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     

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.     

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.     

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.     

Somatic recombination rather than uniparental disomy suggested as another mechanism by which genetic imprinting may play a role in the etiology of Prader-Willi syndrome

Summary Six Prader-Willi syndrome (PWS) patients with normal karyotypes and their parents were analyzed to determine the nature of the molecular aberrations present in the proximal region of 15q and to determine the parental origin of the aberrant chromosome 15. In addition, the likehood that uniparental disomy plays a significant role in the etiology of PWS patients with normal karyotypes was studied. Restriction fragment length polymorphisms (RFLPs) recognized by seven probes [pML34 (D15S9), pTD3-21, pCGS0.9, pCGS1.1 (D15S10), IR4.3 (D15S11), IR10.1 (DS15S12), p189-1 (D15S13), IR39 (D15S18), and CMW-1 (D15S24)] mapping to the Prader-Willi chromosome region (PWCR) and an additional two probes [pMS1-14 (D15S1); the cDNA of neuromedin B] mapping elsewhere on chromosome 15 were analyzed in the six PWS patients and their parents. Copy number of each locus within the PWCR was determined by densitometry. Molecular rearrangements of the proximal region of 15q were observed in all of the six probands and the origin of the aberrant chromosome 15 when determined was consistently paternal in origin. While data obtained from our six patients does not support the mechanism of disomy, results obtained from three of the six patients show more complex rearrangements hypothesized to have resulted from somatic recombination. These rearrangements have resulted in acquired homozygosity and the lack of a paternal allele at various loci within the PWCR. The presence of only a maternal contribution at certain loci as the result of somatic recombination may be another mechanism by which genetic imprinting plays a role in the presentation of the PWS phenotype.     

The Mouse Pink-Eyed Dilution Gene: Association with Hypopigmentation in Prader-Willi and Angelman Syndromes and with Human OCA2

Mutations at the mouse pink-eyed dilution locus, p, cause hypopigmentation. We have cloned the mouse p gene cDNA and the cDNA of its human counterpart, P. The region of mouse chromosome 7 containing the p locus is syntenic with human chromosome 15q11-q13, a region associated with Prader-Willi syndrome (PWS) and Angelman syndrome (AS), both of which involve profound imprinting effects. PWS patients lack sequences of paternal origin from 15q, whereas AS patients lack a maternal copy of an essential region from 15q. However, the critical regions for these syndromes are much smaller than the chromosomal region commonly deleted that often includes the P gene. Hypopigmentation in PWS and AS patients is correlated with deletions of one copy of the human P gene that is highly homologous with its mouse counterpart. A subset of PWS and AS patients also have OCA2. These patients lack one copy of the P gene in the context of a PWS or AS deletion, with a mutation in the remaining chromosomal homologue of the P gene. Mutations in both homologues of the P gene of OCA2 patients who do not have PWS or AS have also been detected.     

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.     

A de novo unbalanced reciprocal translocation identified as paternal in origin in the Prader-Willi syndrome

Summary Interstitial cytogenetic deletions involving the paternally derived chromosome 15q11–13 have been described in patients with the Prader-Willi syndrome (PWS). We report a child with PWS and a de novo unbalanced karyotype –45,XY,–9,–15,+der(9)t(9;15)(q34;q13). Molecular studies with the DNA probe pML34 confirmed that only a single Prader Willi critical region (PWCR: 15q11.2-q12) copy was present. Hybridisation of patient and parental DNA with the multi-allelic probe CMW1, which maps to pter-15q13, showed that the chromosome involved in the translocation was paternal in origin. This is the first example of a paternally-derived PWCR allele loss caused by an unbalanced translocation that has arisen de novo.     

Chromosome 15 uniparental disomy is not frequent in Angelman syndrome.

Genetic imprinting has been implicated in the etiology of two clinically distinct but cytogenetically indistinguishable disorders--Angelman syndrome (AS) and Prader-Willi syndrome (PWS). This hypothesis is derived from two lines of evidence. First, while the molecular extents of de novo cytogenetic deletions of chromosome 15q11q13 in AS and PWS patients are the same, the deletions originate from different parental chromosomes. In AS, the deletion occurs in the maternally inherited chromosome 15, while in PWS the deletion is found in the paternally inherited chromosome 15. The second line of evidence comes from the deletion of an abnormal parental contribution of 15q11q13 in PWS patients without a cytogenetic and molecular deletion. These patients have two maternal copies and no paternal copy of 15q11q13 (maternal uniparental disomy) instead of one copy from each parent. By qualitative hybridization with chromosome 15q11q13 specific DNA markers, we have now examined DNA samples from 10 AS patients (at least seven of which are familial cases) with no cytogenetic or molecular deletion of chromosome 15q11q13. Inheritance of one maternal copy and one paternal copy of 15q11q13 was observed in each family, suggesting that paternal uniparental disomy of 15q11q13 is not responsible for expression of the AS phenotype in these patients.     

