🧬 PART FOUR DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING

534  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING carried the mutation but his mother did not. This scenario—a child with a recessive disorder for which only one parent is heterozygous—is commonly the circumstance behind the discovery of UPIDs that would otherwise have been without clinical effect, and it is sometimes referred to as the “unmasking” of a recessive gene. The other typical route to recognition of harmless UPDs is through the incidental discovery of long continuous/ contiguous stretches of homozygosity on single nucleotide polymorphism (SNP) microarray or genomic sequencing. The state of iso- or heterodisomy can allow an inference as to the site of the initial chromosomal error. Isodisomy for an entire chromosome typically reflects a meiosis II nondisjunction (in the absence of recombination) or a mitotic error (including monosomy rescue). In contrast, heterodisomy for an entire chromosome is due to nondisjunction at meiosis I. More commonly, recombination at meiosis I results in the coexistence of partial heterodisomy and partial isodisomy for the same chromosome pair. For example, a crossover at meiosis I in the distal long arm, followed by meiosis I nondisjunction, could lead to a disomic gamete isodisomic for distal long arm and heterodisomic for proximal long arm (Figure 19–1a, lower right). If the nondisjunction were at meiosis II, the isodisomy and heterodisomy would be the other way around, involving the proximal and distal segments, respectively (Figure 19–1a, lower left). Recognizing some forms of UPD can be achieved on SNP array, and we discuss this below. EPIGENETICS AND IMPRINTING In epigenetic variation, a core consideration is that a phenotype may differ according to whether a DNA sequence is active, or inactive but with the DNA sequence itself remaining unchanged. Our focus is on the activity or non-activity of a gene (or Table 19–1.  Imprinting Disorders due to UPD or Copy Number Variants IMPRINTING DISORDER LOCUS UPD DEL / DUP Transient neonatal diabetes 6q24 pat dup(6q)(pat) Silver-Russell syndrome 7 11p15.5 mat mat dup(mat) dup(11p)(mat) Beckwith-Wiedemann syndrome 11p15.5 pat dup(11p)(mat) Temple syndrome* 14q32 mat del(14q)(pat) Kagami syndrome 14q32 pat del(14q)(mat) Prader-Willi syndrome** 15q11q13 mat del(15q)(pat) Angelman syndrome 15q11q13 pat del(15q)(mat) Pseudohypoparathyroidism type 1B 20q13 pat del(20q)(mat) Mulchandani-Bhoj-Conlin syndrome 20q11q13 mat *Paternally inherited loss-of-function variants in the DLK1 gene at 14q32.2 cause central precocious puberty (Dauber et al. 2017). **Maternally inherited loss-of-function variants in the MAGEL2 gene at 15q11.2 cause Schaaf-Yang syndrome UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  535 Figure 19–1.  Uniparental Heterodisomy and Uniparental Isodisomy. (a) The distinction between uniparental heterodisomy and uniparental isodisomy. The four parental homologs are shown in different patterns. In the child with hetero disomy, the two homologs are different. In iso disomy, they are identical. Meiotic crossing-over can lead to segmental iso/heterodisomy, and the pattern can reveal whether the initial nondisjunction had been at meiosis I or II (see text). (b) The molecular picture of a child with paternal uniparental isodisomy 1. The markers run from D1S468 at the top of chromosome 1 down to D1S2836 at the bottom. Both the child’s chromosome 1 haplotypes are the same, and the same as one of his father’s no. 1 chromosomes. He has no chromosome 1 from his mother. (The arrow points to the position of the TRKA locus. Homozygosity for an abnormal TRKA allele was the cause of his having the recessive condition congenital insensitivity to pain, which had led to his ascertainment.) Source: From Y Miura et al., Complete paternal uniparental isodisomy for chromosome 1 revealed by mutation analyses of the TRKA (NTRK1) gene encoding a receptor tyrosine kinase for nerve growth factor in a patient with congenital insensitivity to pain with anhidrosis, Hum Genet 107: 205–209, 2000. Courtesy Y Indo, and with the permission of Springer-Verlag.

536  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING chromosomal segment), according to the parental origin of the chromosome upon which the gene (or segment) is located. Thus, a chromosomal segment can receive an “epigenetic mark”—or is “imprinted”—as it is transmitted from parent to child, depending upon whether it is the mother or the father who had contributed that chromosomal segment; and this determines whether this segment will be genetically active or not active (“silent”). This is spoken of as a “parent-of-origin” effect. The major physical basis of this epigenetic effect is due to methylation of the DNA (i.e., a methyl group attached to cytosine bases), modification of the histone scaffolding of chromatin, and to the actions of noncoding RNAs, which severally or separately can then prevent the expression pattern of the relevant gene(s). There are certain chromosome segments (in sum, only a small fraction of the whole genome) that are subject to imprinting. Slightly counterintuitively, imprinting refers to non-activity: An imprinted chromosome segment is silenced, while the non-imprinted chromosome segment is the active one. In the normal setting, with biparental inheritance, imprintable segments (or loci) function monoallelically. That is, it is only the segment of maternal origin, or only the segment of paternal origin, as the case may be, which is genetically active.3 But if both segments originate from one parent, there will be either double the amount (biallelic) of expression or no (nulliallelic) expression, according to the gender of the contributing parent. (Some imprinting is tissue-specific, in which case the aberrant expression is confined to that tissue.) It is this functional imbalance that is the root cause of the phenotypic effect in the UPD syndromes. If a chromosome is not subject to imprinting, UPD does not of itself cause abnormality, other things being equal. The only other factor due to UPD (and specifically UPID) which can lead to defect is homozygosity for a recessive mutation (“isozygosity”), as noted above. Although the list of classic UPD syndromes, as in the introduction above, is not long, imprinting as a process is by no means confined to the “big six” chromosomes 6, 7, 11, 14, 15, and 20. Joshi et al. (2016) analyzed samples from 57 individuals with UPDs for many (not quite all) chromosomes, searching for segments within these chromosomes showing a parent-of-origin methylation bias. These segments allowed a recognition of 77 “differentially methylated regions” (DMRs) (Figure 19–2), of which one or more cluster together to form “imprinting control regions” (ICRs). In turn, each ICR can control the expression of one or several genes. However, it remained an open question as to a possible pathogenic or harmless effect of these DMRs, with some of the cohort being phenotypically normal. Jima et al. (2022) expanded the number of ICRs to around 1500, distributed across all 22 autosomes and predicted to influence the expression of up to 500 genes, comprising about 3% of the genome. These imprinted genes are distributed non-randomly across the genome (Figure 19–3). Despite not being associated with childhood syndromes, it is possible that some of these imprinted loci influence the risk of common chronic diseases; for example, obesity, type 2 diabetes, cancer, and late-onset neurological disease. 3 Apart from imprinting, two other epigenetic mechanisms can lead to expression of only one allele of a gene: X-inactivation, and random monoallelic expression (RME). RME is the mosaic, mitotically stable, inactivation of one allele of an autosomal gene, and it may occur for approximately 2% of all genes (Gendrel et al. 2016). Unlike imprinting, RME involves expression, in a random and clonal fashion, from either the paternal or the maternal allele. Although the role of RME is poorly understood, it may contribute, at the level of transcription, to some of the phenotypes associated with chromosome imbalance, particularly those associated with haploinsufficiency.

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  537 An added complexity is that for some imprinted genes, imprinted (monoallelic) expression is restricted to specific tissues; for example, imprinted expression of UBE3A is limited to the brain. UNIPARENTAL DISOMY FOR A COMPLETE CHROMOSOME In UPD for a complete and intact chromosome, both members of a homologous pair come from the one parent. Four routes to lead to this state are the following (and Figures 19–4 and 19–5): • Gametic complementation • Trisomic rescue • Monosomic rescue • Mitotic error Gametic complementation is mentioned first, as the simplest and classic example, but in truth it must hardly ever be that UPD is the consequence of a meiotic error happening coincidentally in both parents (Park et al. 1998; Shaffer et al. 1998). Trisomy “rescue” or “correction”4 is the mechanism behind most UPD. The cause of the trisomy is a typical meiotic nondisjunction that happened in one of the two conceiving gametes. The rescue process takes place in a cell of the trisomic conceptus at a very early post-zygotic stage (possibly even in the zygote), with one of the trisomic chromosomes being discarded, perhaps due to anaphase lag.5 This enables a cell line within the conceptus to be restored to disomy, but if it is the “wrong” chromosome that is eliminated—that is, purely by chance, the discarded chromosome happens to be the one that came from the normal gamete—the remaining two are from the same parent, and UPD results. In this scenario, the two chromosomes will comprise one of each of the homologs of that parent: thus, uniparental heterodisomy. This would be expected to happen by chance in one-third of such rescues, biparental inheritance being maintained in the other two-thirds (close to these ratios was observed in a large study of UPD(16); Yong et al. 2002). The 46-chromosome cell with UPD that results from this process may be the progenitor of the cells that produce the inner cell mass, which in turn gives rise to the embryo. Any remaining trisomic cells may go on to form the placenta, leading to confined placental mosaicism; or, they may also contribute to the inner cell mass, leading to trisomy/disomy mosaicism of the embryo. Thus, the phenotypes in some UPD states are complicated by the additional effects of compromised placental function due to trisomy, and/or of fetal trisomy mosaicism. Monosomic rescue also comes into play following a nondisjunctional event. If a nullisomic gamete is generated at meiosis, then the conceptus will be monosomic (assuming a normal gamete from the other parent). Mitotic correction then takes place, and this is 4 It might be more accurate to speak of a “failed rescue,” or better a “foiled rescue,” since the end result is an unfortunate one. Or, “mistaken correction.” 5 Studies of human preimplantation embryos (see Chapter 23) have revealed that the two requisite events for trisomy rescue— trisomic conception and post-zygotic chromosome loss— are, individually, common occurrences, and so the phenomenon of trisomic rescue is not seen as improbable. 538  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING Figure 19–2.  Chromosomal Segments Subject to Imprinting. A display of autosomal segments subject to an imprinting effect, from a cohort of 57 cases of UPD. Differentially methylated regions (DMRs) are designated according either to a locus within or very close by that region or by a segment flanked by two loci, with the ↕ arrow between. Loci to the left of each chromosome (in red) are maternally imprinted; those to the right (in blue), paternally. Novel DMRs are boxed. Grayed chromosomes (10, 11, 18, 19) were not represented in the cohort, and thus otherwise known DMRs on these chromosomes are not shown here. Source: From RS Joshi et al., DNA methylation profiling of uniparental disomy subjects provides a map of parental epigenetic bias in the human genome, Am J Hum Genet 99: 555–566, 2016. Courtesy AJ Sharp and G Kirov, and with the permission of Elsevier. UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  539 Figure 19–2.  Continued. 540  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING achieved by replication of the single, normal, homolog received from the other parent. In this case, the UPD will be an isodisomy. The fourth possibility is a mitotic error in an initially normal conception, leading to either trisomy or monosomy. In the case of a trisomy, this is followed soon thereafter by loss, in this cell line, of the nonreplicated trisomic chromosome. In the case of a mitotic nondisjunction resulting in monosomy, the remaining homolog is then duplicated. In both cases, the UPD is isodisomic. Note that each of these four scenarios requires there to be two separate abnormal events, occurring either contemporaneously (the first scenario) or sequentially (the latter three). These errors can be meiotic (the first), meiotic followed by mitotic (second and third), or both mitotic (the fourth). In whichever case, the original abnormality will practically always have been a sporadic event, with no discernible increased risk of recurrence due to having had one affected child; and indeed, to our awareness, as yet not one instance is known of a recurrence of UPD in the setting of normal parental karyotypes. Which of these various states applies in a particular case can be discovered on SNP array. The telling observation is of long stretches of homozygosity (typically >13.5 Mb) on a single chromosome (Papenhausen et al. 2011); and the pattern of homozygosity gives insight into the etiology of the UPD (Figure 19–5). One risk factor is known, and this is increasing maternal age. The link here is that meiotic nondisjunction, the root cause of most UPD, is more prevalent in women of older childbearing age. The meiotic errors noted above as leading to trisomic rescue and monosomic rescue are typically of maternal origin. Mothers of children with a maternal UPD syndrome are older than mothers of children with the same syndrome due to other mechanisms (Nakka et al. 2019). A causative factor for the meiotic error leading to UPD(15) may be (as also in the classic disorder with a maternal-age association, namely Down syndrome) a reduced level of recombination (Robinson et al. 1998). It is worth noting that paternal UPD also has a maternal-age effect, which seeming Figure 19–3.  Imprinted Genes per Chromosomes. Notes: Number of known imprinted genes per chromosome. Unsurprisingly, chromosomes associated with UPD syndromes are enriched for imprinted genes. Source: www.geneimprint.com/site/genes-by-species

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  541 Figure 19–4.  Mechanisms Whereby Complete UPD may be Generated. (a) Gametic complementation, with one parent producing a disomic gamete, and the other a nullisomic gamete. (b) Meiotic nondisjunction in one parent to produce a disomic gamete, with a trisomic conceptus following fertilization, and subsequent mitotic loss of the homolog from the other parent. This is uniparental heterodisomy, from the parent in whom the nondisjunction had taken place. (c) Meiotic nondisjunction in one parent to produce a nullisomic gamete, with monosomic conceptus following fertilization, and subsequent mitotic reduplication of the homolog from the other parent. This is uniparental isodisomy, from the parent who had contributed the normal gamete. The reduplication may produce a free homolog or an isochromosome. (d) Two sequential mitotic errors. *Since most meiotic nondisjunction occurs in maternal gametogenesis, these asterisked gametes can be imagined to be oöcytes, with UPD(mat) and UPD(pat) resulting accordingly. Figure 19–5.  Ways in which UPD Arises. The several routes by which UPD may arise (rows 1–3), and the observations on SNP array that may inform interpretation (rows 4–5). (A and B) Meiosis 1 nondisjunction with postzygotic trisomy rescue: UPD with centromeric heterodisomy ± distal isodisomy. (C and D) Meiosis 2 nondisjunction with postzygotic rescue: UPD with centromeric isodisomy ± distal heterodisomy. (E) Postzygotic monosomy rescue: complete isodisomy.4 Source: From HM Kearney et al., Diagnostic implications of excessive homozygosity detected by SNP-based microarrays: Consanguinity, uniparental disomy, and recessive single-gene mutations, Clin Lab Med 31:595–613, 2011. Courtesy HM Kearney and LK Conlin, and with the permission of Elsevier.

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  543 contradictory statement can be appreciated upon considering the mechanism of monosomic rescue after mostly maternal nondisjunction, this being the usual initiating cause of paternal UPD. Rare mechanisms to generate complete UPD include the following: • Correction of interchange trisomy • Correction of interchange monosomy • Isochromosome formation • Correction of imbalance due to small marker chromosome If one parent carries a reciprocal translocation, asymmetric segregation of the chromosomes may lead to an interchange trisomy (p. 113) at conception, in which the translocation chromosomes, plus one of the normal homologs, are transmitted. Post-zygotic correction by the loss of one homolog restores disomy, but if it is the other parent’s chromosome that is lost, UPD is the consequence. Or, if a nullisomic gamete meets a normal gamete (interchange monosomy), the normal gamete may replicate the homolog in question to restore disomy (just as in monosomy rescue, mentioned above). Liehr (2025a) records more than 100 examples of UPD associated with a Robertsonian translocation, involving UPDs for chromosomes 13, 14, 15, 21, and 22.6 In the case of a parent with a Robertsonian translocation, the most common mechanism leading to UPD is a trisomy rescue after nondisjunction. A monosomic acrocentric chromosome, after nondisjunction from a Robertsonian translocation parent and fertilization with a normal gamete, could replicate as an isochromosome in a monosomy rescue (Berend et al. 2000; McGowan et al. 2002). Complementary isochromosomes (p. 317), of which scarcely a double-digit number have ever been described, can even allow the circumstance of “contraposed UPD”: that is, there may be UPD of the p arm from one parent and UPD of the q arm from the other. Finally, in the setting of a supernumerary small marker chromosome (SMC), there may be a coexisting UPD for the same chromosome from which the SMC was derived (James et al. 1995; Liehr 2025b). SEGMENTAL UNIPARENTAL DISOMY Segmental UPD may be acquired as the consequence of a post-zygotic somatic recombination between the maternal and paternal homolog (Figure 19–6), and in that case it will necessarily be an isodisomy (Kotzot 2008a). An assessment of “long contiguous stretches of homozygosity” may prove a useful means to demonstrate the state (Papenhausen et al. 2011). The UPD segment lies distally, the rest of the chromosome pair having a normal biparental disomy. The classical karyotype is normal. Segmental UPD can have an effect if the particular chromosomal segment incorporates loci subject to imprinting. If the recombination occurs in a cell after the formation of the inner cell mass (which gives rise to the embryo), the segmental UPD will involve only some cells; in other words, there is mosaic segmental UPD. Beckwith-Wiedemann syndrome, Russell-Silver syndrome, UPDs for chromosome 14, and transient neonatal 6 These data include both inherited and de novo Robertsonian translocations, as well as homologous Robertsonian translocations (acrocentric-derived isochromosomes). 544  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING diabetes mellitus are conditions in which segmental UPD may apply. If the segment harbors a recessive allele, “unmasking” of a recessive disorder can be the consequence. If the segmental UPD arises at a later stage of somatic development (thus, mosaic segmental UPD), conversion to homozygosity might affect only a localized tissue such as, for example, Amyere et al. (2013) show with mosaic segmental UPD(1p) in the development of cutaneous glomovenous malformations in carriers of a GLMN mutation, the locus being at 1p22.1. This is very rarely recognized.7 A partial trisomy might have different abnormal phenotypic effects according to the parental origin of the duplicated segment, if that segment is subject to imprinting. A classic example is the dup15q11.2q13.1 (p. 428): Inherited from the father, there is frequently no phenotypic consequence, but when the duplication is transmitted maternally, the child is at high risk of autism. In trisomy for distal 14q, a similar picture of dysmorphology and psychomotor deficit is seen in either paternally or maternally originating 14q trisomy. But low birth weight (sometimes less than 2000 grams for a full-term baby) is a specific observation when the duplicated 14q segment comes from the mother (Georgiades et al. 1998). Figure 19–6.  A Mechanism Whereby Segmental Uniparental (Iso)disomy may be Generated. Notes: In one cell of the early conceptus, the paternal and maternal homologs of a chromosome pair (a) undergo somatic recombination between the short arms (b and c). Segregation at mitosis (d) produces daughter cells with segmental UPD: In one (e), the short-arm distal segments of both chromosomes are now of paternal origin, and in the other (f ), they are both of maternal origin. These cells can then be the source of segmentally UPD tissue in a part of the conceptus.a a  The same mechanism may apply in the setting of somatic mosaicism for a Mendelian condition, as Happle and König (1999) discuss in the case of a boy with a variegated manifestation of the rare skin condition epidermolytic hyperkeratosis of Brocq. 7 UPD can be a factor in some adult-acquired cancers. For example, the well-known V617F mutation in the JAK2 gene at 9p24.1, occurring in bone marrow as a somatic event, may be the initiating cause of myelofibrosis, polycythemia rubra vera, or essential thrombocytosis. As clonal hematopoiesis advances, UPD can convert a lineage to 9p isozygosity, producing a greater V617F “allele burden” and presumably, in consequence, accelerated disease (Hinds et al. 2016).

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  545 ABERRANT IMPRINTING IN A BIPARENTAL SETTING Differential methylation at a particular imprinted locus can be due to (1) loss of imprinting, leading to expression from both alleles, or (2) gain of imprinting, leading to loss of expression. Aberrant imprinting can be further classified according to whether the maternal or paternal allele is affected. When imprinting is lost, a chromosomal segment that is normally imprinted (thus inactive) may lose its imprint and become active. This is “relaxation” (or inappropriate erasure) of the imprint effect, an “epimutation.” To re-emphasize the point, the DNA sequence remains unchanged. Consider Beckwith-Wiedemann syndrome (BWS). In some BWS with normal biparental inheritance of chromosome 11, the IGF-2 (insulin-like growth factor 2) and KCNQ1OT1 loci on distal 11p show biallelic expression; normally, only the paternal alleles should be functional. This overexpression of genes contributes to the overgrowth that is characteristic of the syndrome (as discussed in more detail below). Epimutations may represent random or environmental-driven errors in the establishment or maintenance of an imprint. Mosaicism is typical of epimutations, indicating a post-zygotic etiology likely occurring in the developmental period prior to implantation (Monk et al. 2019).8 An iatrogenic cause of aberrant imprinting may relate to pregnancy following assisted reproductive technology (Cortessis et al. 2018); aspects of the process of artificial ovulation stimulation, or of the embryo’s environment in vitro, may disturb DNA methylation. Other environmental factors that may influence the imprinting process are maternal nutritional status and exposure to chemical pollutants (Monk et al. 2019). UNIPARENTAL DISOMY PHENOTYPES UPD has been seen with every chromosome, and the only category yet to be discovered is UPD(19)mat (Nakka et al., 2019; Liehr 2022). Data from four million people, sourced from direct-to-consumer genetics company 23andMe and the UK Biobank Project, showed 675 instances of UPD across both databases. Where parent of origin was known, maternal-origin UPD was three times as prevalent as paternal (Figure 19–7). Six people had a “double UPD,” when two chromosomes in one individual are inherited uniparentally. The most common UPDs are for chromosomes 1, 4, 16, 21, 22, and X, and it is likely not coincidental that these chromosomes have a sparse complement of imprinted genes and thus are less vulnerable to aberrant imprinting influence (Figure 19–3). In contrast, for cases in the published literature (mostly ascertained clinically), UPDs for chromosomes 6, 7, 11, 14, and 15 predominate. Rates of UPD per chromosome in population-based data mostly align well9 with rates of trisomy in blastocysts and miscarriage samples (Figure 20–12), consistent with a shared origin of meiotic nondisjunction.10 8 The early embryo may be particularly vulnerable to epimutation because its genome does not become active until after the eight-cell stage, prior to which the embryo relies on maternal RNA and protein provided in the oöcyte. The same phenomenon may underlie the high frequency of mitotic chromosome errors in the early embryo (p. 741). 9 But not consistently so. Discrepancies between some blastocyst and UPD data are puzzling. For example, trisomy for chromosomes 18 and 19 are common in blastocysts, but UPD for these chromosomes is very rare. 10 Nakka et al. (2019) also did a genome-wide association study on their data, but identified no inherited genetic factors that might have contributed to UPD. 546  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING For certain chromosomes there is a well-defined UPD phenotype, and we list below some of the UPD syndromes. For most chromosomes, as already mentioned, there is no apparent phenotypic consequence or UPD (other than the potential unmasking of a recessive gene). However, an association between UPDs and subtle phenotypes or common diseases could easily escape recognition. Nakka et al. (2019) tested for phenotypic associations between UPDs in the 23andMe dataset and 208 phenotypes obtained from self-reported survey answers, and obtained a robust signal for just three: an association between UPD(6) and lower weight and shorter height, and between UPD(22) and an increased chance of autism. In the case of UPD arising from incomplete trisomic rescue, additional factors of trisomy of the placenta and/or a residual low-level trisomy of the fetus may contribute to the eventual phenotype. De Pater et al. (1997) note that a fetal trisomic cell line may not be detected unless the possibility of mosaicism is painstakingly pursued, and Benn (1998) uses the expression “occult mosaicism” to denote an unprovable suspicion. Because mosaicism can never be completely excluded, and neither can homozygosity for an unknown recessive mutation, one should generally incline in the direction of accepting that there is an absence of any UPD effect when instances are known both of normal and of abnormal phenotypes, or when the observed abnormalities are inconsistent (Kotzot 1999). The abnormal phenotypes will more likely be due to non-UPD mechanisms. Certain clinical groups might be considered as candidates to harbor UPD. Intrauterine growth retardation (IUGR) is one obvious category. Eggermann et al. (2001) studied 21 patients with pre- and post-natal growth retardation, choosing chromosomes 2, 7, 9, 14, 16, and 20 for analysis, and identified one with UPD(14)mat and one with UPD(20) mat. Another major category is developmental disability and congenital malformation. Figure 19–7.  UPD per Chromosome. Notes: Per chromosome instances of UPD from the 23andMe population database. The stand-out upd(16) mat is obvious. UPDs associated with a more severe clinically phenotype, such as for chromosome 15, are likely to be under-represented in this dataset. Source: From P Nakka et al., Characterization of prevalence and health consequences of uniparental disomy in four million individuals from the general population. Am J Hum Genet 105:921–932, 2019.

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  547 Combining data from three large surveys (Conlin et al. 2010; Bruno et al. 2011; King et al. 2014), more than 8,000 cases in total in which testing employed whole-genome genotyping with SNP arrays and exome sequencing, UPD was identified in one in 325 (six-fold the population UPD frequency). Specific UPDs included, not unexpectedly, chromosomes 6, 7, 11, 14, and 15. UPDs of other chromosomes may have been pathogenic due to unmasking of a recessive gene mutation or occult mosaicism; some may have been incidental findings. Scuffins et al. (2021) arrived at nearly the same frequency of UPD from the study of more than 30,000 clinical exome trios. From 99 instances where whole chromosome UPD was detected, one-third had an imprinting syndrome that explained the phenotype, one-third had an autosomal recessive disorder due to UPD, and in one-third the UPD was either incidental or of uncertain association with the patient phenotype. Concerning a possible contribution to spontaneous abortion, Levy et al. (2014) identified a similarly increased frequency (one in 265) of UPD in miscarriage samples, but a clear causal link could not be assumed. We have already noted the UPD (UPID) effect of reduction to homozygosity of a recessive mutation and the consequential unmasking of the respective Mendelian condition. The list of disorders due to this mechanism continues to grow, and even includes rare examples of two recessive diseases in the one individual when the loci happened to be on the same UPD segment/chromosome (Engel and Antonarakis 2002; Yamazawa et al. 2010; Zeesman et al. 2015). We now list, by individual chromosome, the UPD syndromes, or associations with normality, that are on record.11 We frequently comment that there is no known phenotype due to the UPD per se, and that unmasking of a recessive disorder is often the only consequential effect (and often the route to the diagnosis of UPD). Likewise, we make frequent mention that an undetected residual trisomy might contribute to a phenotype when the UPD mechanism has been trisomy correction. Nevertheless, while recognizing that the classic UPD phenotypes are limited to six chromosomes, imprinted loci are known on almost every chromosome (Figure 19–2), and it thus remains possible that more subtle and/or later-onset phenotypes such as effects on behavior and intelligence, a risk for cancer, and other complex disease predisposition, may have (as yet) escaped notice. The case is not closed. Chromosome 1. Maternal UPD of chromosome 1 may itself have no effect (provided no recessive mutations are unmasked, as exemplified in Miura et al. (2000) and illustrated in Figure 19–1b). Field et al. (1998) made the serendipitous discovery of UPD(1) in a normal diabetic adult in the course of a genetic study of diabetes, as did Miyoshi et al. (2001) in their investigation of two normal persons with anomalous Rh blood grouping results: UPD(1)mat in the former, mosaicism for paternal isodisomy 1 in the latter. More than 70 cases of UPD(1) have been ascertained via the diagnosis of recessive genes (Liehr 2025a), the large number presumably reflecting the size and gene content of chromosome 1. Unmasking of recessive genes, rather than an effect of imprinting, may have been the basis of phenotypic abnormality in a unique case of UPD(1) pat described in Chen et al. (1999). A woman of normal intelligence had a myopathy, short stature, sterility, and deafness. In this case there was a paternal isodisomy, with the chromosome 1 elements present in the form of two isochromosomes, i(1)(p10) and i(1) (q10). Using SNP arrays and whole exome sequencing, Roberts et al. (2012) identified 11 A useful and comprehensive collation of reported cases of UPD is available at T Liehr, Cases with uniparental disomy, https://cs-tl.de/DB/CA/UPD/0-Start.html

