends, which is used to guide end joining. The process could result in structural variant formation. (C) Fork stalling or template switching (Fo STe S) and microhomology-mediated break-induced replication (MMBIR) model is represented. When a replication fork encounters a nick (striking arrowhead) in a template strand, one arm of the fork breaks off and results in a collapsed fork. At the single double-strand end, the 5′ end of the lagging strand (dashed black lines) is resected, giving a 30 overhang. The 3′ single-strand end of lagging-strand template (solid red lines) invades the sister leading-strand DNA (gray lines) guided by regions of microhomology (MH), forming a new replication fork. The 3′ end invasion of the lagging-strand template can reform replication forks on different genomic templates before returning to the original sister chromatid and forming a processive replication fork that completes replication. Each line represents a DNA nucleotide strand. New DNA synthesis is shown by dashed lines. For examples of genomic disorders, segmental duplications, and the size of the deleted or duplicated region, see Table 6.4. (Modified from Carvalho CM, Lupski JR: Mechanisms underlying structural variant formation in genomic disorders, Nat Rev Genet 17:224–238, 2016; Chang HH, Pannunzio NR, Adachi N, et al: Non-homologous DNA end joining and alternative pathways to double-strand break repair, Nat Rev Mol Cell Biol 18:495–506, 2017; Malhotra D, Sebat J: CNVs: harbingers of a rare variant revolution in psychiatric genetics, Cell 148:1223–1241, 2012.) ▶

86 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE 22 parents of probands with the 3q29 deletion syndrome, six carried the ~289-kb inversion within SDA and SDB, and three of the affected proba...

Ch6 · Pt9 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 87 on syndromes involving chromosome 22 to illustrate underlying genomic features of this class of disorders. Deletions and Duplications Involving Chromosome 22q11.2. A particularly common deletion, 1 in 3400 live births, involves deletions at chromosome region 22q11.2 and is referred to as 22q11.2 deletion syndrome (DS), or Di George syndrome, or velocardiofacial syndrome. This clinical syndrome is caused by a deletion of ~3 Mb within 22q11.2 on one copy of chromosome 22. The deletion and other rearrangements of this region shown in Fig. 6.7 are each mediated by NAHR between SDs in the region. Patients show characteristic craniofacial anomalies, intellectual disability, immunodeficiency, and heart defects, likely reflecting haploinsufficiency for one or more of the several dozen genes that are normally found in this region. Because the phenotype is often attributed to deficient copies of multiple, contiguous genes, the term contiguous gene syndrome can be applied to this condition. Among the genes deleted, one of the most well studied is the TBX1 gene, which has been mutated or deleted in as many as 5% of all patients with congenital heart defects, particularly for left-sided outflow tract abnormalities. The reciprocal duplication of 22q11.2 is much rarer and leads to a series of distinct dysmorphic malformations and birth defects called the 22q11.2 duplication syndrome (see Fig. 6.7). Normal 22q11.2 duplication DGS/VCFS Cat-eye syndrome 1 Mb Genes 22q11.2 proximal deletions (DGS/VCFS) 22q11.2 distal deletions 2 3 1 No. copies of 22q11.2 4 3 Mb or C A B Figure 6.7 Chromosomal deletions, duplications, and rearrangements in 22q11.2 mediated by homologous recombination between segmental duplications. (A) Normal karyotypes show two copies of 22q11.2, each containing multiple copies of a family of related segmental duplications within the region (dark blue). In Di George syndrome (DGS) or velocardiofacial syndrome (VCFS), a 3-Mb region is deleted from one homologue, removing ~30 genes; in ~10% of patients, a smaller 1.5-Mb deletion (nested within the larger segment) is deleted. The reciprocal duplication is seen in patients with dup(22)(q 11.2q11.2). Tetrasomy for 22q11.2 is seen in patients with cat-eye syndrome. Note that the duplicated region in the cat-eye syndrome chromosome is in an inverted orientation relative to the duplication seen in dup(22) patients, indicating a more complex genomic rearrangement involving these segmental duplications. (B) Expanded view of the 22q11.2 genomic region, indicating the common DGS/VCFS deletions (red) and more distal deletions (also mediated by recombination involving segmental duplications) that are seen in patients with other phenotypes (orange). Genes in the region (from www.genome.ucsc.edu browser) are indicated above the region. (C) Two-color fluorescence in situ hybridization analysis of proband with DGS, demonstrating deletion of 22q11.2 on one homologue. Green signal is hybridization to a control region in distal 22q. Red signal shows hybridization to a region in proximal 22q that is present on one copy of the chromosome but deleted from the other (arrow). (C, fluorescence in situ hybridization image courtesy Kato T, Kosaka K, Kimura M, et al: Thrombocytopenia in patients with 22q11. 2 deletion syndrome and its association with glycoprotein Ib-β, Genet Med 5:113–119, 2003.) ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 87 on syndromes involving chromosome 22 to illustrate underlying genomic features of this class of disorders. Deletions and Duplications Involv...

Ch6 · Pt10 88 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE The general concepts illustrated for disorders associated with 22q11.2 also apply to many other chromosomal and genomic disorders, some of the most common or more significant of which are summarized in Table 6.4 and Box 6.1. Nonrecurrent Structural Variants Nonrecurrent SVs usually do not have the same size in unrelated individuals. The breakpoints of these rearrangements can be localized to anywhere in the genome and are often characterized by microhomologies, small insertions, or blunt ends. At least 70 genomic disorders have now been shown to be caused by nonrecurrent SVs. Although NHEJ is the presumed mechanism for many of these rearrangements, other mechanisms for nonrecurrent SV formation include DNA replication during the aberrant repair and include fork stalling or template switching (Fo STe S) and microhomologymediated break-induced replication (MMBIR) (see Fig. 6.6B and C). In each of these mechanisms, a stalled replication fork is repaired using microhomology to prime for DNA synthesis. Nonrecurrent Chromosome Abnormalities Whereas the abnormalities just described are mediated by the landscape of specific genomic features in particular chromosomal regions, many other chromosome abnormalities are due to deletions or rearrangements that have no definitive mechanistic basis (see Table 6.1). There are TABLE 6.4 Examples of Genomic Disorders Involving Recombination Between Segmental Duplications Genomic Rearrangement Disorder Location Type Size (Mb) 1q21.1 deletion/ duplication syndrome 1q21.1 Deletion/ duplication ≈0.8 3q29 deletion/ duplication syndrome 3q29 Deletion/ duplication ~1.6 Williams syndrome 7q11.23 Deletion ≈1.6 Prader-Willi/Angelman syndrome 15q11-q 13 Deletion ≈3.5 16p11.2 deletion/ duplication syndrome 16p11.2 Deletion/ duplication ≈0.6 Smith-Magenis syndrome 17p11.2 Deletion ≈3.7 dup(17)(p 11.2p11.2) Duplication Di George syndrome/ velocardiofacial syndrome 22q11.2 Deletion ≈3.0, 1.5 Cat-eye syndrome/22q11.2 duplication syndrome Duplication Azoospermia (AZFc) Yq 11.2 Deletion ≈3.5 Based on Carvalho CM, Lupski JR: Mechanisms underlying structural variant formation in genomic disorders, Nat Rev Genet 17:224–238, 2016; Harel T, Lupski JR: Genomic disorders 20 years on—mechanisms for clinical manifestations, Clin Genet 93:439–449, 2018. BOX 6.1 LESSONS FROM GENOMIC DISORDERS Genomic disorders collectively illustrate a number of concepts of general importance for considering the causes and consequences of chromosomal or genomic imbalance. First, with few exceptions, altered gene dosage for any extensive chromosomal or genomic region is likely to result in a clinical abnormality, the phenotype of which will, in principle, reflect haploinsufficiency for or overexpression of one or more genes encoded within the region. In some cases, the clinical presentation appears to be accounted for by dosage imbalance for just a single gene; in other syndromes, however, the phenotype appears to reflect imbalance for multiple genes across the region. Second, the distribution of these duplication/deletion disorders around the genome appears not to be random because the location of families of SDs, especially in pericentromeric and subtelomeric regions, predisposes particular regions to the unequal recombination events that underlie these syndromes. Third, even patients carrying what appears to be the same chromosomal deletion or duplication can present with a range of variable phenotypes. Although the precise basis for this variability is unknown, it could be due to nongenetic causes, underlying genetic variation in the region on the nondeleted chromosome, or differences elsewhere in the genome among unrelated individuals. many reports of cytogenetically detectable abnormalities in dysmorphic patients involving events such as deletions, duplications, or translocations of one or more chromosomes in the karyotype (see Fig. 5.10). Overall, cytogenetically visible autosomal deletions occur with an estimated incidence of 1 in 7000 live births. Most of these have been seen in only a few patients and are not associated with recognized clinical syndromes. Others, however, are sufficiently common to allow delineation of clearly recognizable syndromes in which a series of patients have similar abnormalities. The defining mechanistic feature of this class of abnormalities is that the underlying chromosomal event is nonrecurrent (see Table 6.1); most of them occur de novo and have highly variable breakpoints in the particular chromosomal region, thus distinguishing them as a class from those discussed in the previous section. Autosomal Deletion Syndromes One long-recognized syndrome is the cri du chat syndrome, in which there is either a terminal or interstitial deletion of part of the short arm of chromosome 5. This deletion syndrome was given its common name because crying infants with this disorder sound like a meowing cat. The facial appearance (Fig. 6.8) is distinctive and includes microcephaly, hypertelorism, epicanthal folds, low-set ears, sometimes with preauricular tags, and micrognathia. The overall incidence of the deletion is estimated to be as high as 1 in 15,000 to 50,000 live births. ▶

88 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE The general concepts illustrated for disorders associated with 22q11.2 also apply to many other chromosomal and genomic disorders, some of th...

Ch6 · Pt11 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 89 Most cases of cri du chat syndrome are sporadic; only 10% to 15% of the patients are the offspring of translocation carriers. The breakpoints and extent of the deleted segment of chromosome 5p are highly variable among different individuals, but the critical region missing in all patients with the phenotype has been identified as band 5p15. Many of the clinical findings have been attributed to haploinsufficiency for a gene or genes within specific regions; the degree of intellectual impairment usually correlates with the size of the deletion, although genomic studies suggest that haploinsufficiency for particular regions within 5p14-p 15 may contribute disproportionately to severe intellectual disability (see Fig. 6.8). Although many large deletions can be appreciated by routine karyotyping, detection of other nonrecurrent deletions requires more detailed analysis by microarrays; this is particularly true for abnormalities involving subtelomeric bands of many chromosomes, which can be difficult to visualize well by routine chromosome banding. For example, one of the most common nonrecurrent abnormalities, the chromosome 1p36 deletion syndrome, has a population incidence of 1 in 5000 and involves a wide range of different breakpoints, all within the terminal 10 Mb of chromosome 1p. Approximately 95% of cases are de novo, and many (e.g., the case illustrated in Fig. 6.8) are not detectable by routine chromosome analysis. Typically, and in contrast to the genomic disorders presented in Table 6.4, the breakpoints are highly variable and reflect a range of different mechanisms, including terminal deletion of the chromosome arm, as seen 15.3 15.2 15.1 14 13.3 13.1 13.2 Position on 5p (Mb) 40 30 20 10 Speech Cat cry Facial phenotype Intellectual disability Intellectual disability Intellectual disability 0 p 36.32 p 36.23 p 36.21 p 36.12 p 35.2 p 35.1 p 34.2 p 33 p 32.2 p 31.3 p 31.1 p 22.2 p 21.3 p 21.1 p 13.2 p 12 -4 -2 -1 0 +1 +2 +4 D E A B C Figure 6.8 Nonrecurrent deletion syndromes. 4p- deletion syndrome is illustrated by two children supported by 4p-supportgroup. org: (A) Kamila’s smile reveals missing teeth. (B) Sadie shows what some describe as a Greek warrior helmet facial phenotype. (C) Brielle lives with Cri du chat syndrome (fivepminus.org); here, illustrating characteristic hypertelorism, short philtrum, and epicanthal folds. (D) Phenotype-karyotype map of chromosome 5p, based on chromosomal microarray analysis of a series of del(5p) patients. (E) Chromosomal microarray analysis of ~5-Mb deletion in band 1p36.3 (red), which is undetectable by conventional karyotyping. (A, B and C, Photographs by Rick Guidotti, Positive Exposure, www.positiveexposure.org; D, based on data from Zhang X, Snijders A, Segraves R, et al: High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative genome hybridization, Am J Hum Genet 76:312–326, 2005; E, courtesy M. Katharine Rudd, Emory Genetics Laboratory, Atlanta, Georgia.) ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 89 Most cases of cri du chat syndrome are sporadic; only 10% to 15% of the patients are the offspring of translocation carriers. The breakpoint...

Ch6 · Pt12 90 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE in 6q deletion (Fig. 6.9), interstitial deletion of a subtelomeric segment, or recombination between copies of repetitive elements, such as Alu or LINE-1 (see Chapter 2). Balanced Translocations With Developmental Phenotypes Reciprocal translocations are relatively common (see Chapter 5). Most are balanced and involve the precise exchange of chromosomal material between nonhomologous chromosomes; as such, they usually do not have an obvious phenotypic effect. However, among the ~1 in 2000 newborns who have a de novo balanced translocation, the risk for a congenital abnormality is empirically elevated several-fold, leading to the suggestion that some balanced translocations involve direct disruption of a gene or genes by one or both of the translocation breakpoints. Detailed analysis of a number of such cases by fluorescence in situ hybridization (FISH), microarrays, and targeted or whole genome sequencing has identified defects in protein-coding or noncoding RNA genes in patients with various phenotypes, ranging from developmental delay to congenital heart defects to autism spectrum disorders. Although the clinical abnormalities in these cases can be ascribed to variants in individual genes located at the site of the translocations, the underlying mechanism in each case is the chromosomal rearrangement itself (see Table 6.1). Segregation of Familial Abnormalities The mechanism of pathogenesis here is distinguished from the mechanism of nondisjunction described earlier in this chapter. In contrast to aneuploidy or uniparental disomy, it is not the process of segregation that is abnormal in these cases; rather, it is the random nature of events during segregation that leads to unbalanced karyotypes and thus to offspring with abnormal phenotypes. In the case of balanced translocations, for example, because the chromosomes involved form a quadrivalent in meiosis, the particular combination of chromosomes transmitted to a given gamete can lead to genomic imbalance (see Fig. 5.11), even though the segregation is itself normal. Another type of familial structural abnormality that illustrates this mechanism involves inversion chromosomes. In this case, segregation of the inverted chromosome and its normal homologue during meiosis is typically uneventful; however, unbalanced gametes can be produced as a result of the process of recombination occurring within the inverted segment, in particular for pericentric inversions (see Fig. 5.12). Different inversion chromosomes carry different risks for abnormal offspring, presumably reflecting both the likelihood that a recombination event will occur within the inverted segment and the likelihood that an unbalanced gamete can lead to viable offspring. This overall risk must be determined empirically for use in genetic counseling. Several well-described inversions illustrate this point. A pericentric inversion of chromosome 3 is one of the few for which sufficient data have been obtained to allow an estimate of the transmission of the inversion chromosome to the offspring of carriers. The inv(3) (p 25q21) originated in a couple from Newfoundland in the early 1800s and has since been reported in a number of families whose ancestors can be traced to the Atlantic provinces of Canada. Carriers of the inv(3) chromosome are normal, but some of their offspring have a characteristic abnormal phenotype associated with the presence of a recombinant chromosome 3, in which there is duplication of the segment distal to 3q21 and deficiency of the segment distal to 3p25. The other predicted unbalanced gamete, with duplication of distal 3p and deficiency of distal 3q, does not lead to viable offspring. The empirical risk for an abnormal pregnancy outcome in inv(3) carriers is greater than 40% and indicates the importance of family chromosome studies to identify carriers and to offer genetic counseling and prenatal diagnosis. Not all pericentric inversions have a risk for abnormal offspring, however. One of the most common inversions Figure 6.9 A deletion at the long-arm terminus of one chromosome 6 as revealed by subtelomeric fluorescence in situ hybridization. Green and red signals reveal intact subtelomeric sequences on the short and long arms of chromosome 6. In this metaphase spread from a patient with congenital abnormalities, we can see the loss of the red signal from one of the chromosome 6 s, consistent with a deletion of materials near the long arm terminus of that chromosome. (Courtesy Charles Lee, The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, United States.) ▶

90 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE in 6q deletion (Fig. 6.9), interstitial deletion of a subtelomeric segment, or recombination between copies of repetitive elements, such as A...

Ch6 · Pt13 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 91 seen in human chromosomes is a small pericentric inversion of chromosome 9, which is present in up to 1% of all individuals. The inv(9)(p 11q12) has no known deleterious effect on carriers and does not appear to be associated with a significant risk for miscarriage or unbalanced offspring; the empirical risk is not different from that of the population at large, and it is therefore generally considered a normal variant. Neurodevelopmental Disorders and Intellectual Disability Next we consider another class of disorders that frequently require a wide range of chromosomal and genomic approaches for the diagnosis, management, and genetic counseling. Neurodevelopmental disorders are highly heterogeneous, encompassing impairments in cognition, communication, behavior, and motor functioning. Broadly considered, the category of neurodevelopmental disorders includes overlapping diagnoses such as intellectual disability (defined as impairment of cognitive and adaptive functions in childhood), autism spectrum disorder (ASD) (see Case 5), and attention-deficit hyperactivity disorder (ADHD). This category can also include various neuropsychiatric conditions such as schizophrenia and bipolar disorder, complex traits of the type that are considered later in Chapter 9. The overall incidence of intellectual disability and developmental delay is estimated to be at least 2% to 3%, whereas ASD affects as many as 1%. Determining the genetic cause of intellectual disability in most patients is a particular challenge, especially in the absence of other clinical clues or information about the specific gene or region of the genome responsible. Especially in sporadic cases without an obvious family history, a precise diagnosis can be helpful for clinical management and genetic counseling. Thus the full range of screening methods must be considered, including karyotyping and chromosomal microarrays, as well as whole exome and whole genome sequencing. Genomic Imbalance in Neurodevelopmental Disorders In large studies comparing diagnostic yield in this patient population, chromosomal microarray analysis detects pathogenic genomic imbalances in ~12% to 16% of cases, approximately fivefold more than G-banded karyotyping alone; on this basis, chromosomal microarrays are considered the first-tier clinical test to identify genomic imbalance in patients with unexplained intellectual disability or ASD. Although an increase in the presence of multiple rare copy number variants is true both for intellectual disability and for ASD, the copy number variants in patients with intellectual disability tend to be larger and to encompass more genes and are more likely of de novo origin than those detected in ASD patients. Several deletion and duplication syndromes, including 3q29 deletion syndrome, 16p11.2 deletion and duplication syndromes, and 22q11.2 deletion and duplication syndromes, are associated with increased risk of neurodevelopmental and neuropsychiatric disorders. For instance, results from genome-wide analysis of rare copy number variants in 1123 ASD families showed a strong association between ASD and de novo 7q11.23 duplications. Many hundreds of genes have been implicated to date, with estimates as high as a thousand or more genes in the genome that, when present in too few or too many copies, can lead to neurodevelopmental disorders. Although screening for the genomic imbalance due to copy number variants is accepted as a diagnostic tool, identifying individual genes and their pathogenic variants remains a significant challenge because of clinical and genetic heterogeneity. Some genes appear to be recurrent targets of variation, accounting for up to several percent of cases; exome sequencing can identify de novo coding variants with likely or proven pathogenicity in ~15% of patients with severe, sporadic nonsyndromic intellectual disability and in cohorts of patients with the diagnosis of ASD. Whole genome sequencing has also identified likely pathogenic variants, either de novo or inherited, in ASD and in intellectual disability. Clinical Heterogeneity and Diagnostic Overlap A particular challenge for understanding neurodevelopmental disorders, their etiology, and their clinical course is the extraordinary degree of clinical heterogeneity, cooccurrence of symptoms, and diagnostic overlap among them. For cases due either to copy number variants or to single-gene variants, the same defect can lead to different clinical diagnoses in different cases and even in different family members—some with intellectual disability, some with ASD, and some with diagnosed psychiatric conditions. This heterogeneity and overlap, even when categorized by genetic/genomic diagnosis rather than clinical diagnosis, suggests the need for further study of genotype/phenotype correlations to meaningfully capture the broad range of phenotypes that might emerge among individuals with the same genetic disorder. One important factor is to analyze the effect of the copy number variant by comparing affected individuals to their unaffected family members (rather than to unrelated individuals in the general population), thus minimizing confounding effects of the wide range of cognitive and behavioral phenotypes observed even in the general population. Mechanisms Causing Genomic Disorders NAHR This mechanism is also known as unequal crossing over that occurs between highly similar copies of SDs (see Fig. 6.6A). Direct copies can result in deletions or duplications, and inverted copies can result in inversions. ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 91 seen in human chromosomes is a small pericentric inversion of chromosome 9, which is present in up to 1% of all individuals. The inv(9)(p 11...

