Genetic Variation In LMP/TAP Gene And The Outcomes Of Hepatitis B Or C Virus Infection In The Chinese Population

Section 1 Genetic variation in LMP/TAP Gene and the Outcomes of Hepatitis B Virus Infection in the Chinese PopulationHepatitis B virus (HBV) infection is a major public health problem worldwide, and which threats the people's health and quality of life.The outcomes of HBV infection are extremely varied,from asymptomatic HBV carriers or cryptic hepatitis,to acute hepatitis,chronic hepatitis,liver cirrhosis or primary hepatocellular carcinoma (HCC).What produces the differences in infection,severity, and outcome? At present,most studies about reasons for this variation focus on viral load,genotype,and genetic divergence due to viral gene mutations.In fact,many investigations suggested that HBV was not a direct cytopathic disease.The injury was mediated by the immune response.The variable patterns and clinical outcomes of the infection were mainly determined by host immunological factors and genetic factors as well as the virological factors.The HBV antigen recognition by cytotoxic CD8+ cells is dependent upon a number of crucial steps in antigen processing, which include the cleavage of antigen peptides by LMP2/LMP7, the transportation into the endoplasmic reticulum by TAP1/TAP2, and the binding to human MHC class-I molecule. In this process, the recognition of HBV antigen peptides, which are derived from intracellular processing and presentation on the liver cell surface by human MHC class-I molecules, will lead to direct HBV elimination by human MHC class- I restricted CD8+ cells. If the virus evades the processing of this antigen presenting pathway, it may cause persistent or chronic infection. Therefore, we speculated that LMP/TAP genotype change (polymorphisms) may be associated with outcome of hepatitis B virus infection.[Objective] The aim of this study was to clarify whether LMP/TAP gene polymorphisms are involved in outcomes of HBV infection.[Methods] A case-control study was conducted to test the hypothesis, including a persistent group of 155 patients with chronic hepatitis B and 36 healthy carriers, a recovered group of 165 individuals spontaneously recovered from HBV infection, and an uninfected group of 278 healthy normal controls matched to the cases by age (±5years), gender, and geographical area. Genotypes of eight polymorphisms of LMP/TAP gene were analyzed by PCR-RFLP. The difference in the distribution of gender, smoking, and drinking between cases and controls was analyzed by usingχ2-test, and the age by One-Way ANOVA. The Hardy–Weinberg equilibrium was tested by usingχ2 test. The haplotype frequencies were estimated from observed positive genotypes by using the PHASE1.0 software. Odds ratios (ORs) of polymorphisms or haplotypes were estimated by using unconditional Logistic regression models. The ORs and 95% confidence intervals (95% CI) were estimated by using unconditional Logistic regression analysis that was adjusted by gender, age, smoking, and drinking. All analyses were performed with SAS software (Version 9.1.3, SAS Institute, Cary, NC). [Results] There was no significant difference in the distributions of age and gender among the recovered group, persistent group, and healthy controls (F=0.36, P=0.697;χ2=2.29, P=0.319), suggesting that our frequency-matching was adequate, but significant difference regarding smoking (χ2=36.89, P<0.001) and drinking (χ2=14.55, P=0.006) was found among the three groups. No significant difference was found between the recovered and persistent groups in age of infection (the time when they were diagnosed as HBsAg positive for the first time).Of the eight polymorphisms, two of which (TAP1 codon 637 and LMP7 codon 145) were observed to have statistically significant association with the outcomes of HBV infection (P<0.05). The two-locus haplotype constructed with two such polymorphisms was analyzed. The frequencies of haplotypes B(Asp-Lys), C(Gly-Gln), and D(Gly-Lys) were found to be increased significantly in persistent group, in comparison with that in healthy controls(OR=2.26, 95% CI=1.62–3.15, P<0.001; OR=2.37, 95% CI=1.69–3.32, P<0.001; OR=4.38, 95% CI=1.78–10.77, P=0.001, respectively). The prevalence of the haplotypes B (Asp-Lys), C (Gly-Gln), and D(Gly-Lys) were also significantly higher in the persistent infectious group than in the recovered group (OR=2.68, 95% CI=1.81–3.98, P<0.001; OR=2.40, 95% CI=1.62–3.55, P<0.001; OR=3.03, 95% CI=1.22–7.55, P=0.017, respectively).[Conclusion] These findings indicated that genetic polymorphisms of LMP/TAP gene might be an important factor in determining the outcomes of HBV infection. Section 2 The association between the genetic polymorphisms of LMP/TAP gene and the outcomes of hepatitis C virus infection in drug usersHepatitis C virus (HCV) infection is the most common bloodborne infection, with estimates of 170 million worldwide. Most (70%～80%) HCV infections persist and about 30% of individuals with persistent infection develop chronic liver disease, including cirrhosis and hepatocellular carcinoma. Environmental, viral and host factors have been associated with the differences in HCV clearance or persistence, and studies have demonstrated that a strong host immune response against HCV favors viral clearance. Thus, variation in genes involved in the immune response may contribute to the ability to clear the virus.LMP2/LMP7 gene which located between human MHC class-II DQB1 and DPB1 loci has been shown to be important in the MHC class-I antigen presentation pathway. Being a multifunctional proteasome, LMP2 and LMP7 play a pivotal role in the degradation of endogenous proteins and influence the spectrum of peptides suited for binding to human MHC class-I molecule. In HCV infection, HCV-specific CD8+ T-cell responses play essential roles in the pathogenesis of liver disease. Transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum, where they are bind the MHC-Ⅰmolecules and presented to the cell surface to cause HCV-specific CD8+ T response. TAP1 and TAP2 are likely to function as heterodimers. TAP1 and TAP2 genes are located within the major histocompatibility complex class II region. Sequence polymorphisms have been reported in genes of humans. Therefore, we speculated that LMP/TAP genotype change (polymorphisms) may be associated with outcome of hepatitis C virus infection.[Objective] The aim of this study was to clarify whether LMP/TAP gene polymorphisms are involved in outcomes of HCV infection.[Methods] The 587 drug users from Nanjing compulsory rehabilitation centers (CRCs) in this study were categorized into HCV-infected group (362 individuals), including a persistent group of 173 patients with HCV persistent infection, a recovered group of 189 individuals spontaneously recovered from HCV infection, and HCV-uninfected group of 225 individuals matched to the cases by age (±5years), gender, and geographical area. Antibodies to HCV antigens were detected by enzyme-linked immunoassay. The presence of viral particles in the serum was determined by reverse-transcriptase polymerase chain reaction (RT-PCR). The genotypes of six polymorphisms of TAP gene were analyzed by ARMS-PCR method, and genotypes of two polymorphisms of LMP gene were determined by PCR-RFLP. The difference in the distribution of gender and drug using method between cases and controls was analyzed by usingχ2-test, and the age by One-Way ANOVA. The Hardy–Weinberg equilibrium was tested by usingχ2 test. The haplotype frequencies were estimated from observed positive genotypes by using the PHASE2.0 software. Odds ratios (ORs) of polymorphisms or haplotypes were estimated by using unconditional Logistic regression models. All models included terms for study design variables (i.e. gender, age, and method of drug use). The P-value reported was two-sided and values of P<0.05 were considered statistically significant. All analyses were performed with SPSS13.0 software.[Results] The distributions of selected characteristics between HCV-infected patients and controls are summarized in Table 3. The mean age (±SD, years) was 32.63±6.18 for persistent group, 32.43±6.15 for recovered group, and 31.81±8.09 for controls. There was no significant difference in the distributions of age and gender among the recovered group, persistent group, and healthy controls (F=0.783, P=0.457;χ2=0.896, P=0.639), suggesting that our frequency-matching was adequate, but significant difference regarding drug using method (χ2=149.813, P<0.001) was found among the three groups.Of the eight polymorphisms, one of which (TAP2-379) was observed to have statistically significant association with recovered HCV infection (P<0.05), three of which (TAP1-637, TAP2-379, and LMP7-145) were observed to have statistically significant association with persistent HCV infection (P<0.05), and two of which (TAP1-637 and LMP7-145) was observed to have statistically significant association with outcomes of HCV infection (P<0.05). The extended haplotype constructed with TAP polymorphisms was analyzed among the healthy control, recovered and persistent groups. Only TAP2 haplotype H (Ile-Ala-Arg-Thr) and K (Ile- Thr-Arg-Thr) were shown to present significant difference between the recovered and control groups (OR=2.43, 95%CI=1.29-4.58, P=0.006; OR=2.69, 95%CI=1.06-6.80, P=0.037). TAP1 haplotype B (Ile-Gly), D (Val-Gly), and TAP2 haplotype E (Val-Thr-Arg-Thr), H (Ile-Ala-Arg-Thr) were shown to present significant differences between the persistent and control groups (OR=3.81, 95%CI=1.92-7.58, P<0.001; OR=1.91, 95%CI=1.20-3.02, P=0.006; OR=2.08, 95%CI=1.09-3.98, P=0.027; OR=2.38, 95%CI=1.31-4.32, P=0.004). The frequency of TAP1 haplotype B (Ile-Gly) or D (Val-Gly) was found increased significantly in persistent group, in comparison with that in recovered group (OR=2.05, 95%CI=1.19-3.54, P=0.010; OR=1.58, 95%CI=1.04-2.41, P=0.034).[Conclusion] These findings suggest a potential role of TAP2 gene as a candidate gene for susceptibility to HCV infection. And the genetic polymorphisms of TAP1/LMP7 gene may be associated with outcomes of HCV infection.