cytidylyl-3--5--guanosine and Angelman-Syndrome

Topics: Angelman Syndrome; Dinucleoside Phosphates; DNA Methylation; Enhancer Elements, Genetic; Female; Genomic Imprinting; Humans; Male; Models, Genetic; Prader-Willi Syndrome; Restriction Mapping

A deletion of 15q11-q13 and uniparental disomy 15 lead to Prader-Labhart-Willi syndrome (PWS) or Angelman syndrome (AS) because this region contains genes expressed exclusively from the paternal (PWS) or maternal (AS) chromosome, respectively. DNA methylation plays a role in the control of imprinted gene expression, but so far only a few 5'-CG-3' dinucleotides within the recognition sites of the methylation sensitive enzymes have been studied. As part of a study on DNA methylation patterns in the human genome, we have applied the bisulfite protocol of genomic sequencing to study all 5'-CG-3' dinucleotides around exon 1 of SNRPN and at the D15S63 locus, which contains a start site for alternative SNRPN transcripts possibly involved in imprint switching during gametogenesis. At least 17 PCR products derived from single chromosomes of normal individuals as well as PWS and AS patients have been sequenced. We have found that cytosine residues outside 5'-CG-3' dinucleotides are always unmethylated. However, > 96% of all of the 23 5'-CG-3' dinucleotides around SNRPN exon 1 are methylated on the maternal chromosome and completely devoid of methylation on the paternal chromosome. This finding is in contrast to the D15S63 locus, where only the two Cfol/Hhal sites are methylated on the maternal chromosome at the same frequency as seen for the SNRPN segment. At the other five 5'-CG-3' dinucleotides, differential methylation is less pronounced, i.e. 45-70% on the maternal chromosome and 5-14% on the paternal chromosome. The differences between SNRPN and D15S63 methylation may reflect different biological functions of the alternative SNRPN transcripts. The systematic investigation of 5'-CG-3' methylation patterns as reported here will provide the basis for a PCR-based methylation test to diagnose PWS and AS.

Topics: 5-Methylcytosine; Angelman Syndrome; Autoantigens; Chromosomes, Human, Pair 15; Cloning, Molecular; CpG Islands; Cytosine; Dinucleoside Phosphates; DNA Methylation; Female; Genomic Imprinting; Humans; Male; Plasmids; Polymerase Chain Reaction; Prader-Willi Syndrome; Promoter Regions, Genetic; Restriction Mapping; Ribonucleoproteins, Small Nuclear; Sequence Analysis, DNA; Sequence Deletion; snRNP Core Proteins; Sulfites

To determine the molecular basis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS), we have isolated new transcripts from chromosome 15q11-q13. Two novel transcripts located within 300 kilobases telomeric to the small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN) were paternally expressed in cultured cells, along with SNRPN, defining a large imprinted transcriptional domain. In three PWS patients (two sibs), small deletions remove a differentially methylated CpG island containing a newly described 5' exon alpha of SNRPN, and cause loss of expression for the three imprinted transcripts and altered methylation over hundreds of kilobases. The smallest PWS deletion is familial and asymptomatic with maternal transmission. Our data imply the presence of a paternal imprinting control region near exon alpha.

Topics: Angelman Syndrome; Autoantigens; Base Sequence; Chromosomes, Human, Pair 15; Dinucleoside Phosphates; Fathers; Genomic Imprinting; Humans; Molecular Sequence Data; Prader-Willi Syndrome; Ribonucleoproteins, Small Nuclear; Sequence Deletion; snRNP Core Proteins