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 T6-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 T6-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T5-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 T6-repeat resulting in a T5 sequence. Only one case, a male patient, expressed the mutated WTX gene while being wild type for all other investigated genes.


CONCLUSIONS
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.
Scheel
Silvio K
SK
Pathologisches Institut der Ludwig-Maximilians-Universität München, Thalkirchner Strasse 36, 80337 Munich, Germany.
Porzner
Marc
M
Pfeiffer
Sabine
S
Ormanns
Steffen
S
Kirchner
Thomas
T
Jung
Andreas
A
D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000368:Aged; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000556:secondary*; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D016368:Frameshift Mutation / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D017491:Genes, APC; D005838:Genotype; D006801:Humans; D008297:Male; D053842:Microsatellite Instability; D018895:Microsatellite Repeats; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011379:Prognosis; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D051176:beta Catenin / Q000235:genetics
C517118:AMER1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D004273:DNA, Neoplasm; D025521:Tumor Suppressor Proteins; D051176:beta Catenin; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf