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.     

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.     

The tyrosinase-positive oculocutaneous albinism gene shows locus homogeneity on chromosome 15q11-q13 and evidence of multiple mutations in southern African negroids.

Tyrosinase-positive oculocutaneous albinism (ty-pos OCA) is an autosomal recessive disorder of the melanin pigmentary system. South African ty-pos OCA individuals occur with two distinct phenotypes, with or without darkly pigmented patches (ephelides, or dendritic freckles) on exposed areas of the skin. These phenotypes are concordant within families, suggesting that there may be more than one mutation at the ty-pos OCA locus. Linkage studies carried out in 41 families have shown linkage between markers in the Prader-Willi/Angelman syndrome (PWS/AS) region on chromosome 15q11-q13 and ty-pos OCA. Analysis showed no obligatory crossovers between the alleles at the D15S12 locus and ty-pos OCA, suggesting that the D15S12 locus is very close to or part of the disease locus, which is postulated to be the human homologue, P, of the mouse pink-eyed dilution gene, p. Unlike caucasoid "ty-pos OCA" individuals, negroid ty-pos OCA individuals do not show any evidence of locus heterogeneity. Studies of allelic association between the polymorphic alleles detected at the D15S12 locus and ephelus status suggest that there was a single major mutation giving rise to ty-pos OCA without ephelides. There may, however, be two major mutations causing ty-pos OCA with ephelides, one associated with D15S12 allele 1 and the other associated with D15S12 allele 2. The two loci, GABRA5 and D15S24, flanking D15S12, are both hypervariable, and many different haplotypes were observed with the alleles at the three loci on both ty-pos OCA-associated chromosomes and "normal" chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

A targeted deletion upstream of <Emphasis Type="Italic">Snrpn</Emphasis> does not result in an imprinting defect

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the disturbance of imprinted gene expression within human chromosome 15q11–q13. Some cases of PWS and AS are caused by microdeletions near the SNRPN gene that disrupt a regulatory element termed the imprinting center (IC). The IC has two functional components; an element at the promoter of SNRPN involved in PWS (PWS-IC) and an element 35 kilobases (kb) upstream of SNRPN involved in AS (AS-IC). To further understand the function of the IC, we sought to create a mouse model for AS-IC mutations. We have generated two deletions at a location analogous to that of the human AS-IC. Neither deletion produced an imprinting defect as indicated by DNA methylation and gene expression analyses. These results indicate that no elements critical for AS-IC function in mouse reside within the 12.8-kb deleted region and suggest that the specific location of the AS-IC is not conserved between human and mouse. Camilynn I. Brannan was Deceased     

A modified MS-PCR approach to diagnose patients with Prader-Willi and Angelman syndrome

Prader-Willi (PWS) and Angelman (AS) syndromes are clinically distinct neurodevelopmental genetic diseases with multiple phenotypic manifestations. They are one of the most common genetic syndromes caused by non-Mendelian inheritance in the form of genomic imprinting, and can be attributable to the loss of gene expression due to imprinting within the chromosomal region 15q11-q13. Clinical diagnosis of PWS and AS is challenging, and the use of molecular and cytomolecular studies is recommended to help in determining the diagnosis of these conditions. The methylation analysis is a sensible approach; however there are several techniques for this purpose, such as the methylation-sensitive polymerase chain reaction (MS-PCR). This study aims to optimize the MS-PCR assay for the diagnosis of potential PWS and AS patients using DNA modified by sodium bisulfite. We used the MS-PCR technique of PCR described by Kosaki et al. (1997) adapted with betaine. All different concentrations of betaine used to amplify the methylated and unmethylated chromosomal region 15q11-q13 on the gene SNRPN showed amplification results, which increased proportionally to the concentration of betaine. The methylation analysis is a technically robust and reproducible screening method for PWS and AS. The MS-PCR assures a faster, cheaper and more efficient method for the primary diagnosis of the SNRPN gene in cases with PWS and AS, and may detect all of the three associated genetic abnormalities: deletion, uniparental disomy or imprinting errors.     