548  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING maternal UPID for all of chromosome 1 in an infant with severe combined immune deficiency and isozygosity for a maternally inherited CD45 mutation. Of additional interest in this case, seven other homozygous variants were detected that were predicted to be pathogenic, but the child was apparently without symptoms. Chromosome 1p harbors the maternally imprinted tumor suppressor gene DIRAS3 (Bildik et al. 2022), suggesting (no more than that) a possible elevated tumor risk in those with maternal UPD(1). Chromosome 2. More than 70 cases of UPD(2) have been reported (Liehr 2025a). Most were ascertained through the unmasking of recessive genes, but with multiple cases of maternal UPD(2) and paternal UPD(2) also identified in healthy individuals during the course of paternity testing. In five patients with UPD(2)mat, the recurrent observations included intrauterine and post-natal growth retardation (four of five cases), atypical bronchopulmonary dysplasia/hypoplasia (three cases), and hypospadias (two cases) (Shaffer et al. 1997; Wolstenholme et al. 2001b). Isozygosity for a recessive mutation, in this case the ABCA12 gene located at 2q34 that is the basis of severe harlequin ichthyosis, was the result of trisomic rescue in a case reported by Castiglia et al. (2009), an interpretation underpinned by the observation of non-mosaic trisomy 2 at chorionic villus sampling. In an example of the use and challenges of exome sequencing, Carmichael et al. (2013) describe a girl with UPD(2) and a complex phenotype comprising skeletal and renal dysplasia, immune deficiencies, growth failure, retinal degeneration, and ovarian insufficiency. Exome sequencing identified homozygosity for 18 potentially pathogenic variants, yet none proven to be causal. Chromosome 3. Over 20 cases of UPD(3) have been reported. Paternal UPD(3) was identified as an incidental finding in a healthy patient who was genotyped as part of a linkage study (Xiao et al. 2006). Bu et al. (2021) diagnosed UPD(3)pat at prenatal diagnosis, amniocentesis and SNP array having been performed due to advanced maternal age of 46 years; follow-up to age 18 months showed the child to be healthy with normal development. Maternal UPD(3) has been reported as unmasking the recessive phenotypes of GM1 gangliosidosis (King et al. 2014), Fanconi-Bickel syndrome (Hoffman et al. 2007), and dystrophic epidermolysis bullosa (Fassihi et al. 2006); but in none of these cases was there any evidence of an additional phenotype that might be specific to maternal UPD(3). Chromosome 4. UPD(4)mat, isodisomic or heterodisomic, may be another of the UPDs without a phenotype per se. Over 25 cases have been reported, including by Liu et al. (2015), who detected complete upid(4)mat by prenatal SNP array, performed for increased risk of Down syndrome; the child was reported to be healthy at age one year. Where a phenotype has been present, in all reports to date the clinical presentations are explicable on the basis of the unmasking of recessive alleles (Spena et al. 2004; Cottrell et al. 2012; Ding et al. 2012). Cottrell et al. (2012) propose a sequence whereby UPD led to a case of (autosomal recessive) limb-girdle muscular dystrophy type 2E (Figure 19–8). Paternal isodisomy for all of chromosome 4 led to a mild form of maple syrup urine disease in an otherwise well 21-year-old, due to homozygosity for a paternally inherited mutation in the PPM1K gene (Oyarzabal et al. 2013). Middleton et al. (2006) report a patient with major depression who was genotyped as part of a research study and who had upid(4)mat as a presumed incidental finding. Upid(4)mat may also have been an incidental finding in the child with mild intellectual disability in Palumbo et al. (2015b), although possibly the upid(4)mat unmasked a recessive gene for intellectual disability. Chromosome 5. UPD(5) is rare, with fewer than 20 cases reported, but there is no evidence of an effect of the UPD per se. Maternal UPD(5) in a patient with the skin disease UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  549 Netherton syndrome (Lin et al. 2007) and paternal isodisomy for chromosome 5 in a child with spinal muscular atrophy (Brzustowicz et al. 1994) were presumably simply the cause of the reduction to homozygosity of the respective recessive genes. Chromosome 6. The defining feature of transient neonatal diabetes mellitus (TNDM) is hyperglycemia requiring treatment with insulin, with a gradual resolution to normal glucose metabolism in the first few months of life, although with a risk subsequently for non-insulin-dependent diabetes in adult life. About two-thirds of patients with TNDM have aberrations at the TNDM region at 6q24 causing overexpression of two imprinted genes, PLAGL1 and HYMAI (Docherty et al. 2013). The three proposed mechanisms, occurring in approximately equal proportions, are UPD(6)pat (partial or complete), maternal hypomethylation of the differentially methylated region (DMR) at 6q24, and paternally inherited duplication of 6q24, this latter accounting for all familial cases (one example due to a familial insertion involving this segment is in Temple et al. 1996). Docherty et al. noted an apparent increase in the incidence of congenital abnormalities in the TNDM patients with UPD, compared to the other two categories. But UPD(6)pat can also be without apparent effect, as witness an otherwise normal girl with thalassemia whose family was being studied to find a donor for marrow transplantation and who turned out to have paternal UPID(6) (Bittencourt et al. 1997). Hypomethylation of multiple imprinted loci is a related disorder that presents with TNDM accompanied by variable manifestations of other imprinting disorders, such as intrauterine growth retardation, macroglossia, heart defects, and developmental delay. The underlying mechanism is not UPD, but rather autosomal recessive mutations in ZFP57 that result in hypomethylation of maternally methylated loci (Boonen et al. 2013). A separate and apparently sporadic entity is the multi-locus imprinting disturbance (MLID) that is observed in a minority of patients with imprinting defects. Figure 19–8.  A Sequence of Events Leading to Pathogenic UPD. Notes: The mother in question was heterozygous for the recessive gene causing limb-girdle muscular dystrophy type 2E, SGCB. Her child, homozygous for SGCB, was diagnosed with this muscular dystrophy. Source: From CE Cottrell et al., Maternal uniparental disomy of chromosome 4 in a patient with limb-girdle muscular dystrophy 2E confirmed by SNP array technology, Clin Genet 81:578–583, 2012.

550  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING MLID is characterized by disruptions at multiple loci across the genome including, but not limited to, those associated with known imprinting disorders. Most people with MLIDs have clinical features of BWS, Silver-Russell syndrome, or TNDM. Eggerman et al. (2022) review candidate genes for which variants in the maternal genome may contribute to this phenotype. These include hypomorphic variants in NLRP7, a gene of particular interest because women with biallelic inactivating NLRP7 variants are affected by recurrent hydatidiform mole (p. 623). No consistent phenotype otherwise has been associated with UPD(6)mat, although intrauterine growth retardation and prematurity have been noted in the majority of reported cases, and post-natal short stature in about half (Li et al. 2024a). In keeping with this observation, Mackay et al. (2022) identified UPD(6)mat in 7/4721 referrals for Silver-Russell syndrome testing. Chromosome 7. Silver-Russell syndrome (SRS) has as its major feature intrauterine and post-natal growth retardation, often with a concomitant limb asymmetry. Genetic causes include maternal UPD(7) (~7%–10%), and 11p15 epimutation and structural 11p aberrations (see “Chromosome 11” below); SRS due to UPD(7)mat presents with more speech and language difficulty but less incidence of congenital abnormality (Wakeling et al. 2010; Lin et al. 2021a). Executive function appears to be spared, with one small study finding a mean full-scale IQ of 101 in three individuals with SRS due to UPD(7)mat (Burgevin et al. 2023). The specific loci responsible for UPD(7) imprinting have not been identified, but one or more genes in the MEST imprinted region at 7q32.2 are strongly implicated12 (Carrera et al. 2016). There is a maternal-age association: Mean maternal age is five years older in SRS due to UPD(7)mat than 11p15 epimutation (Lin et al. 2021a); most are consequential upon “trisomy rescue” from an initial maternal meiotic nondisjunction (Chantot-Bastaraud et al. 2017). Two cases are recorded of SRS in the setting of a maternal reciprocal translocation involving chromosome 7 (Dupont et al. 2002; Behnecke et al. 2012). In both instances, the conception was probably an interchange trisomy, with subsequent loss of the paternal chromosome 7 producing the balanced state but with a maternal UPHD 7. As for paternal UPID 7, Liehr et al. (2025a) collated five cases that were identified following a diagnosis of cystic fibrosis (CF). We have seen a similar example, which was in fact the only instance of a child being born with CF from more than 10,000 women who had screened negative for CF carrier status (Archibald et al. 2014). Another such case was a woman of normal linear growth and a normal intellect, and it was only because she had a recessive condition with its locus on chromosome 7 (congenital chloride diarrhea) that she had been investigated (Höglund et al. 1994). Apart from unmasking of recessive genes, there does not appear to be a phenotype associated with paternal UPD(7). Chromosome 8. More than 25 cases of UPD(8) have been reported. UPD(8)pat is apparently without any phenotypic effect, and one may suppose that this reflects a lack of imprinted genes on this chromosome. Yu et al. (2022) diagnosed a fetus with paternal UPD(8) after an NIPT result showing high probability of trisomy 8. The pregnancy continued after amniocentesis showed paternal UPD(8) with a normal karyotype, and the resultant child was healthy at three-year follow-up. Benlian et al. (1996) had made the fortuitous discovery in an otherwise normal child with lipoprotein lipase deficiency, a 12 Microdeletions of 7q32.2 on the paternal allele involving MEST also cause a Silver-Russell syndrome-like phenotype (Vincent et al. 2022).

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  551 recessive condition for which the locus maps to 8p22. Karanjawala et al. (2000) discovered maternal isodisomy 8 by chance in a man participating in a research study. Chromosome 9. UPD(9) has been reported in over 15 cases, most ascertained through the diagnosis of recessive diseases. Maternal UPD(9) itself appears to be without effect, as evidenced by its identification in a healthy woman who presented with recurrent miscarriage and was found to have a karyotype 46,XX,i(9)(p10),i(9)(q10) (Björck et al. 1999). Homozygosity due to upid(9) at these loci has been reported in children with the corresponding recessive disease: SURF-1 with Leigh syndrome, and FOXE with syndromic congenital hypothyroidism (Tiranti et al. 1999; Castanet et al. 2010). Chromosome 10. A little over 10 cases of UPD(10) are on record. Maternal UPD(10) appears to be without effect of itself, and it is only pathogenic when a recessive disease is unmasked, the latter including familial lymphophagocytic histiocytosis and mitochondrial DNA depletion syndrome (Jones et al. 1995; Al-Jasmi et al. 2008; Nogueira et al. 2013). Jones et al. (1995) detected uphd(10)mat in a healthy 8-month-old infant ascertained after the prenatal diagnosis of confined placental mosaicism for trisomy 10. In one case of uphd(10)mat with concomitant trisomy 10 mosaicism, it was presumably the trisomy rather than the UPD that caused a severe phenotype (Hahnemann et al. 2005). Chromosome 11. There are growth regulation loci in 11p15 that are expressed monoallelically according to the parent of origin of the allele (Figure 19–9). These include the paternally expressed genes IGF2 and KCNQ1OT1, and the maternally expressed genes H19 and CDKN1C. IGF2 and H19 are located within one of two “differentially methylated regions”13 (DMR1), such that IGF2 is only expressed from the paternal allele and H19 only from the maternal allele. Similarly, KCNQ1OT1 (paternal expression) and CDKN1C (maternal expression) are under the control of the second region, DMR2 (Manipalviratn et al. 2009; Weksberg et al. 2010). Perturbation of these regions and genes can lead to two syndromes of opposite growth disorder: BWS, of which overgrowth and hemihyperplasia are characteristic, and SRS, in which growth retardation and hemihypoplasia are key features. UPD(11), either segmental or for the whole chromosome, has been reported only in a mosaic form. Beckwith-Wiedemann Syndrome. In BWS, the striking clinical picture is that of overgrowth of tissues and organs. Mosaic segmental UPD(11p15)pat is the basis of ~20% of sporadically occurring BWS. Thus, in UPD(11p)pat, IGF2 and KCNQ1OT1 are expressed biallelically, and H19 and CDKN1C are silenced (“nulliallelic”). That BWS patients with paternal UPD always show mosaicism14 indicates a mitotic origin and suggests that non-mosaic paternal UPD(11) is an embryonic lethal. Brioude et al. (2018) provide an international consensus statement outlining an approach to clinical and molecular diagnosis of BWS, along with tumor surveillance targeted by molecular subgroup. BWS due to 11p15 epimutation, affecting in particular the DMR2, has a particular association with in vitro fertilization (IVF) (Amor and Halliday 2008; Carli et al. 2022). The UPD(11p) subtype of BWS (UPD for the whole 11 short arm) carries a high risk of Wilms tumor and hepatoblastoma, and smaller risk of exomphalos (Brioude et al. 13 There is a multiplicity of nomenclature of these regions. DMR1 and DMR2 may be referred to as Imprinting Control Regions 1 and 2, ICR1 and ICR2, or simply as Imprinting Centre 1 and 2, IC1 and IC2. DMR1 is also known as H19 DMR, and the telomeric cluster; and DMR2 is also known as KvDMR1, KCNQ1OT1 DMR, LIT1 DMR, and the centromeric cluster. 14 Using diagnostic SNP array, we have detected mosaic segmental UPD(11p)pat in individuals with no clinical features of BWS. We presume that in these cases the mosaic UPD(11p) is restricted to tissues that do not contribute to the BWS phenotype. 552  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING 2018). Hemihyperplasia is a clinical indicator of this category, and those tissues with the greater fraction of UPD(11p) cells typically show a correspondingly greater degree of overgrowth. Itoh et al. (2000) describe a child with BWS having a normal adrenal gland on the right, and a very enlarged one on the left: 30% of cells in the right gland had UPD(11)pat compared with 88% on the left. Epigenetic mechanisms exist due to other than UPD, noted in the section below on “Genetic Counseling” and as outlined in Figure 19–10 and Table 19–2. The phenotype of a rare BWS patient with mosaic UPD(11)pat for the whole of chromosome 11 did not differ from that of segmental UPD(11)pat (Dutly et al. 1998), and it may be that phenotype severity in BWS is due to the proportion and distribution of affected cells rather than the size of the UPD segment (Romanelli et al. 2011). Silver-Russell Syndrome. SRS due to 11p anomaly can be considered the countertype to BWS, both clinically and at the molecular level (Schönherr et al. 2007). In SRS due to UPD(11p)mat, or to 11p “epimutation” (hypomethylation of DMR1), the maternally active gene H19 functions biallelically, whereas IGF2 is under-expressed (Horike et al. 2009). Isolated hemihypoplasia, with shorter limbs on one side, is an observation with an epimutation (Zeschnigk et al. 2008). Mosaic UPD(11p)mat appears to be a particularly rare cause of SRS, with only four reports (Bullman et al. 2008; Luk et al. 2016; Pignata et al. 2021).15 Wilms Tumor. In a study of 437 (nonsyndromic) Wilms tumor patients, Scott et al. (2008a) showed, in 13 of them, 11p15 abnormalities of the same sort that may be seen in BWS: UPD(11p), epimutations, a microinsertion, and a microdeletion in DMR1. In Figure 19–9.  The Genomic Landscape at 11p15.5. Source: From T Eggermann and D Prawitt, Further understanding of paternal uniparental disomy in Beckwith-Wiedemann syndrome, Expert Rev Endocrinol Metab 17:513–521, 2022. Courtesy T Eggermann, and with the permission of Taylor and Francis Ltd, www.tandfonline.com. 15 Pignata et al. (2021) proposed that the rarity of UPD(11p)mat might be due to negative selection for cells with this genotype, in contrast to the growth advantage afforded by the much more common UPD(11p)pat.

