title: Multiplex SNaPshot genotyping for detecting loss of heterozygosity in the mismatch-repair genes MLH1 and MSH2 in microsatellite-unstable tumors.
abstract: BACKGROUND
  In the workup of patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC), detection of loss of heterozygosity (LOH) could help pinpoint the mismatch-repair (MMR) gene carrying the germline mutation, but analysis of microsatellite markers has proved unreliable for this purpose. We developed a simple, low-cost method based on single-nucleotide polymorphism (SNP) genotyping and capillary electrophoresis for the assessment of LOH at 2 MMR loci simultaneously.
  
  
  METHODS
  We used the Applied Biosystems SNaPshot Multiplex Kit with meticulously selected primers to assess 14 common SNPs in MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)] and optimized the protocol for DNA isolated from peripheral blood and fresh/frozen or archival microsatellite-unstable tumors from patients with confirmed (n = 42) or suspected (n = 25) HNPCC. The 42 tumors from patients with confirmed MLH1 or MSH2 germline mutations were used to validate the method's diagnostic accuracy against results obtained with DNA sequencing or multiplex ligation-dependent probe amplification.
  
  
  RESULTS
  The SNaPshot assay provided better detection of certain SNPs than DNA sequencing. The MLH1 and MSH2 SNP marker sets were informative in 82% and 76% of the 67 cases analyzed, respectively. The new assay displayed 100% specificity for detecting LOH and predicted the location of the germline mutation in 40% of the cases (54% of those involving MLH1, 22% in MSH2).
  
  
  CONCLUSIONS
  Our SNP-based method for detecting LOH in MLH1 and MSH2 is simple to perform with instruments available in most clinical genetics laboratories. It can be a valuable addition to protocols now used to guide mutational screening of patients with suspected HNPCC.
authors.affiliation: Laboratory of Cancer Genetics, Cancer Research Institute of Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: Maria
authors.initials: M
authors.lastname: Bujalkova
authors.forename: Katarina
authors.initials: K
authors.lastname: Zavodna
authors.forename: Tomas
authors.initials: T
authors.lastname: Krivulcik
authors.forename: Denisa
authors.initials: D
authors.lastname: Ilencikova
authors.forename: Brigitte
authors.initials: B
authors.lastname: Wolf
authors.forename: Michal
authors.initials: M
authors.lastname: Kovac
authors.forename: Judith
authors.initials: J
authors.lastname: Karner-Hanusch
authors.forename: Karl
authors.initials: K
authors.lastname: Heinimann
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Marra
authors.forename: Josef
authors.initials: J
authors.lastname: Jiricny
authors.forename: Zdena
authors.initials: Z
authors.lastname: Bartosova
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D005838:Genotype; D006801:Humans; D019656:Loss of Heterozygosity; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein