【作者】 高金平；

【导师】 张学军； 杨森；

【作者基本信息】 安徽医科大学 ， 皮肤病与性病学， 2013， 博士

【摘要】 研究背景大疱性类天疱疮（Bullous pemphigoid，BP）是一种慢性自身免疫性表皮下大疱病。好发于老年人，儿童罕见，临床表现为皮肤疱壁紧张的浆液性水疱或大疱，尼氏征阴性，多散布在四肢的屈侧、下肢以及腰部等处。BP的发病率低，欧洲人群BP的年发病率大约为1/10万-7/10万。在中国人群中，BP病例约占门诊病例的0.02%左右。在年龄大于80岁的老年人群体中，BP的年发病率明显增多，大约为150-330/100万。罹患类天疱疮的1年内的死亡率大约为20%-40%，但在中国人群的死亡率（12%）低于欧洲人群。迄今为止，BP的病因和发病机制尚不明确，自身免疫反应机制被认为是导致疾病发生的主要原因，其致病过程包括补体的激活、炎症细胞的聚集、释放蛋白水解酶以及直接干扰自身抗原的粘附功能等步骤。BP发病过程中，体液免疫与细胞免疫介导过程皆需要B淋巴细胞与CD4+Th细胞的共同参与。自身抗原被抗原递呈细胞捕获、处理，同时结合到主要组织相容性复合体II类分子而暴露在细胞的表面。T细胞通过受体介导的识别抗原，激发各种细胞因子的释放，促进B细胞产生抗体。鉴于这种免疫机制，HLA基因可能是BP发病机制中最重要的遗传易感性因素。通过候选基因的方法发现MHC区域HLA-II类基因，以及非MHC基因与BP的相关性。在不同地域的高加索人群中，均发现HLA-DQB1*0301与BP具有显著相关性。然而，不同种族和地域的遗传背景差异可能是造成遗传异质性的主要因素。印度人群同样发现HLA-DQB1*0301等位基因与BP相关，但在中国和日本人群中却截然不同。其次是从BP可能的发病机制中，筛选如COL17A1基因、IGHV基因、以及细胞因子基因，探讨与BP的遗传易感性。BP可能是一种由多个因素共同作用而导致的复杂性疾病。当前对复杂疾病遗传学研究的主要策略包括如连锁分析研究、功能候选基因关联研究等方法都存在一定的局限性。因此，开展BP的GWAS研究，对BP遗传学基础以及发病机制的认识具有重要意义。目的利用全基因组关联分析研究方法在全基因组范围内搜寻与BP关联的遗传变异，鉴定出中国汉族人群BP的易感基因/位点；构建中国人群BP病例与对照的全基因组关联分析数据库。方法根据样本收集的标准，从遗传资源协作网医院收集BP患者和正常对照样本，分成2个各自独立中国南方人群和北方人群样本。利用HumanOmniZhongHua-8全基因组SNP分型芯片分别对来自中国北方的91例BP患者和575例正常对照，以及中国南方138例BP病例和991例对照进行SNPs分型；对两组人群的SNP分型数据，分别经过严格的质控后进行联合Meta分析以及单倍型分析。结果1)经过多种严格的数据质控后，本课题组构建了中国汉族北方与南方人群，包括229例BP病例和1566例正常对照801,392个常染色体SNPs的大疱性类天疱疮GWAS数据库。2)通过对中国汉族北方人群和南方人群GWAS数据Meta分析结果发现1个新的易感位点1q43（rs72766895，P=6.38×10-9，OR=3.14）与BP显著相关；该区域内的PLD5基因在功能学可能与BP发病过程中的发病机制相关，提示PLD5基因为BP的易感基因。3)发现2个与BP相关联的提示易感位点，分别位于18q21.31(SNP rs117788424,P=1.38×10^-7，OR=2.51)和2p25.3区域内(SNP rs12988270，P=1.96×10^-7，OR=2.52)，该LD区域内相关基因的功能学证据提示18q21.31和2p25.3为BP的遗传易感区域。4)对既往报道的HLA基因与BP的相关性进行验证，进一步证实HLA与BP的相关性，同时发现位于HLA-II基因区域存在风险性的单倍型(P=3.29×10^-8, OR=1.92)。结论本课题在中国汉族人群中开展BP的GWAS研究，首次在非MHC区域发现一个新的与疾病相关易感位点，两个提示易感位点；通过单倍型分析发现与BP相关的HLA-II基因区域风险性单倍型，并进一步证实HLA与BP的相关性;首次构建第一个中国汉族人群大疱性类天疱疮病例与正常对照的全基因组关联分析数据库。本研究在强调免疫因素在BP发病过程中重要性的同时，还提示与BP致病通路相关的遗传变异对疾病的影响。本研究将加深对BP的遗传学基础的认识，同时为BP的发病机制研究提供新的证据。更多还原