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  553 bilateral Wilms tumor, post-zygotic somatic hypermethylation of DMR1 is the predominant mechanism, observed in 60% of cases (Murphy et al. 2023).16 Chromosome 12. Fewer than 10 cases of UPD(12) have been described, typically associated with autosomal recessive disease. Maternal isodisomy for chromosome 12 resulted in the transmission of type 3 von Willebrand disease (Boisseau et al. 2011) and vitamin D-resistant rickets (Tamura et al. 2015), whereas paternal isodisomy 12 was the Figure 19–10.  The Role of Chromosome 11 in Beckwith-Wiedemann Syndrome. Notes: The no. 11 chromosomes in different chromosomal bases of Beckwith-Wiedemann syndrome (BWS). The maternal homolog is shown pink, the paternal homolog is blue, and the BWS critical region at 11p15 is shown orange. (a) The normal state of biparental inheritance of intact no. 15 chromosomes. (b) Paternal duplication of distal 11p. (c) Maternal reciprocal translocation disrupting the BWS critical region, with the other chromosome of the translocation shown in green. (d) Mosaic segmental paternal UPD of 11p, showing the chromosome 11 pairs of the two cell lines. The pair on the left shows paternal UPD for distal 11p (the blue segments). 16 The next most common mechanism, found in approximately 30%, is germline variants in cancer predisposition genes, such as WT1. Table 19–2.  Different Causes of Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) Syndromes (see also Figure 19–10) GENETIC FORM FRACTIONS (%) BWS SRS Gain/loss of methylation at IC1 5 (gain) 35–50 (loss) Loss of methylation at IC2 50 Uniparental disomy 20 (UPD(11)pat) 5–10 (UPD(7)mat) Large duplication (DMR1 + 2) <1 (paternal) 1–2 (maternal) Smaller CNV 2–5 <1 Inversion, translocation <1 <1 CDKN1C variants 5 (paternal, loss-of-function) <1 (maternal, gain-of-function) Unknown 15 40 Notes: Fractions (rounded) indicate relative frequencies; these data may be influenced by the clinical index of suspicion. False-negative results for methylation testing and UPD testing may occur due to mosaicism. DMR1 and -2, differentially methylated regions 1 and 2. DMR1 gain of methylation causes overexpression of IGF2 and non-expression of H19. DMR2 loss of methylation causes overexpression of KCNQ1OT1 and non-expression of CDKN1C (and see text). In a minority of cases with imprinting changes at DMR1 and/or DMR2, the imprinting change has been shown to be due to a copy number variant at 11p15 (Baskin et al. 2014). 554  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING cause of isolated sulfite oxidase deficiency (Cho et al. 2013b). Shirzadeh et al. (2018) diagnosed phenylketonuria and a homozygous PAH variant in the child of first-cousin Iranian parents; yet, despite the parental consanguinity, the mother did not carry the PAH variant, and the cause was upid(12)pat in the child. In none of these instances was there evidence of an additional phenotype attributable to the UPD. Chromosome 13. Neither maternal nor paternal UPD(13), iso- or heterodisomy, appears to have any effect upon the phenotype (Berend et al. 1999; Soler et al. 2000). A unique example of familial UPD(13), paternal and maternal, emphasizes this point: A normal mother with presumed 45,XX,i(13q)pat had a normal child with 45,XY,i(13q) mat (Slater et al. 1995). She may have been the result of monosomic rescue, and her son due to trisomic rescue! Other examples of i(13q)mat and i(13q)pat are on record, all with normal phenotype. Maternal isodisomy for chromosome 13 has been seen in autosomal recessive GJB2-associated deafness (Alvarez et al. 2003). Chromosome 14. Chromosome 14 contains an imprinted locus at 14q32, and UPD(14) produces different syndromes according to the paternal or maternal basis of the disomy. Either may be seen in the setting of a normal karyotype, or with a Robertsonian translocation (or “acrocentric isochromosome”). A balanced 45,der(13;14) Robertsonian translocation may reflect correction of an initially 46,der(13;14),+14 conception, while the 45,der(14;14) case might in fact result from a 45,–14 conception which then corrected by reduplication of the single chromosome 14 to give an i(14q) with isodisomy. Isodisomy may be present in the setting of a normal karyotype, and it may thus be less rare than is appreciated (Chu et al. 2004). Kagami-Ogata Syndrome. Paternal UPD(14) is the more severe of the UPD(14)s, with obstetric complication (polyhydramnios and premature labor), a particular pattern of malformation, growth retardation, and mild–moderate intellectual disability (Ogata et al. 2016: Prasasya et al. 2020). The main contributor to phenotype is overexpression of the paternally expressed gene RTL1 from a differentially methylated locus at chromosome 14q32.2 (Kagami et al. 2015). A similar but sometimes milder phenotype results from microdeletions or epimutations affecting the maternal allele at this locus (Higashiyama et al. 2022; Sirera Sirera et al. 2023). The bell-shaped thorax (Figure 19–11), reminiscent of Jeune syndrome, is a particular clinical pointer, and it has been observed at 23-week ultrasonography; this anatomy may improve during childhood in those who survive (Kagami et al. 2015). Smith et al. (2024) describe a 35-year-old man with UPD(14)pat, the only reported adult with the condition. In addition to skeletal abnormalities, his phenotype was characterized by intellectual disability, macrocephaly, facial dysmorphism, and obesity. Temple Syndrome. Maternal UPD(14), or more specifically maternal UPD at the 14q32 imprinted locus, causes Temple syndrome, which can also result either from paternal deletions at 14q32 or from an epimutation (hypomethylation at key loci within 14q32) (Ioannides et al. 2014). The syndrome was first recognized in a male patient who had inherited a balanced Robertsonian t(13;14) from his mother (Temple et al. 1991), and is characterized by pre- and post-natal growth retardation, early hypotonia, small hands and feet, early puberty, subtle dysmorphism, mildly reduced intellectual ability, and, in about half of patients, obesity. There is phenotypic overlap with SRS, and Mackay et al. (2022) identified 14q32 methylation changes in 70 out of 4,721 referrals for SRS diagnostic testing. Mitter et al. (2006) point out the overlap with the Prader-Willi phenotype, and there is a biological basis for this similarity: Loss of expression of paternally expressed genes at the Prader-Willi syndrome locus at 15q11q13 leads to upregulation of maternally expressed genes at 14q32 (Stelzer et al. 2014).

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  555 Chromosome 15. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are the two UPD(15) syndromes. In spite of their common causations, they are very different clinically. It may be an oversimplification, but equally a useful perspective, to think of these as each being caused by absent activity of a particular single genetic segment—the PWS region and the AS region, respectively—within 15q11q13. The chromosomal region of interest is illustrated in Figure 19–12. Prader-Willi syndrome is a contiguous gene syndrome, with the phenotype being due to loss of transcription of several genes and RNA transcripts on the paternal chromosome 15. Among these, deficiency of a particular cluster of small nucleolar RNA genes (snoRNAs) called SNORD 116 (present in 24 copies) is responsible for the key features of PWS (Tan et al. 2020). Different components of the PWS phenotype are therefore mediated via perturbed functioning of different genetic targets of these snoRNAs. Another RNA transcript from the paternal chromosome 15, IPW, downregulates transcription of maternally expressed genes at the 14q32 imprinted region, providing an explanation for the similarity in phenotypes between PWS and maternal UPD(14), as just mentioned above.17 The clinical syndrome of hyperphagia with obesity, neurobehavioral disability, small hands and feet, and hypogenitalism is well known, and indeed recorded from classical times. Timely diagnosis can temper the obesity. Angelman syndrome is due to absent activity of a single gene, UBE3A, on the maternal chromosome 15. SNORD 116 and UBE3A lie in close proximity on 15q11q13, and both are under the influence of an imprinting control center (IC): From centromeric 17 Also of relevance is the single-exon gene MAGEL2, which is expressed from the paternal allele. Truncating variants in the paternal allele of MAGEL2 cause Schaff-Yang syndrome, which shares with PWS the features of neonatal hypotonia and intellectual disability, but also has joint contractures. Cognitive impairment in Schaff-Yang syndrome is often more severe than in PWS, or in patients with whole gene deletions of MAGEL2. One possible explanation lies in MAGEL2 being a single-exon gene, so that stop codons should not be subject to nonsense-mediated decay such that proteins may have a harmful gain-of-function effect (Schubert and Schaaf 2025). Figure 19–11.  The Chest Anatomy in Kagami-Ogata Syndrome. Notes: Chest X-ray of a child with Kagami-Ogata syndrome, in this case due to an epimutation in the 14q32.2 imprinted region. The thorax is narrow in its upper and mid parts, and flaring below. The ribs have an increased “coat-hanger” angle. Source: From T Ogata and M Kagami, Kagami-Ogata syndrome: A clinically recognizable upd(14)pat and related disorder affecting the chromosome 14q32.2 imprinted region, J Hum Genet 61:87–94, 2016. Courtesy T Ogata, and with the permission of Nature Publishing Group. Figure 19–12.  The Regions and Loci of Interest within the Segment 15q11.2q13.3. Notes: AS, Angelman syndrome; BP, (numbered) breakpoint; PWS, Prader-Willi syndrome; T1D, T2D, type 1, type 2 deletion. Blue, red, and green shading indicates, respectively, PWS-related, AS-related, and non-imprinted loci. IC, imprinting center: The blue IC segment is the PWS-IC, influencing the blue-coded loci in the PWS region; the red IC segment is the AS-IC, influencing the red-coded loci in the AS region. Source: From DJ Driscoll et al., Prader-Willi syndrome, GeneReviews 2016 (updated 2024). Courtesy DJ Driscoll, and with the permission of the University of Washington.

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  557 to telomeric on the chromosome, the order is IC, SNORD 116, and the UBE3A gene (Figure 19–12). Frequent laughing and stereotypic limb movements are characteristic, along with absence or near-absence of speech. The absence of gene activity in PWS and AS is due either to the loss or to the non-functioning of this PWS/AS region on one chromosome 15 homolog. Loss is most commonly due to a simple interstitial deletion (“classical deletion”). Low-copy repeats on either side of the region can come together and set the stage for nonallelic homologous recombination during meiosis, leading to deletion of the PWS/AS region.18 Whether the phenotype comes to be PWS or AS depends upon which parent contributed the deleted chromosome. Non-functioning of (structurally normal) genes within 15q11q13 is due to the imprint status. This is most commonly the consequence of UPD(15), with the phenotype determined according to the parent of origin of the disomic pair of chromosomes. A rare cause is failure of or damage to the chromosome 15 IC. Study of these IC-damaged cases has cast much light on the processes of molecular pathogenesis in PWS and AS, and so the length of the commentaries that follow is quite out of proportion to their frequencies. In the case of AS, mutation in the UBE3A gene is a further category of mechanism. The 15q11q13 Imprinting Center. Normal persons have one paternally imprinted chromosome 15 and one maternally imprinted chromosome 15. The imprinting state of a chromosome 15 is set and reset as it is transmitted down the generations, according to the sex of the transmitting parent. This resetting—an “epigenetic modification”—is dictated during gametogenesis from the cis-acting IC, with the methylation of genes comprising, in large part at least, the crux of the process. The IC is bipartite with a centromeric element, the AS-IC, and 35 kb distant a telomeric element, the PWS-IC, this latter including exon 1 of SNRPN. Interaction between these two elements directs the process. In maternal gametogenesis, the AS-IC has responsibility for initiating a paternal→maternal switch on the chromosome 15 that the mother herself had received from her father. The chromosome 15 she got from her mother retains a maternal imprint. With an active AS-IC, the UBE3A gene, lying approximately 1 Mb distant, is free to function in the embryo to which this ovum gives rise. Vice versa, paternal gametogenesis serves to effect a maternal→paternal switch, or to retain a paternal status, on the chromosome 15 that the sperm contributes to the embryo. In consequence, a number of genes under its aegis are able to function, in part at least, by being demethylated. The gene activity of UBE3A is prevented. These epigenetic modifications operate only in cis, and so the maternal and paternal chromosomes continue to function autonomously, with their different repertoires of expression, during the life of the individual. A scheme for the various molecular defects of PWS and AS is presented in Figure 19–13. Table 19–3 sets out the test results for the different types of PWS and AS. Classical Deletion. This is the most frequent basis of the two syndromes, accounting for ~70% of both PWS and AS (Horsthemke and Buiting 2006). The deletion removes 5.9 Mb (class I) or 5.0 Mb (class II) within 15q11q13, encompassing the PWS and the AS genetic elements and including the IC (types 1 and 2 deletions in Figure 19–12). There is one common distal breakpoint (BP3) and two variable proximal deletion breakpoint 18 Atypical deletions also occur, characterized by unique breakpoints and phenotypes that can be milder or more severe than typical PWS, depending on the size of the deletion. 558  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING Figure 19–13.  An outline of the different genetic forms of Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Notes: The PWS/AS critical region of chromosome 15 is depicted. A bipartite imprinting center with AS and PWS components (AIC and PIC) controls, in cis, the activity of a set of PWS genes and the UBE3A gene. A switched-on IC and an actively functioning gene are shown in unbroken line; a switched-off IC and an inactivated gene are shown in dashed outline. A mutated UBE3A gene is shown starred and with a dotted outline. (1) Normally, the UBE3A gene is transcribed only from the maternal chromosome (mat), and the PWS genes only from the paternal chromosome (pat), with each chromosome thus functioning appropriately for its parent of origin. In PWS there is nonfunctioning of the PWS genes because: (2) the PWS genes have been removed by a typical large deletion from the paternal chromosome; (3) both chromosomes are of maternal origin; (4) a microdeletion of, or mutation in, the PIC has fixed a maternal imprint status on the paternal chromosome. In AS there is nonfunctioning of the UBE3A gene because: (5) the UBE3A gene has been removed by a typical large deletion from the maternal chromosome; (6) both chromosomes are of paternal origin; (7) a microdeletion of, or mutation in, the AIC has fixed a paternal imprint status on the maternal chromosome; (8) there is a mutation in the UBE3A gene on the maternal chromosome. A further category (9) is not shown, comprising the 10%–15% in which no genetic defect can be shown. Approximate percentages of each PWS/AS category are indicated; in another ~10% of AS, no genetic defect can be identified. patM, a maternally functioning chromosome of paternal origin; matP, a paternally functioning chromosome of maternal origin. Table 19–3.  Categories of Prader-Willi and Angelman Syndromes DELETION ON MICROARRAY LOH ON SNP MICROARRAY METHYLATION PATTERN OF NO. 15s PARENTAL ORIGINS OF NO. 15s UBE3A GENE Prader-Willi syndrome Classical deletion + + Mat Bi UPD(15)mat N ± Mat Mat Imprinting defect N N Mat Bi Imprinting center microdeletion* N N Mat Bi Angelman syndrome Classical deletion + + Pat Bi Deleted UPD(15)pat N ± Pat Pat Intact Imprinting defect N N Pat Bi Intact Imprinting center microdeletion* N N Pat Bi Intact UBE3A variant N N Bi Bi Mutated Notes: The assessment of genetic category of Prader-Willi and Angelman syndromes according to results of molecular testing. *Detection of imprinting center microdeletion requires specialized testing, most commonly the methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) kit from MRC Holland. LOH = loss of heterozygosity. A normal chromosome microarray result is indicated by N, an abnormal result by +. An inconsistent result is shown as ± (LOH will be seen in all UPD due to meiosis II error or UPD of post-zygotic origin, but will only be seen in UPD due to meiosis I error when there has also been recombination). Bi = biparental; Mat = maternal; Pat = paternal. “Intact” means that the DNA sequence of the gene is normal, but its function is epigenetically compromised.