Ch6 · Pt14 92 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE NAHR involves crossing over between two paralogous copies and can occur both in meiosis and mitosis at a lower frequency. The positions, homology, and size of the copies impact the rate of NAHR events. Regions of the genome that possess tandemly arranged SDs are more prone to rearrangements. The rate of NAHR varies between SD pairs in the genome, ranging from 2.32 × 10–5 to 8.74 × 10–7. NHEJ This mechanism results in simple, blunt copy number variant endpoints that can have short homologies at the junctions (one to three nucleotides), and unlike NAHR, extensive sequence homology is not required (see Fig. 6.6B). NHEJ can result in an aberrant repair and structural variation of the genome if ligation between double-strand breaks that are not a part of the same lesion occurs. It is possible to observe small deletions or the insertion of random nucleotides at the breakpoint junctions. NHEJ is error prone. The breakpoints of SVs formed by NHEJ are frequently observed within mobile elements, such as SINEs and LINEs. MMBIR Replication-based repair mechanisms are important when single-strand breaks during the DNA replication process result in collapsed replication forks (see Fig. 6.6C). Variants that occur as a result of this mechanism differ in size and sequence complexity. In addition to microhomology-mediated rearrangements, MMBIR mediated by inverted SDs and coupled with NHEJ can result in complex rearrangements with DUP-TRP/ INV-DUP. DISORDERS ASSOCIATED WITH GENOMIC IMPRINTING For some disorders the expression of the disease phenotype depends on whether the mutant allele or abnormal chromosome has been inherited from the father or from the mother. As we introduced in Chapter 3, such parentof-origin effects are the result of genomic imprinting. The effect of genomic imprinting on inheritance patterns in pedigrees will be discussed in Chapter 7. Here, we focus on the relevance of imprinting to clinical cytogenetics, as many imprinting effects come to light because of chromosome abnormalities. Evidence of genomic imprinting has been obtained for a number of chromosomes or chromosomal regions throughout the genome, as revealed by comparing phenotypes of individuals carrying the same cytogenetic abnormality affecting either the maternal or paternal homologue. Although estimates vary, it is likely that as many as several hundred genes in the human genome show imprinting effects. Some regions contain a single imprinted gene; others contain clusters of multiple imprinted genes, spanning in some cases well over 1 Mb along a chromosome. The hallmark of imprinted genes that distinguishes them from other autosomal loci is that only one allele, either maternal or paternal, is expressed in the relevant tissue. The effect of such mechanisms on the clinical phenotype will necessarily depend on whether a variant (SNV and CNV) is present on the maternal or paternal homologue. Among the best-studied examples of the role of genomic imprinting in human disease are PraderWilli syndrome (Case 38) and Angelman syndrome, and we discuss these next to illustrate the genetic and genomic features of imprinting conditions. An additional example is Beckwith-Wiedemann syndrome. Prader-Willi and Angelman Syndromes Prader-Willi syndrome is a relatively common syndrome characterized by neonatal hypotonia followed by obesity, excessive and indiscriminate eating habits, small hands and feet, short stature, hypogonadism, and intellectual disability (Fig. 6.10). Prader-Willi syndrome results from the absence of a paternally expressed imprinted gene or genes. In ~70% of cases of the syndrome there is a cytogenetic deletion of the proximal long arm of chromosome 15 (15q11.2-q 13); the deletion is mediated by recombination involving SDs that flank a region of approximately 5 to 6 Mb and in that sense is mechanistically similar to other genomic disorders described earlier (see Table 6.4). However, within this region lies a smaller interval that contains a number of monoallelically expressed genes, some of which are normally expressed only from the paternal copy and others of which are expressed only from the maternal copy. In Prader-Willi syndrome, the deletion is found only on chromosome 15 inherited from the patient’s father (Table 6.5). Thus the genomes of these patients have genomic information in 15q11.2-q 13 that derives only from their mothers, and the syndrome results from the loss of expression of one or more of the normally paternally expressed genes in the region. Notably, the low-copy repeats that flank the PraderWilli and Angelman syndrome regions have also been implicated in other disorders, including duplication or triplication of the region or inverted duplication of chromosome 15. This underscores that although imprinting is responsible for the inheritance and specific clinical findings in Prader-Willi and Angelman syndromes, the underlying mechanism of all these disorders involves unequal recombination of the SDs in the region. In contrast, in most patients with the rare Angelman syndrome, which is characterized by unusual facial appearance, short stature, severe intellectual disability, spasticity, and seizures, there is a deletion of the same chromosomal region, but now on the chromosome 15 ▶

92 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE NAHR involves crossing over between two paralogous copies and can occur both in meiosis and mitosis at a lower frequency. The positions, homo...

Ch6 · Pt15 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 93 inherited from the mother. Patients with Angelman syndrome therefore have genetic information in 15q11.2q 13 derived only from their fathers. This unusual circumstance demonstrates strikingly that the parental origin of genetic material (in this case, in a segment of chromosome 15) can have a profound effect on the clinical expression of a defect. Some patients with Prader-Willi syndrome do not have cytogenetically detectable deletions; instead, they have two cytogenetically normal chromosome 15s, both q 12 cen IC snoRNA gene cluster 15q11-13 deletions (PWS/AS) UBE3A PWS region (paternal AS region (maternal tel q 13.2 q 14 q 15.2 q 21.1 q 21.3 q 22.2 q 22.32 q 23 q 24.2 q 25.1 q 25.3 q 26.2 -4 -2 -1 0 +1 15 D C A B)) Figure 6.10 (A) Angelman syndrome (AS) (angelman.org) is represented by Jasper, whose smile reveals his widely spaced teeth and large lower jaw. (B) Prader-Willi syndrome (PWS) (pwsausa.org) is represented by Oaklyn, whose face shows almond-shaped eyes and narrow distance between the temple. (C) Chromosomal microarray detection of ~5-Mb deletion in 15q11.2-q 13.1 (red). (D) Schematic of the 15q11.2-q 13 region. The PWS region (shaded in blue) contains a series of imprinted genes (blue) that are expressed only from the paternal copy. The AS region (shaded in pink) contains two imprinted genes that are expressed only from the maternal copy, including the UBE3A gene, which is imprinted in the central nervous system, and variants in which can cause AS. The region is flanked by nonimprinted genes (purple) that are expressed from both maternal and paternal copies. Common deletions of the PWS/AS region, caused by recombination between pairs of segmental duplications, are shown in green at the bottom. Smaller deletions of the imprinting center (IC; orange) and of a subset of genes in the small nucleolar RNA (snoRNA) gene cluster can also lead to PWS. cen, Centromere; tel, telomere. (Photograph by Rick Guidotti, Positive Exposure, www.positiveexposure.org; C, courtesy M. Katharine Rudd, Emory Genetics Laboratory, Atlanta, Georgia; D, modified from Gene Reviews. Available from www.ncbi.nlm.nih.gov/books/NBK1116/. Copyright © University of Washington.) ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 93 inherited from the mother. Patients with Angelman syndrome therefore have genetic information in 15q11.2q 13 derived only from their fathers...

Ch6 · Pt16 94 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE of which were inherited from the mother (see Table 6.5). This situation illustrates uniparental disomy, introduced previously in this chapter in the section on abnormal chromosome segregation. A smaller percentage of patients with Angelman syndrome also have uniparental disomy, but in their case, with two intact chromosome 15s of paternal origin (see Table 6.5). These patients add further emphasis that, although genomic imprinting is responsible for bringing such cases to clinical attention, the underlying defect in a proportion of cases is one of chromosome segregation, not one of imprinting per se, which is completely normal in these cases. Primary defects in the imprinting process are seen, however, in a few patients with Prader-Willi syndrome and Angelman syndrome, who have abnormalities in the imprinting center itself. As a result, the switch from female to male imprinting during spermatogenesis or from male to female imprinting during oogenesis (see Fig. 3.12) fails to occur. Fertilization by a sperm carrying an abnormally persistent female imprint would produce a child with Prader-Willi syndrome; fertilization of an egg that bears an inappropriately persistent male imprint would result in Angelman syndrome (see Table 6.5). There is evidence that the major features of the Prader-Willi and Angelman syndrome phenotypes can be accounted for by defects at particular genes within the imprinted region. Variants in the maternal copy of a single gene, the ubiquitin-protein ligase E3A gene (UBE3A), have been found to cause Angelman syndrome (see Table 6.5). The UBE3A gene is located within the 15q11.2-q 13 imprinted region and is normally expressed only from the maternal allele in the central nervous system. Maternally inherited single-gene variants in UBE3A account for ~10% of Angelman syndrome cases. In Prader-Willi syndrome, several patients have been described with deletions of a much smaller region on the paternally inherited chromosome 15, specifically implicating the noncoding small nucleolar RNA (snoRNA)116 gene cluster in the etiology of the syndrome. THE SEX CHROMOSOMES AND THEIR ABNORMALITIES The X and Y chromosomes have long attracted interest because they differ between the sexes, have their own specific patterns of inheritance, and are involved in primary sex determination. They are structurally distinct and subject to different forms of genetic regulation, yet they pair in male meiosis. For all these reasons they require special attention. In this section we review the structure of the sex chromosomes, control of the sex determination, and abnormalities of sex development. The Structure of the Sex Chromosomes The X Chromosome One of the chromosomes involved in sex determination is the X chromosome. In 2020, the first telomere-to-telomere assembly of X was finished. There are ~900 genes, many of which are only found on the X. However, genes in pseudoautosomal regions are found on both the X and Y. Males are usually affected by X-linked diseases, e.g. Ornithine transcarbamylase deficiency (Fig. 6.11). Due to X-inactivation, X-linked traits, may appear differently in males and females. X Chromosome Inactivation The principle of X inactivation is that in somatic cells in normal females (but not in normal males), one X chromosome is inactivated early in development, thus equalizing the expression of X-linked genes in the two sexes (see Chapter 3). In normal female development, because the choice of which X chromosome is to be inactivated is a random one that is then maintained clonally, females are mosaic with respect to X-linked gene expression (see Fig. 3.14). There are many epigenetic features, including gene expression, chromatin state, noncoding RNA, DNA replication timing, histone variants, and histone modifications, that distinguish the active and inactive X chromosomes in somatic cells (Table 6.6). These features can be useful diagnostically for identifying the inactive X chromosome(s) in clinical material. In patients with extra X chromosomes (whether male or female), any X chromosome in excess of one is inactivated. Thus all diploid somatic cells in both males and females have a single active X chromosome, regardless of the total number of X or Y chromosomes present. The X chromosome contains ~900 genes, but not all of these are subject to inactivation. Notably, the genes that continue to be expressed, at least to some degree, TABLE 6.5 Genomic Mechanisms Causing Prader-Willi and Angelman Syndromes Mechanism Prader-Willi Syndrome Angelman Syndrome 15q11.2-q 13 deletion ≈70–75% (paternal) ≈75% (maternal) Uniparental disomy ≈25–30% (maternal) ≈1–2% (paternal) Imprinting defects (without an imprinting centre deletion) ≈1% ≈3% Imprinting centre deletion ≈10–15% of patients with an imprinting defect ≈10–15% of patients with an imprinting defect Gene variants Rare (small deletions within snoRNA gene cluster) ≈5–10% (UBE3A variants) Unidentified <1% ≈10–15% snoRNA, Small nucleolar RNA. Data from Beygo J, Buiting K, Ramsden SC, et al: Update of the EMQN/ACGS best practice guidelines for molecular analysis of Prader-Willi and Angelman syndromes, Eur J Hum Genet 27:1326–1340, 2019. ▶

