Human Chromosome 15q11-q13 Research Articles

Genetic mapping of putative Chrna7 and Luzp2 neuronal transcriptional enhancers due to impact of a transgene-insertion and 6.8 Mb deletion in a mouse model of Prader-Willi and Angelman syndromes

BackgroundPrader-Willi and Angelman syndrome (PWS and AS) patients typically have an ~5 Mb deletion of human chromosome 15q11-q13, of opposite parental origin. A mouse model of PWS and AS has a transgenic insertion-deletion (TgPWS/TgAS) of chromosome 7B/C subsequent to paternal or maternal inheritance, respectively. In this study, we define the deletion endpoints and examine the impact on expression of flanking genes.ResultsUsing molecular and cytological methods we demonstrate that 13 imprinted and 11 non-imprinted genes are included in the TgPWS/TgAS deletion. Normal expression levels were found in TgPWS brain for genes extending 9.1- or 5.6-Mb centromeric or telomeric of the deletion, respectively. Our molecular cytological studies map the proximal deletion breakpoint between the Luzp2 and Siglec-H loci, and we show that overall mRNA levels of Luzp2 in TgPWS and TgAS brain are significantly reduced by 17%. Intriguingly, 5' Chrna7 shows 1.7-fold decreased levels in TgPWS and TgAS brain whereas there is a ≥15-fold increase in expression in neonatal liver and spleen of these mouse models. By isolating a Chrna7-Tg fusion transcript from TgAS mice, we mapped the telomeric deletion breakpoint in Chrna7 intron 4.ConclusionBased on the extent of the deletion, TgPWS/TgAS mice are models for PWS/AS class I deletions. Other than for the first gene promoters immediately outside the deletion, since genes extending 5.6–9.1 Mb away from each end of the deletion show normal expression levels in TgPWS brain, this indicates that the transgene array does not induce silencing and there are no additional linked rearrangements. Using gene expression, non-coding conserved sequence (NCCS) and synteny data, we have genetically mapped a putative Luzp2 neuronal enhancer responsible for ~33% of allelic transcriptional activity. The Chrna7 results are explained by hypothesizing loss of an essential neuronal transcriptional enhancer required for ~80% of allelic Chrna7 promoter activity, while the Chrna7 promoter is upregulated in B lymphocytes by the transgene immunoglobulin enhancer. The mapping of a putative Chrna7 neuronal enhancer inside the deletion has significant implications for understanding the transcriptional regulation of this schizophrenia-susceptibility candidate gene.

Unique maternal deletion of 15q in a patient with some symptoms of Prader‐Willi syndrome

Human chromosome 15q11-q13 is a critical region for Prader-Willi syndrome (PWS) and Angelman syndrome (AS) and most of the genes are under the condition of imprinting mechanism. PWS results from the loss of expression of paternally expressed genes and AS of maternally expressed genes. In this study molecular studies about a patient with congenital anomalies and mental retardation are analyzed. Highly polymorphic microsatellite markers were analyzed by PCR. These markers exist within 15q11-q13 and distal to 15q13. Only the maternal D15S986 locus within 15q11-q13 was deleted and other markers were biallelic. The result of maternal small region deletion in this patient is different from the typical PWS with paternal chromosome deletion and it suggests that nearby the deleted region there exists a gene (genes) which is not imprinted but needs biallelic expression.

Narrowed abrogation of the Angelman syndrome critical interval on human chromosome 15 does not interfere with epigenotype maintenance in somatic cells

Human chromosome 15q11-q13 involves a striking imprinted gene cluster of more than 2 Mb that is concomitant with multiple neurological disorders manifested by Prader-Willi syndrome (PWS) and Angelman syndrome (AS). PWS and AS patients with imprinting mutation have microdeletions, which share a 4.3 kb short region of overlap (SRO) at the 5' end of the paternal SNURF-SNRPN gene in PWS, or on the maternal allele, which shares a 880 bp SRO located at the 35 kb upstream of the SNURF-SNRPN promoter in AS. Recent studies have revealed an essential role of PWS-SRO in the postzygotic maintenance of the appropriate epigenotype on the paternal chromosome. For AS-SRO, however, there is insufficient experimental evidence exists to determine the direct functions. Here we show that the complete deletion of AS-SRO does not cause any anomalies of imprinted gene expression or DNA methylation on the mutated human chromosome 15, further supporting the idea that AS-SRO is dispensable for post implantation imprint maintenance. This implies that AS-SRO is not essential for the robust epigenotype preservation in somatic cells.