560  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING regions (BP1, BP2), due to duplicons at these sites. Nonallelic homologous recombination between the distal and one of the two proximal duplicons then causes the deletions (Ji et al. 2000). The behavioral phenotype is a little worse with the class I BP1–BP3 deletion than the class II BP2–BP3 deletion (Bittel et al. 2006a). Larger deletions are infrequent, and they are associated with a more severe phenotype (Sahoo et al. 2007). If the deletion occurs on a paternally originating chromosome, it will cause the PWS phenotype to develop;19 and vice versa, a maternal deletion produces AS. In a sense, there is an “unmasking of the silent elements” on the other chromosome. As well as the crucial PWS and AS genetic elements, a number of other loci may be deleted, and so the expression “contiguous gene syndrome” is not inappropriate, albeit having a somewhat different sense from its usage elsewhere in this book. One of the least important of these other loci is the P gene that contributes to normal pigmentation, and so children with PWS and AS due to classical deletion typically have fairer complexions than do their siblings.20 Mosaicism may lead to a milder phenotype (Golden et al. 1999; Tekin et al. 2000).21 Prader-Willi and Angelman Syndromes due to Deletion, Associated with Uncommon Rearrangement. Loss of the PW/AS region can be due to transmission of an unbalanced translocation or an inversion involving chromosome 15. The male carrier of a balanced reciprocal translocation in which one breakpoint is in the region of 15q13 can transmit an unbalanced complement to produce a deletion PWS child (Hultén et al. 1991; Smeets et al. 1992), and the female carrier can have a child with deletion AS (Stalker and Williams 1998). There may be an additional effect from the concomitant imbalance involving the other chromosome of a translocation, such as the case in Torisu et al. (2004): a child who displayed features both of AS and the 1p36 deletion syndrome due to a tertiary monosomy for these two segments, the mother being a balanced translocation carrier. A handful of PWS cases have been due to a Y;15 translocation with breakpoints in Yp and at 15q12q13, deleting the PWS region having the karyotype 45,X,der(Y),t(Y;15) (Vickers et al. 1994). A grandmother heterozygous for an inverted insertion of chromosome 15 had a PWS grandchild through her carrier son, and an AS grandchild through her carrier daughter (Collinson et al. 2004). Loss or disruption of the PW/AS region can be due to a de novo rearrangement (Dang et al. 2016; Schüle et al. 2005). Disruption may affect topologically associating domains (TAD) of chromosome 15: Lei et al. (2019) described a 13-year-old boy with typical PWS and a balanced reciprocal translocation t(15;19)(q11.2;q13.3), with the breakpoint mapping to within SNORD gene cluster but not disrupting coding genes or the snoRNAs. Expression of the paternally expressed genes downstream of the breakpoint was abolished. Uniparental Disomy and Prader-Willi Syndrome. About one-third of PWS is due to UPD (Horsthemke and Buiting 2006). The cytogenetic study typically shows a normal 46,XX or 46,XY karyotype. Both chromosomes 15 come from the mother, and so neither of the PWS critical regions is expressed. This functional lack causes the PWS phenotype. In most (80% or more), the UPD had its origin in a maternal meiosis I error. 19 An aide-mémoire: Prader-Willi due to Paternal deletion. 20 An additional copy of this gene leads to hyperpigmentation (Akahoshi et al. 2001). This is a good example of a simple dosage effect: One copy of the P gene = pale skin, two copies = normal pigmentation, three copies = hyperpigmentation. 21 Mosaicism for the typical 15q11q13 deletion has never been proven (Beygo et al. 2019), and it is likely that all mosaic deletions have atypical breakpoints.

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  561 A maternal-age effect is clear: mothers of PWS children with UPD are, on average, five years older than mothers of PWS children with a deletion (Butler et al. 2019). The phenotype is very similar to classical deletion PWS, although the facies may be less “typical” with the UPD form of PWS, learning and behavior problems are somewhat less prominent, and some of the minor manifestations are less likely to occur; in consequence, diagnosis may be delayed in comparison to deletion PWS (Mahmoud et al. 2021; Rosenberg et al. 2022; Mao et al. 2024). The UPD form of PWS is particularly associated with a psychiatric phenotype, typically presenting in young adulthood and characterized by a fluctuating psychosis and bipolar mood disorder (Yang et al. 2013). In rare instances, trisomy rescue is incomplete, resulting in a mosaic karyotype in which cells have either trisomy 15 or UPD (Olander et al. 2000). The phenotype is typically severe, presumably reflecting a more severe neurological impact of trisomy 15. In rare instances, UPD may be mosaic, arising from rescue of a post-fertilization error and resulting in an incomplete PWS phenotype (Morandi et al. 2015). Uniparental Disomy and Angelman Syndrome. Only 1%–2% of AS is due to UPD. As with PWS due to UPD, the karyotype is normal 46,XX or 46,XY. Both chromosomes 15 are from the father, and neither chromosome expresses the AS critical region. Most cases involve a post-zygotic origin of the extra paternal chromosome, resulting in isodisomy for the entire chromosome that is readily recognizable as such on SNP microarray. This probably follows the “correction” of monosomy 15 due to a nullisomic ovum (as outlined above) and, as with PWS, is likely a maternal-age effect. Very few AS children born to mothers younger than age 35 years have UPD, but those born to mothers age 35 years or older have about equal numbers due to deletion and UPD (Ginsburg et al. 2000). A few are due to a paternal second meiotic error (Robinson et al. 2000). In parallel with the observations in UPD PWS noted above, the phenotype in AS due to UPD is not quite as severe as in the deletion form, with these children showing a lesser frequency of seizures and some having a few words (Keute et al. 2021). But it remains true that the disability is severe. Fujimoto et al. (2023) report an apparently unique example of mosaicism for paternal isodisomy 15 and a normal cell line, in a 6-year-old boy with a mild AS phenotype. The abnormality may have arisen in a chromosomally normal embryo with an early mitotic error generating a trisomy 15 cell line, followed by trisomy rescue to generate the UPD(15)pat cell line. Prader-Willi and Angelman Syndromes due to Uniparental Disomy, Associated with Chromosome 15 Rearrangement. Uniparental disomy can result from a variety of rearrangements involving chromosome 15. The male carrier of a reciprocal translocation involving chromosome 15 could transmit a disomic 15 spermatocyte from 3:1 nondisjunction, with the maternal chromosome 15 then being lost, and have a child with UPD AS; and vice versa, the female carrier could have a PWS child (Calounova et al. 2006; Heidemann et al. 2010). Similarly, a familial nonhomologous Robertsonian translocation in which one of the component chromosomes is a no. 15 giving a trisomic 15 conception, and with post-zygotic loss of the chromosome 15 from the other parent, would lead to UPD(15) with either PWS or AS, according to the sex of the carrier parent (Tsai et al. 2004). The same thing could happen if the translocation were de novo. A maternally originating de novo homologous der(15;15) (which may actually be a 15q isochromosome), with no chromosome 15 contributed from the father, would cause PWS (Robinson et al. 1994); and vice versa, AS would result from a paternal isochromosome 15q (Tonk et al. 1996). Smith et al. (1994) describe AS from asymmetric segregation of a paternal 8;15 translocation (Figure 12–4). The heterozygous father passed on

562  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING his der(8) and his normal chromosome 15 (thus, paternal UPD), and there was absence of a maternal chromosome 15. Some PWS children with a 47,+idic(15) karyotype may actually have UPD of the two intact chromosomes 15, and the small idic(15) is a phenotypically irrelevant relic of the original process of abnormal chromosomal behavior (Robinson et al. 1993). Imprinting Center Defects. A very small group of PWS and AS patients, ~1% and 3%, respectively, have normal biparental inheritance and no classical deletion, but a uniparental pattern of methylation and gene expression (Horsthemke and Buiting 2006). Most of these cases reflect abnormal function of the IC, while a minority, around 10%–15%, have an actual IC microdeletion. The latter category can be strongly suspected when there is a positive family history, while in the former, sporadic occurrence has been universally observed. Whether PWS or AS is seen depends upon which component of the IC is deleted or nonfunctional. • Imprinting Center Defect: Functional. Buiting et al. (2003) analyzed 44 PWS and 76 AS patients with a failure of IC functioning, an IC deletion or point mutation having been excluded; these aberrant epigenetic states are referred to as epimutations.22 All cases were sporadic. Some shared with an unaffected sibling the 15q11q13 haplotype on their paternal (PWS) or maternal (AS) chromosome, supporting the presumption of a de novo defect. With PWS, the basis of the epimutation may be a failure to erase the maternal imprint, as an act of omission. Thus, for example, the father of such a PWS child passes on his maternal chromosome 15 with its maternal imprint still in place, and the child inherits two maternally imprinted no. 15 chromosomes. In AS, the typical scenario may be the imposition of an anomalous imprint status. This can be thought of as an act of commission: The mother inappropriately applies a paternal imprint to the chromosome 15, or fails to reset her paternal chromosome 15 that she passes to the child; or (since some maternal epimutations are mosaic) the error may occur post-zygotically. If the error is incomplete, a milder AS phenotype may be seen (Le Fevre et al. 2017; Baker et al. 2022). AS due to an imprinting defect, with loss of methylation of the maternal allele, may have an association with subfertility and artificial reproductive technology (Manipalviratn et al. 2009). If the association is indeed causal, the biological basis may be in the subfertility per se, or due to the superovulation treatment as part of IVF protocol, which leads to a failure to acquire normal UBE3A activation status in the ovum. • Imprinting Center Defect: Microdeletion. Microdeletions of the IC, generally of kilobase size, remove one or other of its major component parts, either the PWS-IC or the AS-IC. The inability to reset an appropriate imprint status leads to the “fixation of an ancestral epigenotype” (Saitoh et al. 1997). Only a handful of cases have been identified worldwide (Horsthemke and Buiting 2006; Hassan and Butler 2016). Their particular importance to the counselor lies in the high recurrence risk if a parent is heterozygous: The mode of inheritance is essentially sex-influenced (the 22 The word mutation is normally taken to indicate that there is a change in the DNA sequence (from the Latin mutare, to change). By definition, no such change has occurred in an epimutation. But there has been a change in the functioning of the DNA. UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  563 parent’s sex, that is) autosomal dominant, with a 50% risk for the heterozygous father (for PWS) or the heterozygous mother (for AS), according to which component part of the IC is deleted. In some cases, the IC microdeletion is de novo, or a consequence of germline mosaicism in the father or the mother (Beygo et al. 2019). In Prader-Willi syndrome due to IC microdeletion, the father would have received the deletion on his mother’s chromosome 15. He is normal, since an erased paternal imprint on his maternal chromosome is, naturally, correct. The deletion could have originated in his mother, or antecedent to her, provided transmission had been exclusively matrilineal. But when he passes this chromosome 15 with its fixed maternal epigenotype to a child of his, with the maternal→paternal imprint switch unable to function, the child has, effectively, a functional maternal UPD(15). Such a family is illustrated in Ming et al. (2000). Of 10 children, all of them normal and with normal karyotypes on standard cytogenetics, four inherited an IC microdeletion, presumably from their deceased mother (their father was proven not to have the deletion). Two of these children were male, and each went on to have, in the next generation, a child with PWS: an example of “grandmatrilineal inheritance.” In Angelman syndrome due to IC microdeletion, the scenario is essentially the obverse of the above. A microdeletion on the maternal chromosome 15 removes the AS-IC. The defect may have arisen de novo from the maternal grandfather of the AS child, or alternatively, there could have been patrilineal transmission of the mutation, harmlessly, for any number of previous generations. Transmission from the grandfather to the mother would be without phenotypic consequence, since a paternally originating chromosome 15 would in any event have its AS-IC inactivated. But in oögenesis in the mother, the normal paternal→maternal switch on the abnormal chromosome cannot be effected (thus “fixation” of the ancestral paternal epigenotype). If the child receives this chromosome 15 from the mother, both homologs carry a paternal imprint. In consequence, the child has AS. Two such Japanese families, independently ascertained and reported, had exactly the same 1.487 Mb deletion and may well have represented distant branches from the same, presumably male, ancestor (Sato et al. 2007). Angelman Syndrome due to UBE3A Gene Variant. Classical point mutation, affecting the UBE3A (ubiquitin protein ligase 3A) gene, is a key contributor to AS etiopathogenesis (Abaied et al. 2010). The UBE3A protein regulates the balance of protein synthesis and degradation at synapses, thereby playing a critical role in synaptic plasticity, learning and memory (Greer et al. 2010). This gene is expressed from both parental chromosomes in some tissues, but, in the brain from only the maternal chromosome. The (normal) paternal allele does not function in embryonic brain, or at least in particular parts of the brain. Thus, if the maternal gene is mutated, there is no UBE3A expression, and in consequence brain development is compromised (Rougeulle and Lalande 1998). About 70% of inherited “non-deletion non-UPD non-IC” AS is due to UBE3A mutation of maternal origin. The severity of phenotype in the mutation form falls between the deletion and UPD cases (Abaied et al. 2010). Multigenerational transmission may be seen, with the revealing observation that AS children are born only to carrier daughters of carrier males (Figure 19–14). The mutation transmitted by the father has no effect in his child, since this chromosome 15 region would in any event carry a paternal imprint and be silenced. Intragenic deletions within the UBE3A gene are a rare basis of AS (Aguilera et al. 2017).

564  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING Angelman Syndrome with No Deletion, No Uniparental Disomy, No Imprinting Defect, and No UBE3A Variant. In some 10%–15% of AS, no genetic defect can be found (Beygo et al. 2019). There is a normal karyotype with no deletion demonstrable on FISH, normal methylation analysis (at least on the sampled tissues), biparental inheritance, and an apparently intact UBE3A gene. There may be an epigenetic influence whereby a normal UBE3A gene on the maternal chromosome fails to activate normally during embryogenesis. Or there may be some other AS genetic basis, as yet unknown. Chromosome 16. This is one of the more commonly seen UPDs, and it is almost always due to correction of trisomy 16 of maternal meiotic origin. Thus, it is typically a maternal UPHD or UPHID. It has been difficult to separate out the effects of the UPD and of a placental insufficiency due to confined placental mosaicism for trisomy 16, this typically being the route by which the UPD comes to be recognized following chorionic villus sampling; and in addition, a possible residual occult fetal trisomy mosaicism always remains as a potential confounder. Opinions differ. Yong et al. (2003) showed in a large series of mosaic trisomy 16 discovered at prenatal diagnosis that the degree of fetal growth restriction, and probably the malformation rate, was greater in those with UPD(16)mat than in those with biparental inheritance, thus suggesting a role of the UPD per se. Imprinting of the ZNF597 locus at 16p13.3 has been proposed as a mechanism underpinning some phenotypic features of UPD(16)mat (Yamazawa et al. 2021). Phenotypic overlap with Silver-Russell syndrome may be seen, and Inoue et al. (2019) found two patients with upd(16)mat in a series of 94 patients who met diagnostic criteria for SRS. On the other hand, Scheuvens et al. (2017) suggest that UPD(16)mat is of itself without phenotype, and may serve merely as a biomarker for an underlying trisomy 16 mosaicism. As for paternal UPD(16), it seems probable that it has no clinical consequences (Zhang et al. 2021b). Figure 19–14.  Familial Angelman Syndrome. Notes: A family with inherited Angelman syndrome, due to a UBE3A mutation, reported in Moncla et al. (1999). Filled symbol, Angelman syndrome; bull’s-eye symbol, mutation carrier, demonstrated or inferred; N, demonstrated noncarrier. Note that all the affected children are born to carrier mothers, but that these mothers are related to each other through the male line. Some normal children have been proven to be noncarriers with molecular testing (N in symbol), but the reader can also determine that any unaffected child of a potential carrier mother, such as IV:1 and 2, the children of III:4, or V:9, the sibling of an affected child, cannot be carriers. An inherited imprinting center mutation could present a similar pedigree. UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  565 The usual rare risk from isozygosity for a recessive gene applies, as exemplified in Wattanasirichaigoon et al. (2008), who report a child with hydrops fetalis due to hemoglobin Bart’s, consequential upon UPD(16)mat. Hamvas et al. (2009) reported three infants with paternal isodisomy 16 resulting in surfactant deficiency due to ABCA3 mutation, but none of whom exhibited a non-pulmonary phenotype. Chromosome 17. Several cases of complete UPD(17)mat have been described. One 46,XY child was normal, ascertainment having been via the discovery of trisomy 17 mosaicism at amniocentesis (Genuardi et al. 1999). A recessive etiology was suspected in a girl with developmental delay, microcephaly, and seizures, in whom upd(17)mat was identified by exome sequencing, although no definite causative mutation was found (King et al. 2014). UPD(17)pat has been seen in the setting of a number of the recessive phenotypes. Lebre et al. (2009) identified the UPD in an infant with cystinosis, a recessively inherited multi-organ storage disease, the locus of which is on chromosome 17p and this segment being in isodisomic state in the child. Similarly, Natsuga et al. (2010) and Verebi et al. (2023) have seen cases of junctional epidermolysis bullosa and of limb-girdle muscular dystrophy (the respective genes on no. 17) due to UPD(17)pat. Chromosome 18. Given the frequency of trisomy 18 at conception, it is surprising that UPD for the entire chromosome 18 is so rare. Paternal UPD(18) has been unmasked through the diagnoses of autosomal recessive deafness due to LOXHD1 variants (Morgan et al. 2018) and Dyggve-Melchior-Clausen syndrome, a rare recessive syndrome of skeletal dysplasia and intellectual disability (López-Garrido et al. 2022). Kariminejad et al. (2011) present an extraordinary example of segmental maternal UPD of 18p and segmental paternal UPD of 18q in a girl whose consanguineous parents both carried a pericentric inversion inv(18)(p11.31q21.33). The healthy child received two recombinant chromosomes 18—from the mother a derivative chromosome 18 with dup(18p)/del(18q), and from the father a derivative chromosome 18 with dup(18q)/ del(18p). Chromosome 19. It took until 2018 for the first cases of UPD(19) to be identified. Paternal isodisomy for chromosome 19 was reported in a pair of monochorionic diamniotic twins who presented with dysmorphic feature, hypotonia, and global developmental delay (Yeung et al. 2018). Loss of imprinting for known imprinted genes on chromosome 19 was detected, and this was the probable cause of the phenotype. The second report of UPD(19)pat was a 17-month-old boy in whom upid(19)pat was detected after he had presented with a homozygous variant in the gene EXOSC5, leading to an autosomal recessive disorder of cerebellar ataxia brain abnormalities and cardiac conduction defects (Slavotinek et al. 2020). Apart from developmental delay, his phenotype was not a strong match for the twins described by Yeung et al., and the possible delineation of a UPD(19) syndrome will require the ascertainment of additional cases. UPD(19)mat is yet to be reported, with or without a phenotype. Chromosome 20. Eggermann et al. (2001) reviewed three reported cases of UPD(20), one paternal and two maternal, with a major malformation phenotype in the former and growth retardation the common observation in the latter two. But it took until 2016 for maternal UPD(20) to be confirmed as a bona fide imprinting syndrome, following the diagnosis of eight new cases due in large part to improved ascertainment via SNP microarray (Mulchandani et al. 2016). The condition has since been referred to as Mulchandani-Bhoj-Conlin syndrome. There is phenotypic overlap with Silver-Russell syndrome, and UPD(20)mat has been detected in about 0.2% of children referred for SRS molecular testing (Hjortshøj et al. 2020; Mackay et al. 2022). Neverthelss, Hjortshøj

566  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING and colleagues note that while individuals with UPD(20)mat share with SRS the features of pre- and post-natal growth retardation and prominent feeding difficulties, they do not fulfill the consensus SRS criteria. Dysmorphic features are common but without consistent pattern, and hypotonia and developmental delay are present in about half of cases. The predominant mechanism is trisomy rescue after maternal meiosis II nondisjunction. Pseudohypoparathyroidism type 1b (PHP1b) is caused by imprinting defects in GNAS, which, in certain tissues such as renal proximal tubules, is predominantly expressed from the maternal allele. Hence, symptoms of PHP1b would be expected to accompany paternal UPD(20), and indeed ~20%23 of patients with sporadic PHP1b have UPD(20)pat, either involving the whole chromosome, or more commonly as a segmental mitotic error (Fernández-Rebollo et al. 2010; Bastepe et al. 2011). Colson et al. (2019) suggest that early-onset obesity, resolving after six years, may be an additional UPD(20)pat feature. More commonly, PHP1b follows an autosomal dominant pattern of inheritance, and these cases are caused by deletions that disrupt imprinting the GNAS DMRs on the maternal allele. Chromosome 21. UPD(21), maternal and paternal, have been ascertained in healthy individuals and appears to be without effect (Engel and Antonarakis 2002). Both UPDs have been described in individuals with a der(21;21)(q10;q10). Chromosome 22. More than 25 cases of UPD(22) have been reported, in most cases unmasking autosomal recessive diseases. Maternal UPD(22) has generally not, of itself, been causally associated with any defect (Kotzot 1999; Engel and Antonarakis 2002). Intrauterine growth retardation, if present, may more likely reflect the influence of a trisomic 22 placenta or low-level “occult” mosaicism of the fetus (Balmer et al. 1999; Bryan et al. 2002). A der(22;22)(q10;10q) has been reported in association with both maternal and paternal UPD(22), typically having presented with recurrent miscarriage in otherwise healthy individuals (Liehr et al. 2025a), and Ouldim et al. (2008) report the unexpected transmission of a der(22;22)(q10;10q) from a father to (normal) son (and see p. 208). An instance of homozygosity for a PLA2G6 mutation, leading to neurodegeneration with brain iron accumulation in monozygous twins from an IVF conception, was due to paternal UPD(22) (Tello et al. 2017). Chromosome X. Neither UPD(X)mat nor UPD(X)pat appears to have any consequence in the 46,XX person, with the usual exception of homozygosity for a recessive mutation. An example is a 3-year-old girl with a typical fragile X phenotype, homozygous for a fully expanded FMR1 allele (288 CGG repeats) due to maternal UPID of the X chromosome, her mother carrying a pre-mutation allele (Kim et al. 2020). However, there may be a subtly different neuropsychological phenotype according to the parent of origin in monosomy X (self-evidently a uniparental condition). In a British study, 80 girls with Turner syndrome underwent behavioral evaluation, 55 of whom were 45,XM and 25 were 45,XP. The 45,XP girls were more socially adept and more articulate than the 45,XM girls. Speculatively, this may represent the effect of an imprintable X-borne “locus for social cognition” that is functional on the X chromosome transmitted from a father, and nonfunctional on the X from a mother (Skuse et al. 1997). Autism, which is a male-susceptible condition, is associated with 45,XM in the case of autistic girls with Turner 23 The other 80% of cases of sporadic PHP1b have a GNAS methylation defect without underlying genetic cause (epimutation).

UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  567 syndrome (Donnelly et al. 2000). In terms of response to growth hormone, it makes no difference whether the child is 45,XM or 45,XP (Tsezou et al. 1999). UPD(X)pat offers the intriguing scenario of father-to-son transmission of an X-linked gene. A 24,XY gamete from a hemizygous father could produce a 47,XXY zygote, which could subsequently lose the maternally contributed X; or the ovum could be nullisomic X, with sex chromosomal complementation producing 46,XY. Either mechanism could explain the observations in a family with the X-linked form of ectodermal dysplasia, as presented in Ferrier et al. (2009). Chromosome Y. Self-evidently, all karyotypes 47,XYY are UPD(Y)pat. UNIPARENTAL DISOMY FOR THE ENTIRE CHROMOSOMAL COMPLEMENT Non-mosaic paternal uniparental disomy (UPDpat) for the full diploid complement— all 46 chromosomes are of paternal origin—produces the placental disorder of complete hydatidiform mole. When, in addition to a double set of paternally derived chromosomes, there is also a haploid maternal set (triploidy, with a total chromosome count of 69), a partial hydatidiform mole results. Hydatidiform mole is discussed in more detail in Chapter 20. Complete UPDmat of an oöcyte following failure of a premeiotic or of a meiotic cell division leads to benign cystic ovarian teratoma, an unusual tumor of the ovary in which several embryonic tissues may be represented (Miura et al. 1999). Mosaicism for Genome-Wide Uniparental Disomy.  Abnormal cytogenetic events around the time of fertilization—such as two sperm entering an ovum to produce a zygote with three pronuclei, or an ovum undergoing a mitosis and then cell lines of different (but diploid) genetic constitution being produced—can be the basis of a mosaicism for genome-wide UPD. This can be UPDmat/biparental or UPDpat/biparental mosaicism, and it may be confined to the placenta or also involve the fetus. Complete UPDpat/normal mosaicism in the placenta (androgenetic/biparental mosaicism) leads to the histological phenotype of mesenchymal dysplasia (p. 673). If the UPDpat line also involves the fetus, a complex clinical picture may be observed, comprising features of different paternal UPD syndromes such as BWS, AS, and transient neonatal diabetes. About 20 such cases have been reported (Jawahir-Schonauer et al. 2022). Malignant and nonmalignant cystic lesions, and a cystic placenta, are frequently present (Inbar-Feigenberg et al. 2013; Christesen et al. 2020); and there is the additional possibility of unmasking an autosomal recessive disease carried by the father (Ohtsuka et al. 2015). The tout ensemble of observation presumably reflects the cellular distribution of the UPD tissue. Darcy et al. (2015) reported a 6-month-old girl who was mosaic for a complete UPDpat cell line and a Down syndrome cell line, her phenotype comprising features of both conditions. Complete UPDmat/normal mosaicism is very rare, with only three cases reported (Bens et al. 2017). A consistent phenotype is yet to emerge, but might be expected to comprise mixed features of SRS, Temple syndrome, PWS, and UPD(20)mat syndrome. Two unique cases cast light on how aberrant chromosomal behavior in the perizygotic period can lead to UPD-related pathology. A 46,XX/46,XY male child described in