94 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE of which were inherited from the mother (see Table 6.5). This situation illustrates uniparental disomy, introduced previously in this chapter...

Ch6 · Pt17 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 95 Ichthyosis, X-linked Placental steroid sulfatase deficiency Kallmann syndrome Chondrodysplasia punctata, X-linked recessive Hypophosphatemia Aicardi syndrome Hypomagnesemia, X-linked Retinoschisis Adrenal hypoplasia Glycerol kinase deficiency Duchenne muscular dystrophy (DMD) Becker muscular dystrophy (BMD) Ornithine transcarbamylase (OTC) deficiency Norrie disease Retinitis pigmentosa-2 Alport-like hereditary nephritis Allelic disorders 13 12.2 12.1 11 11.1 11.21 11.22 11.23 11.3 11.4 21.1 21.2 p q 21.3 22.1 22.2 22.3 21.1 21.2 21.3 22.1 22.2 22.3 23 24 25 26 27 28 Figure 6.11 The X chromosome structure and disorders associated with the p arm. (Adapted from Morey C, Avner P: Genetics and epigenetics of the X chromosome, Ann NY Acad Sci 1214(1), E18–E33, 2010; review is available: Migeon BR: X-linked diseases: susceptible females, Genet Med 22:1156–1174, 2020.) TABLE 6.6 Epigenetic and Chromosomal Features of X Chromosome Inactivation in Somatic Cells Feature Active X Inactive X Gene expression Yes; similar to male X Most genes silenced; ≈15% expressed to some degree Chromatin state Euchromatin Facultative heterochromatin; Barr body Noncoding RNA XIST gene silenced XIST RNA expressed from Xi only; associates with Barr body DNA replication timing Synchronous with autosomes Late-replicating in S phase Histone variants Similar to autosomes and male X Enriched for macro H2A Histone modifications Similar to autosomes and male X Enriched for heterochromatin marks; deficient in euchromatin marks Xi, Inactive X. from the inactive X are not distributed randomly along the X chromosome; many more genes “escape” inactivation on distal Xp (as many as 50%) than on Xq (just a few percent). This finding has important implications for genetic counseling in cases of a partial X chromosome aneuploidy because imbalance for genes on Xp may have greater clinical significance than imbalance for genes on Xq, where the effect is largely mitigated by X inactivation. Patterns of X Inactivation. X inactivation is normally random in female somatic cells and leads to mosaicism for two cell populations expressing alleles from one or the other X. Where examined, most females have approximately equal proportions of cells expressing alleles from the maternal or paternal X (i.e., ~50:50), and ~90% of phenotypically normal females fall within a distribution that extends from ~25:75 to ~75:25 (Fig. 6.12). Such a distribution presumably reflects the ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 95 Ichthyosis, X-linked Placental steroid sulfatase deficiency Kallmann syndrome Chondrodysplasia punctata, X-linked recessive Hypophosphatemia...

Ch6 · Pt18 96 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE expected range of outcomes for a random event (i.e., the choice of which X will be the inactive X) involving a relatively small number of cells during early embryogenesis. For individuals who are carriers for X-linked single-gene disorders (see Chapter 7), this X inactivation ratio can influence the clinical phenotype, depending on what proportion of cells in relevant tissues or cell types express the deleterious allele on the active X. 46,XX Nonrandom inactivation of abnormal X Abnormal X Nonrandom inactivation of normal X Balanced Nonrandom inactivation of der(X) Unbalanced X; autosome translocations X Xi Xi X X Xi abn X X Xi der(X) der(A) X Xi der(X) 90% of females Proportion of females 0.10 0.05 0.00 95:5 75:25 50:50 25:75 5:95 X inactivation ratio A B Figure 6.12 X chromosome inactivation in karyotypes with normal or abnormal X chromosomes or X;autosome translocations. (A) Normal female cells (46,XX) undergo random X inactivation, resulting in a mosaic of two cell populations (left) in which either the paternal or maternal X is the inactive X (Xi, indicated by shaded box). In phenotypically normal females, the ratio of the two cell populations has a mode at 50:50, but with variation observed in the population (right), some with an excess of cells expressing alleles from the paternal X and others with an excess of cells expressing alleles from the maternal X. (B) Individuals carrying a structurally abnormal X (abn X) or X;autosome translocation in a balanced or unbalanced state show nonrandom X inactivation in which virtually all cells have the same X inactive. The other cell population is inviable or at a growth disadvantage because of genetic imbalance and is thus underrepresented or absent. der(X) and der(A) represent the two derivatives of the X;autosome translocation. (B, Data from AmosLandfraf JM, Cottle A, Plenge RM, et al: X chromosome inactivation patterns of 1005 phenotypically unaffected females, Am J Hum Genet 79:493–499, 2006; review is available: Fang H, Disteche CM, Berletch JB: X inactivation and escape: epigenetic and structural features, Front Cell Dev Biol 219, 2019.) ▶

96 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE expected range of outcomes for a random event (i.e., the choice of which X will be the inactive X) involving a relatively small number of cel...

Ch6 · Pt19 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 97 However, there are exceptions to the distribution expected for random X inactivation when the karyotype involves a structurally abnormal X chromosome. For example, in nearly all patients with unbalanced structural abnormalities of an X chromosome (including deletions, duplications, and isochromosomes), the structurally abnormal chromosome is always the inactive X. Because the initial inactivation event early in embryonic development is likely random, the patterns observed after birth probably reflect secondary selection against genetically unbalanced cells that are invisible (see Fig. 6.12). Because of this preferential inactivation of the abnormal X, such X chromosome anomalies have less of an impact on phenotype than unbalanced abnormalities of similar size or gene content involving autosomes. Nonrandom inactivation is also observed in most cases of X;autosome translocations (see Fig. 6.12). If such a translocation is balanced, the normal X chromosome is preferentially inactivated, and the two parts of the translocated chromosome remain active, again likely reflecting selection against cells in which critical autosomal genes have been inactivated. In the unbalanced offspring of a balanced carrier, however, only the translocation product carrying the X inactivation center is present, and this chromosome is invariably inactivated; the normal X is always active. These nonrandom patterns of inactivation have the general effect of minimizing, but not always eliminating, the clinical consequences of the particular chromosomal defect. Because patterns of X inactivation are strongly correlated with clinical outcome, determination of an individual’s X inactivation pattern by cytologic or molecular analysis (see Table 6.6) is indicated in all cases involving X;autosome translocations. The X Inactivation Center. Inactivation of an X chromosome depends on the presence of the X inactivation center region (XIC) on that chromosome, whether it is a normal X chromosome or a structurally abnormal X (see Chapter 3). Detailed analysis of structurally abnormal, inactivated X chromosomes led to the identification of the XIC within an ~800-kb candidate region in proximal Xq, in band Xq 13.2 (Fig. 6.13), which coordinates many, if not all, of the critical steps necessary to initiate and promulgate the silenced chromatin state along the near-entirety of the X chosen to become the inactive X. As introduced in Chapter 3, this complex series of events requires a noncoding RNA gene, XIST, that appears to be a key master regulatory locus for the onset of X inactivation. Two additional noncoding RNA genes, DXZ4 and FIRRE are in the interval and have been implicated in various aspects of the development and maintenance of XIC. X-Linked Intellectual Disability A long-appreciated aspect of intellectual disability is the excess of males in the affected population, and a large number of variants, microdeletions, or duplications causing X-linked intellectual disability have been documented. The collective incidence of such X-linked defects has been estimated to be as high as 1 in 500 to 1000 live births. The most common cause of X-linked intellectual disability is a variant in the FMR1 gene in males with fragile X syndrome (Case 17). However, nearly 100 other X-linked genes have been implicated in X-linked intellectual disability, mostly on the basis of large family studies. Chromosomal microarray analysis has identified presumptive causal copy number variants and insertion-deletions in a further 10% of such families. In addition, exome sequencing efforts summarized in the preceding section to identify de novo changes in patients with intellectual disability have revealed an excess of such variants on the X chromosome. The Y Chromosome The structure of the Y chromosome and its role in sex development has been determined at both the molecular and genomic levels (Fig. 6.14). In male meiosis, the X and Y chromosomes normally pair by segments at the ends of their short arms (see Chapter 2) and undergo recombination in that region. The pairing segment includes the pseudoautosomal region of the X and Y chromosomes, so-called because the X- and Y-linked copies of this region are essentially identical to one another and undergo homologous recombination in meiosis I, like pairs of autosomes. (A second, smaller pseudoautosomal segment is located at the distal ends of Xq and Yq [Fig. 6.15].) By comparison with autosomes and the X chromosome, the Y chromosome is relatively gene poor (see Fig. 2.7) and contains fewer than 100 genes (some of which belong to multigene families), specifying only ~2 dozen distinct proteins. Notably, the functions of a high proportion of these genes are restricted to gonadal and genital development. Near the pseudoautosomal boundary on the Y chromosome lies the SRY gene (sex-determining region on the Y). It is present in many males with an otherwise normal 46,XX karyotype and is deleted or mutated in a proportion of females with an otherwise normal 46,XY karyotype, thus strongly implicating SRY in normal male sex determination. SRY is expressed only briefly early in development in cells of the germinal ridge just before differentiation of the testis. SRY encodes a DNAbinding protein that is likely to be a transcription factor, which up-regulates a key autosomal gene, SOX9, in the ambipotent gonad, leading ultimately to testes differentiation. Although there is clear evidence demonstrating the critical role of SRY in normal male sexual development, the presence or absence of SRY does not explain all cases of abnormal sex determination. Other genes are involved in the sex determination pathway and are discussed later in this chapter. ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 97 However, there are exceptions to the distribution expected for random X inactivation when the karyotype involves a structurally abnormal X c...

