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Mutation analysis of the Smad2 gene in human colon cancers using genomic DNA and intron primers
S Takenoshita1, M Tani, A Mogi
1First Department of Surgery, Gunma University School of Medicine, Japan.
Abstract:
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.
Insights
Mutations in the Smad2 gene
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Smad2 is a key mediator of TGF-beta signaling in mammals.
- Smad2 mutations are implicated in colon and lung cancers.
- Previous studies defined the human Smad2 gene structure.
Purpose of the Study:
- To screen for Smad2 gene mutations in colorectal cancers.
- To investigate the role of Smad2 in cancer development.
- To analyze deletions in the polypyrimidine tract of Smad2.
Main Methods:
- PCR-SSCP (Polymerase Chain Reaction - Single-Strand Conformation Polymorphism) method.
- Screening of genomic DNA from sporadic colorectal cancers.
- Analysis of colon cancer cell lines.
Main Results:
- No mutations were found within the Smad2 exons.
- Two of 60 colorectal cancers showed deletions in the polypyrimidine tract preceding exon 4.
- Deletions were also found in colon cancer cell lines, particularly those with microsatellite instability.
- These deletions affected pre-mRNA splicing but not mature Smad2 mRNA splicing in these cases.
Conclusions:
- The polypyrimidine tract in the splicing acceptor site is a potential mutation target in mismatch repair-deficient tumors.
- Smad2 gene mutations may not be a primary driver in all sporadic colorectal cancers.
- Further research is needed to understand the functional consequences of these deletions.