Genomic Imprinting
As discussed in Chapter 6, imprinted genes are expressed from only one parental allele— that is, although two copies of the gene are present in the cell, only one copy is expressed. Which copy is expressed depends on the parent of origin and is determined by DNA methylation marks. The allele that is expressed is unmethylated, and the allele that is silenced is methylated. Although only a small percentage of human genes undergo genomic imprinting, many of these genes are critical regulators of growth and development, and therefore disruption of their normal monoallelic expression results in disorders that often impact both intrauterine and postnatal growth and neurodevelopment. The majority of imprinted genes are found in clusters, called imprinted domains, in specific chromosome regions (i.e., 15q11- 13 and 11p15). Each imprinted domain is controlled by one or more independent imprinting control regions that regulate in cis the expression of target imprinted genes within the domain. More than 120 imprinted genes have been identified across the human genome (Fig. 1). Epigenetic changes that impact imprinting centers (ICs) and result in transcriptional silencing of a gene that is normally active are referred to as epimutations.
Fig1. Ideograms of human imprinted genes. Ideograms were generated using http:// www.dna- rainbow.org/ ideograms/ . An ideogram of each human chromosome known to have an imprinted gene based on the imprinted gene catalogue (http:// igc.otago.ac.nz) and GeneImprint portal (http:// www.geneimprint.com) is shown. Imprinted genes are listed on each ideogram if they were designated as imprinted in both of the aforementioned human imprinted gene catalogs. Blue genes are paternally expressed, red genes are maternally expressed, black genes have unknown parent- of- origin expression, gray genes have parental expression that is isoform dependent. Bold genes are implicated in growth, underlined genes play roles in neurodevelopment. Genes in italic have no reported function in growth or neurodevelopment.
The first human disorders recognized to result from genomic imprinting were Prader- Willi syndrome and Angelman syndrome. These two neurodevelopmental disorders result from the absence of paternally or maternally expressed genes, respectively, in the chromosome 15q11- 13 imprinted region (which contains a cluster of imprinted genes). One of the characteristics of imprinting disorders is molecular heterogeneity in that there are several different mechanisms, including epigenetic and/ or genetic, that can disrupt gene expression. This is seen with both Prader- Willi and Angelman syndromes, which can occur due to chromo some deletions, uniparental disomy (two copies of a single chromosome from one parent), imprinting defects (i.e., epimutation), and pathogenic sequence variants (UBE3A in Angelman syndrome).
Other examples of paired human imprinting dis orders are Beckwith- Wiedemann and Russell- Silver syndromes, which are two clinically opposite growth disorders that result from dysregulation of imprinted genes in the chromosome 11p15 region. Beckwith- Wiedemann syndrome is characterized by overgrowth, whereas Russell- Silver syndrome is characterized by intrauterine growth restriction and postnatal growth deficiency. Beckwith- Wiedemann syndrome is also associated with an increased risk for the development of embryonal tumors. The chromosome 11p15 region contains a cluster of imprinted genes that are organized into two distinct imprinted domains, each with its own imprinting control region: the IC1 domain in the telomeric region and the IC2 domain in the centromeric region. The IC1 domain contains the IGF2 and H19 genes, and the IC2 domain contains the CDKN1C, KCNQ1, and KCNQ10T1 genes. The genes in these regions undergo parent- of- origin imprinting such that typically IC1 is methylated on the paternally derived chromosome resulting in IGF2 expression (promotes cell growth and proliferation) and silencing of H19. On the maternally derived chromosome IC2 is methylated, resulting in silencing of KCNQ10T1 and expression of KCNQ1 and CDKN1C (negative regulator of cell proliferation). Opposite molecular alterations at IC1 and IC2 lead to an imbalance of growth- promoting and/ or growth- suppressing genes in this region, either resulting in overgrowth (Beckwith- Wiedemann) or undergrowth (Russell- Silver). Therefore these conditions are mirror images of each other both clinically and molecularly (Fig. 2). Sometimes these two conditions can be seen in the same family when the underlying etiology is a chromosome duplication/ deletion that is transmitted through a male versus a female due to parent- of- origin– specific imprinting of the chromosome 11p15 region (Fig. 3). The molecular mechanisms that cause these conditions are complex, and similar to the chromosome 15q11- 13- related disorders include epigenetic and/ or genetic alterations: cytogenetic aberrations, uniparental disomy, loss or gain of methylation at ICs (i.e., epimutation), and pathogenic sequence variants (CDKN1C in Beckwith- Wiedemann syndrome).
