Mutation analysis of the Smad2 gene in human colon cancers using genomic DNA and intron primers.

In mammals, one of the Mad homologues, Smad2, was reported to be a mediator of TGF-beta signaling, and was found mutated in some cases of colon and lung cancers. To extend the analysis of this gene, we previously investigated the genomic organization of the human Smad2 gene and defined the structure of 12 exons and flanking introns. In this study, we designed 11 sets of intron-based primers to examine the entire coding region of the Smad2 gene. By the PCR-SSCP method using these primers, we screened genomic DNA sequences of colorectal cancers for mutations of the Smad2 gene. Though there was no mutation within all exons of the Smad2 gene, two of 60 sporadic colorectal cancers displayed deletions in the polypyrimidine tract preceding exon 4. Deletions of this region were also detected in colon cancer cell lines, and were clustered within cells exhibiting microsatellite instability. Deletions in the polypyrimidine tract had various effects on pre-mRNA splicing, but had no effect on the splicing of the Smad2 gene in these cases. However, our data support the idea that the polypyrimidine tract in the splicing acceptor site is a target of mutations in mismatch repair-deficient tumors.