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Disease Markers
Volume 26, Issue 1, Pages 19-26
http://dx.doi.org/10.3233/DMA-2009-0600

Rapid and Accurate Approach for Screening of Microsatellite Unstable Tumours Using Quasimonomorphic Mononucleotide Repeats and Denaturating High Performance Liquid Chromatography (DHPLC)

Gašper Berginc and Damjan Glavač

Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia

Received 20 February 2009; Accepted 20 February 2009

Copyright © 2009 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

MSI analysis is becoming increasingly important for the detection of both hereditary non-polyposis colorectal cancer and sporadic primary colorectal tumours with MSI high phenotype. The Bethesda panel of five microsatellite markers has been proposed to provide uniform criteria for MSI analysis. Here we report on an MSI analysis approach using quasimonomorphic mononucleotide repeats and denaturating high performance liquid chromatography (DHPLC). We analysed 595 newly diagnosed colorectal tumours and 145 normal samples. Microsatellite markers BAT-25, BAT-26, NR-21, NR-22, and NR-27 were amplified in multiplex reaction and analysed using DHPLC and capillary electrophoresis (CE). DHPLC conditions for analysis of MSI multiplex assay were evaluated and tested. Analysis and cross-examination of the results obtained from 96 samples using DHPLC and capillary electrophoresis showed the same sensitivity and specificity of the two approaches for detecting MSI-H tumours. Using our new approach we showed that the tested markers are quasimonomorphic in a 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. Forty-three (7.2%) new MSI-H tumours were identified, of which 84% showed instability in all 5 tested markers. Overall, we developed a high-throughput, robust, accurate and cost-effective approach for the detection of MSI-H tumours.