乙肝易感基因的关联分析及其功能研究

发布时间：2018-03-17 18:31

本文选题：乙肝病毒　切入点：感染　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：乙肝病毒(Hepatitis B virus,HBV)感染是一种严重影响人类健康的疾病。据估计,全世界目前约有2.4亿HBV感染者。双生子研究发现,宿主的遗传组成是影响乙肝相关性肝病的重要因素之一。最近,全基因组关联性研究(Genome-wide association study,GWAS)报道了STAT 基因多态性与乙肝感染及其相关性肝癌显著关联,在功能上该基因参与了机体的免疫反应过程,揭示STAT4是乙肝相关性疾病的一个候选基因。在本论文的第一部分内容,我们探讨了在中国汉族人群中STAT4基因多态性与乙肝相关性肝病的关联性。我们首先基于HapMap数据库挑选出位于STAT4上的13个SNP位点,并在3033个个体中对它们进行基因分型。我们发现rs7574865位点与乙肝病毒感染(OR= 1.15,95%CI = 1.00-1.31,P=0.046),以及病毒清除显著关联(OR= 1.17,95%CI= 1.02-1.33,P=0.028)。为了增加该结论的可靠性,我们从数据库上发表的文章中分别收集了 8944个慢性乙肝病毒感染者(Chronic hepatitis B virus infection,CHBVI)患者,8451 个健康人群和2081个病毒清除者进行Meta分析,同样发现rs7574865位点和HBV感染(固定效应模型和随机效应模型下:pooled OR =1.14,pooled P = 3.8 ×10-5)与病毒清除(固定效应模型下:pooled OR = 1.20,pooled P=0.002;随机效应模型下:pooled OR =1.25,pooled P = 0.044)有着显著的关联关系。进一步的单倍型分析发现,由 rs8179673,rs7574865,rs4274624,rs11889341 和 rs10168266 组成的单倍型CTCTT,不仅与 HBV 感染显著关联(OR = 0.87,95%CI = 0.76-0.99,P = 0.022),而且还与病毒清除有明显相关(OR = 0.86,95%CI = 0.75-0.99,P= 0.018)。利用生物信息学方法对这个单倍型内的SNP位点进行功能预测,发现它们均可能与STAT4基因表达调节有关,但这需要进一步的功能验证。综上,我们的研究首次证明了在中国汉族人群中STAT4不仅与HBV感染相关,而且还与感染后病毒的清除也显著关联。然而,通过GWAS发现的SNP位点,到目前为止也只能解释疾病遗传的一小部分,提示还有许多与疾病相关的基因有待进一步发现。在本论文的第二部分,我们采用了新的研究策略来寻找与HBV感染相关的易感位点。我们首先在300对同胞对和3087个病例-对照人群样本中进行遗传关联性研究,发现了位于31个基因上的36个SNP位点在两个样本人群中均名义上与HBV感染相关,且方向一致。对于这些基因,我们挑选SEC24D作为候选基因进一步研究,主要原因为:第一,来自乙肝病毒感染的原代肝细胞表达谱分析发现,SEC24D表达水平随着病毒感染时间的推移而不断升高(P=4.0×10-4);第二,SEC24D基因多态性位点rs76459466显著与乙肝病毒感染风险呈负相关(ORmeta = 0.82,Pmeta= 2.0 ×10-3);遗传关联分析结果和表达数据提示我们SEC24D可能是与HBV感染相关的一个潜在易感基因。基于此,我们设计了体外实验从细胞学上探讨该基因与HBV感染的相关性。结果发现,当SEC24D表达水平升高时,HBV感染相关标记物显著下降;而当抑制该基因的表达水平时,HBV感染相关标记物显著升高;提示SEC24D基因起到抵抗乙肝病毒复制的作用。另外,在土拨鼠动物模型中,我们发现在病毒感染组中,肝细胞SEC24D基因表达水平显著低于非感染组(P=2.0 × 10-3),支持了上述细胞学研究的结果。综上所述,我们认为SEC24D是新的与HBV感染相关的一个潜在易感基因。总而言之,我们不但首次在中国汉族人群中验证了 STAT4基因多态性与HBV感染显著相关,而且还发现其与病毒清除也存在明显关联。此外,我们还利用新的遗传研究手段,采用整合功能基因组学的方法发现了新的与HBV感染相关的基因SEC24D。但是,这些发现还需要进一步功能学实验,以深入其内在机制。
[Abstract]:Hepatitis B virus (Hepatitis B, virus, HBV) infection is a serious disease affecting human health. It is estimated that the world currently has about 240 million people infected with HBV. The twin study found that host genetic composition is one of the important factors affecting the liver. Recently, whole genome association studies (Genome-wide association study, GWAS) reported that the STAT gene polymorphism and hepatitis B infection and associated hepatocellular carcinoma was significantly associated in function, the genes involved in the immune response of the organism, reveals that STAT4 is a candidate for the base of hepatitis B related diseases. In the first part of this thesis, we investigate the China in Han population STAT4 gene polymorphism and the liver Association. We firstly based on HapMap from a database of 13 SNP sites located in STAT4, which 3033 individuals were genotyped. They found the site of rs7574865 and hepatitis B virus infection (OR= 1.15,95%CI = 1.00-1.31, P=0.046), and viral clearance was significantly associated (OR= 1.17,95%CI= 1.02-1.33, P=0.028). In order to increase the reliability of the conclusion, we published from database articles were collected from 8944 patients with chronic hepatitis B virus infection (Chronic hepatitis B virus infection, CHBVI) patients, 8451 healthy people and 2081 virus clearance were analyzed by Meta, rs7574865 and HBV also found that the sites of infection (fixed