Evaluation of enzymatic mutation detectiontrade mark in hereditary nonpolyposis colorectal cancer.

Abstract

In hereditary nonpolyposis colorectal cancer (HNPCC), the majority of reported mutations are dispersed throughout the 35 exons of the two principal susceptibility genes, MLH1 and MSH2, and because of this complexity, rapid mutation screening methods are required. The aim of this study was to evaluate the sensitivity of the Enzymatic Mutation Detection (EMD) assay in HNPCC using genomic DNA samples with known gene alterations in MLH1 and MSH2. The EMD assay relies upon the enzyme T4 Endonuclease VII recognizing and cleaving DNA mismatches, created when a PCR product containing a sequence 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 detect 62 of the 68 sequence variants (91%) with the majority showing strong cleavage products. One of the advantages of the EMD assay over other mutation screening techniques is that larger fragments can be analyzed in a single assay. No specialized equipment is required and one set of primers is sufficient 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; 227-228delAG) were also detected in HNPCC patients screened using this method.