The tyrosinase-positive oculocutaneous albinism locus maps to chromosome 15q11.2-q12.

Tyrosinase-positive oculocutaneous albinism (ty-pos OCA), an autosomal recessive disorder of the melanin biosynthetic pathway, is the most common type of albinism occurring worldwide. In southern African Bantu-speaking negroids it has an overall prevalence of about 1/3,900. Since the basic biochemical defect is unknown, a linkage study with candidate loci, candidate chromosomal regions, and random loci was undertaken. The ty-pos OCA locus was found to be linked to two arbitrary loci, D15S10 and D15S13, in the Prader-Willi/Angelman chromosomal region on chromosome 15q11.2-q12. The pink-eyed dilute locus, p, on mouse chromosome 7, maps close to a region of homology on human chromosome 15q, and we postulate that the ty-pos OCA and p loci are homologous.     

The Mouse Region Syntenic for Human Spinal Muscular Atrophy Lies within theLgn1Critical Interval and Contains Multiple Copies ofNaipExon 5

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval forLgn1,a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacteriumLegionella pneumophila.We have generated a mouse YAC contig across theLgn1/Smainterval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to theLgn1critical region, making these two genes candidates for theLgn1phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy ofNaipexon 5. Identification of theLgn1gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.     

Mosaic loss of 15q11q13 in a patient with hypomelanosis of Ito: is there a role for the P gene?

We report a patient with mental retardation, behavioral disturbances, and pigmentary anomalies, consistent with the phenotype of hypomelanosis of Ito (HMI), and in whom cytogenetic analysis revealed mosaicism for an unbalanced translocation. His karyotype is 45, XY,–7, –15,+der(7)(7;15)t(q34;ql3)/46, XY. He is therefore monosomic for 7q34 to qter and 15pter to q13 in the cells containing the translocation. The human homolog (P) of the p gene (the product of the mouse pink-eyed dilution locus) maps to 15q11q13. Loss of this locus is believed to be associated with abnormalities of pigmentation, such as the hypopigmentation seen in patients with deletions of 15q11q13, and the Prader-Willi and Angelman syndromes. Mutations within the P gene have also been associated with tyrosinase-positive (type II) oculocutaneous albinism. Using fluorescence in situ hybridization, we confirmed that our patient is deleted for one copy of a P gene probe in the cells with the unbalanced translocation, and for loci within the region critical for the Prader-Willi/Angelman syndromes. Although hypomelanosis of Ito is a heterogeneous disorder, we postulate that, in our case and potentially in others, this phenotype may result directly from the loss of specific pigmentation genes.     

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.     

Methylation-specific multiplex ligation-dependent probe amplification analysis of subjects with chromosome 15 abnormalities

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders caused by loss of expression of imprinted genes from the 15q11-q13 region. They arise from similar defects in the region but differ in parent of origin. There are two recognized typical 15q11-q13 deletions depending on size and several diagnostic assays are available but each has limitations. We evaluated the usefulness of a methylation-specific multiplex ligation-dependent probe amplification (MLPA) kit consisting of 43 probes to detect copy number changes and methylation status in the region. We used the MLPA kit to genotype 82 subjects with chromosome 15 abnormalities (62 PWS, 10 AS and 10 individuals with other chromosome 15 abnormalities) and 13 with normal cytogenetic findings. We developed an algorithm for MLPA probe analysis which correctly identified methylation abnormalities associated with PWS and AS and accurately determined copy number in previously assigned genetic subtypes including microdeletions of the imprinting center. Furthermore, MLPA analysis identified copy number changes in those with distal 15q deletions and ring 15s. MLPA is a relatively simple, cost-effective technique found to be useful and accurate for methylation status, copy number and analysis of genetic subtype in PWS and AS, as well as other chromosome 15 abnormalities.     

