Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based detection of microsatellite instabilities in coding DNA sequences: a novel approach to identify DNA-mismatch repair-deficient cancer cells.
BACKGROUND
Inherited defects in the DNA mismatch repair system lead to increased loss or gain of repeat units in microsatellites, commonly referred to as microsatellite instability (MSI). MSIs in coding regions of critical genes contribute to the pathogenesis of DNA-mismatch repair-deficient cancers, particularly those associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). MSI typing is therefore increasingly used to guide the molecular diagnosis of HNPCC.


METHODS
We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify MSIs in mononucleotide repeats within the coding sequences of genes relevant to the pathogenesis of MSI+ neoplastic lesions. After a primer extension reaction of PCR products encompassing the microsatellites, the molecular masses of the extension products were determined by MALDI-TOF-MS.


RESULTS
MSIs were detected by MALDI-TOF-MS in the GART, AC1, TGFBR2, MSH3, and MSH6 genes in neoplastic tissues and MSI+ colorectal cancer cell lines but not in MSI- control tissues. The analysis of peak-integral ratios in a single spectrum of the peaks representing insertions or deletions compared with the full-length microsatellites allowed relative quantification of MSIs. MALDI-TOF-MS-based genotyping results were confirmed by conventional DNA sequencing and electrophoresis.


CONCLUSIONS
Because of its reliability, short run times, and low costs, this semiquantitative procedure represents an effective alternative, in particular for diagnostic high-throughput typing of MSIs in neoplastic lesions.
Bonk
Thomas
T
Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander Universität Erlangen-Nürnberg, Fahrstrasse 17, Germany.
Humeny
Andreas
A
Gebert
Johannes
J
Sutter
Christian
C
von Knebel Doeberitz
Magnus
M
Becker
Cord-Michael
CM
D020137:Base Pair Mismatch; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured