Identification of four highly conserved genes between breakpoint hotspots BP1 and BP2 of the Prader-Willi/Angelman syndromes deletion region that have undergone evolutionary transposition mediated by flanking duplicons

Prader-Willi and Angelman syndromes (PWS and AS) typically result from an approximately 4-Mb deletion of human chromosome 15q11-q13, with clustered breakpoints (BP) at either of two proximal sites (BP1 and BP2) and one distal site (BP3). HERC2 and other duplicons map to these BP regions, with the 2-Mb PWS/AS imprinted domain just distal of BP2. Previously, the presence of genes and their imprinted status have not been examined between BP1 and BP2. Here, we identify two known (CYFIP1 and GCP5) and two novel (NIPA1 and NIPA2) genes in this region in human and their orthologs in mouse chromosome 7C. These genes are expressed from a broad range of tissues and are nonimprinted, as they are expressed in cells derived from normal individuals, patients with PWS or AS, and the corresponding mouse models. However, replication-timing studies in the mouse reveal that they are located in a genomic domain showing asynchronous replication, a feature typically ascribed to monoallelically expressed loci. The novel genes NIPA1 and NIPA2 each encode putative polypeptides with nine transmembrane domains, suggesting function as receptors or as transporters. Phylogenetic analyses show that NIPA1 and NIPA2 are highly conserved in vertebrate species, with ancestral members in invertebrates and plants. Intriguingly, evolutionary studies show conservation of the four-gene cassette between BP1 and BP2 in human, including NIPA1/2, CYFIP1, and GCP5, and proximity to the Herc2 gene in both mouse and Fugu. These observations support a model in which duplications of the HERC2 gene at BP3 in primates first flanked the four-gene cassette, with subsequent transposition of these four unique genes by a HERC2 duplicon-mediated process to form the BP1-BP2 region. Duplicons therefore appear to mediate genomic fluidity in both disease and evolutionary processes.     

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.     

Retrotransposed genes such as Frat3 in the mouse Chromosome 7C Prader-Willi syndrome region acquire the imprinted status of their insertion site

Prader-Willi syndrome (PWS) results from loss of function of a 1.0- to 1.5-Mb domain of imprinted, paternally expressed genes in human Chromosome (Chr) 15q11-q13. The loss of imprinted gene expression in the homologous region in mouse Chr 7C leads to a similar neonatal PWS phenotype. Several protein-coding genes in the human PWS region are intronless, possibly arising by retrotransposition. Here we present evidence for continued acquisition of genes by the mouse PWS region during evolution. Bioinformatic analyses identified a BAC containing four genes, Mkrn3, Magel2, Ndn, Frat3, and the Atp5l-ps1 pseudogene, the latter two genes derived from recent L1-mediated retrotransposition. Analyses of eight overlapping BACs indicate that these genes are clustered within 120 kb in two inbred strains, in the order tel–Atp5l-ps1–Frat3–Mkrn3–Magel2–Ndn–cen. Imprinting analyses show that Frat3 is differentially methylated and expressed solely from the paternal allele in a transgenic mouse model of Angelman syndrome, with no expression from the maternal allele in a mouse model of PWS. Loss of Frat3 expression may, therefore, contribute to the phenotype of mouse models of PWS. The identification of five intronless genes in a small genomic interval suggests that this region is prone to retroposition in germ cells or their zygotic and embryonic cell precursors, and that it allows the subsequent functional expression of these foreign sequences. The recent evolutionary acquisition of genes that adopt the same imprint as older, flanking genes indicates that the newly acquired genes become `innocent bystanders' of a primary epigenetic signal causing imprinting in the PWS domain. Received: 22 May, 2001 / Accepted: 16 July 2001     

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.     

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.     

Analysis of DEXI/Dexi refines the organization of the mouse 7C and human 15q11-->q13 imprinting clusters

Identification of imprinted genes in the Prader-Willi/Angelman syndrome deletion region is complicated by the presence of large flanking repeats. While inactive copies of DEXI are located within the repeats, we have now localized the active DEXI gene to 15q11-->q13 outside the PWS/AS deletion and Dexi to mouse chromosome 16, suggesting complex evolution of this genomic region in both species.     

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ～3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ～2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ～750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

A 210-kb segment of tandem repeats and retroelements located between imprinted subdomains of mouse distal chromosome 7.

Mammalian genes subject to genomic imprinting often form clusters and are regulated by long-range mechanisms. The distal imprinted domain of mouse chromosome 7 is orthologous to the Beckwith-Wiedemann syndrome domain in human chromosome 11p15.5 and contains at least 13 imprinted genes. This domain consists of two subdomains, which are respectively regulated by an imprinting center. We here report the finished-quality sequence of a 0.6-Mb region encompassing the more centromeric subdomain. The sequence contains four imprinted genes (Ascl2/Mash2, Ins2, Igf2 and H19) and reveals previously unidentified CpG islands and tandem repeats, which may be features of imprinted genes. Most interestingly, a unique 210-kb segment consisting almost exclusively of tandem repeats and retroelements is identified. This segment, located between Th and Ins2, has features of heterochromatin-forming DNA and is highly methylated at CpG sites. The segment exhibits asynchronous replication on the parental chromosomes, a feature of the imprinted domains. We propose that this repeat segment could serve either as a boundary between the two subdomains or as a target for epigenetic chromatin modifications that regulate imprinting.     

Imprinting mechanisms and genes involved in Prader-Willi and Angelman syndromes

Prader-Willi (PWS) and Angelman (AS) syndromes illustrate a disease paradigm of genomic imprinting, an epigenetic modification of DNA that results in parent-of-origin specific expression during embryogenesis and in the adult. From genetic data, at least two imprinted genes may be required for the classical PWS phenotype, whereas AS probably involves a single imprinted gene, and rare familial forms of both disorders involve imprinting mutations. In addition, the nonimprinted P gene is associated with pigmentation disorders in PWS, AS and oculocutaneous albinism. Identification of new genes, delineation of small deletions in unique patients, and direct screening for imprinted sequences, should soon identify candidate genes for PWS and AS. The mechanism of imprinting involves DNA methylation and replication timing, and appears to include multiple imprinted genes within a large imprinted domain. Imprinting of these genes may be regulated in cis, by an imprinting control element (ICE). Future studies can be expected to unravel the gene identities and imprinting mechanisms involved in these fascinating disorders; ultimately it may be possible to reactivate imprinted gene expression as a therapeutic approach.     

Prader-Willi syndrome: clinical genetics, cytogenetics and molecular biology

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder that arises from lack of expression of paternally inherited genes known to be imprinted and located in the chromosome 15q11-q13 region. PWS is considered the most common syndromal cause of life-threatening obesity and is estimated at 1 in 10,000 to 20,000 individuals. A de novo paternally derived chromosome 15q11-q13 deletion is the cause of PWS in about 70% of cases, and maternal disomy 15 accounts for about 25% of cases. The remaining cases of PWS result either from genomic imprinting defects (microdeletions or epimutations) of the imprinting centre in the 15q11-q13 region or from chromosome 15 translocations. Here, we describe the clinical presentation of PWS, review the current understanding of causative cytogenetic and molecular genetic mechanisms, and discuss future directions for research.     

Lack of Pwcr1/MBII-85 snoRNA is critical for neonatal lethality in Prader–Willi syndrome mouse models

Prader–Willi syndrome (PWS) is a neurobehavioral disorder caused by the lack of paternal expression of imprinted genes in the human chromosome region 15q11–13. Recent studies of rare human translocation patients narrowed the PWS critical genes to a 121-kb region containing PWCR1/HBII-85 and HBII-438 snoRNA genes. The existing mouse models of PWS that lack the expression of multiple genes, including Snrpn, Ube3a, and many intronic snoRNA genes, are characterized by 80%–100% neonatal lethality. To define the candidate region for PWS-like phenotypes in mice, we analyzed the expression of several genetic elements in mice carrying the large radiation-induced p^30PUb deletion that includes the p locus. Mice having inherited this deletion from either parent develop normally into adulthood. By Northern blot and RT-PCR assays of brain tissue, we found that Pwcr1/MBII-85 snoRNAs are expressed normally, while MBII-52 snoRNAs are not expressed when the deletion is paternally inherited. Mapping of the distal deletion breakpoint indicated that the p^30PUb deletion includes the entire MBII-52 snoRNA gene cluster and three previously unmapped EST sequences. The lack of expression of these elements in mice with a paternal p^30PUb deletion indicates that they are not critical for the neonatal lethality observed in PWS mouse models. In addition, we identified MBII-436, the mouse homolog of the HBII-436 snoRNA, confirmed its imprinting status, and mapped it outside of the p^30PUb deletion. Taking together all available data, we conclude that the lack of Pwcr1/MBII-85 snoRNA expression is the most likely cause for the neonatal lethality in PWS model mice.     

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.     

Unique Retrotransposon LINE-1 Distribution at the Prader–Willi Angelman Syndrome Locus

We analyzed the distribution of long interspersed nuclear elements (LINE)-1 (L1) along mouse autosomes at a 1-Mb scale, and found a unique combination of high density and strand asymmetry of L1 elements at the imprinted Prader–Willi syndrome/Angelman syndrome (PWS/AS) locus on mouse chromosome 7. This L1 signature overlaps the paternally expressed domain of the locus, excluding the maternally expressed Ube3a gene, and is conserved in rat and human. Unlike the PWS/AS locus, other instances of high L1 density and strand asymmetry in the mouse are not associated with imprinted regions and are not evolutionarily conserved in human. The evolutionary conservation of the L1 signature at the PWS/AS locus despite differences in composition of L1 elements between rodent and human, requires a mechanism for active perpetuation of L1 asymmetry during bursts of L1 activity, and indicates a possible functional role for L1 elements at this locus. Aside from the PWS/AS locus, rodents have a far greater correlation of L1 densities between DNA strands than do humans; we provide evidence that this difference in interstrand correlation between the two taxa is due largely to the difference in average age of the dominant L1 families. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence-ready 1-Mb YAC, BAC and cosmid contigs covering the distal imprinted region of mouse chromosome 7.

We have constructed approximately 1-Mb contigs of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) and cosmid clones covering the imprinted region in mouse chromosome band 7F4/F5. This region is syntenic to human chromosome 11p15.5, which is associated with Beckwith-Wiedemann syndrome (BWS) and certain childhood and adult tumors. These contigs provide the basis for genomic sequencing, identification of genes and their regulatory elements, and functional studies in transgenic and knockout mice, which should be of help to understand not only the mechanisms of imprinting but also the molecular events involved in the genesis of BWS and tumors.