Long-Range PCR Facilitates the Identification ofPMS2-Specific Mutations

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Mutations within the DNA mismatch repair gene, “postmeiotic segregation increased 2” (PMS2), have been associated with a predisposition to hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome). The presence of a large family of highly homologousPMS2pseudogenes has made previous attempts to sequencePMS2very difficult. Here, we describe a novel method that utilizes long-range PCR as a way to preferentially amplifyPMS2and not the pseudogenes. A second, exon-specific, amplification from diluted long-range products enables us to obtain a clean sequence that shows no evidence of pseudogene contamination. This method has been used to screen a cohort of patients whose tumors were negative for the PMS2 protein by immunohistochemistry and had not shown any mutations within theMLH1gene. Sequencing of thePMS2gene from 30 colorectal and 11 endometrial cancer patients identified 10 novel sequence changes as well as 17 sequence changes that had previously been identified. In total, putative pathologic mutations were detected in 11 of the 41 families. Among these were five novel mutations, c.705+1G>T, c.736_741del6ins11, c.862_863del, c.1688G>T, and c.2007-1G>A. We conclude thatPMS2mutation detection in selected Lynch syndrome and Lynch syndrome-like patients is both feasible and desirable. Hum Mutat 27(5), 490-495, 2006. Published 2006 Wiley-Liss, Inc.

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