Abstract for presentation at 11th International Congress of Human Genetics

DHPLC based method using quasimonomorphic mononucleotide repeats and pentaplex PCR for rapid and accurate MSI analysis in colorectal cancer

  • Gasper Berginc, Laboratory of Molecular Genetics, Institute of Pathology, Medical Faculty, Slovenia
  • Prof Damjan Glavac, Laboratory of Molecular Genetics, Institute of Pathology, Medical Faculty, Slovenia

    • Microsatellite instability (MSI) is a phenomenon characterized by small deletions or insertions within short tandem repeats in tumour DNA compared to matching normal DNA. MSI analysis is very important tool for detection of hereditary non-polyposis colorectal cancer and MSI high sporadic primary colorectal tumours. Set of two mononucleotide and three dinucleotide microsatellite markers was proposed by National Cancer Institute Workshop in 1997 to provide uniform criteria for MSI analysis. In 2002 the guidelines were revised and an exclusive use of mononucleotide markers was proposed due to their quasimonomorphic nature. Use of five quasimonomorphic mononucleotide markers eliminates ultimate need for analysis of germline DNA corresponding to tumour DNA. Here we report a method for MSI analysis using denaturating high performance liquid chromatography (DHPLC). We analysed 595 newly diagnosed colorectal tumours and 145 normal samples. Five quasimonomorphic mononucleotide microsatellite markers BAT-25, BAT-26, NR-21, NR-22, and NR-27 were amplified in a single multiplex PCR reaction and analysed using DHPLC and capillary electrophoresis. Analysis and cross-examination of results obtained from 96 samples using DHPLC and capillary electrophoresis showed the same sensitivity and specificity of both methods for detection of MSI-H tumours. Using our new method we have shown that tested markers are quasimonomorphic in Slovenian population with frequencies of polymorphisms 0,07%, 1,4%, 2,1%, 1,4%, and 1,4% for BAT-25, BAT-26, NR-21, NR-22, and NR-27 respectively. 43 (7,2%) new MSI-H tumours were identified among which 84% showed instability in all 5 tested markers and 16% showed instability in 4 of 5 tested markers. Overall, we developed a high-throughput, robust, accurate and cost-effective method for detection of MSI-H tumours in only nine minutes using DHPLC. Method is suitable for large-scale studies as well as for daily use with smaller number of samples.

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