【作者】 潘丽萍；

【导师】 刘英；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2012， 博士

【摘要】 研究背景乙型肝炎病毒(hepatitis B virus, HBV)感染是全世界重要的公共卫生问题。中国是乙肝表面抗原的高度暴露区,乙肝表面抗原携带者约有1亿,每年约有30万人死于乙肝感染相关疾病。疫苗接种是目前预防乙肝的有效措施。然而,约有5-10%的个体在接种疫苗后不能产生保护性抗体。个体对疫苗的应答差异为乙肝预防带米了困难。目前多项研究表明宿主遗传因素与乙肝疫苗免疫低/无应答之间存在关联。本研究采用病例对照的研究设计,分别应用基于功能基因候选的关联研究方法和全基因组关联研究方法,从遗传学角度分析乙肝疫苗免疫低/无应答的易感基因/位点。研究方法收集完成乙肝疫苗基础免疫的北京和山东成人初免人群。以北京214例初免高应答者(anti-HBs≥1000mIU/ml)和107例初免低应答者(anti-HBs:10-99mIU/ml)为受试者,采用聚合酶链反应-限制性片段长度多态性(Polymerase Chain Reaction-Restriction Fragment Length Polymorphism, PCR-RFLP),序列特异性引物-聚合酶链反应(Polymerase Chain Reaction with Sequence-Specific Primers, PCR-SSP),及TaqMan(?)探针(Probe-based Real-time Polymerase Chain Reaction)方法,分析细胞因子及其受体基因的10个单核苷酸多态性(single nucleotide polymorphism, SNP)位点与乙肝疫苗免疫低应答的相关性。以北京214例初免高应答者和107例初免低应答者为初筛阶段受试者,以山东1090例初免高应答者和636例初免低应答者为验证阶段受试者,应用Sequenom(?)质谱(iPLEX MassARRAY system)和TaqMan(?)探针方法分析位于T细胞辅助受体及共刺激分子基因的111个SNP位点与乙肝疫苗免疫低应答的相关性。以山东78例再免无应答者为病例组(初免无应答者接受第二轮基础免疫后,表面抗体水平仍低于10mIU/ml的个体),依据年龄、性别比例随机选择山东113例初免高应答者作为对照组,应用Illumina_HumanOmniExpress SNP芯片,在全基因组水平上分析与乙肝疫苗免疫无应答相关的SNP位点。后续以山东1122例初免高应答者和374例初免无应答者作为第一组验证受试者(验证Ⅰ),以北京214例初免高应答者和46例初免无应答者作为第二组验证受试者(验证Ⅱ),应用TaqMan(?)探针法对全基因组关联研究结果进行验证。研究结果1.细胞因子及其受体基因的多态性与乙肝疫苗免疫低应答的相关性以北京成人214例初免高应答者为对照,107例初免低应答者为病例进行关联研究。结果表明：位于IL12B基因启动子区的rs17860508位点,其劣势等位基因CTCTAA的频率分布在初免低应答组中显著高于初免高应答组(53.3%vs.44.6%, P=0.039,OR=1.41,95%CI=1.00-1.99)。经Logistic回归调整混杂因素后,rs17860508位点的基因型频率分布在初免高应答和初免低应答组中的差异有统计学意义(P=0.033,OR=1.84,95%CI=1.05-3.21)。同时携带IL12B基因rs17860508危险基因型(CTCTAA/CTCTA)和IL12A基因rs2243115危险基因型(TT)的个体,其频率分布在初免低应答组中显著高于初免高应答组(P=0.008,OR=2.19,95%CI=1.23-3.93)。2.T细胞辅助受体和共刺激分子基因多态性与乙肝疫苗免疫低应答的相关性在初筛阶段(北京成人214例初免高应答者和107例初免低应答者),单位点分析发现位于CD3Z基因的rs12133337*C和位于OX40L基因的rs10912564*T与乙肝疫苗免疫低应答相关(P=0.008,OR=2.02,95%CI=1.15-3.53；P=0.019,OR=2.60.95%CI=1.07-6.37)。位于CD3Z基因的rs10918706*T与乙肝疫苗免疫高应答相关(P=0.041,OR=0.68,95%CI=0.46-1.00)。后续在验证阶段(山东成人1090例初免高应答者和636例初免低应答者)对以上3个SNP位点进行验证,发现位于CD3Z基因的rsl2133337*C仍然与乙肝疫苗免疫低应答相关(P=0.033,OR:1.28,95%CI=1.01-1.61)。分层分析显示,在体重指数(Body Mass Index,BMI)低于25kg/m2的初筛和验证阶段受试者中,rs12133337*C与乙肝疫苗免疫低应答的关联性更加明显(初筛阶段：P=0.003；验证阶段：P=0.002)。3.全基因组关联研究结果在初筛阶段(山东108例再免无应答者为病例,77例初免高应答者为对照),单位点分析发现：经过FDR_BH多重检验后,位于HLA-DR区域的27个SNP位点与乙肝疫苗再免无应答相关(P-多重校正<0.05)。其中4个SNP(rs477515,rs3763316, rs28366298, rs13204672)与其余23个SNP之间存在连锁不平衡(Linkage disequilibrium,LD)。后续在验证阶段(验证Ⅰ：山东1122例初免高应答者和374例初免无应答者：验证Ⅱ：北京214例初免高应答者和46例初免无应答者)对以上4个SNP位点进行验证分析,发现在两组验证中该4个SNP均与乙肝疫苗初免无应答相关(验证Ⅰ：P=2.82e-010～9.44e-07；验证Ⅱ：P=1.58e-04～0.048)。结论1.IL12B基因rs17860508位点的劣势等位基因CTCTAA是乙肝疫苗免疫低应答的危险因素。同时携带IL12B基因rs17860508位点危险基因型CTCTAA/CTCTAA和IL12A基因rs2243115位点危险基因型TT更容易产生乙肝疫苗免疫低应答。2.CD3Z基因rs12133337位点的劣势等位基因C是乙肝疫苗免疫低应答的危险因素。低水平的体重指数(BMI)可能增强rs12133337位点对免疫应答的影响。3.HLA-DR区域的4个SNP(rs477515,rs28366298,rs3763316,rs13204672)均与乙肝疫苗免疫无应答相关；单倍型CACA是乙肝疫苗免疫应答的保护因素；HLA-DR区域是乙肝疫苗免疫无应答的易感区域。更多还原

【Abstract】 BackgroundsInfection with hepatitis B virus is (HBV) is a public health problem that seriously threatens human life. Approximately93million Chinese people are carriers of HBV, and300,000deaths occur every year in China as a result of HBV infection. Recombinant vaccines against HBV have been used since the1980s and are an effective method of preventing HBV infection and transmission. However,5-10%of healthy adults fail to produce protective levels of antibody against the hepatitis B vaccine (anti-HBs). The differed outcomes of immune response to hepatitis B vaccination bring out difficulties in HBV prevention. Up to now, several studies have documented that genetic polymorphisms were associated with immune response to hepatitis B vaccination. In the present study, case-control study was used to evaluate the potential SNPs association with immune response to hepatitis B vaccination.MethodsIndividuals from Beijing and Shandong Province were administered three doses of hepatitis B vaccine at0,1, and6months. In Beijing population, a total of10single nucleotide polymorphisms distributed in6cytokine and cytokine receptor genes (TNFRSF1A, IL12A, IL12B, IFNG, IL4, and IL10) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), polymerase chain reaction with sequence-specific primers (PCR-SSP), and TaqMan-MGB probe-based real-time polymerase chain reaction, among214high-responders (anti-HBs>1,000mIU/ml) and107low-responders (anti-HBs:10-99mIU/ml) to three dose of hepatitis B vaccine.A total of111single nucleotide polymorphisms (SNPs) in17genes that encodes T cell co-receptors and costimulatory molecules, were analyzed using the iPLEX MassARRAY system, among214high-responders and107low-responders to three dose of hepatitis B vaccine. The SNPs with P values<0.05were selected for follow-up analysis with the TaqMan-MGB or TaqMan-BHQ probe-based real-time polymerase chain reaction in Shandong population, which included1090high-responders and636-low responders to three dose of hepatitis B vaccine.A Genome Wide Association Study (GWAS) of non-response to hepatitis B vaccination was performed using Illumina_HumanOmniExpress SNP Array among78non-responders to six dose of hepatitis B vaccine and113high responders to three dose of hepatitis B vaccine. The SNPs with P-correction<0.05(FDR_BH multiple correction) were selected for follow-up analysis with the TaqMan-MGB or TaqMan-BHQ probe-based real-time polymerase chain reaction among high responders and non-responders to three dose of hepatitis B vaccine in Shandong (1122vs.374) and Beijing (214vs.46) population.Results1. Association of polymorphisms in cytokine and cytokine receptor genes with immune response to hepatitis B vaccinationThe minor allele’CTCTAA; of rs17860508in the IL12B gene was associated with a low response to hepatitis B vaccination (P=0.039, odds ratio=1.41,95%confidence interval=1.00-1.99). In addition, a significant gene-gene interaction was found:the frequency of the combined genotypes (IL12A rs2243115’TT’and IL12B rs17860508’CTCTAA/CTCTAA’) was significantly higher in the low-response group than in the high-response group(P=0.008, odds ratio=2.19,95%confidence interval=1.23-3.93).2. Association of polymorphisms in genes that encode T cell co-receptors and costimulatory molecules with immune response to hepatitis B vaccinationThree SNPs (rs12133337and rs10918706in the CD3Z gene, rs10912564in the OX40L gene) were associated significantly with the immune response to hepatitis B vaccination in Beijing population (P=0.008,0.041, and0.019, respectively). Confirmation study of these three SNPs was performed in Shandong population, the minor allele’C’of rs12133337continued to show an association with a lower response to hepatitis B vaccination (P=0.033, odds radio=1.28,95%confidence interval=1.01-1.61). Furthermore, in the stratified analysis for both the Beijing and Shandong populations, the association of the minor allele’C’ of rs12133337with a lower response to hepatitis B vaccination was more prominent after individuals who were overweight or obese (body mass index≤25kg/m2) were excluded (Beijing population:P=0.003; Shandong population:P=0.002).3. GWAS of non-response to hepatitis B vaccinationTwenty-seven SNPs in HLA-DR region were associated with non-response to booster hepatitis B vaccination after FDR_BH multiple corrections (P<0.05). Linkage disequilibrium analysis showed that other23SNPs were tagged by4SNPs (rs477515, rs3763316, rs28366298, rs13204672). Confirmation studies of these four SNPs were performed in Shandong and Beijing primary immune populations. These four SNPs were also associated with non-response to hepatitis B vaccination in both the Shandong and Beijing populations (Shandong population:P=2.82e-010-9.44e-07; Beijing population: P=1.58e-04-0.048).Conclusions1. The minor allele’CTCTAA’of rsl7860508in the IL12B gene is associated with susceptibility to a low response to hepatitis B vaccination. In addition, interaction between rs2243115in IL12A and rs17860508in IL12B enhances the risk of a low response to the hepatitis B vaccination.2. Polymorphism (rsl2133337) in the CD3Z gene might affect the immune response to hepatitis B vaccination, and that a lower BMI might increase the contribution of the polymorphism to immunity to hepatitis B vaccination.3. Four SNPs (rs477515, rs28366298, rs3763316, rsl3204672) located in HLA-DR region were significantly associated with non-response to hepatitis B vaccination; Haplotype’CACA’was a protective factor in immune response to hepatitis B vaccination; Polymorphisms in the HLA-DR region might affect the immune response to hepatitis B vaccination.更多还原