title: Assay validation for identification of hereditary nonpolyposis colon cancer-causing mutations in mismatch repair genes MLH1, MSH2, and MSH6.
abstract: Hereditary nonpolyposis colon cancer (HNPCC, Online Mendelian Inheritance in Man (OMIM) 114500) is an autosomal dominant disorder that is genetically heterogeneous because of underlying mutations in mismatch repair genes, primarily MLH1, MSH2, and MSH6. One challenge to correctly diagnosing HNPCC is that the large size of the causative genes makes identification of mutations both labor intensive and expensive. We evaluated the usefulness of denaturing high performance liquid chromatography (DHPLC) for scanning mismatch repair genes (MLH1, MSH2, and MSH6) for point mutations, small deletions, and insertions. Our assay consisted of 51 sets of primers designed to amplify all exons of these genes. All polymerase chain reaction reactions were amplified simultaneously using the same reaction conditions in a 96-well format. The amplified products were analyzed by DHPLC across a range of optimum temperatures for partial fragment denaturation based on the melting profile of each specific fragment. DNA specimens from 23 previously studied HNPCC patients were analyzed by DHPLC, and all mutations were correctly identified and confirmed by sequence analysis. Here, we present our validation studies of the DHPLC platform for HNPCC mutation analysis and compare its merits with other scanning technologies. This approach provides greater sensitivity and more directed molecular analysis for clinical testing in HNPCC.
authors.affiliation: Diagnostic Sequencing Laboratory, Medical Genetics Laboratories, Baylor College of Medicine-NAB 2015, One Baylor Plaza, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. mhegde@bcm.tmc.edu
authors.forename: Madhuri
authors.initials: M
authors.lastname: Hegde
authors.forename: Maria
authors.initials: M
authors.lastname: Blazo
authors.forename: Belinda
authors.initials: B
authors.lastname: Chong
authors.forename: Tom
authors.initials: T
authors.lastname: Prior
authors.forename: Carolyn
authors.initials: C
authors.lastname: Richards
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004252:DNA Mutational Analysis; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D011110:Polymorphism, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein