A 28-kb deletion spanning D15S63 (PW71) in five families: a rare neutral variant?

Methylation analysis with probe PW71 (D15S63) is an established procedure to test patients suspected of having Prader-Willi syndrome or Angelman syndrome. Using this test, we have identified a 28-kb deletion spanning D15S63 in five independent families. Sequence analysis revealed identical breakpoints in all the families. The haplotype data are compatible with a common ancestral origin of the deletion in at least two families. The deletion was not found in 1, 000 unrelated controls. Although the deletion maps within the imprinting-center region, neither maternal nor paternal inheritance of the deletion appears to affect imprinting in proximal 15q. We conclude that the deletion is a rare neutral variant that can lead to false-positive results in the PW71-methylation test.     

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.     

Prader-Willi-Syndrom und Angelman-Syndrom

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurogenetic disorders caused by the loss of function of imprinted genes in the chromosomal region 15q11q13. An approximately 2 Mb region inside 15q11q13 is subject to genomic imprinting. As a consequence the maternal and paternal copies in this region are different in DNA methylation and gene expression. The most frequent genetic lesions in both disorders are an interstitial de novo deletion of the chromosomal region 15q11q13, uniparental disomy 15, an imprinting defect or, in the case of AS, a mutation of the UBE3A gene. Microdeletions in a small number of patients with PWS and AS with an imprinting defect have led to the identification of the chromosome 15 imprinting centre (IC) upstream of the SNURF-SNRPN gene, which acts in cis to regulate imprinting in the whole 15q imprinted domain. The IC consists of two critical elements: one in the more centromeric part which is deleted in patients with AS and which is thought to be responsible for the establishment of imprinting in the female germ line, and one in the more telomeric part which is deleted in patients with PWS and which is required to maintain the paternal imprint.     

Parent-specific complementary patterns of histone H3 lysine 9 and H3 lysine 4 methylation at the Prader-Willi syndrome imprinting center

The Prader-Willi syndrome (PWS)/Angelman syndrome (AS) region, on human chromosome 15q11-q13, exemplifies coordinate control of imprinted gene expression over a large chromosomal domain. Establishment of the paternal state of the region requires the PWS imprinting center (PWS-IC); establishment of the maternal state requires the AS-IC. Cytosine methylation of the PWS-IC, which occurs during oogenesis in mice, occurs only after fertilization in humans, so this modification cannot be the gametic imprint for the PWS/AS region in humans. Here, we demonstrate that the PWS-IC shows parent-specific complementary patterns of H3 lysine 9 (Lys9) and H3 lysine 4 (Lys4) methylation. H3 Lys9 is methylated on the maternal copy of the PWS-IC, and H3 Lys4 is methylated on the paternal copy. We suggest that H3 Lys9 methylation is a candidate maternal gametic imprint for this region, and we show how changes in chromatin packaging during the life cycle of mammals provide a means of erasing such an imprint in the male germline.     

Sporadic imprinting defects in Prader-Willi syndrome and Angelman syndrome: implications for imprint-switch models, genetic counseling, and prenatal diagnosis.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.     

Disruption of the bipartite imprinting center in a family with Angelman syndrome

Imprinting in 15q11-q13 is controlled by a bipartite imprinting center (IC), which maps to the SNURF-SNRPN locus. Deletions of the exon 1 region impair the establishment or maintenance of the paternal imprint and can cause Prader-Willi syndrome (PWS). Deletions of a region 35 kb upstream of exon 1 impair maternal imprinting and can cause Angelman syndrome (AS). So far, in all affected sibs with an imprinting defect, an inherited IC deletion was identified. We report on two sibs with AS who do not have an IC deletion but instead have a 1-1.5 Mb inversion separating the two IC elements. The inversion is transmitted silently through the male germline but impairs maternal imprinting after transmission through the female germline. Our findings suggest that the close proximity and/or the correct orientation of the two IC elements are/is necessary for the establishment of a maternal imprint.     

