Rapid and accurate approach for screening of microsatellite
unstable tumours using quasimonomorphic mononucleotide repeats and denaturating
high performance liquid chromatography (DHPLC)
Affiliations: Department of Molecular Genetics, Institute of
Pathology, Faculty of Medicine, University of Ljubljana, Korytkova 2, 1000
Ljubljana, Slovenia
Note: [] Corresponding author: Prof. Damjan Glavač, PhD, Department
of Molecular Genetics, Institute of Pathology, Faculty of Medicine, Korytkova
2, 1000 Ljubljana, Slovenia. Tel.: +386 1 5437180; Fax: +386 1 543 7181;
E-mail: damjan.glavac@mf.uni-lj.si
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.
