Bernhard Horsthemke

Imprinting in Prader–Willi and Angelman syndromes

Imprinted genes are marked in the germline and retain molecular memory of their parental origin, resulting in allelic expression differences during development. Abnormalities in imprinted inheritance occur in several genetic diseases and cancer, and are exemplified by the diverse genetic defects involving chromosome 15q11-q13 in Prader-Willi (PWS) and Angelman (AS) syndromes. PWS involves loss of function of multiple paternally expressed genes, while mutations in a single gene, UBE3A, which is subject to spatially restricted imprinting, occur in some AS patients. Identification of mutations in the imprinting process in PWS and AS has led to a definition of an imprinting center (IC), involving the promoter (in PWS) or an alternative transcript of the SNRPN gene (in AS). The IC regulates initiation of imprint switching for all genes in a 2 Mb imprinted domain during gametogenesis. Imprinting mutations define a novel mechanism of genetic disease because they have no direct effect in the affected patient but, rather, it is the parental germline effect of an IC mutation that leads to disease in the offspring.

Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples

Recent case reports have suggested that infertility treatment with intracytoplasmic sperm injection (ICSI) may increase the risk of imprinting defects leading to Angelman syndrome (AS). Although imprinting defects account for only 4% of patients with AS, we have found four cases among 16 AS patients born to subfertile couples, who conceived with or without infertility treatment (25%; relative risk (RR) 6.25; 95% confidence interval (CI) 1.68 to 16.00). The risk in untreated couples with time to pregnancy (TTP) exceeding 2 years was identical to that of those treated by ICSI or by hormonal stimulation alone (RR 6.25; 95% CI 0.70 to 22.57). It was twice as high in couples who had received treatment and also had TTP >2 years (RR 12.5; 95% CI 1.40 to 45.13). Our findings suggest that imprinting defects and subfertility may have a common cause, and that superovulation rather than ICSI may further increase the risk of conceiving a child with an imprinting defect.

Mechanisms of imprinting of the Prader-Willi/Angelman region.

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurodevelopmental disorders, each caused by several genetic and epigenetic mechanisms involving the proximal long arm of chromosome 15. Lack of a functional paternal copy of 15q11–q13 causes PWS; lack of a functional maternal copy of UBE3A, a gene within 15q11–q13, causes AS. This region of chromosome 15 contains a number of imprinted genes that are coordinately regulated by an imprinting center (PWS/AS‐IC) that contains two functional elements, the PWS‐SRO and the AS‐SRO. A chromosome lacking the PWS‐SRO has the maternal state of gene activity and epigenetic modification after either maternal or paternal transmission; a chromosome lacking the AS‐SRO but containing the PWS‐SRO has the paternal state of gene activity and epigenetic modification after either maternal or paternal transmission. The maternal state of chromosome 15q11–q13 is associated with methylation of the PWS‐SRO, while the paternal state is associated with lack of methylation of the PWS‐SRO. Although most models of PWS/AS region imprinting assume that the PWS‐SRO is methylated during oogenesis and that this methylation of the maternal PWS‐SRO is maintained after fertilization, several lines of evidence suggest that the maternal PWS‐SRO is in fact not methylated until after fertilization. Imprinting defects affecting the PWS/AS region can arise from failure to demethylate the PWS‐SRO in the male germ line, from failure to methylate the maternal PWS‐SRO, or from failure to maintain PWS‐SRO methylation after fertilization.

Imprinting defects on human chromosome 15

The Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurogenetic diseases that are caused by the loss of function of imprinted genes on the proximal long arm of human chromosome 15. In a few percent of patients with PWS and AS, the disease is due to aberrant imprinting and gene silencing. In patients with PWS and an imprinting defect, the paternal chromosome carries a maternal imprint. In patients with AS and an imprinting defect, the maternal chromosome carries a paternal imprint. Imprinting defects offer a unique opportunity to identify some of the factors and mechanisms involved in imprint erasure, resetting and maintenance. In approximately 10% of cases the imprinting defects are caused by a microdeletion affecting the 5′ end of the SNURF-SNRPN locus. These deletions define the 15q imprinting center (IC), which regulates imprinting in the whole domain. These findings have been confirmed and extended in knock-out and transgenic mice. In the majority of patients with an imprinting defect, the incorrect imprint has arisen without a DNA sequence change, possibly as the result of stochastic errors of the imprinting process or the effect of exogenous factors.

