Study On Genetic Association Of SNPs In The Coding Region Of Câ…¡TA Gene With The Outcome Of Chronic HBV Infection And Preliminary Functional Analysis On The Different Haplotypes Of Câ…¡TA

Backgroud/AimsChronic Hepatitis B Virus (HBV) infection is associated with a variety of clinical phaenotypes, and its mechanism is not still illuminated. Beside the role of HBV, host hereditary susceptibility is an important factor associated with the outcome of HBV infection. The expression of HLA class II antigen influences directly host immune response to HBV. CⅡTA is a switching factor of mastering the expression of HLA class II antigen. It is able to control host immune function by mastering the expression of HLA class II antigen, accordingly influencing the occurrence, development and outcome of infectious diseases, autoimmune disease, graft rejection and tumor. The study of disease association is the most frequently used in the study of hereditary susceptibility of HBV infection. In foreign studies, the single nucleotide polymorphisms (SNPs) were discovered in human CⅡTA gene, and associated with the susceptibility to autoimmune diseases. In our previous study, four SNPs were discovered in the promoter IV of CⅡTA gene, and eight non- homonymy SNPs in its coding region. The two SNPs at C30799G (Ala500Gly) and C19170G (Leu45Val) sites with higher allele frequencies in the coding region were selected to be analyzed among patients with chronic HBV infection, investigating whether gene polymorphisms in CⅡTA coding region are or not associated with the different clinical phenotypes of chronic HBV infection, and the preliminary functional analysis being studied on the different haplotypes of CⅡTA.MethodThe two SNPs at C30799G (Ala500Gly) and C19170G (Leu45Val) sites of CⅡTA gene coding region were genotyped using sequence-specific primer PCR(PCR-SSP), and the distribution of allele and genotype frequencies were investigated among 627 patients with chronic HBV infection and 101 healthy non-HBV infected blood donors. The cDNA fragments of three different CⅡTA haplotypes were prepared by inducing one or two single nucleotide mutation of wild type recombinant plasmid EBS-NPL-CⅡTA cDNA, which are correspond to two non-homonymy single nucleotide polymorphism (SNP) sites in the coding region of human CⅡTA gene, using overlap extension PCR site-directed mutagenesis technology. And the eukaryotic expression vectors were constructed for three different haplotypes cDNA of human CⅡTA gene. Hela cells without HLA class II antigen expression itself were transfected respectively with eukaryotic expression vectors containing four different haplotypes cDNA of human CⅡTA gene. CⅡTA mRNA and HLA class II antigen (HLA-DR,DP,DQ) were respectively detected by RT-PCR and indirect cell immunofluorescence technique and flow cytometry in the untransfected and transfected Hela cells.Results:1. The frequencies of C allele and CC genetype at 30799 site were significantly higher among patients with hepatocellular carcinoma than those among patients with liver cirrhosis(χ~2=4.861,4.993; P=0.027,0.025). Logistic regression analysis indicated that there were significantly different with the genotype frequencies at 30799 site in the patients between liver cirrhosis and hepatocellular carcinoma(OR 0.557;95%CI,0.334-0.930;P=0.025), and the main differences were observed among C recessive model(OR 0.491;95%CI,0.269-0.898;P=0.021).2. The frequencies of G allele and genetypes carring G allele (GG and GC) at 19170 site were significantly higher among patients with liver cirrhosis than those among nonprogressed liver diseases including asymptomatic carriers and patients with chronic hepatitis B(χ~2=7.128,6.464; P=0.008,0.011). Logistic regression analysis indicated that there were significantly different with the allele frequencies at 19170 site in the patients between liver cirrhosis and nonprogressed liver diseases (OR 0.742;95%CI,0.552-0.998;P=0.048), and the main differences were observed among G dominant model(OR 0.581;95%CI,0.353-0.954;P=0.032).3. The cDNA fragments of three different human CⅡTA haplotypes were successfully constructed, and the eukaryotic expression vectors containing three different haplotypes cDNA of human CⅡTA gene were obtained.4. No expression of CⅡTA mRNA and HLA class II antigen(HLA-DR,DP,DQ) were observed on original Hela cells and empty vector transfected Hela cells. CⅡTA mRNA expression was emerged, and the expression of HLA class II antigen (HLA-DR,DP,DQ) were observed in the Hela cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA.5. There were not significantly different with the levels of HLA class II antigen (HLA-DR,DP,DQ) expression among Hela cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P >0.05).Conclusions:1. The polymorphism at 30799 site of CⅡTA gene was associated with hepatocellular carcinoma in chronic HBV infection, and CC genotype was associated with significantly increased risk of hepatocellular carcinoma. The polymorphism at 19170 site of CⅡTA was associated with liver cirrhosis in chronic HBV infection, and the G allele was associated with significantly increased risk of liver cirrhosis.2. The eukaryotic expression vectors containing three different haplotypes cDNA of human CⅡTA gene were successfully constructed, and were essential for the following study on the function of four haplotypes.3. CⅡTA is a switching factor of mastering the expression of HLA class II antigen in Hela cells, and no expression of HLAⅡantigen in Hela Cells is associated with the lack of CⅡTA expression.4. The two SNP at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of CⅡTA gene did not influence capability of CⅡTA trans-activating HLA class II gene expression.