Evaluation of enzymatic mutation detection (TM) in hereditary nonpolyposiscolorectal cancer

In hereditary nonpolyposis colorectal cancer (HNPCC), the majority of repor ted mutations are dispersed throughout the 35 exons of the two principal su sceptibility genes, MLH1 and MSH2, and because of this complexity, rapid mu tation screening methods are required. The aim of this study was to evaluat e the sensitivity of the Enzymatic Mutation Detection(TM) (EMD(TM)) assay i n HNPCC using genomic DNA samples with known gene alterations in MLH1 and M SH2. The EMD assay relies upon the enzyme T4 Endonuclease VII recognizing a nd cleaving DNA mismatches, created when a PCR product containing a sequenc e alteration is hybridized with a wild type probe. A total of 68 different sequence variants from 30 exons were analyzed. The EMD assay was able to de tect 62 of the 68 sequence variants (91%) with the majority showing strong cleavage products. One of the advantages of the EMD assay over other mutati on screening techniques is that larger fragments can be analyzed in a singl e assay. No specialized equipment is required and one set of primers is suf ficient for radioactive detection of the cleavage products. This method can be adapted to use fluorescent dye-labelled primers and may be automated to detect mutations accurately and rapidly in a large number of samples. One new MLH1 mutation (418delA) and two novel MSH2 mutations (1A>C; 227-228delA G) were also detected in HNPCC patients screened using this method. Hum Mut at 16:61-67, 2000. (C) 2000 Wiley-Liss, Inc.