The human retinoblastoma gene is imprinted.

Genomic imprinting is an epigenetic process leading to parent-of-origin–specific DNA methylation and gene expression. To date, ∼60 imprinted human genes are known. Based on genome-wide methylation analysis of a patient with multiple imprinting defects, we have identified a differentially methylated CpG island in intron 2 of the retinoblastoma (RB1) gene on chromosome 13. The CpG island is part of a 5′-truncated, processed pseudogene derived from the KIAA0649 gene on chromosome 9 and corresponds to two small CpG islands in the open reading frame of the ancestral gene. It is methylated on the maternal chromosome 13 and acts as a weak promoter for an alternative RB1 transcript on the paternal chromosome 13. In four other KIAA0649 pseudogene copies, which are located on chromosome 22, the two CpG islands have deteriorated and the CpG dinucleotides are fully methylated. By analysing allelic RB1 transcript levels in blood cells, as well as in hypermethylated and 5-aza-2′-deoxycytidine–treated lymphoblastoid cells, we have found that differential methylation of the CpG island skews RB1 gene expression in favor of the maternal allele. Thus, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.

Exclusion of the C/D box snoRNA gene cluster HBII-52 from a major role in Prader–Willi syndrome

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurogenetic disorders caused by the loss of function of imprinted genes in 15q11–q13. The maternally expressed UBE3A gene is affected in AS. Four protein-encoding genes (MKRN3, MAGEL2, NDN and SNURF-SNRPN) and several small nucleolar (sno) RNA genes (HBII-13, HBII-436, HBII-85, HBII-438A,xa0HBII-438B and HBII-52) are expressed from the paternal chromosome only but their contribution to PWS is unclear. To examine the role of the HBII-52 snoRNA genes, we have reinvestigated an AS family with a submicroscopic deletion spanning UBE3A and flanking sequences. By fine mapping of the centromeric deletion breakpoint in this family, we have found that the deletion affects all of the 47 HBII-52 genes. Since the complete loss of the HBII-52 genes in family members who carry the deletion on their paternal chromosome is not associated with an obvious clinical phenotype, we conclude that HBII-52 snoRNA genes do not play a major role in PWS. However, we cannot exclude the possibility that the loss of HBII-52 has a phenotypic effect when accompanied by the loss of function of other genes in 15q11–q13.

A paternal deletion of MKRN3 , MAGEL2 and NDN does not result in Prader–Willi syndrome

The Prader–Willi syndrome (PWS) is caused by a 5–6u2009Mbp de novo deletion on the paternal chromosome 15, maternal uniparental disomy 15 or an imprinting defect. All three lesions lead to the lack of expression of imprinted genes that are active on the paternal chromosome only: MKRN3, MAGEL2, NDN, C15orf2, SNURF-SNRPN and more than 70 C/D box snoRNA genes (SNORDs). The contribution to PWS of any of these genes is unknown, because no single gene mutation has been described so far. We report on two patients with PWS who have an atypical deletion on the paternal chromosome that does not include MKRN3, MAGEL2 and NDN. In one of these patients, NDN has a normal DNA methylation pattern and is expressed. In another patient, the paternal alleles of these genes are deleted as the result of an unbalanced translocation 45,X,der(X)t(X;15)(q28;q11.2). This patient is obese and mentally retarded, but does not have PWS. We conclude that a deficiency of MKRN3, MAGEL2 and NDN is not sufficient to cause PWS.

