Preliminary Functional Study On The Different Haplotypes Of CⅡTA Promoter Ⅳ And Analysis On Their Genetic Association With Outcome Of HBV Infection

Backgroud/Aims:Hepatitis B Virus(HBV) infection is associated with a variety of clinical phaenotypes, and its mechanism remains unclear.Beside the role of virus,host hereditary susceptibility is an important factor associated with the outcome of HBV infection.The expression of MHC classâ…¡molecule influences directly host immune response to HBV.Câ…¡TA is a switching factor of mastering the expression of MHC classâ…¡antigen.It is able to control host immune function by mastering the expression of MHC classâ…¡molecule,and accordingly to influence the occurrence,development and outcome of infectious diseases,autoimmune diseases,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 Câ…¡TA gene,and associated with the susceptibility to autoimmune diseases.In our previous study,four SNPs were discovered in the promoterâ…£of Câ…¡TA gene,and eight non- homonymy SNPs in its coding region.The two SNPs at C-944G and C-1350T sites with higher allele frequencies in the promoterâ…£region were selected to be analyzed among patients with HBV infection, investigating whether gene polymorphism in promoterâ…£region are or not associated with the different clinical phenotypes of HBV infection.And the preliminary functional analysis was studied on the different haplotypes of Câ…¡TA promoterâ…£.Method:The two SNPs at C-944G and C-1350T sites of Câ…¡TA gene promoterâ…£region were genotyped using tetra-primer ARMS-PCR,and the distribution of allele and genotype frequencies were investigated among 1420 HBV infected population and 125 healthy non-HBV infected blood donors.The DNA fragments of four different Câ…¡TA promoterâ…£haplotypes were prepared.And the expression vectors containing four different haplotypes DNA of human Câ…¡TA promoterâ…£were constructed.These recombinant plasmids were then cotransfected with pRL-TK plasmid into HepG2 cells,luciferase activity was measured with Dual-Luciferase Reporter Assay System and Fluoroskan Ascent Fluorimetry.Results:1.Analysis on the association between polymorphism of Câ…¡TA promoterâ…£and persistent HBV infectionThere were significantly decreased frequencies of-1350T and -944G allele,and increased frequencies of-1350C and -944C allele in persistent HBV infected subjects, compared to spontaneously recovered subjects(X²= 18.1,18.0;P=6.3×10^-5,6.3×10^-5).There were significantly decreased frequency of TG haplotype and increased frequency of CC haplotype in persistent HBV infected subjects,compared to spontaneously recovered subjects(X²= 17.3,P=7.9×10^-5).On the basis of unconditional logistic regression analysis with adjustment for age and sex,there were significant difference between spontaneously recovered and persistent HBV infected subjects in the distribution of four group genotypes(OR 0.507;95%CI, 0.401-0.642;p= 6.9×10-8),and the difference was mainly observed between CC/CC and TG/TG genotypes(OR0.503;95%CI,0.394-0.641;P=1.7×10-8).Subjects bearing the CC/CC homozygote had an increased susceptibility to persistent HBV infection compared to those bearing TG/TG genotypes.The frequencies of haplotypes and genotypes in blood donors were between persistent HBV infected subjects and spontaneously recovered subjects.2.Analysis on the association between polymorphism of Câ…¡TA promoterâ…£and different outcomes of HBV infectionThere were significant difference with the distribution of four haplotypes among AsC, CHB and LC(P＜0.001).There were significantly decreased frequency of CC haplotype and increased frequency of CG and TG haplotype in LC patients,compared to AsC.And significantly lower frequency of CC haplotype and higher frequency of TG and CG haplotype in CHB patients especially TG than AsC.Meanwhile,compared to CHB,there were significantly decreased frequency of CC and TG haplotypes and increased frequency of CG haplotype in LC patients. 3.Functional analysis of Câ…¡TA promoterâ…£haplotypesThe inserted Câ…¡TA promoterâ…£DNA fragment(-1382～-913nt) did not influence the luciferase activity of pGL3-basic vector,but was able to significantly decrease the luciferase activity of pGL3-Promoter vector.There were significantly different with the activities of inserted Câ…¡TA promoterâ…£DNA fragment among four haplotypes.The mean relative luciferase activities(RLA) of TG haplotype(0.558±0.023) were significantly higher than those of CG haplotype(0.464±0.016),TC haplotype(0.463±0.015 ) and CC haplotype(0.302±0.016).The RLA of CG and TC haplotype were all significantly higher than those of CC haplotype(P=0.000),but there were not significant difference with the RLA between CG and TC haplotype(P=0.892).Conclusions:1.Functional analysis on Câ…¡TA promoterâ…£haplotypes showed that the DNA fragment of Câ…¡TA promoterâ…£(-1382～-913nt) containing two SNPs(-1350C/T and -944G/C) had no activity of promoter,but had activity of attenuator.The significant difference was observed with the activities of inserted Câ…¡TA promoterâ…£DNA fragment among four haplotypes.The activity of Câ…¡TA promoterâ…£was influenced by the polymorhisms of the two SNPs.2.Our results of analysis on the haplotypes of Câ…¡TA gene promoterâ…£showed that the polymorphism of Câ…¡TA gene promoterâ…£was significantly associated with the outcomes of HBV infection.These findings indicated that the haplotype CC and CC/CC genotype were associated with persistent HBV infection,but the haplotype TG and TG/TG genotype were associated with HBV clearance,CG and the genotypes containing CG haplotype(CG/CG,CG/CCå’ŒCG/TG) were associated with significantly increased risk of liver cirrhosis.