DCC Gene Point Mutation And Loss Of Heterozygosity Of Sporadic Colorectal Carcinoma In Guangxi Region

Background and objective: Colorectal carcinoma is one of the most common gastrointestinal tumors. Its incidence is just less than lung carcinoma in some metropolis such as Shanghai of China. The pathogenesis of colorectal carcinoma is not completely understood. It is reported that colorectal carcinoma is involved in a procedure of multi-gene changes. These genes include oncogenes,tumor suppressor genes,and DNA mismatch repair genes,and so on. They play an important role in cell proliferation and differentiation. They can be changed by some carcinogens such as radiation,smoking and so on. The changes of these genes such as mutation,amplification,rearrangement,deletion and methylation can cause gene dysfunctions,and then led to cancer. DCC gene is one of the largest tumor suppressor genes. Controversy about the role of DCC gene in colorectal carcinoma always exists. Some scholars suppose that DCC gene is nothing to do with colorectal carcinoma. But others argue that DCC gene is closely related to distant metastasis and prognosis of colorectal carcinoma. The purpose of this study is to explore the pathogenesis of the sporadic colorectal carcinoma by analyzing DCC gene mutation and loss of heterozygosity in Guangxi.Methods: DNA was extracted from tumor tissue and normal mucosa using phenol/chloroform protocol and DNA extraction kit. DNA from blood of 20 healthy individuals was extracted by using a DNA extraction kit (Sangon Biotech Co.,Ltm). Exon4,exon28,exon29,VNTR fragments and MspI fragments of DCC gene were amplified by PCR. DNA point mutations were analyzed by using SSCP and DNA sequencing methods. LOH of VNTR fragment were analyzed by using polyacrylamide gel electrophoresis and silver stain. PCR Products were digested with MspI enzyme to analyze the LOH.Results: Codon201 (C>G) was found in 66 of 73 patients with sporadic colorectal cancer,accounting for 90.4%. Among of them,18 cases were homozygotes,and 48 cases were heterozygotes. 20 healthy individuals were heterozygotes of Codon201 (C>G). A polymorphism(NT₀10966:g.1459443A>C)was found at intron4-nt 8. The polymorphism rate was 1.4%. No abnormal of the exon28 and exon29 was detected. LOH of VNTR fragments was observed in 11 of 70 cases(15.7%). LOH of MspI fragments was detected in 7 of 24 informative cases,accounting for 29.2%. DCC gene mutations and VNTR-LOH were independent of the patients'age,the tumor's site,depth,pathological type,grade,lymph node metastasis and Dukes stage by statistical analysis (P> 0.05). And MspI-LOH was not correlated with the patients'age,the tumor's site,depth,pathological type,grade,lymph node metastasis (P> 0.05),but it was related to the Dukes staging (P <0.05).Conclusion:codon201(C>G) was found in sporadic colorectal carcinoma in Guangxi. But it was also found in all of 20 healthy individuals,meaning that codon201 was a normal polymorphism,and it could not be served as a biological marker of colorectal cancer. The polymorphism ( NT₀10966: g.1459443A>C)at Intron 4- nt 8 was rare. LOH of the VNTR fragment was not related with colorectal cancer P>0.05. LOH of MspI fragments was related with Dukes stage,which perhaps meaned that LOH of MspI fragments of DCC gene might be involved in the later stage of colorectal cancer(P<0.05).