本文選題：乙型肝炎病毒 + 全基因組關(guān)聯(lián)研究　； 參考：《南京醫(yī)科大學(xué)》2013年博士論文

【摘要】：乙型肝炎病毒(hepatitis B virus, HBV)感染是全球重大公共衛(wèi)生問(wèn)題之一，目前全球約20億人感染過(guò)HBV，我國(guó)約7-8億人感染過(guò)HBV。免疫力正常的成人感染HBV后，超過(guò)90%可以自發(fā)清除病毒；然而低于10%的感染者會(huì)發(fā)展為慢性HBV感染，其機(jī)制不明。慢性HBV感染包括無(wú)癥狀攜帶和慢性乙型肝炎。慢性HBV感染與肝細(xì)胞癌(hepatocellular carcinoma, HCC)的發(fā)生關(guān)系密切，控制HBV感染將有助于降低HCC的發(fā)病率。HBV的慢性感染率在不同種族間也有較大差異，亞裔人群HBV慢性感染率較高，亞裔后代遷移到HBV低感染率的地區(qū)，HBV慢性感染率仍然顯著高于當(dāng)?shù)厝�，提示遺傳因素可能影響慢性HBV感染的易感性。雙生子和家系研究也提示慢性HBV感染的轉(zhuǎn)歸與遺傳因素密切相關(guān)。因此，篩選具有遺傳易感性的高危個(gè)體，并對(duì)其采取針對(duì)性的預(yù)防措施或治療手段，可能會(huì)有效的控制慢性HBV感染。 單核苷酸多態(tài)性(single nucleotide polymorhphisms, SNPs)是人類(lèi)最常見(jiàn)的遺傳變異，是指在人群中頻率大于1%的單個(gè)核苷酸的變異，包括轉(zhuǎn)換、顛換、缺失和插入。全基因組關(guān)聯(lián)研究(genome-wide association study, GWAS)采用高通量的基因分型平臺(tái)，檢測(cè)大樣本病例和對(duì)照的全基因組水平上的SNPs，并輔以多個(gè)獨(dú)立的人群研究進(jìn)行后續(xù)驗(yàn)證，最終得到與該疾病關(guān)聯(lián)最為密切的遺傳易感SNPs。2009年和2011年，日本先后開(kāi)展了兩個(gè)慢性乙型肝炎的GWAS，最終識(shí)別出位于HLA-DP/DQ上的SNPs與慢性乙型肝炎相關(guān)。但是這兩個(gè)GWAS的對(duì)照缺乏HBV感染的暴露信息，因此所發(fā)現(xiàn)的SNPs與慢性乙型肝炎的強(qiáng)關(guān)聯(lián)程度可能有混雜因素影響。2012年，日本和韓國(guó)還共同開(kāi)展了慢性HBV感染的GWAS，盡管對(duì)照是明確的HBV清除者，但是初篩階段的樣本量很小，僅不足200對(duì)病例/對(duì)照，統(tǒng)計(jì)學(xué)把握度較低，不足以系統(tǒng)篩選易感SNPs。此外，這三個(gè)GWAS識(shí)別出的慢性HBV感染的易感位點(diǎn)都是位于HLA-DP/DQ上，僅能解釋慢性HBV感染的小部分易感機(jī)制；因此有必要開(kāi)展一項(xiàng)設(shè)計(jì)優(yōu)良的大樣本的GWAS，以系統(tǒng)研究慢性HBV感染的遺傳易感機(jī)制。 本研究中的病例是HBV攜帶者，指HBsAg陽(yáng)性、HBV核心抗體(HBV coreantibody, HBcAb)陽(yáng)性；對(duì)照是HBV清除者，指HBsAg陰性、HBV表面抗體(HBVsurface antibody, HBsAb)和HBcAb陽(yáng)性。所有研究對(duì)象均排除丙型肝炎病毒(hepatitis C virus, HCV)感染，即HCV抗體陰性。本研究采用的是三階段病例對(duì)照研究設(shè)計(jì)：初篩階段的1000例病例來(lái)自上海，包括500例HCC的HBV攜帶者和500例非HCC的HBV攜帶者；對(duì)照是來(lái)自張家港地區(qū)的983例HBV清除者。一期驗(yàn)證是來(lái)自蘇南地區(qū)的1248例病例和1248例對(duì)照（來(lái)自張家港和常州地區(qū)），二期驗(yàn)證是來(lái)自蘇中地區(qū)的1000例病例和1803例對(duì)照（來(lái)自泰州和南通地區(qū)）。 本研究全基因組篩選階段1000例病例的基因型分析采用美國(guó)Illumina公司Omni Express芯片檢測(cè)，983例對(duì)照的基因型分析采用Illumina公司的Omni中華芯片檢測(cè)，兩張芯片重疊的SNPs（595,310個(gè)SNPs）進(jìn)入質(zhì)量控制。通過(guò)對(duì)研究變量(SNPs)和研究對(duì)象這兩方面的質(zhì)量控制后，，共951例病例（包括478例HCC的慢性HBV攜帶者和473例非HCC的慢性HBV攜帶者）和937例對(duì)照、490,610個(gè)SNPs位點(diǎn)進(jìn)入后續(xù)分析，采用logistic回歸模型計(jì)算SNPs相加模型的關(guān)聯(lián)P值、關(guān)聯(lián)強(qiáng)度比值比(odds ratio, OR)值以及95%可信區(qū)間(confidence interval, CI)。滿(mǎn)足以下要求的位點(diǎn)進(jìn)入一期驗(yàn)證：1)病例（951例）與對(duì)照（937例）關(guān)聯(lián)P值≤1.0×10-5；2) HCC的病例（478例）與非HCC的病例（473例）與對(duì)照（937例）分別分析后關(guān)聯(lián)P值≤1.0×10-3；3)同時(shí)滿(mǎn)足上述兩條標(biāo)準(zhǔn)的位點(diǎn)中具有高連鎖不平衡(linkage disequilibrium, LD)(r20.8)的位點(diǎn)，則納入P值最低的位點(diǎn)。一期驗(yàn)證的位點(diǎn)結(jié)果滿(mǎn)足P≤0.05時(shí)將進(jìn)入二期驗(yàn)證。 最終本研究發(fā)現(xiàn)了兩個(gè)新的慢性HBV感染易感SNPs：位于6p21.33區(qū)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區(qū)域的rs4821116（位于UBE2L3上, OR=0.81,95%CI=0.75-0.87,P=1.69×10-9）。此外，我們還證實(shí)了日本人群GWAS識(shí)別出的位于6p21.32區(qū)域的rs7453920（位于HLA-DQ上）與慢性HBV感染相關(guān)(OR=0.54,95%CI=0.48-0.61, P=8.54×10-25)。HLA-DQ屬于HLA II類(lèi)分子，與HBV抗原遞呈密切相關(guān)，rs7453920可能影響機(jī)體對(duì)HBV的適應(yīng)性免疫應(yīng)答；HLA-C屬于HLA I類(lèi)分子，rs3130542可能會(huì)影響抗HBV感染的細(xì)胞免疫途徑。UBE2L3被報(bào)道與一些自身免疫性疾病相關(guān)，但與HBV感染相關(guān)的研究未見(jiàn)報(bào)道，其可能是HBV慢性感染新的易感基因，為闡明慢性HBV感染機(jī)制提供了新的思路。 我們進(jìn)一步分析了本研究發(fā)現(xiàn)的3個(gè)慢性HBV感染相關(guān)SNPs是否與HCC發(fā)生風(fēng)險(xiǎn)存在關(guān)聯(lián)。在初篩階段的478例HCC病例與473例非HCC病例中，3個(gè)SNPs與HCC均不存在顯著關(guān)聯(lián)。此外，我們?cè)?300例獨(dú)立的HBV陽(yáng)性HCC病例中檢測(cè)了這3個(gè)SNPs的基因型，以驗(yàn)證階段的HBV攜帶者作為對(duì)照，也沒(méi)有發(fā)現(xiàn)這3個(gè)SNPs與HCC存在顯著關(guān)聯(lián)。將上述兩部分結(jié)果合并后，3個(gè)SNPs與HCC仍不存在顯著關(guān)聯(lián)(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)。本研究發(fā)現(xiàn)的3個(gè)SNPs與慢性HBV感染相關(guān)，與HCC風(fēng)險(xiǎn)均不相關(guān)，提示這三個(gè)SNPs可能影響HBV感染慢性化過(guò)程，但并不參與慢性HBV感染向HCC進(jìn)展的過(guò)程。 本研究首次發(fā)現(xiàn)了位于染色體6p21.33和22q11.21區(qū)域的兩個(gè)新的慢性HBV感染易感區(qū)域，同時(shí)驗(yàn)證了6p21.32區(qū)域與慢性HBV感染易感性的關(guān)聯(lián)。本研究結(jié)果對(duì)識(shí)別和評(píng)價(jià)慢性HBV感染的遺傳易感因素，指導(dǎo)慢性HBV感染的機(jī)制研究具有重要理論價(jià)值。本研究發(fā)現(xiàn)的慢性HBV感染的遺傳易感標(biāo)志物有望對(duì)HBV暴露高危人群的篩檢及預(yù)防提供幫助。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R512.62

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