SITE AND SEQUENCE-SPECIFIC DNA METHYLATION IN THE NEUROFIBROMATOSIS (NF1) GENE INCLUDES C5839T - THE SITE OF THE RECURRENT SUBSTITUTION MUTATION IN EXON-31

CPG dinucleotides provide hotspots for transitional mutations in a var iety of genes, some leading to genetic diseases in humans, Although th is phenomenon is attributed to cytosine methylation at such sites, dir ect and specific observations of CpG methylation at the sites of recur rent mutations are lacking, We have used a bisulfite genomic sequencin g method to analyze DNA methylation within three representative exons from the neurofibromatosis type 1 (NF1) gene, well recognized for its high frequency of spontaneous mutations, We observed that the cytosine methylation within NF1 exons 28, 29, and 31 is restricted to CpG dinu cleotides, including the CpG dinucleotide present at the site of the r ecurrent NF1 mutation (C5839T; also referred to as R1947X). At several sites, clone-specific methylation differences were also observed. Our results provide experimental evidence for the hypothesis that methyla table CpGs in the NF1 gene contribute to spontaneous germline mutation s associated with this gene, by showing that DNA methylation does occu r at all CpGs contained within these representative NF1 exons. As well , the DNA methylation seen at the common mutation site in exon 31 may explain why this site is frequently mutated, Methylation-dependent mut agenesis may also provide a basis for some somatic (second hit) mutati ons which disable the normal allele and result in the development of N F1 associated symptoms.