SNURF-SNRPN and UBE3A transcript levels in patients with Angelman syndrome

The imprinted domain on human chromosome 15 consists of two oppositely imprinted gene clusters, which are under the control of an imprinting center (IC). The paternally expressed SNURF-SNRPN gene hosts several snoRNA genes and overlaps the UBE3A gene, which is encoded on the opposite strand, expressed — at least in brain cells — from the maternal chromosome only, and affected in patients with Angelman syndrome (AS). In contrast to SNURF-SNRPN, imprinted expression of UBE3A is not regulated by a 5′ differentially methylated region. Here we report that splice forms of the SNURF-SNRPN transcript overlapping UBE3A in an antisense orientation are present in brain but barely detectable in blood. In contrast, splice forms that do not overlap with UBE3A are of similar abundance in brain and blood. The tissue distribution of the splice forms parallels that of the snoRNAs encoded in the respective parts of the SNURF-SNRPN transcript. Using a quantitative PCR assay, we have found that the ratio of SNURF-SNRPN/UBE3A transcript levels is increased in blood cells of AS patients with an imprinting defect, but not in AS patients with a UBE3A mutation or an unknown defect. Our findings are compatible with the assumption that imprinted UBE3A expression is regulated through the SNURF-SNRPN sense-UBE3A antisense transcript.

Low frequency of imprinting defects in ICSI children born small for gestational age

Although there is an increased frequency of low birth weight after assisted reproduction, the mechanisms underlying this association are unclear. We have proposed that some of the children conceived by intracytoplasmic sperm injection (ICSI) with low birth weight might have an epimutation (faulty methylation pattern) in one of the imprinted genes involved in fetal growth control, eg, KCNQ1OT1, PEG1, PEG3, GTL2, IGF2/H19 and PLAGL1. Using bisulfite DNA sequencing and sequence-based quantitative methylation analysis (SeQMA), we determined the methylation pattern of these genes in buccal smears from 19 ICSI children born small for gestational age (SGA, birth weight <3rd percentile) and from 29 term-born normal weight children after spontaneous conception. We detected clear hypermethylation of KCNQ1OT1 and borderline hypermethylation of PEG1 in one and the same ICSI child. The other children and the parents of the affected child have normal methylation patterns. Imprinting defects appear to be a rare finding in ICSI children born SGA. Methylation of the paternal KCNQ1OT1 and PEG1 alleles may be a previously unrecognized cause of SGA. The epimutations found in the SGA child, whose father had oligozoospermia, probably result from an imprint erasure defect in the paternal germ line and therefore appear to be linked to the fertility problem of the father and not to in vitro fertilization/ICSI.

Identification of cis- and trans-acting factors possibly modifying the risk of epimutations on chromosome 15.

In the majority of patients with a chromosome 15 imprinting defect (ID) causing Prader–Willi syndrome (PWS) or Angelman syndrome (AS), the defect is a primary epimutation that occurred spontaneously in the absence of a DNA mutation. We have investigated whether common DNA sequence variants in the bipartite imprinting centre (IC) are associated with an increased susceptibility to imprinting defects. We have determined the haplotype structure of the IC and found that the two IC elements called ‘PWS-SRO’ and ‘AS-SRO’ lie on separate haplotype blocks. To identify susceptible IC sequence variants, we have used the transmission disequilibrium test. While we did not observe preferential transmission of a paternal allele or haplotype in 41 PWS-ID trios, we found a trend for preferential maternal transmission of one AS-SRO haplotype (H-AS3) in 48 AS-ID trios (P=0.058) and could identify two sequence variants in H-AS3 that are responsible for this effect. We also obtained tentative evidence that homozygosity for the 677C>T variant of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene on chromosome 1 might increase the risk of a maternal imprinting defect: the frequency of the TT genotype was significantly higher in the mothers of the AS patients with an imprinting defect than in the patients fathers or the general population (P=0.028). Our findings suggest that women with the IC haplotype H-AS3 or homozygosity for the MTHFR 677C>T variant may have an increased risk of conceiving a child with an imprinting defect, although the absolute risk is low.