Ch6 · Pt20 98 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE One or more genes on the long arm of the Y chromosome appear to be important for spermatogenesis because deletions of these regions, AZFa, AZFb, and AZFc, termed azoospermia factors (AZF), lead to low sperm count, ranging from cases of nonobstructive azoospermia (no sperm detectable in semen) to severe oligospermia (<5 million/m L; normal range, 20–40 million/m L). De novo deletions of AZFc arise in ~1 in 4000 males and account for ~12% of azoospermic males and ~6% of males with severe oligospermia. The Control of Sex Determination The process of sex determination can be thought of as occurring in distinct but interrelated steps: Establishment of chromosomal sex (i.e., XY or XX) at the time of fertilization Initiation of alternate pathways to differentiation of one or the other gonadal sex, as determined normally by the presence or absence of the testis-determining gene (SRY) Continuation of sex-specific differentiation of internal and external sexual organs Especially after puberty, development of distinctive secondary sexual characteristics to create the corresponding phenotypic sex, as a male or female Whereas the sex chromosomes play a determining role in specifying chromosomal and gonadal sex, a number of genes located on both the sex chromosomes and the autosomes are involved in sex determination and subsequent sexual differentiation. In most instances, the role of these genes has come to light as a result of patients with various conditions known as disorders of sex development (DSD), and many of these are discussed later in this chapter. XIC Normal X (with XIC) Abnormal X (XIC absent) X inactivation XIST ncRNA from Xi Spreading along Xi Epigenetic silencing of most genes on Xi Monoallelic gene expression Biallelic gene expression No X inactivation Xi A B Figure 6.13 X chromosome inactivation and dependence on X inactivation center (XIC). (A) On normal X chromosomes, XIC lies within an ~800-kb candidate region in Xq 13.2 that contains a number of noncoding RNA (ncRNA) genes, including XIST, the master X inactivation control gene. In early development in XX embryos, the XIST RNA spreads along the length of one X, which will become the inactive X (Xi), with epigenetic silencing of most genes on that X chromosome, resulting in monoallelic expression of most, but not all X-linked genes. (B) On structurally abnormal X chromosomes that lack the XIC, X inactivation cannot occur and genes present on the abnormal X are expressed biallelically. Although a fairly large abnormal X is shown here for illustrative purposes, in fact only very small such fragments are observed in female patients, who invariably display significant congenital anomalies, suggesting that biallelic expression of larger numbers of X-linked genes is inconsistent with normal development and is likely inviable. ▶

98 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE One or more genes on the long arm of the Y chromosome appear to be important for spermatogenesis because deletions of these regions, AZFa, AZ...

Ch6 · Pt21 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 99 Embryology of the Reproductive System By the sixth week of development in both sexes, the primordial germ cells have migrated from their earlier extraembryonic location to the paired genital ridges, where they are surrounded by the sex cords to form a pair of primitive gonads. Up to this time, the developing gonad is ambipotent, regardless of whether it is chromosomally XX or XY (Fig. 6.16). Development into an ovary or a testis is determined by the coordinated action of a sequence of genes in finely balanced pathways that lead to ovarian development when no Y chromosome is present but tip to the side of testicular development when a Y is present. Under normal circumstances, the ovarian pathway is followed unless the SRY gene diverts development into the male pathway. In the absence of the SRY gene, the gonad begins to differentiate to form an ovary, beginning as early as the eighth week of gestation and continuing for several weeks; the cortex develops, the medulla regresses, and oogonia begin to develop within follicles (see Fig. 6.16). Beginning at approximately the third month, the oogonia enter meiosis I, but (as described in Chapter 2) this process is arrested at dictyotene until ovulation occurs many years later. In the presence of the SRY gene, however, the medullary tissue forms typical testes with seminiferous tubules and Leydig cells that, under the stimulation of chorionic gonadotropin from the placenta, become capable of androgen secretion (see Fig. 6.16). Spermatogonia, derived from the primordial germ cells by successive mitoses, line the walls of the seminiferous tubules where they reside together with supporting Sertoli cells, awaiting the onset of puberty to begin spermatogenesis. In the early embryo, the external genitalia consist of a genital tubercle, paired labioscrotal swellings, and paired urethral folds. From this undifferentiated state, male external genitalia develop under the influence of androgens, beginning at around 12 weeks of gestation. In the absence of a testis (or, more specifically, in the absence of androgens), female external genitalia are formed regardless of whether an ovary is present. Sex Chromosomal Aneuploidy and Aberration The most common sex chromosome abnormalities involve aneuploidy for the X and/or Y chromosomes. The phenotypes associated with these chromosomal defects are, in general, less severe than those associated with comparable autosomal disorders because, as discussed earlier, X chromosome inactivation, as well as the low gene content of the Y, minimize the clinical consequences of sex chromosome imbalance. By far the most common sex chromosome defects in liveborn infants and in fetuses are the trisomic types (XXY, XXX, and XYY), but all three are rare in spontaneous abortions. For instance, the incidence of Klinefelter syndrome (XXY) is estimated to be 1 in 650 male births, the incidence of triple X syndrome (XXX) is estimated to be 1 in 1000 females, and the incidence rate of XYY syndrome is p q SRY region present in XX testicular DSD region deleted in XY gonadal dysgenesis regions deleted in azoospermia USP9Y DDX3Y AZFa DAZ genes AZFc AZFb 11.3 11.2 11.21 11.22 11.23 12 Centromere Yp pseudoautosomal ▶

region Yq pseudoautosomal region Heterochromatic region Figure 6.14 The Y chromosome in sex determination and in disorders of sex development (DSDs). Individual genes and regions implicated in sex de...

Ch6 · Pt22 100 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE 1 in 1000 males (Table 6.7). In contrast, monosomy for the X (Turner syndrome Case 47) is less frequent in liveborn infants but is the most common chromosome anomaly reported in spontaneous abortions (see Table 6.7 and Table 5.2). Klinefelter Syndrome (47,XXY). The incidence of Klinefelter syndrome (Fig. 6.17; Table 6.8) is estimated to be as high as 1 in 650 male births. Approximately half the cases result from nondisjunction in paternal meiosis 11.3 11.2 11.1 11.1 11.21 11.22 11.23 12 Y X Yp and Yq PARs RPS4Y/RPS4X ZFY/ZFX Distal Yp 11.2/Xq 21.3 PRKY/PRKX AMGY/AMGX Proximal Yp 112/Xq 21.3 DFFRY/DFFRX XGPY/XG Yq 1L21/Xp 22.3 SMCY/SMCX+HY and pro Yq 1L22/Xq 28 13 12 11 11.1 11.1 11.2 11.21 11.22 11.23 11.3 11.4 21.1 21.2 21.3 22.1 22.2 22.3 21.1 21.2 21.3 22.1 22.2 22.3 23 24 25 26 27 28 Figure 6.15 The human genome X and Y homology chart. (Adapted from Affara N, Bishop C, Brown W, et al: Report of the second international workshop on Y chromosome mapping 1995, Cytogenet Cell Genet 73:33–76, 1996.) Ambipotent gonad (TDF/SRY) Testis Ovary (AZF genes) Paramesonephric duct Mesonephric duct Y chromosome present Y chromosome absent Spermatogenesis Female internal/external genitalia Male internal/external genitalia Androgens Figure 6.16 Scheme of developmental events in sex determination and differentiation of the male and female gonads from the ambipotent gonad. See text for discussion. TABLE 6.7 Incidence of Sex Chromosome Abnormalities Sex Disorder Karyotype Approximate Incidence Male Klinefelter syndrome 47,XXY 1/650 males 48,XXXY 1/25,000 males Others (48,XXYY; 49,XXXYY; mosaics) 1/10,000 males 47,XYY syndrome 47,XYY 1/1000 males Other X or Y chromosome abnormalities 1/1500 males XX testicular DSD 46,XX 1/20,000 males Overall incidence: 1/300 males Female Turner syndrome 45,X 1/4000 females 46,X,i(Xq) 1/50,000 females Others (deletions, mosaics) 1/15,000 females Trisomy X 47,XXX 1/1000 females Other X chromosome abnormalities 1/3000 females XY gonadal dysgenesis 46,XY 1/20,000 females Androgen insensitivity syndrome 46,XY 1/20,000 females Overall incidence: 1/650 females DSD, Disorder of sex development. Data updated from Robinson A, Linden MG, Bender BG: Prenatal diagnosis of sex chromosome abnormalities. In Milunsky A, ed: Genetic disorders of the fetus, ed 4, Baltimore, 1998, Johns Hopkins University Press, pp 249–285; Kanakis GA, Nieschlag E: Klinefelter syndrome: more than hypogonadism, Metabolism 86: 135–144, 2018; Cui X, Cui Y, Shi L, Luan J, Zhou X, & Han J: A basic understanding of Turner syndrome: incidence, complications, diagnosis, and treatment, Intractable Rare Dis Res 7(4): 223–228, 2018. ▶

100 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE 1 in 1000 males (Table 6.7). In contrast, monosomy for the X (Turner syndrome Case 47) is less frequent in liveborn infants but is the most...