Fig2. Opposite imprinting alterations on 11p15 can cause opposite phenotypes. Schematic representation of imprinting regulation at imprinting center 1 (IC1) in the chromosome 11p15 region. The highlighted box (middle) represents normal expression in which IC1 is methylated on the paternally derived chromosome and unmethylated on the maternally derived chromosome, resulting in expression of insulin- like growth factor 2 (IGF2) only from the paternal allele. (Top) Loss of methylation at IC1 on the paternal allele results in silencing of IGF2; suppression of IGF2 results in reduced growth and causes Russell- Silver syndrome (RSS). (Bottom) Gain of methylation at IC1 on the maternal allele results in activation of IGF2, which promotes growth and causes Beckwith- Wiedemann syndrome (BWS). Loss and gain of methylation at IC2 (not shown here) can also lead to BWS and RSS.
Fig3. Pedigree of a family in which a chromosome 11p15 duplication is segregating; different phenotypes determined by parent- of- origin– specific imprinting. Individual II- 1 has a diagnosis of Beckwith- Wiedemann syndrome, which is determined to be due to a de novo chromosome duplication of chromosome 11p15 encompassing imprinting center 1 (IC1) on her paternally derived chromosome 11. She therefore has two copies of paternally imprinted genes in this region and one copy of maternally imprinted genes, which leads to relative hypermethylation of IC1. When she passes this chromosome duplication on to her children, the parental imprints will be erased and replaced with maternal imprints. Therefore her daughter (III- 2) who inherits the chromosome 11p15 duplication will have two copies of maternally imprinted genes in this region and one copy of paternally imprinted genes, which leads to relative hypomethylation of IC1. This is associated with Russell- Silver syndrome.
Whereas imprinting disorders generally result from disturbed methylation in cis at one imprinted locus, there are also reports of individuals with multilocus imprinting disorders (MLID) in which there is aberrant methylation of multiple imprinted loci. Individuals with MLID can present with features specific for a single imprinting disorder or overlapping features of multiple imprinting disorders. MLID can be observed in children conceived via ART or caused by pathogenic variants in the patient’s genome (e.g., ZFP57), or pathogenic variants in maternal effect genes such as NLRP5 or PAD16, which encode proteins that impact imprinted loci in trans (see Epigenetics in Development, earlier).
Pathogenic variants in maternal effect genes cause variable imprint dysregulation at multiple imprinted loci resulting in a broad range of clinical presentations, including infertility and adverse reproductive outcomes such as hydatidiform moles, recurrent miscarriages, and one or more imprinting disorders. Therefore, when investigating the etiology of MLID where there is a history of infertility and/ or adverse pregnancy outcomes, one must consider testing not only the proband but also the proband ’s mother.
Another consideration in the differential diagnosis of overlapping features of multiple imprinting disorders in the same individual is genome- wide paternal isodisomy. While genome- wide uniparental paternal disomy is not associated with a viable pregnancy, mosaicism for genome- wide paternal isodisomy has been reported in several individuals with overlapping features of multiple imprinting disorders; specifically, conditions resulting from uniparental disomy of imprinted chromosome regions (6q24, 11p15, 14q32, 15q11, 20q13). Genome- wide paternal uniparental disomy is typically characterized by mosaicism for paternal uniparental and biparental cell lineages. Clinical presentation depends on percentage of mosaic cells and location of the uniparental lineage.
Disorders Involving Unstable Repeat Expansions
Epigenetic mechanisms have been shown to play a critical role in the etiology of disorders due to unstable repeat expansions. This has been well established for fragile X syndrome, where the expansion of the FMR1 CGG repeat to a full mutation triggers a cascade of epigenetic events, including methylation of the FMR1 promotor, which leads to reduced or absent production of the fragile X mental retardation protein (FMRP). In males with normal size FMR1 alleles, the FMR1 promotor is unmethylated resulting in an open chromatin conformation that allows access of transcription factors to the FMR1 promoter, leading to transcription of FMRP. The importance of DNA methylation in mediating the expression of FMRP is illustrated by rare cases of males with full FMR1 expansion and nor mal cognition, in whom the FMR1 promoter has been shown to remain unmethylated.