effect model and random effect model: pooled OR = 1.14, pooled = 3.8 * 10-5 P) with viral clearance (fixed effect model: pooled OR = 1.20, pooled P=0.002; random effects model: pooled OR =1.25 pooled, P = 0.044) have a significant relationship. Further haplotype analysis identified by rs8179673, rs7574865, rs4274624, rs11889341 and rs1016826 6 haplotype CTCTT, not only significantly associated with HBV infection (OR = 0.87,95%CI = 0.76-0.99, P = 0.022), but also with viral clearance was significantly correlated (OR = 0.86,95%CI = 0.75-0.99, P= 0.018). Using bioinformatics methods of this haplotype within the SNP locus was predicted, and found that they are likely to STAT4 gene expression regulation, but it needs further verification. In summary, our study has demonstrated that STAT4 is not only associated with HBV infection in China Han population, but also clear and virus infection was significantly associated. However, SNP loci detected by GWAS, so far only explain a small fraction of there are many genetic diseases, suggesting that genes associated with disease remains to be found. In the second part of this paper, we adopted a new strategy to find related to HBV infection and a susceptible We first point. In 300 sib pairs and 3087 case-control genetic association studies of population samples, found in the 36 SNP loci of 31 genes in a sample of two people were nominally associated with HBV infection, and the same direction. For these genes, we choose SEC24D as a further study the candidate gene, main reasons: first, primary hepatocyte expression analysis found that hepatitis B virus infection, the expression level of SEC24D increased continuously with the infection time (P=4.0 * 10-4); second, SEC24D gene polymorphism sites were rs76459466 and hepatitis B virus infection was inversely related to the risk (ORmeta = 0.82. Pmeta= 2 x 10-3); genetic correlation analysis results and expression data indicates that SEC24D may be related to HBV infection and a potential susceptible gene. Based on this, we designed in vitro cytological study The correlation between the gene and HBV infection. The results showed that the expression level of SEC24D, HBV infection markers decreased significantly; while inhibiting the expression level of the gene, HBV infection markers were significantly increased; suggesting that the SEC24D gene plays a role in the resistance of hepatitis B virus replication. In addition, the woodchuck animal model we found that, in the virus infection group, the liver cells SEC24D gene expression level was significantly lower than the non infection group (P=2.0 * 10-3), support the cytological results. In summary, we believe that the new SEC24D is related to HBV infection and a potential susceptible gene. In short, we not only for the first time in China Han population verify the STAT4 gene polymorphism is significantly correlated with HBV infection, but also found that there are obvious correlation with viral clearance. In addition, we also use genetic research a new method, using the integration function Genomics approaches the discovery of a new gene SEC24D. associated with HBV infection, but these findings also require further functional experiments in order to penetrate into the internal mechanism.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R512.62

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