Establishing the epigenetic status of the Prader-Willi/Angelman imprinting center in the gametes and embryo.

The Prader-Willi/Angelman imprinted domain on human chromosome 15q11-q13 is regulated by an imprinting control center (IC) composed of a sequence around the SNRPN promoter (PWS-SRO) and a sequence located 35 kb upstream (AS-SRO). We have previously hypothesized that the primary imprint is established on AS-SRO, which then confers imprinting on PWS-SRO. Here we examine this hypothesis using a transgene that includes both AS-SRO and PWS-SRO sequences and carries out the entire imprinting process. The epigenetic features of this transgene resemble those previously observed on the endogenous locus, thus allowing analyses in the gametes and early embryo. We demonstrate that the primary imprint is in fact established in the gametes, creating a differentially methylated CpG cluster (DMR) on AS-SRO, presumably by an adjacent de novo signal (DNS). The DMR and DNS bind specific proteins: an allele-discrimination protein (ADP) and a de novo methylation protein, respectively. ADP, being a maternal protein, is involved in both the establishment of DMR in the gametes and in its maintenance through implantation when methylation of PWS-SRO on the maternal allele takes place. Importantly, while the AS-SRO is required in the gametes to confer methylation on PWS-SRO, it is dispensable later in development.

Control elements within the PWS/AS imprinting box and their function in the imprinting process.

A cluster of imprinted genes on human chromosome 15q11-q13 (the PWS/AS domain) and its ortholog on mouse chromosome 7c is believed to be regulated by an imprinting control center. Although minideletions in this region in Angelman syndrome (AS) and Prader-Willi syndrome (PWS) patients revealed that two elements, shortest deletion regions of overlap in AS families and PWS families (AS-SRO and PWS-SRO), respectively, constitute the IC, the molecular mechanism that governs this regional control remains obscure. To understand how this imprinting center works, a mouse model was sought. The striking similarity between the human and mouse sequences allowed the generation of a minitransgene (AS-SMP) composed of AS-SRO and the Snrpn minimal promoter (SMP) the mouse ortholog of PWS-SRO. This minitransgene carries out, in a highly reliable and reproducible manner, all steps of the imprinting process. In an attempt to decipher the molecular mechanism of the imprinting process, we generated and tested for imprinting five minitransgenes based on AS-SMP, in which various parts of the 160 bp SMP were deleted. These experiments revealed a set of five cis elements that carry out the various steps of the imprinting process. This set includes: (i). two copies of a de novo methylation signal (DNS) that establish the maternal imprint during oogenesis; (ii). an allele discrimination signal that establishes the paternal imprint; and (iii). two elements that act together to maintain the paternal imprint. Two functionally redundant sets of the five elements were found on the respective endogenous mouse sequence explaining the previously published contradictory results of targeted deletion experiments. Together with the fact that all five elements bind specific proteins that are presumably the factors acting in trans in the imprinting process, our observations set the stage for a comprehensive study of the molecular mechanism involved in the control of the imprinting process.

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.

Prader–Willi syndrome transcripts are expressed in phenotypically significant regions of the developing mouse brain

Prader–Willi syndrome (PWS) is a neurobehavioral disorder that is due to the loss of multiple, paternally-expressed, imprinted genes on human chromosome 15q11-q13. The candidate genes are conserved in mice and are located in an orthologous region on mouse 7C. Several mouse models in which one or more of the PWS candidate genes are silenced do not recapitulate the full PWS phenotype. We examined the expression patterns of the mouse orthologues of PWS candidate genes throughout the development of the brain regions most implicated in PWS, including the hypothalamus, pituitary, forebrain and hindbrain. Snrpn, Ipw and Ndn are widely expressed at high levels throughout the mouse brain, whereas Magel2, Mkrn3 and the snoRNA MBII-85 are preferentially expressed in specific brain regions. Magel2 is most specifically expressed in developing hypothalamus, the region of the brain implicated in PWS hyperphagia and obesity. Although Snrpn, Ipw and MBII-85 are putatively transcribed from the same promoter, the transcripts are differentially detected in neural tissues. The analysis of expression patterns of murine orthologues of human disease genes is valuable for identifying sites of gene expression that correlate with disease phenotype.

Atp10a, the mouse ortholog of the human imprinted ATP10A gene, escapes genomic imprinting

The mouse Atp10a gene is located at the border of an imprinted domain distal to the p-locus on mouse chromosome 7. The localization of Atp10a neighboring the maternally expressed gene Ube3a in the imprinted domain and an unusual inheritance pattern of the obesity phenotype with a p-locus deletion have suggested that Atp10a might be imprinted and associated with body fat. Recently, its human ortholog, ATP10A, was identified as the second imprinted gene with maternal expression in the human chromosome 15q11–q13 imprinted domain. To elucidate the imprinting status of Atp10a, we performed expression analysis in various tissues from reciprocal crosses between C57BL/6 and PWK (divergent strains of Mus musculus) mice. The results revealed that Atp10a was biallelically expressed in all tissues examined. Furthermore, there was no differential methylation in the CpG island and no antisense transcripts of the gene. These findings suggest that the mouse Atp10a gene escapes genomic imprinting.

Genomic inversions of human chromosome 15q11-q13 in mothers of Angelman syndrome patients with class II (BP2/3) deletions.

Parental submicroscopic genomic inversions have recently been demonstrated to be present in several genomic disorders. These inversions are genomic polymorphisms that facilitate misalignment and abnormal recombination between flanking segmental duplications. Angelman syndrome (AS; MIM 105830) is associated with specific abnormalities of chromosome 15q11-q13, with about 70% of cases being mother-of-origin 4 Mb deletions. We present here evidence that some mothers of AS patients with deletions of the 15q11-q13 region have a heterozygous inversion involving the region that is deleted in the affected offspring. The inversion was detected in the mothers of four of six AS cases with the breakpoint 2-3 (BP2/3) 15q11-q13 deletion, but not in seven mothers of AS due to paternal uniparental disomy (UPD) 15. We have identified variable inversion breakpoints within BP segmental duplications in the inverted AS mothers, as well as in AS deleted patients. Interestingly, the BP2-BP3 region is inverted in the mouse draft genome sequence with respect to the human draft sequence. The BP2-BP3 chromosome 15q11-q13 inversion was detected in four of 44 subjects (9%) of the general population (P<0.004). The BP2/3 inversion should be an intermediate estate that facilitates the occurrence of 15q11-q13 BP2/3 deletions in the offspring.

Predominant maternal expression of the mouse Atp10c in hippocampus and olfactory bulb.

The human chromosome 15q11-q13 region is one of the most intriguing imprinted domains, and the abnormalities inherited are associated with neurological disorders including Prader-Willi syndrome (PWS), Angelman syndrome (AS) and autism. Recently we have identified a novel maternally expressed gene, ATP10C, that encodes a putative aminophospholipid translocase within this critical region, 200 kb distal to UBE3A in an imprinted domain on human chromosome 15. ATP10C, with UBE3A, displayed tissue-specific imprinting with predominant expression of the maternal allele in the brain. In this study, we demonstrated that the mouse homologue, Atp10c/pfatp, showed tissue-specific maternal expression in the hippocampus and olfactory bulb, which overlapped the region of imprinted Ube3a expression. These data suggest that the imprinted transcript of Atp10c in the specific region of CNS may be associated with neurological disorders including AS and autism.

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.

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.

Establishment and maintenance of DNA methylation patterns in mouse Ndn: implications for maintenance of imprinting in target genes of the imprinting center.

