MUTATIONAL ANALYSIS OF THE HMLH1 GENE USING AN AUTOMATED 2-DIMENSIONAL DNA TYPING SYSTEM

Searching for mutations in DNA mismatch repair genes is important not only for presymptomatic diagnosis, but also for documenting the spectr um of mutations among families carrying predispositions to hereditary nonpolyposis colorectal cancer (HNPCC). We utilized an automated two-d imensional DNA typing system for mutational analysis of the hMLH1 gene and established optimal conditions for application of the technique t o analysis of hMLH1. This approach enabled us to visualize 21 spots co vering all 19 coding exons on a single gel and to envisage whether and where: any mutations existed. All mutations that we had detected prev iously by other means in a panel of HNPCC patients and in one patient with sporadic endometrial cancer were also detectable by this method. Furthermore, using the 2-D system, we screened the entire coding regio ns of the hMLH1 gene in DNAs isolated from affected individuals belong ing to two large HNPCC kindreds and four HNPCC-like kindreds, and from four patients with multiple primary cancers as well as eight sporadic colorectal cancers with replication error (RER)-positive phenotypes. We detected novel germline mutations in one HNPCC proband and one spor adic colorectal cancer with the RER-positive phenotype and one polymor phism in two HNPCC-like kindreds. This new diagnostic method is applic able to mutation of analysis of any disease causing gene, and it offer s a major improvement over current approaches. (C) 1997 Wiley-Liss, In c.