Genetic Polymorphisms Of Chemokine CXCL10 Associated With Chronic Hepatitis B Infection

Background Chronic Hepatitis B virus infection(HBV)is a highly prevalent infectious disease caused by the interaction among viral factors,environmental factors and host factors.It is a multi-factor and multi-gene disease that seriously affects human health.An estimated 250 million people worldwide are currently chronically infected with HBV.Clinical outcomes in HBV infected vary from spontaneous recovery(SR)to persistent infection and subsequently progress to cirrhosis and even hepatocellular carcinoma(HCC)due to recurrent inflammatory fibrosis.Although there are important discoveries revealed by previous studies that host genetic factors play a crucial role in the establishment of complete molecular pathways underlying the pathogenesis of HBV infection.Further exploration of the disease-related-genetic susceptibility genes may be a protential target for the treatment of chronic persistent HBV infection.Chemokine(C-X-C motif)ligand 10(CXCL10)has recently been shown to be a candidate gene significantly associated with inflammatory liver disease,participating in HBV virus clearance and inflammation through the recruitment effect Th1 lymphocytes,involving in the body’s immune response process.Therefore,the single nucleotide polymorphisms(SNP)in this gene may be involved in the pathogenesis of HBV Infection and drug treatment response.However,the association between the genetic variation of CXCL10 gene and the susceptibility to HBV infection in Chinese Han adults and children,the genotype-related differences in HBV pathogenicity,and even the underlying mechanism and clinical drug treatment response,remain unclear.Part 1 The association analysis of the promoter regional genetic variation of the susceptibility gene CXCL10 with Hepatitis B virus infectionObjective: This study aimed to investigate the impact of the naturally occurred sequence variation in the CXCL10 gene on chronic HBV infection in both adults and children groups.Methods: The sequence variants in the CXCL10 gene were genotyped,and a two-stage case-control study,including adults and independent children in Chinese Han population,was performed to assess the susceptibility of the variants to the HBV infection.Results:1.A total of 1048 adult case-control samples were enrolled in the first stage,including 518 controls and 530 cases.The results of case-control analysis showed that the age and sex of the Adult group were matched(P>0.05);while,there were significant differences in age(P<0.001),sex(P=0.004),HBs Ag-positive mothers(P<0.05)and the vaccination(P<0.05)between the case-control groups in children group.2.In the first phase of association,SNPs were associated with persistent HBV infection in adults.Logistic regression analysis was performed after adjusting for age and sex.The results showed that differences in the frequency distribution of rs4256246 genotype and allele in this case-control group were statistically significant(P<0.05).Compared with genotype GG,the risk for individuals who carrying genotype AA had an increased risk of HBV infection after adjusting for age and sex(OR=1.58,95% CI:1.04-2.42,P=0.033,respectively).As for rs4508917(A>G),individuals carrying rs4508917 AA genotype had a higher probablity to get HBV infection in Chinese Han adults after adjusting for age and sex(OR= 1.51,95% CI: 1.03-2.22,P = 0.036).After stratification by age and sex,we further compared the genotype frequencies of the two SNPs,rs4508917 and rs4256246.Compared with the rs4508917 GG genotype,the AG and AA genotypes had a higher risk of HBV infection in the higher age group(>40)(P=0.037,P=0.041,respectively).Correspondingly,in stratification analysis for rs4256246,the results were also significant in subjects older than 40 years(OR=1.73,95%CI: 1.08-2.78,P=0.024).For the association analysis between variants and HBV viral load,a significant correlation was found between the genotype AG of rs4508917 and HBV viral load(degree of replication)among patients >40 years(P=0.004).In addition,rs4508917 AA genotype showed an increased risk of abnormal HBV viral load.3.In the second phase of validation,a total of 627 children case-control samples were enrolled,including 274 controls and 353 cases.SNP rs4508917 was associated with HBV infection in children.Variant rs4508917 in CXCL10 promoter region was identified to increase HBV persistent infection susceptibility(OR= 1.81,95% : 1.13-2.92,P= 0.014)and HBV breakthrough infection in children(OR=4.95,95% CI-1.45-16.90,P= 0.011).No statistically significant was found after adjusting for age and sex.Conclusion: The genotype of rs4508917 in the CXCL10 gene promoter may contribute to the risk of persistent HBV infection not only in adults,but also in children.Part 2 Functional Analysis of Variants in the Promoter Region of CXCL10 GeneObjective: To preliminarily explore the biological mechanism of SNP involved in the pathogenesis of HBV infection.Methods: Functional analyses were conducted to verify the biological significances of the associated genetic variation.The serum expression of CXCL10 level was detected by enzyme-linked immunosorbent assay(ELISA).And positive variation on the transcription activity of CXCL10 gene was tested by Dual-Luciferase Reporter Assays.Results:1.The serum level of CXCL10 in chronic hepatitis BCHB patients was higher than that in control group(P= 0.014)conducted by ELISA.With the increased gene dose of A allele,serum CXCL10 level showed a higher expression and the difference was statistically significant(P =0.013).2.Dual-Luciferase reporter assay indicated that the direct influence of rs4508917 polymorphism A allele might increase the promoter activity of CXCL10.Conclusion:In this study,the luciferase reporter transcriptional activity loading at rs4508917 locus suggests that this novel polymorphism may regulate the CXCL10 gene expression.Besides,we showed that the CXCL10 protein level is increased in sera of CHB patients and the rs4508917 AA genotype is associated with higher CXCL10 transcription,therefore providing in vivo functional evidence of association of this risky variant.Part 3 Association between the Susceptibility Gene CXCL10 variant rs4508917 and the Response to Nucleoside Analogues TreatmentObjective: To explore the association of the HBV Susceptibility gene CXCL10 SNPs to the nucleoside analogue(NAs)Treatment response.Methods: We performed an analysis of dynamic change of HBV viral load in 62 patients with CHB at 3,6,and 9 weeks during the antiviral therapy.Results: We observed that the patients with rs4508917 AA or AG genotype had a limited HBV viral load suppression in response to NAs treatment than those with rs4508917 GG genotype.There was a similar trend toward rs4256246 AA or GA genotype in response to NAs treatment.Conclusion: rs4508917 AA or AG genotype had a limited HBV viral load suppression in response to NAs treatment.