Ndn is located on chromosome 7C, an imprinted region of the mouse genome. Imprinting of Ndn and adjacent paternally expressed genes is regulated by a regional imprinting control element known as the imprinting center (IC). An IC also controls imprint resetting of target genes in the region of conserved synteny on human chromosome 15q11-q13, which is deleted or rearranged in the neurodevelopmental disorder Prader-Willi syndrome. Epigenetic modifications such as DNA methylation, which occur in gametes and can be stably propagated, are presumed to establish and maintain the imprint in target genes of the IC. While most DNA becomes substantially demethylated by the blastocyst stage, some imprinted genes have regions that escape global demethylation and may maintain the imprint. We have now analyzed the methylation of 39 CpG dinucleotide sequences in the 5' end of Ndn by sodium bisulfite sequencing in gametes and in preimplantation and adult tissues. While sperm DNA is completely unmethylated across this region, oocyte DNA is partially methylated. A distinctive but unstable maternal methylation pattern persists until the morula stage and is lost in the blastocyst stage, where low levels of methylation are present on most DNA strands of either parental origin. The methylation pattern is then substantially remodeled, and fewer than half of maternally derived DNA strands in adult brain resemble the oocyte pattern. We postulate that for Ndn, DNA methylation may initially preserve a gametic imprint during preimplantation development, but other epigenetic events may maintain the imprint later in embryonic development.

Association of acetylated histones with paternally expressed genes in the Prader--Willi deletion region.

Imprinted genes within the Prader-Willi/Angelman syndrome region of human chromosome 15q11-q13 are regulated by a mechanism involving allele-specific DNA methylation. Since transcriptional regulation by DNA methylation involves histone deacetylation, we explored whether differences in histone acetylation exist between the two parental alleles of SNRPN and other paternally expressed genes in the region by using a chromatin immunoprecipitation assay with antibodies against acetylated histones H3 and H4. SNRPN exon 1, which is methylated on the silent maternal allele, was associated with acetylated histones on the expressed paternal allele only. SNRPN intron 7, which is methylated on the paternal allele, was not associated with acetylated histones on either allele. The paternally expressed genes NDN, IPW, PWCR1 and MAGEL2 were not associated with acetylated histones on either allele. Treatment of the lymphoblastoid cells with trichostatin A, a histone deacetylase inhibitor, did not result in any changes to SNRPN expression or association of acetylated histones with exon 1. Treatment with 5-aza-deoxycytidine (5-aza-dC), which inhibits DNA methylation, resulted in activation of SNRPN expression from the maternal allele, but was not accompanied by acetylation of histones. Our finding of allele-specific association of acetylated histones with the SNRPN exon 1 region, which encompasses the imprinting center, suggests that histone acetylation at this site may be important for regulation of SNRPN and of other paternally expressed genes in the region. On the silent allele, 5-aza-dC treatment altered SNRPN expression, but not association with acetylated histones, suggesting that histone acetylation is a secondary event in the process of gene reactivation by CpG demethylation.

The imprinting box of the Prader-Willi/Angelman syndrome domain.

A subset of mammalian genes is monoallelically expressed in a parent-of-origin manner. These genes are subject to an imprinting process that epigenetically marks alleles according to their parental origin during gametogenesis. Imprinted genes can be organized in clusters as exemplified by the 2-Mb domain on human chromosome 15q11-q13 and its mouse orthologue on chromosome 7c (ref. 1). Loss of this 2-Mb domain on the paternal or maternal allele results in two neurogenetic disorders, Prader-Willi syndrome (PWS) or Angelman syndrome (AS), respectively. Microdeletions on the paternal allele share a 4.3-kb short region of overlap (SRO), which includes the SNRPN promoter/exon1, cause PWS and silence paternally expressed genes. Microdeletions on the maternal allele share a 0.88-kb SRO located 35 kb upstream to the SNRPN promoter, cause AS and alleviate repression of genes on the maternal allele. Individuals carrying both AS and PWS deletions on the paternal allele show a PWS phenotype and genotype. These observations suggest that cis elements within the AS-SRO and PWS-SRO constitute an imprinting box that regulates the entire domain on both chromosomes. Here we show that a minitransgene composed of a 200-bp Snrpn promoter/exon1 and a 1-kb sequence located approximately 35 kb upstream to the SNRPN promoter confer imprinting as judged by differential methylation, parent-of-origin-specific transcription and asynchronous replication.

Identification of Novel Imprinted Transcripts in the Prader-Willi Syndrome and Angelman Syndrome Deletion Region: Further Evidence for Regional Imprinting Control

Deletions and other abnormalities of human chromosome 15q11-q13 are associated with two developmental disorders, Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Loss of expression of imprinted, paternally expressed genes has been implicated in PWS. However, the number of imprinted genes that contribute to PWS, and the range over which the imprinting signal acts to silence one copy of the gene in a parent-of-origin-specific manner, are unknown. To identify additional imprinted genes that could contribute to the PWS phenotype and to understand the regional control of imprinting in 15q11-q13, we have constructed an imprinted transcript map of the PWS-AS deletion interval. The imprinting status of 22 expressed sequence tags derived from the radiation-hybrid human transcript maps or physical maps was determined in a reverse transcriptase-PCR assay and correlated with the position of the transcripts on the physical map. Seven new paternally expressed transcripts localize to an approximately 1.5-Mb domain surrounding the SNRPN-associated imprinting center, which already includes four imprinted, paternally expressed genes. All other tested new transcripts in the deletion region were expressed from both alleles. A domain of exclusive paternal expression surrounding the imprinting center suggests strong regional control of the imprinting process. This study provides the means for further investigation of additional genes that cause or modify the phenotypes associated with rearrangements of 15q11-q13.

Autistic Disorder and Chromosome 15q11–q13: Construction and Analysis of a BAC/PAC Contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2–10/10,000 individuals. Chromosome 15q11–q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the γ-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11–q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11–q13.

A 5-kb imprinting center deletion in a family with Angelman syndrome reduces the shortest region of deletion overlap to 880 bp.

Imprinting on human chromosome 15q11-q13 is controlled by a bipartite imprinting center (IC) that maps to the SNRPN locus. Deletions of the IC result in an imprinting defect and Prader-Willi syndrome or Angelman syndrome (AS). We have now identified a 5-kb IC deletion in an English AS patient (AS-LO); this represents the smallest microdeletion found in AS and narrows down the shortest region of deletion overlap to 880 bp.

Disruption of the mouse necdin gene results in early post-natal lethality.

Prader-Willi syndrome (PWS) is a neurobehavioural disorder characterized by neonatal respiratory depression, hypotonia and failure to thrive in infancy, followed by hyperphagia and obesity among other symptoms. PWS is caused by the loss of one or more paternally expressed genes on chromosome 15q11-q13, which can be due to gene deletions, maternal uniparental disomy or mutations disrupting the imprinting mechanism. Imprinted genes mapped to this region include SNRPN (refs 3,4), ZNF127 (ref. 5), IPW (ref. 6) and NDN (which encodes the DNA-binding protein necdin; refs 7,8,9,10). The mouse homologues of these genes map to mouse chromosome 7 in a region syntenic with human chromosome 15q11-q13 (refs 7,11). Imprinting of the human genes is under the control of an imprinting center (IC), a long-range, cis-acting element located in the 5' region of SNRPN (ref. 12). A related control element was isolated in the mouse Snrpn genomic region which, when deleted on the paternally inherited chromosome, resulted in the loss of expression of all four genes and early post-natal lethality. To determine the possible contribution of Ndn to the PWS phenotype, we generated Ndn mutant mice. Heterozygous mice inheriting the mutated maternal allele were indistinguishable from their wild-type littermates. Mice carrying a paternally inherited Ndn deletion allele demonstrated early post-natal lethality. This is the first example of a single gene being responsible for phenotypes associated with PWS.

Towards a molecular understanding of Prader-Willi and Angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurological disorders that map to human chromosome 15q11-q13 and involve perturbations of imprinted gene expression. PWS is caused by a deficiency of paternal gene expression and AS is caused by a deficiency of maternal gene expression. Experiments in the last year have focused on molecular analysis of the human chromosomal region as well as the homologous region on central mouse chromosome 7. New transcripts and exons have been identified and the epigenetic status of the PWS/AS region in mice and humans has been examined. The imprinting center that is hypothesized to control the switch between the maternal and paternal epigenotypes has also been characterized in greater detail and a mouse model that deletes the homologous element demonstrates a conservation in imprinting center function between mice and humans. In addition, analysis of non-deletion AS patients has revealed that UBE3A intragenic mutations are found in a significant number of cases. However, both human patients and mouse model systems indicate that other genes may also contribute to the AS phenotype. Thus, although much has been learned in the last year, considerable information is still required before these complex syndromes are fully understood.