Localization of the gene encoding the GABAA receptor β3 subunit to the Angelman/Prader-Willi region of human chromosome 15

Deletions of the proximal long arm of chromosome 15 (bands 15q11q13) are found in the majority of patients with two distinct genetic disorders, Angelman syndrome (AS) and Prader-Willi syndrome (PWS). The deleted regions in the two syndromes, defined cytogenetically and by using cloned DNA probes, are similar. However, deletions in AS occur on the maternally inherited chromosome 15, and deletions in PWS occur on the paternally derived chromosome 15. This observation has led to the suggestion that one or more genes in this region show differential expression dependent on parental origin (genetic imprinting). No genes of known function have previously been mapped to this region. We show here that the gene encoding the GABAA (gamma-aminobutyric acid) receptor beta 3 subunit maps to the AS/PWS region. Deletion of this gene (GABRB3) was found in AS and PWS patients with interstitial cytogenetic deletions. Evidence of beta 3 gene deletion was also found in an AS patient with an unbalanced 13;15 translocation but not in a PWS patient with an unbalanced 9;15 translocation. The localization of this receptor gene to the AS/PWS region suggests a possible role of the inhibitory neurotransmitter GABA in the pathogenesis of one or both of these syndromes.     

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.     

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.     

Genomic structure and chromosomal localization of the mouse gene Punc

The mouse gene Punc encodes a member of the immunoglobulin superfamily of cell surface proteins. It is highly expressed in the developing embryo in nervous system and limb buds. At mid-gestation, however, expression levels of Punc decrease sharply. To allow investigation of such a regulatory mechanism, the genomic locus encompassing the Punc gene was cloned, characterized, and mapped. Fluorescent in situ hybridization was used to determine the chromosomal location of the Punc gene of mouse and human. Mouse Punc maps to Chromosome (Chr) 9 in the region D-E1, whereas the human PUNC gene is localized to Chr 15 at 15q22.3-23, a region known to be syntenic to mouse 9D-E1. The human PUNC gene therefore maps close to a genetic locus that is linked to Bardet-Biedl Syndrome, an autosomal recessive human disorder. Confirmation for the location of human PUNC was obtained through sequence relationships between mouse Punc cDNA, human PUNC cDNA, genomic sequence upstream of the murine Punc gene, and human STS markers that had been previously mapped on Chr 15. The STS sequence WI-14920 is in fact derived from the 3′-untranslated region of the human PUNC gene. WI-14920 had been placed at 228cR from the top of the Chr 15 linkage group, which provided positional information for the human PUNC gene at high resolution. Thus, this study identifies PUNC as the gene corresponding to a previously anonymous marker and serves as a basis to investigate its role in genetic disorders. Received: 8 July 1998 / Accepted: 14 October 1998     

Increasing the complexity: new genes and new types of albinism

Albinism is a rare genetic condition globally characterized by a number of specific deficits in the visual system, resulting in poor vision, in association with a variable hypopigmentation phenotype. This lack or reduction in pigment might affect the eyes, skin, and hair (oculocutaneous albinism, OCA), or only the eyes (ocular albinism, OA). In addition, there are several syndromic forms of albinism (e.g. Hermansky–Pudlak and Chediak–Higashi syndromes, HPS and CHS, respectively) in which the described hypopigmented and visual phenotypes coexist with more severe pathological alterations. Recently, a locus has been mapped to the 4q24 human chromosomal region and thus represents an additional genetic cause of OCA, termed OCA5, while the gene is eventually identified. In addition, two new genes have been identified as causing OCA when mutated: SLC24A5 and C10orf11, and hence designated as OCA6 and OCA7, respectively. This consensus review, involving all laboratories that have reported these new genes, aims to update and agree upon the current gene nomenclature and types of albinism, while providing additional insights from the function of these new genes in pigment cells.     

Assignment of the gene for neuroendocrine protein 7B2 (SGNE1 locus) to mouse chromosome region 2[E3-F3] and to human chromosome region 15q11-q15

The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNE1) was localized to the E3-F3 region of mouse chromosome 2 and to the q11-q15 region of human chromosome 15. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNE1 in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 15. Clinically, the human SGNE1 DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 15q11-15q13 region.