Ch6 · Pt23 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 101 I because of a failure of normal Xp/Yp recombination in the pseudoautosomal region. Among cases of maternal origin, most result from errors in maternal meiosis I; maternal age is increased in such cases. Approximately 15% of Klinefelter patients have mosaic karyotypes, most commonly 46,XY/47,XXY. As a group, such mosaic patients have variable phenotypes, and some may have normal testicular development. Patients with Klinefelter syndrome have a several-fold increased risk for learning difficulties, especially in reading, that may require educational intervention. Language difficulties may lead to shyness, unassertiveness, apparent immaturity, and an increased risk for depression. In adulthood, persistent androgen deficiency may result in decreased muscle tone, a loss of libido, and decreased bone mineral density (Box 6.2). The Mechanism of Sex Reversal Disorders of sex development (DSD) consists of 46,XY karyotype and 46,XX karyotype. The overall incidence of DSDs associated with a 46,XY karyotype is ~0.6 per million females. Although a number of cytogenetic or single-gene defects have been demonstrated, many B A Figure 6.17 Phenotype of males with 47,XXY Klinefelter syndrome. The patients are tall and thin and have relatively long legs. They appear physically normal until puberty, when signs of hypogonadism become obvious. Puberty occurs at a normal age, but the testes remain small, and secondary sexual characteristics remain underdeveloped. Note narrow shoulders and chest. Gynecomastia is a feature of some Klinefelter males and is visible in the 16-year-old patient in (A) (A, From Jones KL, Jones MC, del Campo M: Smith’s recognizable patterns of human malformation, ed 7, Philadelphia, 2013, WB Saunders; B, from Grumbach MM, Hughes IA, Conte FA: Disorders of sex differentiation. In Larsen PR, Kronenberg HM, Melmed S, et al, eds: Williams textbook of endocrinology, ed 10, Philadelphia, 2003, WB Saunders.) ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 101 I because of a failure of normal Xp/Yp recombination in the pseudoautosomal region. Among cases of maternal origin, most result from errors...

Ch6 · Pt24 102 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE such cases remain unexplained. Approximately 15% of patients with 46,XY complete gonadal dysgenesis (CGD) have deletions or variants in the SRY gene that interfere with the normal male pathway. However, most females with a 46,XY karyotype have an apparently normal SRY gene. The DAX1 gene in Xp 21.3 encodes a transcription factor that plays a dosage-sensitive role in the determination of gonadal sex, implying a tightly regulated interaction between DAX1 and SRY. Although production of SRY at a critical point in early development normally leads to testis formation, an excess of DAX1 resulting from duplication of the gene can apparently suppress the normal male-determining function of SRY, leading to ovarian development. A key master gene in gonadal development and the target of SRY signaling is the SOX9 gene on chromosome 17. SOX9 is normally expressed early in development in the genital ridge and is required for normal testis formation. Variants in one copy of the SOX9 gene, typically associated with a skeletal malformation disorder called campomelic dysplasia, lead to complete gonadal dysgenesis in ~75% of 46,XY cases (Table 6.9). In the absence of one copy of the SOX9 gene, testes fail to form, and the ovarian pathway is followed instead. The phenotype of these patients suggests that the critical step for the male pathway is sufficient SOX9 expression to drive the formation of testes, normally after up-regulation by the SRY gene. In 46,XY CGD, with either a variant in SRY or a variant in SOX9, the levels of SOX9 expression remain too low for testis differentiation, allowing ovarian differentiation to ensue. As many as 10% of patients with a range of 46,XY DSD phenotypes carry variants in the NR5A1 gene, which encodes a transcriptional regulator of a number of genes, including SOX9 and DAX1. These variants are associated with inadequate androgenization of external genitalia, leading to ambiguous genitalia, partial gonadal dysgenesis, and absent or rudimentary müllerian structures. The second type of DSDs is a series of phenotypes known as the 46,XX testicular DSDs (previously termed XX sex reversal), which are characterized by the presence of male external genitalia in individuals with an apparently normal 46,XX karyotype. The overall incidence is ~1 in 20,000. Most patients have a normal male appearance at birth and are not diagnosed until puberty because of small testes, gynecomastia, and infertility, despite otherwise normal-appearing male genitalia and pubic hair TABLE 6.8 Features of Sex Chromosome Aneuploidy Conditions Feature 47,XXY Klinefelter Syndrome 47,XYY 47,XXX Trisomy X 45,X Turner Syndrome Prevalence 1 in 650 male births 1 in 1000 male births 1 in 1000 female births 1 in 2500–4000 female births Clinical phenotype Tall male (see Fig. 6.17 and text) Tall, but otherwise typical male appearance Hypotonia, delayed milestones; language and learning difficulties; tend to be taller than average Short stature, webbed neck, lymphedema; risk for cardiac abnormalities Cognition/ intelligence Verbal IQ reduced to lownormal range; educational difficulties Verbal IQ reduced to lownormal range; language delay; reading difficulties Normal to low-normal range (both verbal and performance IQ decreased) Typically normal, but performance IQ lower than verbal IQ Behavioral phenotype No major disorders; tendency to poor social adjustments, but normal adult relationships Subset with specific behavioral problems likely associated with lower IQ Typically, no behavioral problems; some anxiety and low self-esteem; reduced social skills Typically normal, but impaired social adjustment Sex development/ fertility Hypogonadism, azoospermia, infertility Normal Reduced fertility in some Premature ovarian failure Gonadal dysgenesis, delayed maturation, infertility Variant karyotypes See Table 6.7 48,XXXX; 49,XXXXX Increased severity with additional Xs 46,Xi(Xq); 45,X/46,XX mosaics; other mosaics Summarized from Ross JL, Roeltgen DP, Kushner H, et al: Behavioral and social phenotypes in boys with 47,XYY syndrome or 47,XXY Klinefelter syndrome, Pediatrics 129:769–778, 2012; Pinsker JE: Turner syndrome: updating the paradigm of clinical care, J Clin Endocrinol Metab 97:E994-E1003, 2012; and AXYS, http: www.genetic.org; Skuse D, Printzlau F, Wolstencroft J: Sex chromosome aneuploidies, Handb Clin Neurol 147: 355–376, 2018. BOX 6.2 DISORDERS OF GONADAL DEVELOPMENT Gonadal dysgenesis refers to a progressive loss of germ cells, typically leading to underdeveloped and dysfunctional (streak) gonads, with consequent failure to develop mature secondary sex characteristics. Complete gonadal dysgenesis (CGD) – as in the case of XX males (now formally designated 46,XX testicular CGD) or XY females (now formally designated 46,XY CGD) – is characterized by normal-appearing external genitalia of the opposite chromosomal sex. Cases with ambiguous external genitalia are said to have partial gonadal dysgenesis. Various types of gonadal dysgenesis, their clinical phenotypes, and genetic causes are summarized in Table 6.10. ▶

102 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE such cases remain unexplained. Approximately 15% of patients with 46,XY complete gonadal dysgenesis (CGD) have deletions or variants in the...

Ch6 · Pt25 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 103 TABLE 6.9 Examples of Genes Involved in Disorders of Sex Development Gene Location Genetic Abnormality Phenotypic Sex, Disorder 46,XY Karyotype SRY Yp 11.3 SRY variant Female, XY gonadal dysgenesis DAX1 (NR0B1) Xp 21.3 DAX1 gene duplication Female, XY gonadal dysgenesis SOX9 17q24 SOX9 variant Female, XY gonadal dysgenesis, with campomelic dysplasia NR5A1 9q33 NRSA1 variant Ambiguous genitalia, XY partial gonadal dysgenesis WNT4 1p35 WNT4 gene duplication Ambiguous genitalia, cryptorchidism AR Xq 12 AR variant Female, complete or partial androgen insensitivity syndrome 46,XX Karyotype SRY Yp 11.3 SRY gene translocated to X Male, XX (ovo)testicular DSD SOX3 Xq 27.1 SOX3 gene duplication Male, XX testicular DSD SOX9 17q24 SOX9 gene duplication Male, XX testicular DSD CYP21A2 6p21.3 CYP21A2 variant Ambiguous genitalia, virilization, micropenis DSD, Disorder of sex development. Updated from Achermann JC, Hughes IA: Disorders of sex development. In Melmed S, Polonsky KS, Larsen PR, et al, eds: Williams textbook of endocrinology, ed 12, Philadelphia, 2011, WB Saunders, pp 886–934; and Witchel SF: Disorders of sex development, Best Pract Res Clin Obstet Gynaecol 48: 90–102, 2018. TABLE 6.10 Disorders of Sex Development and Their Characteristics Disorder Gonadal Sex Phenotypic Sex Characteristics Sex chromosome DSDs Klinefelter syndrome (47,XXY and variants) Testes (dysgenetic) Male Gonadal dysgenesis; hypogonadism; azoospermia Turner syndrome (45,X) Ovary (streak gonads) Female Gonadal dysgenesis; amenorrhea 46,XX testicular DSD Testes (bilateral) Normal male (≈80%) or ambiguous (≈20%) Most present clinically after puberty with small testes, gynecomastia, azoospermia 46,XX ovotesticular DSD Testicular and ovarian tissue (ovotestis or one of each) Ambiguous Uterus may be present; surgery often required to repair external genitalia; raised as male, female, or intersex 46,XY DSD Testes (dysgenetic) Ambiguous Variable müllerian structures; penoscrotal hypospadias; risk for gonadoblastoma; raised as male or female 46,XY complete gonadal dysgenesis Undeveloped streak gonads; no sperm production Female Normal müllerian structures; risk for gonadoblastoma 46,XY partial gonadal dysgenesis Regressed testes Variable (male, female, or ambiguous) Ambiguous external genitalia with or without müllerian structures; raised as male, female, or intersex 45,X/46,XY mixed gonadal dysgenesis Asymmetric (dysgenetic testis and streak gonad) Variable (male, female, or ambiguous) Variable phenotype, ranging from a typical (short) male to Turner syndrome female; risk for gonadoblastoma DSD, Disorder of sex development. Summarized from Achermann JC, Hughes IA: Disorders of sex development. In Melmed S, Polonsky KS, Larsen PR, et al, eds: Williams textbook of endocrinology, ed 12, Philadelphia, 2011, WB Saunders, pp 886–934; Pagon RA, Adam MP, Bird TD, et al, eds: Gene Reviews [Internet]. Seattle, 1993–2013, University of Washington, Seattle, http://www.ncbi.nlm.nih.gov/books/NBK1116/ and Witchel SF: Disorders of sex development, Best Pract Res Clin Obstet Gynaecol 48:90–102, 2018. (Table 6.10). As described previously in the section on the Y chromosome, most of these individuals are found to have a copy of a normal SRY gene translocated to an X chromosome as a result of aberrant recombination. Those 46,XX males who lack an SRY gene, however, are a clinically more heterogeneous group. Approxi mately 15% to 20% of such patients are identifiable at birth because of ambiguous genitalia, including penoscrotal hypospadias and cryptorchidism (undescended testes); there are no identifiable müllerian structures, and their gender identity is male. A somewhat smaller percentage of patients, however, have both testicular and ovarian tissue, either as an ovotestis or as a separate ovary and testis, a condition known as 46,XX ovotesticular DSD (formerly called true hermaphroditism). Individuals with either testicular DSD or ovotesticular DSD who lack a translocated SRY gene have been the subject of an intense investigation to identify the responsible genetic causes. Duplications of at least two genes have been described, suggesting that increased levels of transcriptional regulators can overcome the absence of SRY and initiate the testis-specific pathway (see Table 6.9). Both gene duplications and regulatory variants can increase the level of SOX9 expression to bypass the requirement for SRY. Similarly, duplications of the X-linked SOX3 gene, which is very closely related in sequence to the SRY gene, can stimulate increased SOX9 expression, replacing the usual need for SRY (Box 6.3). Virilization of 46,XX Infants: Congenital Adrenal Hyperplasia These patients include those who have 46,XX karyotypes with a normal uterus and ovaries but with ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 103 TABLE 6.9 Examples of Genes Involved in Disorders of Sex Development Gene Location Genetic Abnormality Phenotypic Sex, Disorder 46,XY Karyo...

Ch6 · Pt26 104 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Figure 6.18 Masculinized external genitalia of a 46,XX infant caused by congenital adrenal hyperplasia (virilizing form). See text for discussion. (From Moore KL, Persaud TVN: The developing human: clinically oriented embryology, ed 5, Philadelphia, 1993, WB Saunders.) BOX 6.3 OVARIAN DEVELOPMENT AND MAINTENANCE Ovarian maintenance typically lasts for up to 5 decades in normal females. Loss of normal ovarian function before the age of 40, as seen in ~1% of women, is considered premature ovarian failure (or premature ovarian insufficiency). It has long been thought that two X chromosomes are necessary for ovarian maintenance because 45,X females, despite normal initiation of ovarian development in utero, are characterized by germ cell loss, oocyte degeneration, and ovarian dysgenesis. Further, patients with 47,XXX or with cytogenetic abnormalities involving Xq, as well as carriers of fragile X syndrome (Case 17), frequently show premature ovarian failure. Because many nonoverlapping deletions on Xq show the same effect, this finding may reflect a need for two structurally normal X chromosomes in oogenesis or simply a requirement for multiple X-linked genes. Nearly a dozen specific genes, such as desert hedgehog gene (DHH), have been implicated in familial cases of premature ovarian failure and in various forms of 46,XX gonadal dysgenesis. ambiguous or male external genitalia due to excessive virilization. The majority of such patients have congenital adrenal hyperplasia (CAH), an inherited disorder arising from specific defects in enzymes of the adrenal cortex required for cortisol biosynthesis and resulting in excess androgen production. In addition to being a frequent cause of female virilization, CAH accounts for approximately half of all cases presenting with ambiguous external genitalia. Ovarian development is normal, but excessive production of androgens causes masculinization of the external genitalia, with clitoral enlargement and labial fusion to form a scrotum-like structure (Fig. 6.18). Androgen Insensitivity Syndrome There are several forms of androgen insensitivity that result in incomplete masculinization of 46,XY individuals. Here we illustrate the essential principles by considering the X-linked syndrome known as androgen insensitivity syndrome. As the original name indicates, testes are present either within the abdomen or in the inguinal canal, where they are sometimes mistaken for hernias in infants who otherwise appear to be normal females. Although the testes in these patients secrete androgen normally, end-organ unresponsiveness to androgens results from an absence of androgen receptors in the appropriate target cells. The receptor protein, specified by the normal allele at the X-linked androgen receptor (AR) locus, has the role of forming a complex with testosterone and dihydrotestosterone. If the complex fails to form, the hormone fails to stimulate the transcription of target genes required for differentiation in the male direction. The molecular defect has been determined in many hundreds of cases and ranges from a complete deletion of the AR gene to point variants in the androgen-binding or DNA-binding domains of the androgen receptor protein. Affected individuals are chromosomal males (karyotype 46,XY) who have apparently normal female external genitalia but have a blind vagina (the female reproductive canal that ends in a sac and does not connect to internal genitalia) and no uterus or fallopian tubes. The incidence of androgen insensitivity is ~1 in 10,000 to 20,000 live births, and both complete and partial forms are known, depending on the severity of the genetic defect. In the complete form (Fig. 6.19), axillary and pubic hair are sparse or absent, and breast development occurs at the appropriate age but without menses; primary amenorrhoea is frequently the presenting clinical finding that leads to a diagnosis. TECHNOLOGIES USED IN DIAGNOSTIC TESTING Microarrays, short-read (SR) whole exome sequencing, and whole genome sequencing are the most widely utilized cost-effective diagnostic methods (see Chapter 5). But to find novel variations, SV calling methods from de novo assembled haplotype-resolved genomes using high-coverage sequencing should be utilized. Accurate detection, genotyping, and annotation of SVs are only a few of the difficulties that must be overcome for accurate SV detection in clinical settings. Determining the frequency of the variants in the population is critical to confirm that they occur at a sufficiently low frequency to call pathogenic variants. While it is possible to evaluate ▶

104 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE Figure 6.18 Masculinized external genitalia of a 46,XX infant caused by congenital adrenal hyperplasia (virilizing form). See text for discu...

Ch6 · Pt27 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 105 the frequency of SNVs using reference datasets like gnom AD, this is significantly more challenging for SVs, even though several recent population-scale studies provide much-needed SV assessment and annotation. Despite the fact that there are only a few potential SNVs at each site, the number of potential SVs that could affect each site is significantly greater because of the differences in their size and type. The ability to compare SVs to one another is further complicated by this. Because of this, it’s essential to use advanced methods like long read (LR) sequencing and genomic data from many diverse populations. The significance of selecting the proper SV calling algorithm is another facet of SVs. Using simulated and actual whole genome sequencing datasets, Kosugi and colleagues assessed the performance of 69 existing SR sequencing SV detection algorithms. They concluded that the following algorithms perform better in the deletion or duplication categories: GRIDSS, Lumpy, SVseq 2, Soft SV, Manta, and Wham. Numerous SV algorithms are frequently employed to increase the accuracy of SV calling and overlaps across techniques for all types and size ranges of SV are assessed. These findings imply that careful algorithm selection is necessary for each type and size range of SVs to accurately call SVs. Recent research has demonstrated that SV calling based on LR sequencing data should also consider a similar strategy. An alternate technology is necessary due to the shortcomings of the present cytogenetic techniques, such as chromosomal microarray and SR-based sequencing. Sequencing techniques are quite good at finding SVs, but they struggle to resolve complex regions of the genome precisely, necessitating the use of an orthogonal technique to detect and validate the SVs. Optical mapping is another technique that has been demonstrated to elucidate complex regions in SDs and detect SVs in these regions, some of which are associated with deletion and duplication syndromes. Compared to LR-based sequencing approaches, optical mapping is less expensive and has the capacity to resolve complex SVs, which shows its potential as a diagnostic tool in clinical investigations. Diagnostics depend on the accurate detection of all types of genetic disorders by optical mapping, including deletion and duplication syndromes, aneuploidies, sex chromosomal abnormalities, and disorders involving repeat expansion/contraction. DATABASES OF GENOMIC VARIANTS Name URL Description DECIPHER https://www. deciphergenomics. org/ “The DECIPHER database contains data from 40,078 patients who have given consent for broad data-sharing” NCBIClin Var https://www.ncbi.nlm. nih.gov/clinvar/ “Clin Var is a public archive with free access to reports on the relationships between human variations and phenotypes, with supporting evidence.” OMIM https://omim.org/ “Online Mendelian Inheritance in Man, An Online Catalog of Human Genes and Genetic Disorders” DGV http://dgv.tcag.ca/dgv/ app/home “Database of genomic variants containing variants identified in individuals without any known diseases or disorders” gnom AD https://gnomad. broadinstitute.org/ Genome Aggregation Database Figure 6.19 Phenotype of a 46,XY individual with complete androgen insensitivity syndrome. Note female body contours, breast development, absence of axillary hair, and sparse pubic hair. (Courtesy L. Pinsky, Mc Gill University, Montreal, Canada.) ▶

CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 105 the frequency of SNVs using reference datasets like gnom AD, this is significantly more challenging for SVs, even though several recent pop...

