| ?Objective?1.Exploring the key loci of NTCP and CYP7A1 gene which associate with the clinical outcomes of HBV infection;2.Disscussing the association of the concentration of serum bile acid and the NTCP gene polymorphism?Method?1.Tagger SNPs were identified utilizing direct sequencing and then confirmed by the Haploview 4.2.Rs7154439 has strong linkage disequilibrium(LD)with rs4646285.Finally,rs2296651,rs4646285 and rs7154439 located on the NTCP gene,rs3824260,rs4738687 and rs8192871 located on the CYP7A1 gene were chosen as tagger SNPs.2.1619 subjects were enrolled in our study: 246 patients with chronic hepatitis B(CHB),177 patients with liver cirrhosis(LC),163 patients with HCC,595 patients with HBV immune recovery and 438 healthy controls.Total genomic DNA was isolated from human peripheral blood sample via TIANamp Genomic DNA Kit.Improved multiple ligase detection reaction(i MLDR)was utilized to genotype and the concentration of bile acids was measured by enzymatic cycling method.HBV serum markers were quantified by chemiluminescence microparticle immunoassay(CMIA)and real-time fluorescence quantitative PCR.3.The liver tissue and whole blood samples of HBV-related hepatocellular carcinoma patients were collected.The expression of NTCP in tumor tissues and adjacent tissues was compared by real-time fluorescence quantitative PCR and Western blot.The NTCP expression of rs7154439 mutant and wild types in tumor tissues and adjacent tissues were also compared in our study.?Results?1.Rs2296651 and rs4646285 are the major SNP loci in the exons of NTCP gene.50 healthy controls and 50 patients with chronic hepatitis B were enrolled to the first exon sequencing stage.The results showed that rs2296651 and rs466285 were the major mutants of the NTCP gene in Chinese Han population.2.Association between NTCP SNPs and HBV immune recovery.For the rs2296651,the distribution of genotype frequency was highly different among the HBV infection patients,HBV immune recovery subjects and healthy controls(P=0.000),the frequencies of genotype CT and TT in HBV immune recovery subjects and healthy controls were higher than that in chronic HBV infection patients(P=0.000).The allele frequency of the rs2296651 was also distributed differently among the patients,immune recovery subjects and healthy controls(P=0.000),the frequency of allele T in HBV immune recovery group and healthy control group was higher than that in patients(P=0.000 and P = 0.000,respectively).However,in our study,the distribution of genotype frequency and allele frequency all showed no differences among the case groups(CHB group,LC group and HCC group)(P=0.246).The distribution of rs4646285 genotypes frequency significant differences among the HBV infection patients,immune recovery subjects and healthy controls(P=0.000),whereas it had no statistic differences among the case groups(P=0.246).Comparing to the chronic HBV infection group,the rs4646285 AA was more inclined to appear in the recoverys(P=0.011).However,the frequency of allele A showed no differences among the chronic HBV infection group,recovery and healthy control groups(P=0.389)as well as among the CHB,LC and HCC groups(P=0.424)Rs7154439 genotypes had statistic differences among the chronic HBV infection,immune recovery and healthy control group(P=0.000).Genotype AA appeared more frequently in immune recovery subjects than controls(P=0.000).Comparing to chronic infection patients,the frequency of AA in the immune recovery group increased significantly(P=0.024).However,the frequency of allele A showed no significantly different among not only the chronic infection,recovery and healthy control groups(P=0.438)but also the case groups(P=0.542).3.Haplotype analysis Comparing to the chronic infection group,the frequency of haplotype rs2296651T/rs4646285G/rs7154439 G was significantly higher in immune recovery group and healthy control group(P<0.05).4.Function experiments4.1 Association analyses of the NTCP SNPs with serum TBA For rs2296651,high concentration of TBA in subjects with allele T(mutate type)was observed,compared to the one who didn't carry allele T(P=0.009).Then,we investigated the TBA level of the individual carried allele T or not in CHB group,LC group and HCC group,respectively.We found the subjects with genotype TT or CT of rs2296651 had a significantly higher serum concentration of TBA(P=0.006,P=0.009 and P=0.029,respectively)For rs4646285 and rs7154439,the TBA level showed no significant differences between the one who carried allele A or not(P>0.05).In LC and HCC group,the average concentration of TBA in mutation individual is lower,comparing to the wild types.However,the level of TBA in CHB group exhibited significantly differ between the one with wild type and mutate type(P<0.05).4.2 The differences of NTCP expression between rs7154439 wild-genotype and mutant-genotype. NTCP m RNA levels were lower in subjects carried rs7154439 allele A than those without allele A.Also,the NTCP m RNA levels in the intratumoral tissues was markedly lower than that in the peritumoral tissues.Human NTCP expression was verified by Western blot analysis.The expression of NTCP in tumor tissue was lower than in normal tissue.Nearly no NTCP was detected in rs7154439 mutant liver tissue(intratumoral tissues and peritumoral tissues).5.Association between CYP7A1 SNPs and the clinical outcomes of HBV infection The frequency distribution of rs3824260 TT,TC and CC genotypes were observed no significant difference among groups(P>0.05),however,for men,the frequency of allele C in HBV immune recovery group was significantly higher than in HBV persistent infection group(P<0.05),whereas women carried rs3824260 C allele was more likely to be found in HCC group(P<0.05);Compared with HCC group,the frequency of rs4738687 GG genotype was more likely to be observed in non-HCC group(P<0.05),and the frequency of GG genotype in non-HCC patients was significantly higher than HCC patients under a recessive model,for men,the distribution of GG genotype was significantly different among CHB,LC,HCC groups.However,in female,no significant differences in rs4738687 allele and genotype distributions were found among any groups(P>0.05).There was no significant difference in rs8192871 locus compared the genotype distribution among all groups(P>0.05),so as under the gender stratification(P>0.05).?Conclusion?1.NTCP gene polymorphism is associated with the different clinical outcomes of HBV infection in Chinese Han population: rs2296651,rs4646285 and rs7154439 mutations can promote the immune recovery after HBV infection;2.Patients carried rs2296651 allele T had a higher serum total bile acid,rs7154439 mutation may decrease the expression of NTCP protein,and thus promote the HBV immune recovery;3.CYP7A1 gene polymorphism is associated with the different clinical outcomes of HBV infection in Chinese Han population.Rs3824260 mutation has a certain gender tendency,male patients had a higher detection proportion of mutant alleles,male patients carrying rs3824260 allele C had more opportunities to reach hepatitis B rehabilitation;rs4738687 may be correlated with the occurrence of hepatocellular carcinoma(HCC),especially in male group,rs4738687 GG genotype may be negatively correlated with the occurrence and development of HCC. |
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