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

AbstractBackgroundGenetically, 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 β-CATENIN are frequently mutated. Whereas in CIN CRCsAPC(Adenomatous Polyposis Coli) is affected in most cases, high grade MSI (MSI-H) CRCs frequently display mutations in various genes, like theAPC-,AXIN2- orCTNNBI(β-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 β-CATENIN. As theWTX-gene harbors a short T6-microsatellite in its N-terminal coding region, we hypothesized that frameshift-mutations might occur in MSI-H CRCs in theWTXgene, thus additionally contributing to the stabilization of β-CATENIN in human CRCs.MethodsDNA 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 theAPC-,AXIN2- andCTNNBIgenes were analyzed for genomic alterations by didesoxy-sequencing while theWTXT6-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T5-repeats were subcloned and mutations were validated using didesoxy-sequencing. Furthermore, theKRASand theBRAFproto-oncogenes were analyzed for the most common activating mutations applying pyro-sequencing. mRNA expression ofWTXfrom MSI-H and MSS cases and a panel of colorectal cancer cell lines was investigated using reverse transcription (RT-) PCR and FCE.ResultsIn 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 T6-repeat resulting in a T5sequence. Only one case, a male patient, expressed the mutatedWTXgene while being wild type for all other investigated genes.ConclusionMutations in theWTX-gene might compromise the function of the β-CATENIN destruction complex in only a small fraction of MSI-H CRCs thus contributing to the process of carcinogenesis.