568  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING Strain et al. (1995) with growth asymmetry and aggressive behavior had complete maternal isodisomy in the 46,XX cell line and biparental inheritance in the 46,XY line. It may be that an ovum had completed a mitosis on its own, and then one of its daughter cells received the sperm (for the 46,XY line) while the other underwent endoreduplication (for the 46,XX,UPDmat line): thus, biparental/gynogenetic mosaicism. A diploid sperm may have been the basis of the case in Hsu et al. (2008), in which the pregnancy from an apparently normal IVF embryo ended in intrauterine fetal death, with a portion of the placenta of molar appearance, at 14 weeks. Three separate genetic constitutions could be determined. The molar tissue was androgenetic 46,XX,uphd(pat); the fetus and placenta, both 46,XX, shared the same maternal genome but had different paternal genomes. Any explanation for this circumstance is necessarily complex. GENETIC COUNSELING Uniparental Disomy for Individual Chromosomes No instance of recurrence of full UPD for a particular chromosome, with a 46,XX or 46,XY karyotype, is known, and we assume there to be no discernibly increased recurrence risk. The association with increasing childbearing age is to be noted, but in reality the increase in risk for older mothers would be very small. Segmental Uniparental Disomy Segmental UPD arising post-zygotically and which is karyotypically 46,XX or 46,XY, we presume to imply no increased risk for a future pregnancy. UPD due to rearrangement would have a risk according to the nature of the specific rearrangement. Five Imprinting Syndromes with More Than One Genetic Basis TRANSIENT NEONATAL DIABETES MELLITUS Risks to family members depend on the underlying mechanism, as outlined below. Uniparental Disomy 6.  About one-third of cases are due to paternal uniparental disomy of chromosome 6, a sporadic event with no known risk of recurrence. Paternal Duplication of 6q24.  Duplication of 6q24 can be either de novo or inherited. For de novo deletions, there is presumably a very low risk of recurrence, acknowledging a possibility of gonadal mosaicism. If the father has the 6q24 duplication, then the risk to offspring is 50%. For a female proband with a paternally inherited 6q24 duplication, her children would have a 50% chance of inheriting the duplication but would not be at risk of developing transient neonatal diabetes mellitus (TNDM). Maternal Hypomethylation of the 6q24 Region.  Isolated hypomethylation at the 6q24 DMR is typically a sporadic event with a low risk of recurrence. For TNDM caused by autosomal recessive mutations in ZFP57 (hypomethylation at multiple imprinted loci), the risk to sibs is 25%. UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  569 BECKWITH-WIEDEMANN SYNDROME The considerable majority (>90) of BWS occurs sporadically, including the two more common categories of UPD(11) and epigenetic error. UPD(11) is diagnosed readily by SNP microarray (Keren et al. 2013), but detection of epimutation requires more specialized testing.24 The other categories that may have an important recurrence risk are recognized by an abnormal cytogenetic report and/or by a positive family history (Brioude et al. 2018). Uniparental Disomy 11.  About one-fifth of sporadic cases are due to mosaic segmental paternal UPD of 11p. This category of BWS can be suspected clinically if there is hemihyperplasia. No increased recurrence risk applies in the setting of segmental UPD and a normal karyotype. Epigenetic Error.  In sporadic BWS with biparental disomy, the underlying cause is an epigenetic error (“epimutation”) affecting the ovum or early conceptus. This is the basis of a little over half of all cases. There is biparental inheritance with aberrant methylation on the maternal chromosome of either DMR1 (gain of methylation, ~5% of cases) or DMR2 (loss of methylation, ~50% of cases), the latter combination particularly associated with IVF. No cases of recurrence in this setting are known, and this fits the understanding of a typical post-zygotic generation of the epimutation (Scott et al. 2008b) (but note recurrences in the section below on “Silver-Russell Syndrome,” epimutations of 11p15). Theoretically, there might be a very small increased risk if the same susceptibility factors (subfertility, IVF) were operating. The application of methylation-sensitive MLPA, a test which detects both methylation abnormalities and genomic alterations at 11p15 (Baskin et al. 2014), shows that in a small fraction of cases of BWS diagnosed with an epigenetic error, there is actually an underlying 11p15 copy number variant (a maternal deletion or a paternal duplication). Detection of these CNVs, which may be de novo or inherited, will alter recurrence risk counseling. Molecular testing for chromosome 11p15-associated imprinting disorders is complicated by molecular heterogeneity and the complexity of this region; the reader seeking detailed advice is referred to Shuman et al. (2023). 11p Rearrangement.  Chromosome rearrangements are rare causes of BWS. A balanced reciprocal translocation or an inversion with one breakpoint in distal 11p, if of maternal transmission, may lead to BWS (Li et al. 1998). An unbalanced distal 11p15 duplication, if of paternal origin, leads to double expression of IGF2 in the 11p15 region, and this brings about the growth pattern of BWS (and if of maternal origin, a Silver-Russell growth retardation phenotype results). Functional trisomy of non-imprinted 11p segments, or other imbalance due to a translocation, may contribute to the clinical picture (Han et al. 2006; Russo et al. 2006; South et al. 2008b; Bliek et al. 2009). An extraordinary familial case, in Jurkiewicz et al. (2017) is due to triplication of the 11p15.5 imprinting region: a father with SRS, and his daughter with BWS. The recurrence risks for these various circumstances will depend upon the nature of the rearrangement and the parental karyotypes. Mendelian Mutation.  Autosomal dominant BWS accounts for about 5% of cases, the major locus being CDKN1C. Typically, only the offspring of female heterozygotes 24 UPD(11) and epigenetic errors causing BWS are always mosaic, and therefore may not be present in the sample tested.

570  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING are affected. Careful review of the pedigree in the maternal line is necessary to identify mildly affected individuals, and bearing in mind the amelioration of phenotype with time (Elliott et al. 1994; Hunter and Allanson 1994). Mutations in CDKN1C are associated with a high risk of abdominal wall defects and a very low risk of abdominal tumors (Mussa et al. 2016). One might consider the very rare case of deletion of the differentially methylated region DMR1 also to be in the category of Mendelian mutation; a child receiving this deletion from a mother has BWS, due to consequential biallelic IGF2 expression (Sparago et al. 2004). A rare recessive basis of a maternal susceptibility to have a BWS child resides in the NLRP2 gene, with failure to impose a proper imprint upon ova (Meyer et al. 2009). SILVER-RUSSELL SYNDROME About half of SRS can be traced to an anomaly of chromosome 7 (UPD) or chromosome 11p15 (epimutation), the latter mirroring the mechanism in BWS as outlined above. Both these genetic forms typically imply a low risk of recurrence. The same applies to the SRS-like presentations that have also been reported in individuals with UPD(6)mat, UPD(7)mat and UPD(16)mat, and with epimutations at 14q32 consistent with Temple syndrome (Mackay et al. 2022). Many different copy number variants have also been reported to cause SRS-like presentations, including at deletions at 1q21, 15q26, 17p13.3 and 22q11 (Tümer et al. 2018) and if inherited would carry a chance of recurrence. An international consensus statement for the diagnosis and management of SRS has been published (Wakeling et al. 2017). Chromosome 7.  UPD(7)mat is seen in up to 10% of cases, and the clinical phenotype is particularly associated with speech and language difficulty (Wakeling et al. 2010). Sporadic occurrence has been the universal observation. A paternally inherited deletion of the MEST locus at 7q32.2, such as reported in Vincent et al. (2022), would imply a 50% chance of recurrence of the resulting SRS-like phenotype. Chromosome 11.  Epimutations of 11p15, with hypomethylation of the IGF2/ H19 differentially methylated region (DMR1), comprise the largest single category: According to the stringency of clinical criteria, these account for up to half of all SRS. This category may be associated with conception by IVF (Hattori et al. 2019). Sporadic occurrence is very much the rule, although very rare examples of sibling recurrence and parent-child transmission of 11p15 hypomethylation have been recorded (Bartholdi et al. 2009). Structural rearrangement of 11p15, such as microduplication involving the differentially methylated region DMR2, is a rare cause (Heide et al. 2018). A familial translocation such as the t(11;15)(p15.5;p12) described in Eggermann et al. (2010), in which one segment comprises distal 11p, could lead to either SRS if maternally transmitted, or BWS if from the father. Familial BWS is reported in Lekszas et al. (2019), due to a segregating paternal t(9;11)(p24.3;p15.4), the diagnosis being made in an aunt and her niece. PRADER-WILLI SYNDROME A summary of the different genetic forms of PWS and the associated risks of recurrence is set out in Tables 19–3 and 19–4. Classical Deletion 15q11q13.  The empiric observation of zero recurrences out of some thousands of “trials” underscores the very considerable unlikelihood of significant paternal gonadal mosaicism for the deletion observed in the PWS child. This is the basis of the substantial optimism that can be offered to parents in terms of any further UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  571 pregnancies. A figure of ~0.1% may be a fair one to offer for the risk of recurrence. Nevertheless, the theoretical possibility of paternal gonadal mosaicism, or of a paternal predisposition to undergo chromosome 15 deletion in spermatogenesis (Molina et al. 2011), obliges acknowledgment that the risk is not absolutely zero. If prenatal diagnosis is pursued, chorion villus sampling (CVS) can be offered using microarray for deletions, or the SNRPN methylation test (Buiting et al. 1998). Uniparental Disomy 15, Karyotype 46,XX or 46,XY.  We know of no recorded instance of recurrence of UPD(15)mat PWS in a chromosomally normal couple, and we would otherwise assume, on theoretical grounds, any increased risk in a future pregnancy to be practically negligible, the modest maternal-age effect notwithstanding. Prader-Willi Syndrome Imprinting Center Microdeletion.  The recognition of these cases will require referral to a specialist laboratory. A positive family history, if observed, would oblige the assumption of this category unless or until otherwise proven. Assuming the father carries the genetic defect, the recurrence risk is high, namely 50%. SNRPN methylation testing on CVS can identify an affected pregnancy. The father’s brothers would have a 50% likelihood to be heterozygous (making the assumption that their mother would have carried the mutation), and in that case, these brothers would also have a 50% risk to have a PWS child. Equally, his sisters could be carriers, but their Table 19–4.  Recurrence Risks for Prader-Willi and Angelman Syndromes CATEGORY RELATIVE FREQUENCY (%) RECURRENCE RISK Prader-Willi syndrome Classical deletion 70 Extremely low* UPD(15)mat 25-30 Extremely low* Imprinting center epimutation 1 Extremely low* Imprinting center deletion <0.2 50% (if present in father) 15q translocation/ inversion Rare According to rearrangement Angelman syndrome Classical deletion 75 Extremely low** UPD(15)pat 1-2 Extremely low* Imprinting center epimutation 3 Extremely low* Imprinting center deletion <0.5 50% (if present in mother) UBE3A mutation 5-10 50% (if present in mother) 15q translocation/ inversion Rare According to rearrangement Unknown 10-15 Presumed low Notes: Approximate relative frequencies and recurrence risks to parents having had an affected child, for the different categories of Prader-Willi and Angelman syndromes. *No case yet recorded. **Only two cases in the world recorded (Kokkonen and Leisti 2000; Sánchez et al. 2014) Source: From J Beygo 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.

572  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING children would all be unaffected, and it would only be their sons who might, in the next generation, have the risk for a PWS child. The siblings of the affected child would themselves have no different genetic risk than the general population. The reader should work through the reasoning behind these various risk assessments, even though most counselors will never encounter this actual circumstance in the clinic. Uncommon Cytogenetically Detectable Rearrangement.  The nature of the rearrangement and the parental karyotypes will determine the recurrence risk in each type. A remarkable example, which calls to notice both AS and PWS, is the family depicted in Figure 19–15. The index case had a diagnosis of AS, shown to be due to an unbalanced translocation, 45,XX,der(10)t(10;15)(q26;q13),–15. Upon this having been recognized, family counseling was offered. Although the mother declined testing, her brother, whose partner was pregnant, agreed to be tested, and he was shown to be a balanced carrier. Prenatal diagnosis showed the same unbalanced karyotype as in the proband, but this time, coming from a carrier father, the predicted diagnosis would have been PWS. ANGELMAN SYNDROME A summary of the different genetic forms of AS and the associated risks of recurrence is set out in Tables 19–3 and 19–4. More detail is given in the reviews of Beygo et al. (2019) and Dagli et al. (2021). The clinical diagnosis of AS is sometimes easy (parents have recognized the condition in their child having seen a television program), but at other times more challenging. Of course, if accurate genetic advice is to be given, an accurate clinical diagnosis is crucial. The possibility of phenocopies such as Rett syndrome and Pitt-Hopkins syndrome may need to be considered. The counselor must obtain a detailed family history. A genetic defect could have been transmitted through males for some generations, only causing AS when it had been passed from a daughter of such a male. Figure 19–14 shows a family in which some quite distant relatives, including second cousins once removed and first cousins twice removed, had AS due to an inherited UBE3A mutation. Classical Deletion 15q11q13.  Similarly to PWS, ~70% of AS is due to a de novo interstitial deletion. Recurrence in siblings of a typically sized deletion is extraordinarily rare, and in the two known cases reflected presumed gonadal (Kokkonen and Leisti 2000) or somatic-gonadal (Sánchez et al. 2014) maternal mosaicism.25 Thus, as for classical deletion PWS, we presume a very low—but clearly not quite zero—recurrence risk. There are two recorded examples of deletion AS in cousins that manifestly represented coincidental de novo events in these families, in that different ancestral chromosomes were involved (Connerton-Moyer et al. 1997). The comments on prenatal diagnosis in PWS (above) apply similarly here. Uniparental Disomy 15, Karyotype 46,XX or 46,XY, Parents’ Chromosomes Normal.  AS due to paternal UPD(15) is uncommon. As discussed above, the initial error may actually reflect a maternal-age effect. Interestingly, the AS phenotype may be somewhat milder in UPD(15), and in some children it was only after an electroencephalogram showed typical findings that the diagnosis was suspected (Bottani et al. 1994). But this does not mean that some UPD(15)pat AS children may not be severely affected (Prasad and Wagstaff 1997). No recurrence is on record (Chan et al. 1993), and we assume on theoretical grounds that no usefully measurable increased risk would exist. 25 In neither example were the breakpoints mapped at a molecular level, and the existence of mosaicism (gonadal or somatic) for the classical deletion remains unproven (Beygo et al. 2019). UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  573 Angelman Syndrome Imprinting Center Microdeletion.  Assuming the mother carries the genetic defect, there is a high recurrence risk, namely 50%. SNRPN methylation testing on CVS can identify an affected pregnancy. The possibility of maternal gonadal mosaicism for an IC mutation complicates the picture (Stalker et al. 1998). The siblings of the carrier mother could also be carriers (assuming their father to be heterozygous). However, it would only be the sisters who would have the risk for an AS child. Functional Defect (“Epimutation”) of Angelman Syndrome Imprinting Center.  The previous comments on PWS apply similarly here. All cases of AS due to a functional IC defect have so far been sporadic, but it would be prudent to offer prenatal diagnosis in a subsequent pregnancy (SNRPN methylation testing). Although the numbers are very small (but epimutation AS is rare), there are grounds for supposing there might be a link with infertility/IVF (Manipalviratn et al. 2009). Had there been such a reproductive history, this fact would need to be weighed. UBE3A Mutation.  If the mother carries the mutation, the risk for recurrence is 50%. Maternal mosaicism has been recognized (Malzac et al. 1998; Hosoki et al. Figure 19–15.  A Translocation Predisposing to both Angelman and Prader-Willi Syndromes. Notes: Family tree (above left) shows the index case, arrowed, a child diagnosed with AS, and with an unbalanced translocation 45,XX,der(10)t(10;15)(q26;q13),–15 identified. Her maternal uncle was subsequently shown to be a balanced carrier. Her mother (who had declined testing) is thus an obligate heterozygote, shown as light grey half-symbol, as presumably was one or other of the grandparents of generation I. In a pregnancy (blue diamond) from the uncle, the same unbalanced karyotype as in the proband was seen at amniocentesis. Termination was chosen. Karyotypes show the balanced translocation in the uncle (a), and the unbalanced translocation as in the AS child and similarly in the tested pregnancy (b), resulting from a 3:1 tertiary monosomy. FISH on amniotic fluid cells (image, c), with probes for chromosome 15 centromere (aqua) and SNRPN (red), showed only single signals, consistent with the PWS/AS region being present in single dose, from the maternally-originating (II:4) no. 15, and reflecting absence of the paternally-originating (II:3) tiny der(15). This absence of a paternally-originating PWS/AS region would have determined a PWS phenotype. Source: From P Ranganath et al., Angelman syndrome and prenatally diagnosed Prader-Willi syndrome in first cousins, Am J Med Genet 155A:2788–2790, 2011. Courtesy SR Phadke, and with the permission of John Wiley & Sons.

574  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING 2005), and so non-demonstration (on blood) of the mutation in the mother does not necessarily exclude a genetic risk. It may be appropriate to track the mutation through the patrilineal family in order to be able to offer genetic counseling to female cousins who might be carriers. The reader should study the illustrative pedigree in Figure 19–14. No Genetic Defect Demonstrable.  In a small fraction of AS, ~10%, no cytogenetic or molecular defect, nor UBE3A mutation, is demonstrable. Most of these cases will be phenocopies, and an alternative diagnosis should be sought; but some could conceivably reflect a mutation that has not been able to be detected. The family history, if positive, may compel the assumption of a mutation and thus imply a high recurrence risk. A negative family history might support the inference of a low risk, but it would not allow a definite assumption. If a normal sibling carried the same 15q11q13 haplotype on molecular study, a low-risk scenario would be probable. Expert advice should be sought. Uncommon Cytogenetically Detectable Rearrangement.  The nature of the rearrangement, and the parental karyotypes, will determine the recurrence risk in each type. The rare circumstance of UPD associated with a parental Robertsonian translocation is noted on p. 213. A Simplification for Angelman Syndrome Some parents will not find it easy to come to grips with these various possible causes for their child’s condition even if, in the end, they need only consider the category that applies to themselves. It may be helpful to discuss AS, and the risks of recurrence, in the following terms.26 Let us say that AS is due simply to a lack of the UBE3A protein, a very important protein that is necessary for the brain to grow normally. The gene for UBE3A works only on the chromosome 15 from the mother, while the gene on the father’s chromosome is dormant. There is a switch on the mother’s chromosome that makes this gene work. • If the bit of the maternal chromosome that contains this gene is missing (deletion), or if the mother’s chromosome is replaced by another one from the father (UPD), no UBE3A protein can be made. These two types happen as one-off events. • If the switch fails on the mother’s chromosome, then the gene remains dormant and no protein is made (imprinting center fault). This type can happen one-off, as though the switch “gets stuck” for reasons that we do not well understand. Or, there may be a genetic fault in the actual switch, and in this case the defect could be passed to a subsequent child. • If the UBE3A gene itself is faulty on the mother’s chromosome (mutation), no protein is made, or only an abnormal protein that cannot function. The genetic risk depends on whether the faulty gene started with the child (no increased risk) or if the mother is a carrier (high risk). Note that the mother can be a carrier and still be perfectly normal, since the faulty gene would be the one she got from her father, and so in any event it would have been switched off. • Sometimes the UBE3A gene fails to work, even though the maternal chromosome is normal and has a normal switch. We do not know why this happens (there has been a suggestion that one cause may be if there had been difficulty achieving the pregnancy, either naturally or with IVF). This type is a one-off event. 26 See also the European Journal of Human Genetics Clinical Utility Card (Buiting et al. 2015). UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  575 A common question parents have is whether their normal children might, in the next generation, have an AS child. Or the aunts and uncles of an AS child might want advice about risks to their future children or to their grandchildren. The answers are as follows: • The normal siblings of an AS child have no increased risk for any genetic category, with the possible exception of a familial translocation. Even if the AS child has (or had) a potentially heritable type of UBE3A genetic defect, the fact that the sibs themselves are normal self-evidently declares that they cannot have received it. If they had got the abnormal gene, they would have AS; since they do not have AS, they cannot have the gene. The sex of the siblings is immaterial. • Aunts and uncles have an increased risk for children or grandchildren of theirs only if a heritable type of AS is involved (imprinting center defect, UBE3A mutation). In that case, an uncle could be a carrier, but his children would not be at risk, since the UBE3A gene would be dormant anyway. Daughters of his, however, could have an AS child. A carrier aunt would have a high risk (50%) to have an AS child. But her grandchildren, through her normal sons and daughters, would have no increased risk. Her normal children would have declared themselves, by their very normality, not to have inherited the genetic defect. Uniparental Disomy for the Entire Chromosomal Complement UPD for the entire paternal chromosome set (hydatidiform mole) is associated with an increased recurrence risk; this is discussed in detail in Chapter 20. There is no discernibly increased risk for the recurrence of UPD for the entire maternal chromosome set (ovarian teratoma). Diagnostic Testing for Uniparental Disomy Guidelines from the American College of Genetics and Genomics summarize the indications for diagnostic testing for UPD (Del Gaudio et al. 2020; Table 19–5). SNP microarray is the mainstay. Much UPD (between 50% and 100%, depending on origin of UPD) is detectable by microarray of the index case via the detection of homozygosity. However, a normal SNP in an individual cannot detect UPD originating in meiosis I when there has been no meiotic recombination—these cases can only be detected using patient-parent trios. UPD can also be detected using data from exome or genome sequencing (Yauy et al. 2020; Dong et al. 2021). As for prenatal diagnosis, two scenarios warrant specific mention. First, a small risk27 of UPD applies for non-Robertsonian translocations if one or both chromosomes carry imprinted genes, specifically when there is a risk of 3:1 nondisjunction and where trisomy or monosomy rescue or gamete complementation could occur. Reports of this (very rare) occurrence are listed in Table 19–6. 27 Using data from UPD risk in the setting of Robertsonian translocations (Moradkhani et al. 2019) and data showing segregation in PGT embryos (Zhang et al. 2018; Jia et al. 2023) we estimate this risk to be approximately 1 in 5,000.

576  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING Table 19–5.  Circumstances in which Diagnostic Testing for UPD Should Be Considered PRENATAL TESTING POST-NATAL TESTING 1.  Level II or level III mosaicism for trisomy or monosomy of chromosomes 6, 7, 11, 14, 15, or 20 in amniocentesis or CVS 2.  In the context of preimplantation genetic testing, a pregnancy following transfer of a mosaic embryo with trisomy or monosomy of chromosomes 6, 7, 11, 14, 15, or 20. 3.  Prenatal imaging anomalies consistent with a UPD phenotype. 4.  Familial or de novo balanced Robertsonian translocation or isochromosome involving chromosome 14 or 15 based on CVS or amniocentesis.* 5.  De novo sSMC with no apparent euchromatic material in the fetus. 6.  Non-Robertsonian translocation between an imprinted chromosome with possible 3:1 disjunction that can lead to trisomy or monosomy rescue.** 1.  Patients evaluated for developmental delay and found to have a familial or de novo balanced Robertsonian translocation involving chromosome 14 or 15. 2.  Patients evaluated for developmental delay and found to have a supernumerary structurally abnormal chromosome derived from chromosome 14 or 15. 3.  Patients with homozygosity for a pathogenic variant causing an autosomal recessive disorder when only one parent is a carrier for that variant. 4.  Patients with TNDM and hypomethylation within the 6q24 DMR region. 5.  Patients with clinical suspicion for SRS. 6.  Patients with BWS found to have loss of methylation at IC2 and gain of methylation at IC1 at 11p15. 7.  Patients with clinical findings and physical features suggestive of maternal or paternal UPD(14). 8.  Patients with PWS or AS with abnormal methylation studies who have normal karyotype and microarray results. 9.  Patients with PHP1B who have abnormal methylation studies of the DMRs at the GNAS complex locus with normal karyotype and microarray results. 10.  Female patients who present with unexplained severe manifestations of X-linked conditions and who are found to have homozygosity for a pathogenic variant in an X-linked gene. 11.  Male patients with unexplained father-to-son transmission of an X-linked disorder. *Moradkhani et al. (2019) estimated the risk of UPD of chromosome 14 or 15 in the setting of an inherited Robertsonian translocation to be 0.06%, and argued that prenatal testing is not warranted at this level of risk. **In the setting of a non-Robertsonian translocation, we consider an extremely low risk of ~1 in 5,000 to be fair. sSMC = small supernumerary marker chromosome; TNDM = transient neonatal diabetes mellitus; AS = Angelman syndrome; BWS = Beckwith-Wiedemann syndrome; IC1/2 = imprinting center 1/2; PWS = Prader-Willi syndrome; SRS = Silver-Russell syndrome; DMR = differentially methylated region; PHP1B = Pseudohypoparathyroidism type 1B. Source: Adapted from D Del Gaudio et al., Diagnostic testing for uniparental disomy: a points to consider statement from the American College of Medical Genetics and Genomics, Genet Med 22:1133– 1141, 2020. UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  577 Second, if a parent is a carrier of a Robertsonian translocation involving chromosome 14 or 15, the risk of UPD in a fetus carrying the same translocation is estimated to be ~1 in 1500 (Moradkhani et al. 2019). This risk is doubled when the Robertsonian translocation has arisen de novo (p. 703), but still very small. Reassurance rather than an offer of invasive prenatal diagnosis may be the more appropriate. If prenatal diagnosis is chosen and the fetus has a normal karyotype, the risk is minuscule, with only a single report of a child with complete paternal isodisomy of chromosome 14 and a normal karyotype, whose mother had a rob(13;14) (Potok et al. 2009). Diagnostic Implications of Excessive Homozygosity Detected by SNP-Based Microarrays SNP-based microarrays, in addition to detecting CNVs, also allow the recognition of homozygosity. An excessive degree of homozygosity is most often observed with one or more uninterrupted regions of homozygous SNP alleles, having a copy number state of 2. Such a region is frequently referred to as a “long-contiguous stretch of homozygosity” (LCSH). A LCSH in excess of ~5 Mb is considered to be of potential clinical significance, albeit that excessive homozygosity of itself is not diagnostic of any particular condition and may be benign (Kearney et al. 2011). When LCSH is present on two or more chromosomes, this is assumed to represent regions identical by descent (IBD), implying consanguinity and the concomitant association, therefore, with autosomal recessive disease. The total proportion of LCSH across the genome can be used as a crude measure of degree of parental relationship, as summarized in Table 19–7. A LCSH fraction of above one-eighth should arouse suspicion of incest, but it does not provide information about the specific parental relationship. A LCSH of >12.5% may also reflect, in parents who are first cousins, additional consanguinity in preceding generations. The percentage of LCSH correlates with the risk of autosomal recessive disease, and the coordinates of the specific regions of LCSH can be used to focus the search for candidate autosomal recessive genes. Table 19–6.  Imprinting Syndromes due to Malsegregation of a Parental Translocation TRANSLOCATION PHENOTYPE REFERENCE t(8;15)(p23.3;q11)pat* AS Smith et al. 1994 t(7;16)(q21;q24)mat SRS Dupont et al. 2002 t(8;15)(q24.1;q21.2)mat PWS Calounova et al. 2006 t(2;15)(p11;q11.2)mat PWS Heidemann et al. 2010 t(7;13)(q11.2;q14)mat SRS Behnecke et al. 2012 AS = Angelman syndrome; PWS = Prader-Willi syndrome; SRS = Silver-Russell syndrome. *In this case, the translocation in the father and son was unbalanced, 45,XY,–15,+der(8),t(8;15)(p23.3;q11). 578  DISORDERS ASSOCIATED WITH ABERRANT GENOMIC IMPRINTING LCSH restricted to a single chromosome could be due to either isodisomy or IBD. If LCSH extends along the whole chromosome, this can be assumed to represent isodisomy for that chromosome. When LCSH involves only part of the chromosome, distinguishing IBD from UPD is more complex. Papenhausen et al. (2011) showed that in proven cases of UPD, LCSH always exceeded 13.5 Mb, suggesting that a single Figure 19–16.  UPD Patterns. Patterns of isodisomy and heterodisomy observed in UPD and their associations with the mechanisms of UPD. Table 19–7.  Homozygosity and Consanguinity Correlation Between Degree of Parental Relationship and Percentage of Long Contiguous Stretches of Homozygosity (LCSH) DEGREE OF RELATIONSHIP EXAMPLE PREDICTED LCSH IN CHILD (%) First Parent/child Full siblings 25 Second Uncle/niece Aunt/nephew Grandparent/grandchild 12.5 Third First cousins 6 Fourth First cousins once removed 3 Fifth Second cousins 1.5 UNIPARENTAL DISOMY AND DISORDERS OF IMPRINTING  579 chromosomal LCSH of >13.5 Mb should trigger additional investigation for UPD. In practice, a more conservative threshold of 10 Mb may be used, particularly in the context of an imprinted chromosome. Once UPD is confirmed, the pattern of isodisomy and heterodisomy can be used to infer the origin of the UPD: homozygosity of terminal segments implies meiosis I error, whereas centromeric homozygosity is consistent with meiosis II error (Figure 19–16).