Analysis of hMLH1 and hMSH2 Gene Dosage Alterations in Hereditary Nonpolyposis Colorectal Cancer Patients by Novel Methods

A significant fraction of hereditary nonpolyposis colorectal cancer cases with defective mismatch repair (ie, Lynch syndrome) have large genomic deletions or duplications in the mismatch repair genes, hMLH1 and hMSH2, which can be challenging to detect by traditional methods. For this study, we developed and validated a novel Southern blot analysis method that allows for ascertainment of the extent of the dosage alterations on an exon-by-exon basis and compared this method to a second novel technique, multiplex ligation-dependent probe amplification (MLPA). From a total of 254 patients referred for Lynch syndrome testing, 20 of the 118 MLH1 cases and 42 of the 136 MSH2 cases had large genomic alterations, as detected by Southern blot. MLPA and Southern blot results were concordant with the exception of three major discrepancies: one because of a lack of MLPA probes for the region altered, another because of a point mutation near the MLPA probe ligation site, and another that was unexplained. Compared to Southern blot, MLPA has a shorter turn-around time, the analysis is less costly, less time-consuming, and less labor-intensive, and results are generally clear and unambiguous. However, concerns with MLPA include the presence of false-negatives and -positives because of positioning of probes and DNA variants near the probe ligation site. Overall, both Southern blot and MLPA provide important tools for the complete evaluation of patients with Lynch syndrome.