Identification of germline mutations in the RB1 gene by denaturant gradient gel electrophoresis and polymerase chain reaction direct sequencing.

Germline mutations in the RB1 gene confer hereditary predisposition to retinoblastoma. The majority of these mutations occur de novo and differ from one patient to another. Cytogenetics and Southern blotting were shown to detect less than 15% of constitutional rearrangements. In this study we used the polymerase chain reaction (PCR) combined with denaturant gradient gel electrophoresis (DGGE) to detect point mutations or small deletions and insertions in a pool of 120 unrelated retinoblastoma patients. Partial DGGE analysis of the RB1 gene enabled us to identify sequence alterations generating stop codons, leading to amino acid substitution or affecting splice sites as well as several polymorphisms. Most of the nucleotide changes detected are flanked by direct repeats. The approach described here has proved to be a useful method for the detection of germline mutations in the RB1 gene.