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The RBI gene from 12 human retinoblastoma tumors has been analyzed exon-by-exon with the single-strand conformation polymorphism technique. Mutations were found in all tumors, and one-third of the tumors had independent mutations in both alleles neither of which were found in the germ line, confirming their true sporadic nature. In the remaining two-thirds of the tumors only one mutation was found, consistent with the loss-of-heterozygosity theory of tumorigenesis. Point mutations, the majority of which were C --T transitions, were the most common abnormality and usually resulted in the conversion of an arginine codon to a stop codon. Small deletions were the second most common abnormality and most often created a downstream stop codon as the result of a reading frameshift. Deletions and point mutations also affected splicejunctions. Direct repeats were present at the breakpoint junctions in the majority of deletions, supporting a slipped-mispairing mechanism. Point mutations generally produced DNA sequences which resulted in perfect homology with endogenous sequences which lay within 14 bp.

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We have analyzed the 27 exons and the promoter region of the RB1 gene in familial or sporadic bilateral retinoblastoma by using single-strand conformation polymorphism analysis. For improvement over previous studies, a new set of primers has been designed, which allow for amplification of the coding and splicing sequences only. The positioning of the polymerase chain reaction (PCR) primers was such that the resulting PCR products were of different sizes, which enabled us to analyze two different exons simultaneously and still distinguish between the banding profiles for both (biplex analysis). By using this approach, we were able to identify mutation in 22 new patients, but the overall efficiency of the procedure when we used a single-pass regimen was only 48%. The mutations were small insertions and deletions and point mutations in roughly equal proportions.