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.     

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.     

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 mouse pink-eyed dilution locus: a model for aspects of Prader-Willi syndrome,Angelman syndrome,and a form of hypomelanosis of Ito

The region of mouse Chromosome (Chr) 7 containing the mouse pink-eyed dilution locus, p, is syntenic with human chromosome 15q11–q13, a region associated with three human syndromes, Prader-Willi syndrome (PWS), Angelman syndrome (AS), and a form of hypomelanosis of Ito (HI). Because some mutant alleles of p also share a subset of phenotypes with PWS, AS, and HI, the same gene or genes disrupted by p locus mutations are potentially involved in the phenotypes of PWS, AS, and HI.     

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.     

Combined cytogenetic and molecular analyses for the diagnosis of Prader-Willi/Angelman syndromes

Prader-Willi (PWS) and Angelman (AS) are syndromes of developmental impairment that result from the loss of expression of imprinted genes in the paternal (PWS) or maternal (AS) 15q11-q13 chromosome. Diagnosis on a clinical basis is difficult in newborns and young infants; thus, a suitable molecular test capable of revealing chromosomal abnormalities is required. We used a variety of cytogenetic and molecular approaches, such as, chromosome G banding, fluorescent in situ hybridization, a DNA methylation test, and a set of chromosome 15 DNA polymorphisms to characterize a cohort of 27 PWS patients and 24 suspected AS patients. Molecular analysis enabled the reliable diagnosis of 14 PWS and 7 AS patients, and their classification into four groups: (A) 6 of these 14 PWS subjects (44 %) had deletions of paternal 15q11-q13; (B) 4 of the 7 AS patients had deletions of maternal 15q11-q13; (C) one PWS patient (8 %) had a maternal uniparental disomy (UPD) of chromosome 15; (D) the remaining reliably diagnoses of 7 PWS and 3 AS cases showed abnormal methylation patterns of 15q11-q13 chromosome, but none of the alterations shown by the above groups, although they may have harbored deletions undetected by the markers used. This study highlights the importance of using a combination of cytogenetic and molecular tests for a reliable diagnosis of PWS or AS, and for the identification of genetic alterations.     

A DNA methylation imprint, determined by the sex of the parent, distinguishes the angelman and Prader-Willi syndromes

The Angelman (AS) and Prader-Willi (PWS) syndromes are two clinically distinct disorders that are caused by a differential parental origin of chromosome 15q11-q13 deletions. Both also can result from uniparental disomy (the inheritance of both copies of chromosome 15 from only one parent). Loss of the paternal copy of 15q11-q13, whether by deletion or maternal uniparental disomy, leads to PWS, whereas a maternal deletion or paternal uniparental disomy leads to AS. The differential modification in expression of certain mammalian genes dependent upon parental origin is known as genomic imprinting, and AS and PWS represent the best examples of this phenomenon in humans. Although the molecular mechanisms of genomic imprinting are unknown, DNA methylation has been postulated to play a role in the imprinting process. Using restriction digests with the methyl-sensitive enzymes HpaII and HhaI and probing Southern blots with several genomic and cDNA probes, we have systematically scanned segments of 15q11-q13 for DNA methylation differences between patients with PWS (20 deletion, 20 uniparental disomy) and those with AS (26 deletion, 1 uniparental disomy). The highly evolutionarily conserved cDNA, DN34, identifies distinct differences in DNA methylation of the parental alleles at the D15S9 locus. Thus, DNA methylation may be used as a reliable, postnatal diagnostic tool in these syndromes. Furthermore, our findings demonstrate the first known epigenetic event, dependent on the sex of the parent, for a locus within 15q11-q13. We propose that expression of the gene detected by DN34 is regulated by genomic imprinting and, therefore, that it is a candidate gene for PWS and/or AS.