Many unstable repeat expansion disorders demonstrate anticipation, whereby increased disease severity and decreased age of onset are observed in subsequent generations. The basis of anticipation is the tendency for unstable repeats to undergo expansion when trans mitted from parent to child. It has been proposed that epigenetic factors are involved in both disease pathogenesis and repeat instability. For example, congenital myotonic dystrophy (CDM1) is the most severe form of myotonic dystrophy type 1, a neuromuscular disease caused by the expansion of a CTG repeat in the DMPK gene. CDM1 shows strong genetic anticipation, as well as altered patterns of DNA methylation. Specifically, in individuals with CDM1, cis- regulatory elements upstream and downstream of the DMPK gene are often aberrantly methylated, thereby altering chromatin structure and gene expression at this locus— that is, impairment of these regulatory elements can lead to increased repeat instability providing early evidence for epigenetic involvement in genetic anticipation.
Disorders of the Epigenetic Machinery
Advances in genome sequencing technology have accelerated the discovery of genes involved in mendelian dis orders. Over the last decade, an increasing number of mendelian disorders have been recognized to be caused by sequence variants in genes that are important for maintaining normal epigenetic regulation, including writers, erasers, readers, and chromatin remodelers. Although the majority of these syndromes are caused by loss of function of a single allele (haploinsufficiency) suggesting that these proteins function in a dosage- sensitive manner, both autosomal recessive and X- linked recessive patterns of inheritance are also described. In contrast to classical imprinting disorders that impact imprinted genes in cis, for this group of disorders the epigenetic dysregulation occurs in trans, impacting multiple genomic- wide targets. To date, there are over 80 disorders of the epigenetic machinery that have been identified and likely many more yet to be recognized (Fig. 4). These disorders are characterized by a wide range of multisystem anomalies, with the two most common phenotypic features observed being intellectual disability and growth dysregulation. Several of these dis orders will be discussed later.
Fig4. Mendelian disorders of the epigenetic machinery. Over 70 genes with defined epigenetic domains (reader, writer, eraser, remodeler, middle icons) have been linked to mendelian phenotypes. The majority of genes cause disease in the heterozygous state (filled circle). Enzyme domains (writer, eraser, remodeler) are mutually exclusive in any given factor but many coexist with a reader domain (gray shading). Intellectual disability is seen in the vast majority (blue), as are growth abnormalities (orange). A = genes on autosomes; X = genes on the X chromosome. (Modified from Fahrner JA, Bjornsson HT: Mendelian disorders of the epigenetic machinery: postnatal malleability and therapeutic prospects, Human Molecul Genet 28(2):R254– R264, 2019.)
Disorders of the Epigenetic Machinery: DNA Methylation
There are a small number of genes that regulate DNA methylation marks in contrast to those that regulate histones; these include writers, readers, and erasers. Pathogenic variants in each of these genes are associated with specific phenotypes. Heterozygous germline pathogenic loss- of- function variants in the DNA methyltransferase DNMT3A (a writer) cause Tatton- Brown- Rahman syndrome (TBRS), a nonprogressive neurodevelopmental disorder characterized by increased growth, intellectual disability, and dysmorphic facial features. While constitutional pathogenic variants in DNMT3A cause TBRS, somatically acquired pathogenic variants in DNMT3A are associated with over 20% of acute myeloid leukemia (AML) cases. Notably the same pathogenic variants have been reported in association with both AML and TBRS; however, AML rarely occurs in individuals with TBRS, emphasizing the requirement for multistep deregulation in cancer. Although there is an increased cancer (myeloid neoplasms, including AML) risk above the baseline population risk in individuals with TBRS, this does not meet the threshold for clinical surveillance; therefore cancer screening is not recommended for these individuals. There are other epigenetic regulators associated with mendelian disorders for which somatically acquired pathogenic variants are involved in cancers, including NSD1 and EZH2, which encode two histone methyltransferases (writers). Similarly, the phenotypes associated with germline pathogenic variants in these genes (Sotos syndrome and Weaver syndrome, respectively) have an increased cancer risk above the baseline population risk, which does not meet the threshold for clinical surveillance.
Of interest, pathogenic variants in DNMT1, the maintenance methyltransferase, are associated with two distinct progressive adult- onset neurologic disorders. These are the only adult- onset conditions associated with pathogenic variants in an epigenetic regulator that we currently recognize and likely result from the ongoing loss of the cell’s capacity to maintain critical DNA methylation marks. The specific phenotype is determined by the position of the pathogenic variants in the gene. Hereditary sensory and autonomic neuropathy type 1 with dementia and hearing loss (HSAN1E), associated with variants in exon 20, is a disorder that presents in early adulthood with sensory neuropathy and hearing loss and progresses to dementia. The second syndrome, associated with variants in exon 21 of DNMT1, is called autosomal dominant cerebellar ataxia, deafness and narcolepsy and is characterized by adult- onset of narcolepsy followed by the onset of sensorineural deafness, cerebellar ataxia, and ultimately dementia.
