Mutations in the WTX - gene are found in some high-grade microsatellite instable (MSI-H) colorectal cancers

Background: Genetically, colorectal cancers (CRCs) can be
subdivided into tumors with chromosomal instability (CIN) or
microsatellite instability (MSI). In both types of CRCs genes that
are involved in the degradation of beta-CATENIN are frequently
mutated. Whereas in CIN CRCs APC (Adenomatous Polyposis Coli) is
affected in most cases, high grade MSI (MSI-H) CRCs frequently
display mutations in various genes, like the APC-, AXIN2- or CTNNBI
(beta-CATENIN) gene itself. Recently in Wilms tumors, WTX (Wilms
tumor gene on the X-chromosome) was discovered as another gene
involved in the destruction of beta-CATENIN. As the WTX-gene
harbors a short T(6)-microsatellite in its N-terminal coding
region, we hypothesized that frameshift-mutations might occur in
MSI-H CRCs in the WTX gene, thus additionally contributing to the
stabilization of beta-CATENIN in human CRCs. Methods: DNA was
extracted from 632 formalin-fixed, paraffin-embedded metastatic
CRCs (UICCIV) and analyzed for MSI-H by investigating the stability
of the highly sensitive microsatellite markers BAT25 and BAT26
applying fluorescence capillary electrophoresis (FCE). Then, in the
MSI-H cases, well described mutational hot spot regions from the
APC-, AXIN2- and CTNNBI genes were analyzed for genomic alterations
by didesoxy-sequencing while the WTX T(6)-microsatellite was
analyzed by fragment analysis. Additionally, the PCR products of
T(5)-repeats were subcloned and mutations were validated using
didesoxy-sequencing. Furthermore, the KRAS and the BRAF
proto-oncogenes were analyzed for the most common activating
mutations applying pyro-sequencing. mRNA expression of WTX from
MSI-H and MSS cases and a panel of colorectal cancer cell lines was
investigated using reverse transcription (RT-) PCR and FCE.
Results: In our cohort of 632 metastatic CRCs (UICCIV) we
identified 41 MSI-H cases (6.5%). Two of the 41 MSI-H cases (4.8%)
displayed a frameshift mutation in the T(6)-repeat resulting in a
T(5) sequence. Only one case, a male patient, expressed the mutated
WTX gene while being wild type for all other investigated genes.
Conclusion: Mutations in the WTX-gene might compromise the function
of the beta-CATENIN destruction complex in only a small fraction of
MSI-H CRCs thus contributing to the process of carcinogenesis.