title: Long-range PCR facilitates the identification of PMS2-specific mutations.
abstract: 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 homologous PMS2 pseudogenes has made previous attempts to sequence PMS2 very difficult. Here, we describe a novel method that utilizes long-range PCR as a way to preferentially amplify PMS2 and 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 the MLH1 gene. Sequencing of the PMS2 gene 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 that PMS2 mutation detection in selected Lynch syndrome and Lynch syndrome-like patients is both feasible and desirable.
authors.affiliation: Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Clendenning
authors.forename: Heather
authors.initials: H
authors.lastname: Hampel
authors.forename: Jennifer
authors.initials: J
authors.lastname: LaJeunesse
authors.forename: Annika
authors.initials: A
authors.lastname: Lindblom
authors.forename: Jan
authors.initials: J
authors.lastname: Lockman
authors.forename: Mef
authors.initials: M
authors.lastname: Nilbert
authors.forename: Leigha
authors.initials: L
authors.lastname: Senter
authors.forename: Kaisa
authors.initials: K
authors.lastname: Sotamaa
authors.forename: Albert
authors.initials: A
authors.lastname: de la Chapelle
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000175:diagnosis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D045643:DNA Repair Enzymes / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D006801:Humans; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011544:Pseudogenes
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes