Prader-Willi Syndrome Critical Region Research Articles

Microarray analysis of gene/transcript expression in Prader-Willi syndrome: deletion versus UPD

Background: Prader-Willi syndrome (PWS), the most common genetic cause of marked obesity, is caused by genomic imprinting and loss of expression of paternal genes in the 15q11–q13 region. There is...

A study of the influence of different genotypes on the physical and behavioral phenotypes of children and adults ascertained clinically as having PWS.

A population-based cohort of people with a clinical diagnosis of Prader-Willi syndrome (PWS) was genetically assessed using molecular diagnostic methods and subsequently divided into the following genetic subtypes involving chromosome 15: 'deletion', 'disomy' and genetically negative (referred to as 'PWS-like'). The physical and behavioral characteristics of the three groups were compared in order to evaluate the unique characteristics of the phenotype resulting from loss of expression of imprinted genes at 15q11q13 (PWS vs. PWS-like cases), the possible effect of either haploid insufficiency of non-imprinted genes (deletion cases), or gain of function of imprinted genes (disomy cases) located within the PWS critical region at 15q11q13. In this study, the main differences between probands with either a deletion or disomy are considered, and the possible involvement of contributing genes discussed. The differences within the PWS group proved difficult to quantify. It would appear that haploid insufficiency or gain of function are more subtle contributors than gender-specific genomic imprinting in the production of the PWS phenotype.

The changing purpose of Prader-Willi syndrome clinical diagnostic criteria and proposed revised criteria.

The changing purpose of Prader-Willi syndrome clinical diagnostic criteria and proposed revised criteria.

Identification of a Testis-Specific Gene (C15orf2) in the Prader–Willi Syndrome Region on Chromosome 15

Prader–Willi syndrome (PWS) results from the loss of paternal contributions for a 2-Mb imprinted region on the proximal long arm of human chromosome 15. Hitherto, five paternally active genes have been identified in this region (ZNF127, NDN, MAGEL2, SNURF–SNRPN, and IPW). Here we report the identification of a novel gene in the PWS critical region, which has been designated “chromosome 15 open reading frame 2” (C15orf2). C15orf2 is an intronless gene located between MAGEL2 and SNURF–SNRPN. It is associated with a CpG island, which is methylated in all tissues tested except for germ cells. C15orf2 is transcribed as a 7.5-kb mRNA and contains an open reading frame encoding a predicted 1156-amino-acid protein of unknown function. Transcription of C15orf2 occurs exclusively in the testis, and in adult testis samples, we observed biallelic expression. By zoo-blot analysis, we found related sequences in DNA from other primates, but not in nonprimate DNA. We conclude that C15orf2 may play a role in primate spermatogenesis.

A novel imprinted gene, encoding a RING zinc-finger protein, and overlapping antisense transcript in the Prader-Willi syndrome critical region.

We describe a complex imprinted locus in chromosome 15q11-q13 that encodes two genes, ZNF127 and ZNF127AS. The ZNF127 gene encodes a protein with a RING (C3HC4) zinc-finger and multiple C3H zinc-finger motifs, the former being closely related to a protein from variola major virus, the smallpox etiological agent. These motifs allow prediction of ZNF127 function as a ribonucleoprotein. The intronless ZNF127 gene is expressed ubiquitously, but the entire coding sequence and 5' CpG island overlaps a second gene, ZNF127AS, that is transcribed from the antisense strand with a different transcript size and pattern of expression. Allele-specific analysis shows that ZNF127 is expressed only from the paternal allele. Consistent with this expression pattern, in the brain the ZNF127 5' CpG island is completely unmethylated on the paternal allele but methylated on the maternal allele. Analyses of adult testis, sperm and fetal oocytes demonstrates a gametic methylation imprint with unmethylated paternal germ cells. Recent findings indicate that ZNF127 is part of the coordinately regulated imprinted domain affected in Prader-Willi syndrome patients with imprinting mutations. Therefore, ZNF127 and ZNF127AS are novel imprinted genes that may be associated with some of the clinical features of the polygenic Prader-Willi syndrome.

Imprinting-Mutation Mechanisms in Prader-Willi Syndrome

Microdeletions of a region termed the "imprinting center" (IC) in chromosome 15q11-q13 have been identified in several families with Prader-Willi syndrome (PWS) or Angelman syndrome who show epigenetic inheritance for this region that is consistent with a mutation in the imprinting process. The IC controls resetting of parental imprints in 15q11-q13 during gametogenesis. We have identified a larger series of cases of familial PWS, including one case with a deletion of only 7.5 kb, that narrows the PWS critical region to <4. 3 kb spanning the SNRPN gene CpG island and exon 1. Identification of a strong DNase I hypersensitive site, specific for the paternal allele, and six evolutionarily conserved (human-mouse) sequences that are potential transcription-factor binding sites is consistent with this region defining the SNRPN gene promoter. These findings suggest that promoter elements at SNRPN play a key role in the initiation of imprint switching during spermatogenesis. We also identified three patients with sporadic PWS who have an imprinting mutation (IM) and no detectable mutation in the IC. An inherited 15q11-q13 mutation or a trans-factor gene mutation are unlikely; thus, the disease in these patients may arise from a developmental or stochastic failure to switch the maternal-to-paternal imprint during parental spermatogenesis. These studies allow a better understanding of a novel mechanism of human disease, since the epigenetic effect of an IM in the parental germ line determines the phenotypic effect in the patient.

Maternal disomy and Prader-Willi syndrome consistent with gamete complementation in a case of familial translocation (3;15) (p25;q11.2)

Maternal uniparental disomy (UPD) for chromosome 15 is responsible for an estimated 30% of cases of Prader-Willi syndrome (PWS). We report on an unusual case of maternal disomy 15 in PWS that is most consistent with adjacent-1 segregation of a paternal t(3;15)(p25;q11.2) with simultaneous maternal meiotic nondisjunction for chromosome 15. The patient (J.B.), a 17-year-old white male with PWS, was found to have 47 chromosomes with a supernumerary, paternal der(15) consisting of the short arm and the proximal long arm of chromosome 15, and distal chromosome arm 3p. The t(3;15) was present in the balanced state in the patient's father and a sister. Fluorescent in situ hybridization analysis demonstrated that the PWS critical region resided on the derivative chromosome 3 and that there was no deletion of the PWS region on the normal pair of 15s present in J.B. Methylation analysis at exon alpha of the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) gene showed a pattern characteristic of only the maternal chromosome 15 in J.B. Maternal disomy was confirmed by polymerase chain reaction analysis of microsatellite repeats at the gamma-aminobutyric acid receptor beta3 subunit (GABRB3) locus. A niece (B.B.) with 45 chromosomes and the derivative 3 but without the der(15) demonstrated a phenotype consistent with that reported for haploinsufficiency of distal 3 p. Uniparental disomy associated with unbalanced segregation of non-Robertsonian translocations has been reported previously but has not, to our knowledge, been observed in a case of PWS. Furthermore, our findings are best interpreted as true gamete complementation resulting in maternal UPD 15 and PWS.

Sequencing and Functional Analysis of the SNRPN Promoter: In Vitro Methylation Abolishes Promoter Activity

The gene encoding the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) maps to the Prader-Willi syndrome critical region on chromosome 15 and is expressed preferentially from the paternal allele. A CpG island encompassing the first exon of SNRPN is methylated on the inactive maternal allele. DNA sequence was determined for a cosmid containing the first three exons of SNRPN and extending 20 kb upstream and 15 kb downstream from the CpG island. This region is extremely rich in Alu elements and other repetitive sequences and contains a single CpG island, which includes numerous short direct repeat sequences. Functional analysis of the first exon revealed strong promoter activity for a 260-bp fragment extending 207 bp upstream from the exon. In vitro methylation of this 260-bp fragment abolished promoter activity completely, suggesting that the silencing of the maternal SNRPN allele may be a direct consequence of methylation of the promoter region.

Prader-Willi syndrome in a child with mosaic trisomy 15 and mosaic triplo-X: a molecular analysis.

A 3.3 year old girl with Prader-Willi syndrome (PWS) and mosaicism for two aneuploidies, 47,XXX and 47,XX,+15, is presented. The triplo-X cell line was found in white blood cells and...

Identification of a novel paternally expressed transcript adjacent to snRPN in the Prader-Willi syndrome critical region.

Small nuclear ribonucleoprotein-associated polypeptide N (snRPN) and an anonymous transcript, PAR-5, are two of the paternally expressed transcripts mapped to the Prader-Willi syndrome critical region. Using long-range PCR, we have isolated the genomic interval between snRPN and PAR-5, identified a novel transcript in this region, and termed it PAR-SN. Northern analysis demonstrates that PAR-SN is expressed in brain, skeletal muscle, and heart. Like snRPN and PAR-5, PAR-SN is expressed exclusively from the paternal homolog in cultured lymphoblasts. Sequence analysis of the transcript revealed no significant open reading frame but did include a polymorphic dinucleotide repeat (CA)17.