Methyl- CpG- binding protein 2 (MeCP2), which functions as a reader of DNA methylation marks, has been studied extensively in part because pathogenic variants in this gene cause a well- recognized neurodevelopmental disorder, Rett syndrome, which is characterized by acquired microcephaly, progressive intellectual disability, and loss of motor skills beginning in the first year of life. The majority (90%) of classic Rett syndrome cases are caused by loss- of- function variants in MeCP2, located at Xq28. Those with classic Rett syndrome are generally girls who are heterozygous for the loss- of- function variants. When boys with a pathogenic MeCP2 variant or deletion survive until birth they exhibit a severe infantile encephalopathy with seizures.
As described earlier, the TET family of enzymes acts as erasers of DNA methylation marks. Whereas disorders involving writers and readers of DNA methylation have been known for some time, only recently was the first neurodevelopmental disorder impacting the DNA methylation eraser system described. TET3 deficiency, or Beck- Fahrner syndrome (BEFAHRS), is caused by either mono- and biallelic pathogenic variants in TET3, which encodes methylcytosine dioxygenase and is characterized by highly variable and nonspecific clinical features, including intellectual disability, features of autism, hypotonia, and dysmorphic facial features. This syn drome can be transmitted in an autosomal recessive or autosomal dominant manner.
Disorders of the Epigenetic Machinery: Histones
The number of genes involved in regulating histone modifications is much larger than for DNA methylation and include writers, erasers, readers, and chromatin remodelers. This group of disorders often presents with overlapping phenotypes, which can make them difficult to differentiate clinically. This phenotypic overlap can be attributed to the fact that the downstream targets of the various epigenetic regulators, which include different genes, are all involved in the regulation of a common pathway to brain and organ development. This means that distinguishing individual disorders is clinically very challenging. For example, Sotos and Weaver syndromes are two overgrowth conditions with overlapping features caused by different genes that function as epigenetic writers, NSD1 and EZH2, respectively. In spite of the fact that these two genes have different downstream targets, these conditions can be difficult to differentiate clinically especially in the first year or two of life. An accurate clinical diagnosis is important for anticipating the natural history as well as clarifying recurrence risk. Sotos syndrome is associated with significant intellectual and behavioral problems, whereas Weaver syndrome can have relatively mild or no intellectual deficits. There are also differences with respect to the types of cancers and their respective risks in these two conditions, which is important for anticipatory medical care. With respect to recurrence risks, most cases of Sotos syndrome have a de novo etiology, whereas Weaver syndrome is often familial, with a milder presentation in a parent only recognized after an affected child is born.
Phenotypic overlap can also be seen when pathogenic variants occur in genes that function as part of multiprotein complexes. In this instance the phenotypic overlap results from the fact that regulation of common downstream targets is disrupted. This can be seen in Kabuki syndrome, a neurodevelopmental disorder characterized by growth deficiency, which can be caused by loss of function variants in one of two genes with opposite functions, KMT2D and KDM6A. KMT2D encodes a histone methyltransferase (writer) and KDM6A encodes a histone demethylase (eraser), two proteins that form a complex and have complementary roles in regulating chromatin state and transcriptional activity at a specific set of target genes. KMT2D adds a methylation mark associated with open chromatin (H3K4me3), whereas KDM6A removes a mark associated with closed chromatin (H3K27me3). Both genes facilitate the opening of chromatin and promote gene expression. Disruption of either gene/ protein function will disrupt the balance of open versus closed chromatin at overlapping target genes resulting in the same clinical outcome (i.e., Kabuki syndrome). Identification of the specific genetic etiology is important because pathogenic variants in KMT2D are inherited in an autosomal dominant manner and usually occur de novo, whereas KDM6A is an X- linked recessive gene that can have a high risk of recurrence if inherited from a phenotypically normal carrier mother.
There are also distinct clinical conditions with over lapping phenotypes that are caused by pathogenic variants in different genes within the same multiprotein complex. This can be seen with Coffin- Siris syndrome (CSS) and Nicolaides- Baraitser syndrome (NCBRS), two neurodevelopmental disorders that are caused by pathogenic variants in the ARID1B, SMARCB1, and SMARCA4 genes (CSS) and SMARCA2 gene (NCBRS). These genes are all part of the BAF chromatin remodeling complex. Although these two conditions have over lapping clinical features, attributable to the common downstream targets of the multiprotein complex that includes the respective causative genes, they also have important differences in natural history that require gene- based diagnosis for optimal management.
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