Mutation detection in colorectal cancers : direct sequencing of DNA mismatch repair genes

The detection of unknown mutations has proved complex and time consuming, and this is certainly the case for HNPCC. Germline mutations have been detected in five of the six human DNA mismatch repair genes (in hMLH1, hMSH2, hMSH6, hPMS1, hPMS2, but not hMSH3) in HNPCC patients (1-7) with hMLH1 and hMSH2 being the most frequently affected (8-9). Point mutations resulting in missense, nonsense and frameshift alterations are found in HNPCC, as well as mutations leading to splicing alterations (9). The detection of mutations in these genes has relied upon direct sequencing of genomic DNA. Scanning or prescreening methods have been investigated (12-15), but at this stage direct sequencing is the gold standard by which these methods are generally judged. One caveat however, is that this method does not detect deletions that span exons or entire genes, the frequency of which may be up to 6.5% of HNPCC cases (16). To be rigorous, it would be prudent to include direct sequencing along with Southern analysis. This chapter will focus on the direct sequencing of hMLH1 and hMSH2, although the overall strategy may also be used for the analysis of the other mismatch repair genes.