Ch6 · Pt28 106 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE GENERAL REFERENCES Achermann JC, Hughes IA: Disorders of sex development. In Melmed S, Polonsky KS, Larsen PR, editors: Williams textbook of endocrinology ed 12, Philadelphia, 2011, WB Saunders, pp 886–934. Gardner RJM, Sutherland GR, Shaffer LG: Chromosome abnormalities and genetic counseling, ed 4, Oxford, England, 2012, Oxford University Press. Moore KL, Persaud TVN, Torchia MG: The developing human: clinically oriented embryology, ed 9, Philadelphia, 2013, WB Saunders. REFERENCES FOR SPECIFIC TOPICS 100,000 Genomes Project Pilot Investigators, Smedley D, Smith KR, et al: 100,000 genomes pilot on rare-disease diagnosis in health care – preliminary report, NEJM 385:1868–1880, 2021. Allen EG, Freeman SB, Druschel C, et al: Maternal age and risk for trisomy 21 assessed by the origin of chromosome nondisjunction: a report from the Atlanta and National Down Syndrome Projects, Human Gen 125:41–52, 2009. Bartolomei MS, Ferguson-Smith AC: Mammalian genomic imprinting, Cold Spring Harb Perspect Biol 3:a 002592, 2011. Baxter R, Vilain R: Translational genetics for diagnosis of human disorders of sex development, Annu Rev Genomics Hum Genet 14:371–392, 2013. Berglund A, Johannsen TH, Stochholm K, et al: Incidence, prevalence, diagnostic delay, and clinical presentation of female 46, XY disorders of sex development, J Clin Endocrinol Metab 101:4532– 4540, 2016. Carvalho CM, Lupski JR: Mechanisms underlying structural variant formation in genomic disorders, Nat Rev Genet 17:224–238, 2016. Cassidy SB, Schwartz S, Miller JL, et al: Prader-Willi syndrome, Genet Med 14:10–26, 2012. Chaisson MJ, Sanders AD, Zhao X, et al: Multi-platform discovery of haplotype-resolved structural variation in human genomes, Nat Comm 10:1–16, 2019. Cooper GM, Coe BP, Girirajan S, et al: A copy number variation morbidity map of developmental delay, Nat Genet 43:838–846, 2011. Cui X, Cui Y, Shi L, Luan J, Zhou X, Han J: A basic understanding of Turner syndrome: incidence, complications, diagnosis, and treatment, Intractable Rare Dis Res 7(4):223–228, 2018. de Ligt J, Willemsen H, van Bon BWM, et al: Diagnostic exome sequencing in persons with severe intellectual disability, NEJM 367:1921–1929, 2012. Dittwald P, Gambin T, Szafranski P, et al: NAHR-mediated copynumber variants in a clinical population: mechanistic insights into both genomic disorders and Mendelizing traits, Genome Res 23(9): 1395–1409, 2013. Ellison JW, Rosenfeld JA, Shaffer LG: Genetic basis of intellectual disability, Ann Rev Med 64:441–450, 2013. Fang H, Disteche CM, Berletch JB: X inactivation and escape: epigenetic and structural features, Front Cell Dev Biol:219, 2019. Gajecka M, Mac Kay KL, Shaffer LG: Monosomy 1p36 deletion syndrome, Am J Med Genet Part C Semin Med Genet 145C:346–356, 2007. Gebhardt GS, Devriendt K, Thoelen R, et al: No evidence for a parental inversion polymorphism predisposing to rearrangements at 22q11.2 in the Di George/velocardiofacial syndrome, Eur J Human Gen 11:109–111, 2003. Higgins AW, Alkuraya FS, Bosco AF, et al: Characterization of apparently balanced chromosomal rearrangements from the Developmental Genome Anatomy Project, Am J Hum Genet 82:712–722, 2008. Hughes IA, Davies JD, Bunch TI, et al: Androgen insensitivity syndrome, Lancet 380:1419–1428, 2012. Hughes IA, Houk C, Ahmed SF, et al: Consensus statement on management of intersex disorders, Arch Dis Child 91:554–563, 2006. Huguet G, Ey E, Bourgeron T: The genetic landscapes of autism spectrum disorders, Ann Rev Genomics Hum Genet 14:191–213, 2013. Jiang Y, Yuen RKC, Jin X, et al: Detection of clinically relevant genetic variants in autism spectrum disorder by whole-genome sequencing, Am J Hum Genet 93:1–15, 2013. Kaminsky EB, Kaul V, Paschall J, et al: An evidence-based approach to establish the functional and clinical significance of copy number variants in intellectual and developmental disabilities, Genet Med 13: 777–784, 2011. Kanakis GA, Nieschlag E: Klinefelter syndrome: more than hypogonadism, Metabolism 86:135–144, 2018. Kazazian HH Jr, Moran JV: Mobile DNA in health and disease, NEJM 377:361–370, 2017. Kessler MD, Yerges-Armstrong L, Taub MA, et al: Challenges and disparities in the application of personalized genomic medicine to populations with African ancestry, Nature communications 7(1):1–8, 2016. Korbel JO, Tirosh-Wagner T, Urban AE, et al: The genetic architecture of Down syndrome phenotypes revealed by high-resolution analysis of human segmental trisomies, Proc Natl Acad Sci USA 106:12031–12036, 2009. Kosugi S, Momozawa Y, Liu X, et al: Comprehensive evaluation of structural variation detection algorithms for whole genome sequencing, Genome Biol 20:1–18, 2019. Leggett V, Jacobs P, Nation K, et al: Neurocognitive outcomes of individuals with a sex chromosome trisomy: XXX, XYY, or XXY: a systematic review, Dev Med Child Neurol 52:119–129, 2010. Mabb AM, Judson MC, Zylka MJ, et al: Angelman syndrome: insights into genomic imprinting and neurodevelopmental phenotypes, Trends Neurosci 34:293–303, 2011. Malhotra D, Sebat J: CNVs: harbingers of a rare variant revolution in psychiatric genetics, Cell 148:1223–1241, 2012. Mc Donald-Mc Ginn DM, Sullivan KE, Marino B, et al: 22q11.2 deletion syndrome, Nat Rev Dis Prim 1:1–19, 2015. Miga KH, Koren S, Rhie A, et al: Telomere-to-telomere assembly of a complete human X chromosome, Nature 585:79–84, 2020. Moreno-De-Luca A, Myers SM, Challman TD, et al: Developmental brain dysfunction: revival and expansion of old concepts based on new genetic evidence, Lancet Neurol 12:406–414, 2013. Morris JK, Alberman E, Mutton D, et al: Cytogenetic and epidemiological findings in Down syndrome: England and Wales 1989– 2009, Am J Med Genet A 158A:1151–1157, 2012. Mulle JG: The 3q29 deletion confers >40-fold increase in risk for schizophrenia, Mol Psych 20:1028–1029, 2015. Najmabadi H, Hu H, Garshasbi M, et al: Deep sequencing reveals 50 novel genes for recessive cognitive disorders, Nature 478:57–63, 2011. Rodriguez-Martin B, Alvarez EG, Baez-Ortega A, et al: Pan-cancer analysis of whole genomes identifies driver rearrangements promoted by LINE-1 retrotransposition, Nat Genet 52:306–319, 2020. Sanders SJ, Ercan-Sencicek AG, Hus V, et al: Multiple recurrent de novo CNVs, including duplications of the 7q11. 23 Williams syndrome region, are strongly associated with autism, Neuron 70: 863–885, 2011. Silber SJ: The Y chromosome in the era of intracytoplasmic sperm injection, Fertil Steril 95:2439–2448, 2011. Talkowski ME, Maussion G, Crapper L, et al: Disruption of a large intergenic noncoding RNA in subjects with neurodevelopmental disabilities, Am J Hum Genet 91:1128–1134, 2012. Talkowski ME, Rosenfeld JA, Blumenthal I, et al: Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries, Cell 149:525–537, 2012. Umehara F, Tate G, Itoh K, et al: A novel variant of desert hedgehog in a patient with 46, XY partial gonadal dysgenesis accompanied by minifascicular neuropathy, Am J Hum Genet 67:1302–1305, 2000. Watson CT, Tomas MB, Sharp AJ, et al: The genetics of microdeletion and microduplication syndromes: an update, Ann Rev Gen Hum Genet 15:215–244, 2014. Weischenfeldt J, Symmns O, Spitz F, et al: Phenotypic impact of genomic structural variation: insights from and for human disease, Nat Rev Genet 14:125–138, 2013. Yilmaz F, Gurusamy U, Mosley T, et al: Multi-modal investigation of the schizophrenia-associated 3q29 genomic interval reveals global genetic diversity with unique haplotypes and segments that increase the risk for non-allelic homologous recombination, med Rxiv, 2021. Zarrei M, Mac Donald JR, Merico D, Scherer SW. A copy number variation map of the human genome. Nat Rev Genet. 2015 Mar; 16(3):172–83. doi:10.1038/nrg 3871. Epub 2015 Feb 3. PMID: 25645873. Zufferey F, Sherr EH, Beckmann ND, et al: A 600 kb deletion syndrome at 16p11.2 leads to energy imbalance and neuropsychiatric disorders, J Med Genet 49:660–668, 2013. ▶

106 THOMPSON AND THOMPSON GENETICS AND GENOMICS IN MEDICINE GENERAL REFERENCES Achermann JC, Hughes IA: Disorders of sex development. In Melmed S, Polonsky KS, Larsen PR, editors: Williams textbook of...

Ch6 · Pt29 CHAPTER 6 — THE CHROMOSOMAL AND GENOMIC BASIS OF DISEASE 107 1. In a woman with a 47,XXX karyotype, what types of gametes would theoretically be formed and in what proportions? What are the theoretical karyotypes and phenotypes of her progeny? What are the actual karyotypes and phenotypes of her progeny? 2. Individuals carrying a copy of the inv(9) described in the text are clinically normal. Provide two possible explanations. 3. The birth incidence rates of 47,XXY and 47,XYY males are approximately equal. Is this what you would expect on the basis of the possible origins of the two abnormal karyotypes? Explain. 4. How can a person with an XX karyotype differentiate as a phenotypically normal male? 5. A small centric ring X chromosome that lacks the X inactivation center is observed in a patient with short stature, gonadal dysgenesis, and intellectual disability. Because intellectual disability is not a typical feature Turner syndrome explain its presence, with or without other associated physical anomalies, in individuals with a 46,X,r(X) karyotype. In a prenatal diagnosis involving a different family, a somewhat larger ring that contains the X inactivation center is detected. What phenotype would you predict for the fetus in this pregnancy? 6. A baby girl with ambiguous genitalia is found to have 21-hydroxylase deficiency of the salt-wasting type. What karyotype would you expect to find? What is the disorder? What genetic counseling would you offer to the parents? 7. What are the expected clinical consequences of the following deletions? If the same amount of DNA is deleted in each case, why might the severity of each be different? a. 46,XX,del(13)(pter→p 11.1:) b. 46,XY,del(Y)(pter→q 12:) c. 46,XX,del(5)(p 15) d. 46,XX,del(X)(q 23q26) 8. In genetics clinic, five pregnant women inquire about the risk that their fetus has Down syndrome. What are their risks and why? a. 23-year-old mother of a previous child with trisomy 21 b. 41-year-old mother of a previous child with trisomy 21 c. 27-year-old woman whose niece has Down syndrome d. a woman who is a carrier of a 14;21 Robertsonian translocation e. a woman whose husband is a carrier of a 14;21 Robertsonian translocation 9. A young girl with Down syndrome is karyotyped and found to carry a 21q21q translocation. With use of standard cytogenetic nomenclature, what is her karyotype? 10. Paracentric inversions generally do not raise the problem of imbalance in offspring. Why not? ▶

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🧬 Part 3: Chromosomal and Genomic Basis of Disease English

Chapter 6: The Chromosomal and Genomic Basis of Disease