OBJECTIVETo explore germline mutations of MLH1 in hereditary nonpolyposis colorectal cancer (HNPCC), and to investigate the pathobiology of novel detectable mutations of MLH1.

METHODRNA was extracted from the peripheral blood of 12 patients from 12 different families fulfilling the Amsterdam II Criteria of HNPCC. Germline mutations of MLH1 were determined by RT-PCR with gene specific primers, heat-resistance reverse transcriptase and long-template PCR polymerase, followed by cDNA sequencing analysis. PCR-Genescan analysis was used to further investigate microsatellite instability with a panel of 5 microsatellite markers (BAT26, BAT25, D5S346, D2S123 and Mfd15), along with immunohistochemistry staining to detect the expression of MLH1 protein in the tumor tissues.

RESULTSFour germline mutations were found in 4 patients, 2 of which were previously reported GTT-->GAT mutation at codon 384 of exon 12, and the other two were novel mutations: CGC-->TGC at codon 217 of exon 8 and CCG-->CTG at codon 581 of exon 16. Two tumors with the novel mutations had high frequency microsatellite instability showing more than 2 instable loci (RER + phenotype), and both tumors lost their MLH1 protein expression.

CONCLUSIONThe two novel germline mutations of MLH1 identified in this study, i.e. CGC-->TGC at codon 217 of exon 8 and CCG-->CTG at codon 581 of exon 16, are very likely to have pathological significance.