Multiplex SNaPshot Genotyping for Detecting Loss of Heterozygosity in the Mismatch-Repair Genes MLH1 and MSH2 in Microsatellite-Unstable Tumors

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 d...

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Published in:Clinical chemistry (Baltimore, Md.) Md.), 2008-11, Vol.54 (11), p.1844-1854
Main Authors: Bujalkova, Maria, Zavodna, Katarina, Krivulcik, Tomas, Ilencikova, Denisa, Wolf, Brigitte, Kovac, Michal, Karner-Hanusch, Judith, Heinimann, Karl, Marra, Giancarlo, Jiricny, Josef, Bartosova, Zdena
Format: Article
Language:English
Subjects:
Adaptor Proteins, Signal Transducing - genetics
Analytical, structural and metabolic biochemistry
Base Pair Mismatch - genetics
Base Sequence
Biological and medical sciences
Cancer
Colorectal Neoplasms, Hereditary Nonpolyposis - genetics
DNA Primers
DNA Repair - genetics
E coli
Fundamental and applied biological sciences. Psychology
Genes
Genetic testing
Genotype
Humans
Investigative techniques, diagnostic techniques (general aspects)
Loss of Heterozygosity
Mass spectrometry
Medical sciences
Methods
Mutation
MutL Protein Homolog 1
MutS Homolog 2 Protein - genetics
Nuclear Proteins - genetics
Polymerase Chain Reaction
Polymorphism, Single Nucleotide
Roles
Sensitivity and Specificity
Studies
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Summary: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. 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. 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). 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.
ISSN:0009-9147
1530-8561
DOI:10.1373/clinchem.2008.108902