HBV相关慢加急性肝衰竭的全基因组关联研究及主要位点HLA-DR功能解析

发布时间：2018-05-17 20:08

  本文选题：慢加急性肝衰竭 + 人类白细胞抗原　； 参考：《第三军医大学》2016年博士论文

【摘要】：HBV相关慢加急性肝衰竭(HBV-related acute-on-chronic liver failure,HBV-ACLF)是我国及亚太地区慢性肝病患者常见的危急重症,临床上表现为短期内出现高胆红素血症、凝血酶原时间延长(INR≥1.5),起病迅速,病死率高,缺乏特异治疗手段。目前认为HBV-ACLF的发生主要与宿主遗传因素、病毒以及宿主免疫相互作用有关,但是其确切的发病机制尚不明确。近年来,西方基于酒精性肝硬化的ACLF的研究报道较多,认为酒精性肝硬化相关ACLF患者从急性发作、炎症损伤、免疫失调到系统性炎症反应综合征(systematic inflammatory response syndrome,SIRS)、代偿性抗炎反应综合征(compensatory anti-inflammatory response syndrome,CARS)、再生反应等多环节事件的幅度和次序,决定着患者是炎症消退-康复还是进展到肝外器官衰竭,系统性炎症反应是酒精性肝硬化患者发生ACLF的主要驱动因素。慢性HBV感染者是否发生肝炎发作,发作后是否进展为肝衰竭,由于HBV缺乏慢性感染的动物模型,且ACLF的病因和诱因存在很大的异质性,目前对HBV相关ACLF的肝脏免疫学应答及免疫病理过程仍然知之甚少。既往针对HBV病毒序列变异的研究认为C基因启动子区、前C区和C区变异与乙型肝炎的发作及暴发性肝衰竭相关。但是,同样的前C、C区变异既可以见于不同的病变表型(普通乙型肝炎、肝硬化、肝癌),也可见于无症状携带者。事实上,病毒变异是否导致乙型肝炎重症化的发生和发展,受到宿主因素的制约。乙型肝炎的发生、发展和转归,是病毒(病毒载量、变异、进化)和宿主(遗传异质性、年龄、性别等)通过免疫应答相互作用导致的常见复杂疾病。常见复杂疾病(common complex diseases)通常由许多微效累加基因与环境因素共同作用而决定的。HBV-ACLF的发生、发展,涉及到免疫识别、炎症活化/放大、肝细胞坏死、SIRS、CARS、器官衰竭等多个环节,也属于常见复杂疾病的范畴,其遗传因素也涉及多个微效基因的作用。在复杂疾病遗传易感性研究中,关联研究-连锁不平衡分析方法最为常用,统计效能也远高于家系连锁分析。基于全基因组策略的遗传关联研究,是目前复杂疾病遗传关联研究的主流,与候选基因策略相比其优点在于统计效能有着极大的提高,同时可以避免群体分层偏倚和基因选择的偶然性,可以从全基因组角度获得复杂疾病遗传特征的全局、系统的认识。开展HBV-ACLF表型的全基因组关联研究(genome-wide association study,GWAS),解析其全基因组范围内的宿主遗传位点及遗传关联图谱,有助于认识HBV-ACLF的驱动机制及干预靶点。因此,本课题在建立HBV-ACLF病例大样本、多中心队列的基础上,对其临床特征及炎症因子谱进行了分析,开展了HBV-ACLF的全基因组关联研究,并对主要关联位点HLA-DR进行了精细解析和功能注释,以及阳性关联HLA等位对HBV变异的限制性分析。本研究结果首次从全基因组角度明确了HBV-ACLF的主要遗传位点,提示HLA-DR等位限制性CD4+T细胞途径对HBV-ACLF的驱动作用。本课题的主要研究结果如下:1.共纳入1300例HBV-ACLF患者和2087例HBV无症状携带者进入3-stage的全基因组关联研究,样本来自重庆、北京、泉州、遵义、南宁5个中心。所有患者均无抗病毒史、无既往肝炎发作或肝功能失代偿病史。对1300例HBV-ACLF患者进行临床特征分析,LC-ACLF占60%左右,NLC-ACLF占40%;HBe Ag阴性的HBV-ACLF占60%,HBe Ag阴性并且HBV DNA104 IU/m L的HBV-ACLF占17.1%。与LC-ACLF相比,NLC-ACLF患者表现为更年轻(平均年龄39.5 vs.43.9岁,p=3.14×10-11)、更高的ALT水平(平均值1153 vs.809 IU/L,p=1.36×10-11)、腹水阳性率较低(58.7%vs.88.4%,p=9.25×10~(-35))、起病后更短时间内INR达到1.5及发生脑病(平均值分别19.8vs.24.8天,p=1.36×10-11;27.3 vs.38.1天,p=5.16×10-6)。2.对1013份患者血清样本进行35种细胞因子的多重定量检测,结果显示,国外酒精性肝硬化ACLF研究所关注的重要指标PCT(提示细菌感染)和CRP(提示肝外器官衰竭),在我们HBV-ACLF患者中其浓度低于普通慢性乙型肝炎(CHB)患者。与慢性乙型肝炎轻中度及重度相比,HBV-ACLF患者显著升高的细胞因子有6种:Th1型细胞因子IL-1a、TNF-β,Th2型细胞因子IL-5、IL-10和IL-13,Th17型细胞因子IL-17。3.HBV-ACLF的GWAS初始阶段分析显示全基因组范围内1、6、12号染色体有显著的关联信号(p1×10-5),LC-ACLF和NLC-ACLF两种亚型的共同遗传关联位点位于6号染色体HLA-II区域。经过replication 1a和1b独立样本验证表明HLA-DR区域是主要关联位点,再经过replication 2a和2b验证,明确了rs3129859与HBV-ACLF显著相关(P combine=7.40×10-19,OR=1.83)。4.分层分析显示rs3129859是HBV-ACLF的独立风险因素,与慢性乙型肝炎活动状态和HBV再活化无关。风险等位rs3129859*C与HBV-ACLF临床进程相关,携带风险等位rs3129859*C的ACLF患者和S-CHB患者,在入院28天时INR达到1.5及发生腹水的风险更高(分别有p=3.95×10-4,p=3.03×10-4)。在NLC-ACLF亚组,携带风险等位rs3129859*C的患者,28天死亡率更高(p=0.03)。5.HLA-DRB1*1202等位是HBV-ACLF最显著的风险等位(p=3.94×10-6,OR=2.05)。HLA-DRB1*1202的全球分布与HBV-ACLF地理流行病学趋势一致,在中国(5.9-21.7%)和东南亚地区(6.8-35.3%)等位频率最高。6.HLA-DRβ第28位氨基酸是最显著的关联氨基酸(p=3.03×10-6,OR=1.75)。其次,关联的氨基酸还包括HLA-DRβ蛋白第26、30、32、37、38和85位氨基酸,以及HLA-DQα蛋白第40、47、50、51、53、56、69、76位氨基酸和HLA-DQβ蛋白第45位氨基酸(p0.001)。蛋白结构三维重建显示这些关联氨基酸位于HLA分子抗原结合沟槽内。7.等位单倍型rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301和氨基酸单倍型DRβ-LEHHLLA-DQα-GCVLQdel TL-DQβ-E是风险单倍型,携带风险单倍型的HBV感染者,罹患ACLF的风险更高(additive model,分别p=1.16×10-4,OR=1.94;p=2.43×10-6,OR=2.10)。8.生存分析提示风险等位及风险单倍型与HBV-ACLF临床进程相关。在非肝硬化ACLF亚组,携带风险单倍型rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301的患者入院后28天(p=3.49×10-4)、90天(p=0.003)及180天(p=0.003)的死亡率更高,且肝性脑病发生率更高(p=0.019),氨基酸单倍型也观察到一致的相关性。9.HBV-ACLF关联位点功能注释:e QTL分析显示关联位点rs3129859与HLA-II基因表达相关;GRAIL文献分析显示HLA-DR和HLA-DQ是最合理的关联基因。与其他HBV相关GWAS文献overlap分析,显示HBV-ACLF的遗传关联位点与慢性HBV感染的遗传关联位点有部分重叠。10.通过分析HLA-DRB1*1202风险等位对HBV变异的限制作用,鉴定出12个HBV变异,既表现出HBV-ACLF风险等位HLA-DRB1*1202特异性和肝炎活化特异性,同时也是病毒发生正选择的热点氨基酸位置。表位预测分析发现,这些变异可引发病毒表位漂移。结论:1.我们的数据对于认识HBV-ACLF的自然史有重要意义:相当比例的慢性HBV感染者可在无肝硬化基础的情况下发生ACLF;HBV自然史处于低复制期和e抗原阴性期的患者,发生ACLF的风险不容忽视;NLC-ACLF的疾病进展更为迅速,HBV-ACLF发病年龄(42岁)远低于欧洲酒精性肝硬化ACLF患者(56岁)。2.HBV-ACLF患者血清炎症因子谱分析表明HBV-ACLF患者确实存在剧烈的SIRS和CARS并存局面。HBV-ACLF患者CD4 Th细胞因子通路剧烈活化,有别于慢性乙型肝炎轻中度和重度,也不同于欧洲酒精性肝硬化ACLF患者。3.NLC-ACLF和LC-ACLF亚型具有共同的遗传易感位点,亚型位点、定量性状位点(肝酶、性激素等)与HBV-ACLF位点通路存在交互作用。4.HLA-DR是HBV-ACLF的主要遗传易感位点,风险等位rs3129859*C和HLA-DRB1*1202可作为HBV-ACLF临床预警预后的marker。风险等位有特异限制的病毒变异位点,与肝炎活化、病毒正选择和表位漂移有关。总之,本研究首次在HBV相关慢加急性肝衰竭患者中开展了全基因组关联研究,从全基因组范围内鉴定出HLA-DR是HBV-ACLF的主要关联位点,且关联的风险等位和单倍型与HBV-ACLF临床进程显著相关,提示了HLA-II类分子限制的CD4+T细胞途径在HBV-ACLF的免疫病理过程中的重要作用,同时也表明肝硬化和非肝硬化基础的HBV-ACLF为同质性疾病,支持一个疾病的假说(one disease hypothesis)。
[Abstract]:HBV related chronic acute liver failure (HBV-related acute-on-chronic liver failure, HBV-ACLF) is a common critical disease in the patients with chronic liver disease in China and the Asia Pacific region. The clinical manifestations include hyperbilirubinemia in the short term, prolonged prothrombin time (INR > 1.5), rapid onset, high mortality, and lack of specific treatment. At present H The occurrence of BV-ACLF is mainly related to the host genetic factor, virus and host immune interaction, but its exact pathogenesis is not clear. In recent years, there are many reports on ACLF based on alcoholic cirrhosis in the West. It is considered that alcoholic cirrhosis related ACLF patients are from acute attack, inflammatory injury, immune disorder to systemic inflammation. The response syndrome (systematic inflammatory response syndrome, SIRS), compensatory anti inflammatory response syndrome (compensatory anti-inflammatory response syndrome, CARS), regenerative response, and the magnitude and order of the multiple link events determine that the patient is retreated by inflammation or progressing to extrahepatic organ failure, and the systemic inflammatory response is wine The main driving factors of ACLF in patients with severe cirrhosis. Whether or not chronic HBV infected persons have HBV attack, whether or not they are progressing to liver failure after the seizure, because HBV lacks the animal model of chronic infection, and the causes and causes of ACLF are very heterogeneous. The liver immunological response and immunopathological process of HBV related ACLF are still known to be still known. Very few. Previous studies of HBV virus sequence variation suggest that the C gene promoter region, the anterior C region and the C region variation are associated with the attack of hepatitis B and fulminant liver failure. However, the same pre C, C region variation can be seen in different pathological phenotypes (common hepatitis B, liver hardened, liver cancer), or asymptomatic carriers. In fact, the disease is a disease. Whether the virus variation leads to the occurrence and development of hepatitis B severe and is restricted by host factors. The occurrence, development and prognosis of hepatitis B are common complex diseases caused by the interaction of virus (viral load, variation, evolution) and host (genetic heterogeneity, age, sex, etc.) through immune response. Common complex diseases (common complex) Diseases) the occurrence and development of.HBV-ACLF, which is usually determined by the interaction of many micro effect accumulating genes and environmental factors, involves immune recognition, inflammatory activation / amplification, hepatocyte necrosis, SIRS, CARS, organ failure and so on. It also belongs to the common complex disease domain. Its genetic factors also involve the role of multiple micro genes. In the study of genetic susceptibility to miscellaneous diseases, association studies - linkage disequilibrium analysis is the most common method, and the statistical efficiency is far higher than that of family linkage analysis. Genetic association based on the whole genome strategy is the mainstream of the genetic association of complex diseases, and the advantages of the genetic association are that the statistical efficiency is greatly improved. At the same time, it can avoid the chance of population stratification bias and gene selection. It can obtain the global and systematic understanding of the genetic characteristics of complex diseases from the whole genome angle. The whole genome association study of the HBV-ACLF phenotype (genome-wide association study, GWAS) is used to analyze the host genetic locus and genetic correlation within the whole genome. The combined atlas helps to understand the driving mechanism of HBV-ACLF and the target of intervention. Therefore, on the basis of the establishment of a large sample of HBV-ACLF cases and multi center queues, the clinical features and the spectrum of inflammatory factors are analyzed, and the whole genome association of HBV-ACLF is carried out, and the main associated locus HLA-DR is carefully parsed and functional. Annotations, and the restrictive analysis of HBV variation with positive associated HLA alleles. The results of this study have first identified the main genetic locus of HBV-ACLF from the whole genome, suggesting the driving effect of the HLA-DR allelic restrictive CD4+T cell pathway on HBV-ACLF. The main results of this study are as follows: 1. a total of 1300 patients with HBV-ACLF and 2087 cases of HBV were included. A complete genome association study of asymptomatic carriers entered 3-stage, with samples from 5 centers in Chongqing, Beijing, Quanzhou, Zunyi and Nanning. All patients had no history of antiviral, no previous hepatitis and liver function decompensation. 