Constitutively methylated CpG dinucleotides as mutation hot spots in the retinoblastoma gene (RB1)

A wide spectrum of mutations, ranging from point mutations to large deletions, have been described in the retinoblastoma gene (RB1). Mutations have been found throughout the gene; however, these genetic alterations do not appear to be homogeneously distributed. In particular, a significant proportion of disease-causing mutations results in the premature termination of protein synthesis, and the majority of these mutations occur as C-->T transitions at CpG dinucleotides (CpGs). Such recurrent CpG mutations, including those found in RB1, are likely the result of the deamination of 5-methylcytosine within these CpGs. In the present study, we used the sodiumbisulfite conversion method to detect cytosine methylation in representative exons of RB1. We analyzed DNA from a variety of tissues and specifically targeted CGA codons in RB1, where recurrent premature termination mutations have been reported. We found that DNA methylation within RB1 exons 8, 14, 25, and 27 appeared to be restricted to CpGs, including six CGA codons. Other codons containing methylated cytosines have not been reported to be mutated. Therefore, disease-causing mutations at CpGs in RB1 appear to be determined by several factors, including the constitutive presence of DNA methylation at cytosines within CpGs, the specific codon within which the methylated cytosine is located, and the particular region of the gene within which that codon resides.