CpG dinucleotides provide hotspots for transitional mutations in a variety of 
genes, some leading to genetic diseases in humans. Although this phenomenon is 
attributed to cytosine methylation at such sites, direct and specific 
observations of CpG methylation at the sites of recurrent mutations are lacking. 
We have used a bisulfite genomic sequencing 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 dinucleotides, including the CpG dinucleotide present at the site of the 
recurrent 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 methylatable CpGs in the NF1 gene 
contribute to spontaneous germline mutations associated with this gene, by 
showing that DNA methylation does occur 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 mutagenesis may also provide a basis for some somatic 
(second hit) mutations which disable the normal allele and result in the 
development of NF1 associated symptoms.