1300 cases of HBV-ACLF patients were analyzed, LC-ACLF accounted for 60%, NLC-ACLF accounted for 40%, and HBe Ag negative HBV- ACLF was 60%, HBe Ag was negative and HBV-ACLF of HBV DNA104 IU/m L accounted for 17.1%. compared to LC-ACLF. NLC-ACLF patients showed younger (average age 39.5 vs.43.9, 10-11), higher level (average value 1153, 10-11). R reached 1.5 and the occurrence of encephalopathy (average 19.8vs.24.8 days, p=1.36 x 10-11; 27.3 vs.38.1 days, p=5.16 * 10-6).2. multiple quantitative detection of cytokines in 1013 serum samples. The results showed that the important index of foreign alcoholic liver cirrhosis ACLF research, PCT (suggestive of bacterial infection) and CRP (suggestive of liver organ failure) In our HBV-ACLF patients, their concentration was lower than that of patients with chronic hepatitis B (CHB). Compared with mild to moderate and severe chronic hepatitis B, there were 6 significant cytokines in HBV-ACLF patients: Th1 cytokine IL-1a, TNF- beta, Th2 cytokine IL-5, IL-10 and IL-13, Th17 cytokine IL-17.3.HBV-ACLF. The analysis showed that there was a significant correlation signal (P1 x 10-5) on chromosome 1,6,12 in the whole genome, and the common genetic association loci of the two subtypes of LC-ACLF and NLC-ACLF were located in the HLA-II region of chromosome 6. The independent sample of replication 1a and 1b showed that the HLA-DR region was the main Association site, and then confirmed by replication 2a and 2b. The significant correlation between rs3129859 and HBV-ACLF (P combine=7.40 x 10-19, OR=1.83).4. stratification analysis showed that rs3129859 was an independent risk factor for HBV-ACLF, not related to chronic hepatitis B activity state and HBV reactivation. Risk allele rs3129859*C was associated with HBV-ACLF clinical processes, carrying patients with risk allele and patients, At 28 days, the risk of INR reached 1.5 and the risk of ascites was higher (p=3.95 x 10-4, p=3.03 x 10-4). In the NLC-ACLF subgroup, the 28 day mortality (p=0.03).5.HLA-DRB1*1202 was the most significant risk allele of HBV-ACLF (p= 3.94 x 10-6, OR=2.05).HLA-DRB1*1202 (p= 3.94 x 10-6, OR=2.05).HLA-DRB1*1202. The trend of geographical epidemiology is consistent. The highest.6.HLA-DR beta twenty-eighth - bit amino acids in China (5.9-21.7%) and Southeast Asia (6.8-35.3%) are the most significant associated amino acids (p=3.03 x 10-6, OR=1.75). Secondly, the associated amino acids also include HLA-DR beta protein 26,30,32,37,38 and 85 - bit amino acids, as well as HLA-DQ alpha protein 40,47,50,51,53 56,69,76 amino acid and HLA-DQ beta protein forty-fifth amino acid (p0.001). The three-dimensional reconstruction of protein structure shows that these associated amino acids are located at the.7. allele of the HLA molecular antigen binding groove, the.7. allele rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301 and the amino acid haplotype DR beta -LEHHLLA-DQ a -GCVLQdel TL-DQ beta -E are the risk haplotypes, carrying the risk. The risk of ACLF was higher in haplotype HBV infected people (additive model, p=1.16 x 10-4, OR=1.94; p=2.43 x 10-6, OR=2.10).8. survival analysis suggested that the risk allele and the risk haplotype were related to the HBV-ACLF clinical process. In the non cirrhosis ACLF subgroup, the patients with the risk of the risk of the double type rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301 were hospitalized. The mortality of the 28 days (p=3.49 x 10-4), 90 days (p=0.003) and 180 days (p=0.003) was higher, and the incidence of hepatic encephalopathy was higher (p=0.019). The amino acid haplotype also observed the consistent correlation of.9.HBV-ACLF associated site function annotation: e QTL analysis showed that the associated locus rs3129859 was associated with the HLA-II gene expression; GRAIL literature analysis showed HLA-DR and HLA. -DQ is the most reasonable association gene. Overlap analysis with other HBV related GWAS literature shows that there is partial overlap between the genetic association site of HBV-ACLF and the genetic association loci of chronic HBV infection, and.10. is limited by the analysis of HLA-DRB1*1202 risk alleles, identifying 12 HBV variations, showing both HBV-ACLF risk allele HLA-DRB1*120. 2 specificity and specificity of hepatitis activation are also the location of positive hot amino acids that the virus is selecting. Epitope prediction analysis found that these variations can cause virus epitopes drift. Conclusion: 1. our data are important for the understanding of the natural history of HBV-ACLF: a considerable proportion of chronic HBV infections can be in the absence of liver cirrhosis. ACLF, HBV natural history was at low replication and e antigen negative, the risk of ACLF could not be ignored; NLC-ACLF's disease progressed more rapidly. The age of HBV-ACLF (42 years old) was far below the European alcoholic liver cirrhosis (56 years old) ACLF patients (56 years old), the serum inflammatory factor analysis of.2.HBV-ACLF patients showed that the HBV-ACLF patients did have acute severity. The coexistence of SIRS and CARS in.HBV-ACLF patients with CD4 Th cytokine pathway is strongly activated, different from chronic hepatitis B, moderate and severe, and the.3.NLC-ACLF and LC-ACLF subtypes of ACLF patients in European alcoholic cirrhosis have common genetic susceptibility loci, subtype loci, and quantitative trait loci (liver enzymes, sex hormones, etc.) and HBV-ACLF bits. .4.HLA-DR is the main genetic susceptibility locus of HBV-ACLF, and risk allele rs3129859*C and HLA-DRB1*1202 can be used as the marker. risk allele of HBV-ACLF clinical early warning prognosis, which is related to hepatitis activation, positive virus selection and epitope drift. All genomic association studies have been carried out in patients with acute hepatic failure to identify HLA-DR as the main associated locus of HBV-ACLF, and the associated risk alleles and haplotypes are significantly related to the clinical process of HBV-ACLF, suggesting that the CD4+T cell pathway restricted by the HLA-II class is important in the immune pathological process of HBV-ACLF. It also indicates that HBV-ACLF is a homogenous disease of liver cirrhosis and non cirrhosis. It supports the hypothesis of a disease (one disease hypothesis).
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.62;R575.3
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