A candidate model for angelman syndrome in the mouse

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are well-recognized examples of imprinting in humans. They occur most commonly with paternal and maternal 15q11-13 deletions, but also with maternal and paternal disomy. Both syndromes have also occurred more rarely in association with smaller deletions seemingly causing abnormal imprinting. A putative mouse model of PWS, occurring with maternal duplication (partial maternal disomy) for the homologous region, has been described in a previous paper but, although a second imprinting effect that could have provided a mouse model of AS was found, it appeared to be associated with a slightly different region of the chromosome. Here, we provide evidence that the same region is in fact involved and further demonstrate that animals with paternal duplication for the region exhibit characteristics of AS patients. A mouse model of AS is, therefore, strongly indicated. Received: 15 December 1996 / Accepted: 31 January 1997     

The impact of imprinting: Prader-Willi syndrome resulting from chromosome translocation, recombination, and nondisjunction.

Prader-Willi syndrome (PWS) is most often the result of a deletion of bands q11.2-q13 of the paternally derived chromosome 15, but it also occurs either because of maternal uniparental disomy (UPD) of this region or, rarely, from a methylation imprinting defect. A significant number of cases are due to structural rearrangements of the pericentromeric region of chromosome 15. We report two cases of PWS with UPD in which there was a meiosis I nondisjunction error involving an altered chromosome 15 produced by both a translocation event between the heteromorphic satellite regions of chromosomes 14 and 15 and recombination. In both cases, high-resolution banding of the long arm was normal, and FISH of probes D15S11, SNRPN, D15S10, and GABRB3 indicated no loss of this material. Chromosome heteromorphism analysis showed that each patient had maternal heterodisomy of the chromosome 15 short arm, whereas PCR of microsatellites demonstrated allele-specific maternal isodisomy and heterodisomy of the long arm. SNRPN gene methylation analysis revealed only a maternal imprint in both patients. We suggest that the chromosome structural rearrangements, combined with recombination in these patients, disrupted normal segregation of an imprinted region, resulting in uniparental disomy and PWS.     

Molecular mechanism of angelman syndrome in two large families involves an imprinting mutation.

Patients with Angelman syndrome (AS) and Prader-Willi syndrome with mutations in the imprinting process have biparental inheritance but uniparental DNA methylation and gene expression throughout band 15q11-q13. In several of these patients, microdeletions upstream of the SNRPN gene have been identified, defining an imprinting center (IC) that has been hypothesized to control the imprint switch process in the female and male germlines. We have now identified two large families (AS-O and AS-F) segregating an AS imprinting mutation, including one family originally described in the first genetic linkage of AS to 15q11-q13. This demonstrates that this original linkage is for the 15q11-q13 IC. Affected patients in the AS families have either a 5.5- or a 15-kb microdeletion, one of which narrowed the shortest region of deletion overlap to 1.15 kb in all eight cases. This small region defines a component of the IC involved in AS (ie., the paternal-to-maternal switch element). The presence of an inherited imprinting mutation in multiple unaffected members of these two families, who are at risk for transmitting the mutation to affected children or children of their daughters, raises important genetic counseling issues.     

Molecular diagnosis of the Prader-Willi and Angelman syndromes by detection of parent-of-origin specific DNA methylation in 15q11-13

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are distinct genetic disorders that are caused by a deletion of chromosome region 15q11-13 or by uniparental disomy for chromosome 15. Whereas PWS results from the absence of a paternal copy of 15q11-13, the absence of a maternal copy of 15q11-13 leads to AS. We have found that an MspI/HpaII restriction site at the D15S63 locus in 15q11-13 is methylated on the maternally derived chromosome, but unmethylated on the paternally derived chromosome. Based on this difference, we have devised a rapid diagnostic test for patients suspected of having PWS and AS.     

Epimutations in Prader-Willi and Angelman syndromes: a molecular study of 136 patients with an imprinting defect

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurogenetic disorders that are caused by the loss of function of imprinted genes in 15q11-q13. In a small group of patients, the disease is due to aberrant imprinting and gene silencing. Here, we describe the molecular analysis of 51 patients with PWS and 85 patients with AS who have such a defect. Seven patients with PWS (14%) and eight patients with AS (9%) were found to have an imprinting center (IC) deletion. Sequence analysis of 32 patients with PWS and no IC deletion and 66 patients with AS and no IC deletion did not reveal any point mutation in the critical IC elements. The presence of a faint methylated band in 27% of patients with AS and no IC deletion suggests that these patients are mosaic for an imprinting defect that occurred after fertilization. In patients with AS, the imprinting defect occurred on the chromosome that was inherited from either the maternal grandfather or grandmother; however, in all informative patients with PWS and no IC deletion, the imprinting defect occurred on the chromosome inherited from the paternal grandmother. These data suggest that this imprinting defect results from a failure to erase the maternal imprint during spermatogenesis.     