【Abstract】 Background：Bullous pemphigoid （BP） is a chronic autoimmune blistering diseasecharacterized by subepidermal blistering. BP mainly affects older people and rare inchildren, typically presents with tense, mostly clear, blisters with a negative Nikolskysign and frequently scattered in limbs flexor side, leg and waist. The incidence of BP isvery low, which has been estimated between10and70new cases per1million per yearin central Europe. In Chinese people, it is also quite low, accounting for about0.02%ofoutpatients in a survey. Incidence rises substantially to150–330per1million peopleper year in people older than80years.1-year mortality for patients with BP ranges from20%to40%, which is about two times higher than that mortality rate （12%） in ChineseHan population.So far, the etiology and pathogenesis of BP is unclear. The mechanism by which BPautoantibodies are thought to be pathogenic may be summarized in these steps,including complement activation, recruitment of inflammatory cells, release ofproteolytic enzymes, and direct interference with the adhesion function of theautoantigens. The humoral and cellular immune-mediated process in BP pathogenesisrequires the cooperation between B cells and CD4+Th cells. In patients with BP,self-antigens are caught by antigen-presenting cells, processed, bound to majorhistocompatibility complex class （MHC） II and displayed on the cell surface. Thereceptor-mediated recognition of these epitopes by T cells, triggers the release ofvarious cytokines and, consequently, B cells are stimulated to produce autoantibodies.HLA genes are probably the most significant genetic predisposition factor, due to theirrole in the antigen presentation process. Using the candidate genes approach, it has found the correction among the HLA class IIgene in MHC region, non-MHC genes and BP. Especially, the findings ofHLA-DQB1*0301has a significant correlation with BP. However, the geneticbackground of different ethnic and geographic may be the main factor to result inheterogeneity genetic. The HLA-DQB1*0301allele is also associated with Indianpopulation, but different in Chinese and Japanese populations. In addition, it is also toexplore the genetic susceptibility of BP from the pathogenesis, screening the possiblecandidate genes, such as the COL17A1, IGHV and cytokine genes. BP is a complexdisease caused by multiple factors. The strategy, including such as linkage analysisstudy, candidate gene association studies, may have the limitation to explore the geneticof complex disease. Therefore, to conduct the GWAS of BP will be of great importancein promoting understand of the pathogenesis and genetic basis of BP.Object: To explore BP associated SNPs in whole genome and identify susceptibilitygenes/loci for BP by using GWAS approach and to build the GWAS database of BPcases and controls in Chinese Han population.Method: All subjects in this study were recruited from the collaboration with multiplehospitals in China, which were divided into two separate groups in southern andnorthern Chinese population. Samples in the GWAS stage were genotyped by IlluminaHumanOmniZhongHua-8BeadChips. A total of1795subjects,91BP patients and575normal controls from north of China,138BP cases and991normal controls form southof China, respectively, were genotyped by this BeadChips. After strict quality control，two genotyping data from different groups were combined for Meta-analysis andhaplotype analysis.Results：1)It was the first GWAS study of bullous pemphigoid and had built theGWAS database of801,392autosomal SNPs, including total229BP cases and1566controls in Chinese Han population.2) Through Meta-analysis of the GWAS data from northern and southern Chinese population, we found a new susceptibility loci at1q43（rs72766895，P=6.38×10-9，OR=3.14） significantly associated with BP. In thisLD region, the PLD5gene prompt to be the susceptibility gene of BP, according to thefunctional study on published researches.3) We also Found two suggestivesusceptibility loci associated with BP, which located in18q21.31(SNP rs117788424, P=1.38×10^-7, OR=2.51) and2p25.3(SNP rs12988270, P=1.96×10^-7, OR=2.52).Based on the evidence of genes function study in this LD block, it indicated that18q21.31and2p25.3might be the susceptibility loci associated with BP.4) Replicatedthe previous reports of association between BP and HLA genes, we further confirmedthe correlation between HLA and BP and also found a risk haplotype in the HLA-IIgene region associated with BP.Conclusion：It is the first GWAS of bullous pemphigoid research in Chinese Hanpopulation. We found a new susceptibility locus and two suggestive loci associated withBP at non-MHC region. Through the haplotype analysis, we found a risk haplotype inHLA-II gene region associated with BP, and further confirmed the correlation betweenHLA and BP. We had built the GWAS database of bullous pemphigoid in Chinesedatabase. Not only emphasized the importance of immune factors in the pathogenesis ofBP in this study, but also pay attention to genetic variations associated with pathogenicpathways. This study will be promote us to understand the genetic bases of BP deeply,and also provided new evidence for the pathogenesis research of BP.更多还原