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 mediatorof TGF-beta signaling, and was found mutated in some cases of colon andlung cancers. To extend the analysis of this gene, we previouslyinvestigated the genomic organization of the human Smad2 gene and definedthe structure of 12 exons and flanking introns. In this study, we designed11 sets of intron-based primers to examine the entire coding region of theSmad2 gene. By the PCR-SSCP method using these primers, we screened genomicDNA sequences of colorectal cancers for mutations of the Smad2 gene. Thoughthere was no mutation within all exons of the Smad2 gene, two of 60sporadic colorectal cancers displayed deletions in the polypyrimidine tractpreceding exon 4. Deletions of this region were also detected in coloncancer cell lines, and were clustered within cells exhibitingmicrosatellite instability. Deletions in the polypyrimidine tract hadvarious effects on pre-mRNA splicing, but had no effect on the splicing ofthe Smad2 gene in these cases. However, our data support the idea that thepolypyrimidine tract in the splicing acceptor site is a target of mutationsin mismatch repair-deficient tumors.