中国华东地区慢性乙型肝炎病毒感染的全基因组关联研究

发布时间：2018-05-20 02:14

本文选题：乙型肝炎病毒 + 全基因组关联研究　；参考：《南京医科大学》2013年博士论文

【摘要】：乙型肝炎病毒(hepatitis B virus, HBV)感染是全球重大公共卫生问题之一，目前全球约20亿人感染过HBV，我国约7-8亿人感染过HBV。免疫力正常的成人感染HBV后，超过90%可以自发清除病毒；然而低于10%的感染者会发展为慢性HBV感染，其机制不明。慢性HBV感染包括无症状携带和慢性乙型肝炎。慢性HBV感染与肝细胞癌(hepatocellular carcinoma, HCC)的发生关系密切，控制HBV感染将有助于降低HCC的发病率。HBV的慢性感染率在不同种族间也有较大差异，亚裔人群HBV慢性感染率较高，亚裔后代迁移到HBV低感染率的地区，HBV慢性感染率仍然显著高于当地人，提示遗传因素可能影响慢性HBV感染的易感性。双生子和家系研究也提示慢性HBV感染的转归与遗传因素密切相关。因此，筛选具有遗传易感性的高危个体，并对其采取针对性的预防措施或治疗手段，可能会有效的控制慢性HBV感染。单核苷酸多态性(single nucleotide polymorhphisms, SNPs)是人类最常见的遗传变异，是指在人群中频率大于1%的单个核苷酸的变异，包括转换、颠换、缺失和插入。全基因组关联研究(genome-wide association study, GWAS)采用高通量的基因分型平台，检测大样本病例和对照的全基因组水平上的SNPs，并辅以多个独立的人群研究进行后续验证，最终得到与该疾病关联最为密切的遗传易感SNPs。2009年和2011年，日本先后开展了两个慢性乙型肝炎的GWAS，最终识别出位于HLA-DP/DQ上的SNPs与慢性乙型肝炎相关。但是这两个GWAS的对照缺乏HBV感染的暴露信息，因此所发现的SNPs与慢性乙型肝炎的强关联程度可能有混杂因素影响。2012年，日本和韩国还共同开展了慢性HBV感染的GWAS，尽管对照是明确的HBV清除者，但是初筛阶段的样本量很小，仅不足200对病例/对照，统计学把握度较低，不足以系统筛选易感SNPs。此外，这三个GWAS识别出的慢性HBV感染的易感位点都是位于HLA-DP/DQ上，仅能解释慢性HBV感染的小部分易感机制；因此有必要开展一项设计优良的大样本的GWAS，以系统研究慢性HBV感染的遗传易感机制。本研究中的病例是HBV携带者，指HBsAg阳性、HBV核心抗体(HBV coreantibody, HBcAb)阳性；对照是HBV清除者，指HBsAg阴性、HBV表面抗体(HBVsurface antibody, HBsAb)和HBcAb阳性。所有研究对象均排除丙型肝炎病毒(hepatitis C virus, HCV)感染，即HCV抗体阴性。本研究采用的是三阶段病例对照研究设计：初筛阶段的1000例病例来自上海，包括500例HCC的HBV携带者和500例非HCC的HBV携带者；对照是来自张家港地区的983例HBV清除者。一期验证是来自苏南地区的1248例病例和1248例对照（来自张家港和常州地区），二期验证是来自苏中地区的1000例病例和1803例对照（来自泰州和南通地区）。本研究全基因组筛选阶段1000例病例的基因型分析采用美国Illumina公司Omni Express芯片检测，983例对照的基因型分析采用Illumina公司的Omni中华芯片检测，两张芯片重叠的SNPs（595,310个SNPs）进入质量控制。通过对研究变量(SNPs)和研究对象这两方面的质量控制后，，共951例病例（包括478例HCC的慢性HBV携带者和473例非HCC的慢性HBV携带者）和937例对照、490,610个SNPs位点进入后续分析，采用logistic回归模型计算SNPs相加模型的关联P值、关联强度比值比(odds ratio, OR)值以及95%可信区间(confidence interval, CI)。满足以下要求的位点进入一期验证：1)病例（951例）与对照（937例）关联P值≤1.0×10-5；2) HCC的病例（478例）与非HCC的病例（473例）与对照（937例）分别分析后关联P值≤1.0×10-3；3)同时满足上述两条标准的位点中具有高连锁不平衡(linkage disequilibrium, LD)(r20.8)的位点，则纳入P值最低的位点。一期验证的位点结果满足P≤0.05时将进入二期验证。最终本研究发现了两个新的慢性HBV感染易感SNPs：位于6p21.33区域的rs3130542（位于HLA-C下游, OR=1.39,95%CI=1.26-1.53, P=5.09×10-11）和位于22q11.21区域的rs4821116（位于UBE2L3上, OR=0.81,95%CI=0.75-0.87,P=1.69×10-9）。此外，我们还证实了日本人群GWAS识别出的位于6p21.32区域的rs7453920（位于HLA-DQ上）与慢性HBV感染相关(OR=0.54,95%CI=0.48-0.61, P=8.54×10-25)。HLA-DQ属于HLA II类分子，与HBV抗原递呈密切相关，rs7453920可能影响机体对HBV的适应性免疫应答；HLA-C属于HLA I类分子，rs3130542可能会影响抗HBV感染的细胞免疫途径。UBE2L3被报道与一些自身免疫性疾病相关，但与HBV感染相关的研究未见报道，其可能是HBV慢性感染新的易感基因，为阐明慢性HBV感染机制提供了新的思路。我们进一步分析了本研究发现的3个慢性HBV感染相关SNPs是否与HCC发生风险存在关联。在初筛阶段的478例HCC病例与473例非HCC病例中，3个SNPs与HCC均不存在显著关联。此外，我们在1300例独立的HBV阳性HCC病例中检测了这3个SNPs的基因型，以验证阶段的HBV携带者作为对照，也没有发现这3个SNPs与HCC存在显著关联。将上述两部分结果合并后，3个SNPs与HCC仍不存在显著关联(rs7453920: OR=0.90, P=2.39×10-1; rs3130542:OR=1.09, P=2.19×10-1; rs4821116: OR=1.03, P=6.54×10-1)。本研究发现的3个SNPs与慢性HBV感染相关，与HCC风险均不相关，提示这三个SNPs可能影响HBV感染慢性化过程，但并不参与慢性HBV感染向HCC进展的过程。本研究首次发现了位于染色体6p21.33和22q11.21区域的两个新的慢性HBV感染易感区域，同时验证了6p21.32区域与慢性HBV感染易感性的关联。本研究结果对识别和评价慢性HBV感染的遗传易感因素，指导慢性HBV感染的机制研究具有重要理论价值。本研究发现的慢性HBV感染的遗传易感标志物有望对HBV暴露高危人群的筛检及预防提供帮助。
[Abstract]:Hepatitis B virus (hepatitis B virus, HBV) infection is one of the major public health problems in the world. At present, about 2 billion people in the world have been infected with HBV. About 7-8 billion people in our country have infected HBV with normal HBV., more than 90% can remove the virus spontaneously; however, less than 10% infected people will develop into chronic HBV infection, and the mechanism is unknown. Chronic HBV infection includes asymptomatic and chronic hepatitis B. chronic HBV infection is closely related to the occurrence of hepatocellular carcinoma (HCC). The control of HBV infection will help to reduce the incidence of HCC, and the chronic infection rate of.HBV is also significantly different among the different races. The Asian population has a higher rate of chronic infection, and the Asian descendants are of Asian descendants. The rate of chronic infection of HBV is still significantly higher than that of local people in the region of low HBV infection rate, suggesting that genetic factors may affect the susceptibility to chronic HBV infection. Preventive measures or treatments may be effective in controlling chronic HBV infection.
Single nucleotide polymorhphisms (SNPs) is the most common genetic variation in human beings. It refers to the mutation of a single nucleotide in a population with frequencies greater than 1%, including conversion, transformation, deletion and insertion. The whole genome association study (genome-wide association study, GWAS) uses a high throughput genotyping platform for detection. The SNPs in the whole genome level of the sample and the control, supplemented by a number of independent population studies, was followed up with the most closely related genetic susceptibility to the disease in SNPs.2009 and 2011. In Japan, two GWAS of chronic hepatitis B were carried out, and the SNPs and chronic hepatitis B on HLA-DP/DQ were identified. Inflammation related. But the two GWAS controls lack the exposure information of HBV infection, so the strong association between SNPs and chronic hepatitis B may have mixed factors affecting.2012, and Japan and South Korea also jointly carry out the GWAS of chronic HBV infection, although the control is a clear HBV scavenger, but the initial screening stage is small, only the sample size is small. Less than 200 of cases / controls, the statistics are low, not enough to screen the susceptible SNPs.. The susceptible loci of the chronic HBV infection identified by the three GWAS are all located on the HLA-DP/DQ, which can only explain the small part of the susceptibility to chronic HBV infection; therefore, it is necessary to develop a good design large sample of GWAS for systematic study. Genetic susceptibility to chronic HBV infection.
The cases in this study were HBV carriers, HBsAg positive, HBV core antibody (HBV coreantibody, HBcAb) positive, the control was HBV scavengers, HBsAg negative, HBV surface antibody (HBVsurface antibody, HBsAb) and positive. All the subjects excluded hepatitis C virus infection, that is, the antibody negative. A three stage case-control study was designed: 1000 cases from Shanghai, including 500 HCC HBV carriers and 500 non HCC HBV carriers, and 983 HBV scavengers from the Zhangjiagang region. The first phase was verified by 1248 cases from South of Jiangsu and 1248 controls (from Zhangjiagang and Changzhou). The two phase was verified by 1000 cases from Central Jiangsu and 1803 controls (from Taizhou and Nantong).
The genotypic analysis of 1000 cases in the whole genome screening stage was detected by the Omni Express chip of Illumina company in the United States. The genotype analysis of 983 cases was detected by Omni Chinese chip in Illumina company, and SNPs (595310 SNPs) overlapped by two chips entered the quality control system. The study variable (SNPs) and the research object were two. After quality control, 951 cases (including 478 HCC chronic HBV carriers and 473 non HCC chronic HBV carriers) and 937 controls, 490610 SNPs sites entered the follow-up analysis, and the logistic regression model was used to calculate the associated P value of the SNPs additive model, the ratio of correlation intensity (odds ratio, OR) and 95% confidence interval (confid). Ence interval, CI). The loci meet the following requirements: 1) cases (951 cases) and control (937 cases) associated with P value less than 1 * 10-5; 2) HCC cases (478 cases) and non HCC cases (473 Cases) and the control (937 cases) respectively analysis the P value less than 1 * 10-3; 3) at the same time to meet the two criteria of the high linkage disequilibrium at the same time. The loci of (linkage disequilibrium, LD) (r20.8) were included in the lowest P loci. The first phase verified that the loci result would meet the P phase < 0.05 and would enter the two phase validation.
Finally, two new chronic HBV infections were found to be susceptible to SNPs: rs3130542 located in the 6p21.33 region (downstream of HLA-C, OR=1.39,95%CI=1.26-1.53, P=5.09 x 10-11) and rs4821116 located in the 22q11.21 region (on UBE2L3, OR=0.81,95%CI=0.75-0.87, P=1.69 * 10-9). Rs7453920 (located on HLA-DQ) in the 6p21.32 region is associated with chronic HBV infection (OR=0.54,95%CI=0.48-0.61, P=8.54 x 10-25).HLA-DQ belongs to the II class of HLA, which is closely related to the recursion of HBV antigens. Rs7453920 may affect the adaptive immune response to HBV. The cellular immune pathway.UBE2L3 is reported to be associated with some autoimmune diseases, but the research related to HBV infection has not been reported. It may be a new susceptible gene for chronic HBV infection and provides a new way of thinking for clarifying the mechanism of chronic HBV infection.
We further analyzed whether 3 chronic HBV infections associated with SNPs were associated with the risk of HCC. In the initial screening stage, there were no significant associations between 3 SNPs and HCC in 473 Cases of HCC and 473 non HCC cases. In addition, we detected the genotype of these 3 SNPs in 1300 independent HBV positive HCC cases. There was no significant association between the 3 HBV carriers at the stage of the syndrome, and there was no significant correlation between the 3 SNPs and the HCC. After the combination of the above two parts, there was still no significant association between 3 SNPs and HCC (rs7453920: OR=0.90, P=2.39 * 10-1; rs3130542:OR=1.09, P=2.19 x 10-1; rs4821116: OR, 10-1). V infection is not associated with HCC risk, suggesting that these three SNPs may affect the chronicity of HBV infection, but it is not involved in the progression of chronic HBV infection to HCC.
This study first discovered two new susceptible regions of chronic HBV infection located in the chromosome 6p21.33 and 22q11.21 regions, and verified the association between the 6p21.32 region and the susceptibility to chronic HBV infection. The results of this study have important theoretical value for the identification and evaluation of the genetic susceptibility to chronic HBV infection and the mechanism for guiding the slow HBV infection. The findings suggest that genetic susceptibility markers for chronic HBV infection are expected to help in screening and prevention of high-risk populations exposed to HBV.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62

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