Association Analysis Of The C/T Polymorphism In The 5'-Untranslated Region Of The CD40 Gene In HBV Infection

Hepatitis B virus(HBV)infection is the most common cause of liver diseases worldwide.More than 350 million people worldwide are persistently infected with HBV which show variable clinical outcome,ranging from the asymptomatic and self-limited infection to the chronic hepatitis,cirrhosis and even hepatic-cellular carcinoma.However,the persistence and pathogenic mechanism of the hepatocellular injuries caused by HBV infection are still not entirely clear.It is believed that the individual differences in HBV infection.are determined by comprehensive factors including virological itself,host immune response,genetic background and environment.Furthermore,there is increasing evidence that genetic factors can influence the natural history of HBV infection.CD40 is a member of the tumor necrosis factor receptor family,expressed predominantly on B lymphocytes,monocytes,antigen presenting cell.The ligation of CD40 with its ligand(CD40L)plays an important role in both cellular and humoral immunity.Recently several studies have shown that the -1C/T polymorphism in the 5'-untranslated region(UTR)of the CD40 is associated with the susceptibility or processes of some diseases such as Grave' disease.But up to date there is no study about the single nucleotide polymorphism(SNP)of CD40 in the HBV infection.In this study we detected CD40 5'UTR-1C/T SNP among patients with HBV infection as well as the relation of sCD40 level in the serum aimed to determine the association of SNP of CD40 with HBV infection.PartⅠAssociation analysis of the -1C/T Polymorphism in the 5'-Untranslated Region of the CD40 gene in HBV infectionObjective:In order to investigate the association of CD40 polymorphism with HBV infection,CD40 -1C/T polymorphism in the 5'-untranslated region(UTR)was analyzed in patients with HBV infection.Meanwhile,the effect of the polymorphism of CD40-1C/T on the expression of protein was also detected in vitro.Methods:Patients with chronic hepatitis B(n=132),severe hepatitis(n=35), asymptomatic HBV carriers(n=59)and normal healthy controls were examined (n=181).Genomic DNA were extracted from these subjects and genotypes were determined by polymerase-chain reaction(PCR)-restriction fragment polymorphism (PCR-RFLP).The effect of SNP in the untranslated region of CD40 was assessed by the enhanced green fluorescent protein(EGFP)through fusing CD40 5'UTR with -1C/T into the upstream of coding sequence of EGFP.Result:The frequencies of CC,CT,or TT genotypes were significantly different between HBV carriers and control subjects(P＜0.05)as well as C alleles frequency(50.2%vs 65.6%,P＜0.05).By contrast,the frequency of TT genotype was significantly higher in HBV carriers than in healthy controls(25.4%vs 12.6%).No significant differences was found in the frequencies of the -1C/T polymorphism between the controls and patients with chronic hepatitis B(P＞0.05).Among the three groups(the asymptomatic HBV carriers,chronic hepatitis B and severe hepatitis, respectively),they didn't show any statistical difference in the frequencies of different genotypes and alleles.Compared with the asymptomatic HBV carriers,the frequencies of CC genotype and C allele in patients with chronic hepatitis B or severe hepatitis were higher but they were still lower than the controls.EGFP expression from cos-7 cells transfected with the CD40 5'UTR -1C-EGFP construct was higher than those transfected with the CD40 5'UTR -1T-EGFP construct(P=0.02).Conclution:CD40 5'-UTR -1C/T SNP may be one of the important genetic factors that influence the different outcome after HBV infection.We have proved that CD40 5'-UTR -1C/T SNP affect the efficiency of protein expression in vitro,which may result in the differential expression of CD40 in vivo,leading to influencing the interaction of CD40/CD40L in the end.PartⅡAssociation between the -1C/T Polymorphism in the 5'-Untranslated Region of the CD40 gene and the serum sCD40 in chromic hepatitis BObjective:In order to study the relationship of sCD40 with CD40 -1C/T polymorphism,the serum levels of sCD40 and the CD40 genotypes were detected in patients with chronic hepatitis B.Methods:Serum samples were collected from the patients with chronic hepatitis B(n=64)and matched healthy controls(n=24).The enzyme-linked immunosorbent assay(ELISA)was used for the quantitative detection of sCD40 in the serum.The genotypes were determined by polymerase-chain reaction(PCR)-restriction fragment polymorphism(PCR-RFLP)as mentioned above.Result:The concentration of serum sCD40 in patients with chronic hepatitis B was significantly higher than that of the controls(54.41±37.97 pg/ml vs 35.4±15.82 pg/ml,P＜0.05).There was no significant difference in the levels of serum sCD40 among patients of different CD40 genotypes.Conclusion:The elevated expression of sCD40 suggests that CD40 and its ligand CD40L may be involved in the hepatitis B infection.CD40 -1C/T SNP may not influence the expression of the sCD40.