Enhanced detection of deleterious and other germline mutations of hMSH2 and hMLH1 in Japanese hereditary nonpolyposis colorectal cancer kindreds

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal, dominant ly inherited cancer-prone syndrome. Here, we describe a novel and efficient approach for screening mutations of two major HNPCC susceptibility genes, hMSH2 and hMLH1. The system consists of RNA extraction from whole blood tre ated with the translation inhibitor, followed by long RT-PCR of the entire coding regions combined with direct sequencing. In analysis of 15 kindreds suspicious for HNPCC, 8 samples were subjected to analysis after puromycin treatment and 7 samples mere analyzed without puromycin treatment. Three de leterious mutations were detected in the kindreds with puromycin treatment, while none were observed in those without puromycin. Signals from mutated alleles were enhanced after puromycin treatment and easily distinguished fr om the wild-type allele, achieved by suppression of nonsense-mediated mRNA decay. Furthermore, 12 other mutations were detected in 15 kindreds. The sy stem is considered to be a reliable and useful approach for detecting germl ine mutations of hMSH2 and hMLH1 in HNPCC kindreds. (C) 2000 Academic Press .