Characterization of MLH1 and MSH2 alternative splicing and its relevance to molecular testing of colorectal cancer susceptibility.

The phenomenon of alternative splicing in the DNA mismatch repair genes MLH1 and MSH2 was extensively investigated by coupled reverse transcription-polymerase chain reaction in different human tissues, including 42 mononuclear blood cell samples--31 obtained from familial colon cancer patients or their at-risk relatives and 11 from healthy blood donors--7 normal colonic mucosae, 4 established human cancer cell lines, 8 colorectal tumors, and one sample each of ileum, liver, muscle, thymus, breast, and EBV-transformed lymphoblasts. Several isoforms were observed for each gene. Products of MLH1 alternative splicing included mRNAs lacking alternative exons 6/9, 9, 9/10, 9/10/11, 10/11, 12, 16, and 17. For MSH2, products lacking exons 5, 13, 2 through 7, and 2 through 8 were identified. The levels of expression were found to vary among different samples. All isoforms were found in a relevant fraction (43-100%) of the mononuclear blood cell samples, as well as in other tissues. The splicing variants were also detected in normal colonic mucosa, with the exceptions of the MLH1 -6/9 and -10/11 and the MSH2 -13 isoforms. Germline mutations of MLH1 and MSH2 confer constitutional predisposition to the development of colorectal cancer and other neoplasms. A substantial proportion of the mutations identified so far involve alterations of the normal splicing process. Knowledge of the existence of multiple alternative splicing events, not caused by genomic DNA changes, is important for the evaluation of the results of molecular diagnostic tests based on RNA analysis.