Molecular characterization of two proximal deletion breakpoint regions in both Prader-Willi and Angelman syndrome patients.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation syndromes caused by paternal and maternal deficiencies, respectively, in chromosome 15q11-q13. Approximately 70% of these patients have a large deletion of approximately 4 Mb extending from D15S9 (ML34) through D15S12 (IR10). To further characterize the deletion breakpoints proximal to D15S9, three new polymorphic microsatellite markers were developed that showed observed heterozygosities of 60%-87%. D15S541 and D15S542 were isolated from YAC A124A3 containing the D15S18 (IR39) locus. D15S543 was isolated from a cosmid cloned from the proximal right end of YAC 254B5 containing the D15S9 (ML34) locus. Gene-centromere mapping of these markers, using a panel of ovarian teratomas of known meiotic origin, extended the genetic map of chromosome 15 by 2-3 cM toward the centromere. Analysis of the more proximal S541/S542 markers on 53 Prader-Willi and 33 Angelman deletion patients indicated two classes of patients: 44% (35/80) of the informative patients were deleted for these markers (class I), while 56% (45/80) were not deleted (class II), with no difference between PWS and AS. In contrast, D15S543 was deleted in all informative patients (13/48) or showed the presence of a single allele (in 35/48 patients), suggesting that this marker is deleted in the majority of PWS and AS cases. These results confirm the presence of two common proximal deletion breakpoint regions in both Prader-Willi and Angelman syndromes and are consistent with the same deletion mechanism being responsible for paternal and maternal deletions. One breakpoint region lies between D15S541/S542 and D15S543, with an additional breakpoint region being proximal to D15S541/S542.     

Angelman syndrome in an inbred family

Angelman syndrome (AS) is characterized by severe mental retardation, absent speech, puppet-like movements, inappropriate laughter, epilepsy, and abnormal electroencephalogram. The majority of AS patients ( 65%) have a maternal deficiency within chromosomal region 15q11–q13, caused by maternal deletion or paternal uniparental disomy (UPD). Approximately 35% of AS patients exhibit neither detectable deletion nor UPD, but a subset of these patients have abnormal methylation at several loci in the 15q11–q13 interval. We describe here three patients with Angelman syndrome belonging to an extended inbred family. High resolution chromosome analysis combined with DNA analysis using 14 marker loci from the 15q11-q13 region failed to detect a deletion in any of the three patients. Paternal UPD of chromosome 15 was detected in one case, while the other two patients have abnormal methylation atD15S9, D15S63, andSNRPN. Although the three patients are distantly related, the chromosome 15q11-q13 haplotypes are different, suggesting that independent mutations gave rise to AS in this family.     

An unexpected function of the Prader-Willi syndrome imprinting center in maternal imprinting in mice

Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11-q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of PWS.     

Familial cryptic translocation resulting in Angelman syndrome:implications for imprinting or location of the Angelman gene?

Angelman syndrome (AS) is associated with a loss of maternal genetic information, which typically occurs as a result of a deletion at 15q11-q13 or paternal uniparental disomy of chromosome 15. We report a patient with AS as a result of an unbalanced cryptic translocation whose breakpoint, at 15q11.2, falls within this region. The proband was diagnosed clinically as having Angelman syndrome, but without a detectable cytogenetic deletion, by using high-resolution G-banding. FISH detected a deletion of D15S11 (IR4-3R), with an intact GABRB3 locus. Subsequent studies of the proband's mother and sister detected a cryptic reciprocal translocation between chromosomes 14 and 15 with the breakpoint being between SNRPN and D15S10 (3- 21). The proband was found to have inherited an unbalanced form, being monosomic from 15pter through SNRPN and trisomic for 14pter to 14q11.2. DNA methylation studies showed that the proband had a paternal-only DNA methylation pattern at SNRPN, D15S63 (PW71), and ZNF127. The mother and unaffected sister, both having the balanced translocation, demonstrated normal DNA methylation patterns at all three loci. These data suggest that the gene for AS most likely lies proximal to D15S10, in contrast to the previously published position, although a less likely possibility is that the maternally inherited imprinting center acts in trans in the unaffected balanced translocation carrier sister.     

Asynchronous DNA replication between 15q11.2q12 homologs: cytogenetic evidence for maternal imprinting and delayed replication

DNA replication kinetics of Prader-Willi/Angelman syndrome region of 15q11.2q12 was studied without synchronization in five human amniotic cell and five skin fibroblast strains with a marker 15 chromosome, i.e., 15p+ or der(15), as cytological marker to distinguish between the two homologs. BrdU-33258 Hoechst-Giemsa techniques were used to analyze and compare the late replication patterns in the 15q11.2q12 region between the homologs. Asynchronous replication between the homologs was observed in both amniocytes and fibroblasts. From cells of a marker 15 of known parental origin, the paternal 15q11.2q12 replicated earlier than that of the maternal 15 in 92%–95% of asynchronous metaphases. The remaining 5%–8% of asynchronous metaphases displayed maternal early/paternal late replication. This mosaic pattern of replication in the 15q11.2q12 region may be due to methylation mosaicism of genomic imprinting or a relative lack of self-control of replication. These results provide cytogenetic evidence of maternal imprinting and delayed replication in the 15q11.2q12 region.     

Imprinted methylation and its effect on expression of the mouse Zfp127 gene.

The Zfp127 gene is located on mouse chromosome 7 in an imprinted region that is homologous to the 2-Mb Prader-Willi and Angelman Syndromes region on human chromosome 15q11-q13. Here, we show that the gene is differentially methylated, the maternal allele being methylated and the paternal allele being unmethylated. This maternal methylation is established promptly after fertilization prior to syngamy. We also provide data that demonstrate the significance of methylation in the paternal expression of the gene. The expression of the Zfp127 gene in methyltransferase-deficient mice is significantly higher, suggesting that the gene is biallelically expressed in these mice. The data presented here will help to understand the mechanism by which the monoallelic expression of the entire 2-Mb Prader-Willi and Angelman Syndrome region is regulated.     

Mouse imprinting defect mutations that model Angelman syndrome

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders resulting from deficiency of imprinted gene expression from paternal or maternal chromosome 15q11-15q13, respectively. In humans, expression of the imprinted genes is under control of a bipartite cis-acting imprinting center (IC). Families with deletions causing PWS imprinting defects localize the PWS-IC to 4.3 kb overlapping with SNRPN exon 1. Families with deletions causing AS imprinting defects localize the AS-IC to 880 bp 35 kb upstream of the PWS-IC. We report two mouse mutations resulting in defects similar to that seen in AS patients with deletion of the AS-IC. An insertion/duplication mutation 13 kb upstream of Snrpn exon 1 resulted in lack of methylation at the maternal Snrpn promoter, activation of maternally repressed genes, and decreased expression of paternally repressed genes. The acquisition of a paternal epigenotype on the maternal chromosome in the mutant mice was demonstrated by the ability to rescue the lethality and growth retardation in a mouse model of a PWS imprinting defect. A second mutation, an 80-kb deletion extending upstream of the first mutation, caused a similar imprinting defect with variable penetrance. These results suggest that there is a mouse functional equivalent to the human AS-IC.     

Deletion analysis of the imprinting center region in patients with Angelman syndrome and Prader-Willi syndrome by real-time quantitative PCR

The molecular basis of Angelman syndrome and Prader-Willi syndrome is well established, and genetic testing for these disorders is clinically available. Imprinting abnormalities account for up to 4% of patients with Angelman and Prader-Willi syndromes. Deletions of the imprinting center region are the molecular abnormality observed in a subset of Angelman and Prader-Willi syndrome cases with imprinting defects. Genetic testing of imprinting center deletions in patients with Angelman and Prader-Willi syndrome is not readily available. Such testing is important for the diagnostics of Angelman and Prader-Willi syndrome because it allows for more accurate diagnosis and recurrence risk prediction in families. Here we describe the development, validation, and implementation of a real time quantitative polymerase chain reaction (PCR)-based assay for imprinting center deletion detection in patients with Angelman and Prader-Willi syndrome, which we have incorporated into our genetic testing strategy for these disorders. To date we have tested, on a clinical basis, five patients with either Angelman or Prader-Willi syndrome in whom an imprinting center defect was implicated and found a deletion in one patient that was determined to be familial.