Deletion of small nuclear ribonucleoprotein polypeptide N (SNRPN) in Prader-Willi syndrome detected by fluorescence in situ hybridization: Two sibs with the typical phenotype without a cytogenetic deletion in chromosome 15q

The small nuclear ribonucleoprotein polypeptide N (SNRPN) gene is regarded as one of the candidates for Prader-Willi syndrome (PWS). We describe two sibs with typical PWS presenting deletion of SNRPN detected by fluorescence in situ hybridization (FISH). Neither a cytogenetically detectable 15q12 deletion nor a deletion for the D15S11, D15S10, and GABRB3 cosmid probes were found in either patient. This implies a smaller deletion limited to the PWS critical region. FISH with a SNRPN probe will permit analysis of PWS patients with limited deletions not detectable with other probes.

Molecular diagnosis of Prader‐Willi syndrome: Parent‐of‐origin dependent methylation sites and non‐isotopic detection of (CA)n dinucleotide repeat polymorphisms

We describe our experience in the molecular diagnosis of 22 patients suspected of Prader-Willi syndrome (PWS) using a DNA probe PW71 (D15S63) which detects a parent-of-origin specific methylated site in the PWS critical region. The cause of the syndrome was determined as deletion or uniparental disomy according to the segregation of (CA)n dinucleotide repeat polymorphisms of the PWS/AS region and more distal markers of chromosome 15. In 10 patients the clinical diagnosis was confirmed by this approach, 6 with paternal deletion and 4 with maternal disomy. In one patient, the aberrant methylation pattern that was detected by PW71 could not be confirmed by the segregation of (CA)n, probably due to paternal microdeletion in the PWS critical region which did not include the loci D15S97, D15S113, GABRB3, and GABRA5. This case demonstrates the advantage of the DNA probe PW71 in the diagnosis of PWS.

Sister chromatid exchange in families with Angelman or Prader‐Willi syndrome

Using estimation of numbers of sister chromatid exchanges arising in 15q11q13 as a measure, comparisons of the stability of the Prader-Willi syndrome critical region have been made. The groups studied included probands with Prader-Willi or Angelman syndromes either with or without a cytogenetically visible deletion in 15q11q13, their parents, specifically those parents who had passed on the homologue which had become deleted, and a control group. No significant differences were found between any of the four groups, indicating that there was no increase in the instability of the PWSCR region as measured by sister chromatid exchange.

Small nuclear ribonucleoprotein polypeptide N (SNRPN), an expressed gene in the Prader-Willi syndrome critical region.

Prader-Willi syndrome (PWS) is associated with paternally derived chromosomal deletions in region 15q11-13 or with maternal disomy for chromosome 15. Therefore, loss of the expressed paternal alleles of maternally imprinted genes must be responsible for the PWS phenotype. We have mapped the gene encoding the small nuclear RNA associated polypeptide SmN (SNRPN) to human chromosome 15q12 and a processed pseudogene SNRPNP1 to chromosome region 6pter-p21. Furthermore, SNRPN was mapped to the minimal deletion interval that is critical for PWS. The fact that the mouse Snrpn gene is maternally imprinted in brain suggests that loss of the paternally derived SNRPN allele may be involved in the PWS phenotype.

Replication asynchrony between homologs 15q11.2: Cytogenetic evidence for genomic imprinting

Replication kinetics of the Prader-Willi syndrome critical region (15q11.2) was investigated in seven normal healthy adult females using RBG replication bands. Replication asynchrony between homologs 15q11.2 was identified consistently in about 40% of cells in all individuals. It was limited to the stages in which Xp22, Xp11, Xq13 and Xq24/26 were visible in the late-replicating X chromosome. This asynchrony suggested that replication timing overlapped between 15q11.2 and the early replicating R-bands of the late X chromosome in some cells, and that the difference in replication timing between homologs was probably related to genomic imprinting; the latter has been suggested as a pathogenetic basis of Prader-Willi syndrome. As a result of an analysis of the proportions of asynchronous and synchronous cells in each replication stage, two types of cells were deduced providing 1:1 methylation mosaicism of genomic imprinting was assumed. The first type was composed of cells with normal replication in one homolog and delayed replication in the other. The second type was composed of cells with normal replication in both homologs. Our results provide cytogenetic evidence of methylation mosaicism for mammalian genomic imprinting.