Detection of RB Germline Mutations Using Exon-by-exon Heteroduplex Analysis Compared with SSCP

Purpose: To compare heteroduplex analysis with SSCP and to develop a simple and effective method for mutational screening of RB gene.Materials and Methods: Leukocyte DNA was prepared from 12 unrelated Japanese patients with hereditary retinoblstoma. PCR combined with simultaneous nonisotopic heteroduplex and SSCP analysis was used to screen leukocyte DNA for such mutations, exon-by-exon, without the use of restriction endonu-clease digestion. PCR was conducted using 28 pairs of primers flanking all 27 ex-ons and the promoter region of the RB gene, with PCR products ranging from 159bp to 326bp. Mutations were identified by sequencing.Results: Heterozygous germline mutations were detected in 8 of 12 Japanese patients. The mutations were identified by sequencing as follows; G→C/acceptor of exon 11, T insertion/codon 389, C→T/codon 455, 33bp insertion/codon 455 (C GA), G→T/codon 533, C→T/codon 579, C deletion/codon 674, and C→T/ codon 787.Conclusion: Our results suggest that small RB gene mutations are