中国人Vogt-小柳原田综合征致病基因鉴定及相关机制研究

Vogt-Koyanagi-Harada (VKH) syndrome is one of the most common uveitis entities leading to incurable blindness in China. Though genetic background, environmental factors (especially virus infection) and immune responses have been shown to be involved in the development of VKH syndrome, the precise pathogenesis of the disease is still not clear. Our previous genome-wide association study (GWAS) in VKH syndrome showed that more than 60 genetic variants (polymorphisms) were associated with VKH syndrome. Most of these genetic variants are located in about 20 gene regions, such as endoplasmic reticulum aminopeptidase 1 (ERAP1), autophagy associated gene (ATG) and so on. Six gene regions implicated by this study are involved in the virus antigen processing and presenting, complement system and the regulation of T cell responses. Based on our previous GWAS results, the present project aims to identify the genetic variants in these 6 gene regions which lead to VKH syndrome and explore the mechanisms involved in the development of VKH syndrome. Firstly, the causal genetic variants for VKH syndrome will be identified in a large case-control cohort (1300 patients with VKH syndrome). Secondly, bioinformatics methods and in vitro studies (such as quantitative PCR, Western Blot and so on) will be used to investigate the effect of disease-causing genetic variants on the mRNA expression, protein level and protein structure. Because the three processes (the virus antigen processing and presenting, complement system and the regulation of T cell responses) indicated by our GWAS study are all important in the development of VKH syndrome, we will investigate the level of complement system in patients with VKH syndrome and the effect of complement factors on the T cell responses. Experimental autoimmune uveitis (EAU) is widely accepted as the animal model for VKH syndrome. We will explore the mechanisms of antigen processing and presenting, complement factors and T cell responses in the development of uveitis using EAU model. The project will shed light on the genetic background of VKH syndrome and provide new targets for prevention and treatment of VKH syndrome.

VKH综合征是我国常见致盲性葡萄膜炎，遗传、环境和免疫因素均参与其发生，但确切机制不完全清楚。本项目以课题组全基因相关分析发现的与VKH综合征显著相关的ERAP1等6个基因区为对象，首先利用大样本量（1300例患者）鉴定出参与VKH综合征发生的基因变异，并通过体内、外实验探讨基因变异对mRNA表达、蛋白表达、结构和功能等的影响；然后，以这6个基因区提示的微生物诱导自身免疫反应、补体系统和T细胞免疫反应等VKH综合征发生机制中的三个重要环节为切入点，以VKH综合征患者为对象，检测补体系统的表达及其对T细胞免疫的影响，以期明确补体系统在VKH综合征发生中的作用；以小鼠EAU模型为对象，探讨微生物抗原呈递环节对补体系统和T细胞免疫平衡的影响及其在诱导葡萄膜炎发生中的作用。项目的实施将可能揭示遗传基因背景在VKH综合征发生中的作用，明确该病发生的相关免疫学机制，为VKH综合征防治提供新的靶点。

VKH综合征是我国常见致盲性葡萄膜炎，遗传、环境和免疫因素均参与其发生，但确切机制不完全清楚。本项目以课题组前期全基因相关分析发现为基础，寻找和确定与VKH综合征易感性显著相关基因多态，探讨了VKH综合征与其他不同类型葡萄膜炎遗传背景的异同，基因组甲基化水平以及环境因素，尤其是肠道微生物与遗传背景相互作用参与VKH综合征发病的相关生物学机制，主要研究成果包括以下几个方面：.一、基于前期GWAS研究结果，选择与该病显著相关的ERAP1、ATG-16、C3AR1、C5、IDO、IL-2、NOD1、NOD2、NLRP1、NLRP3、NLRP5和CIITA等12个基因区作为靶点，发现NOD2基因rs3135499位点和NLRP1基因rs80790341与VKH综合征的易感性显著相关，部分解释了VKH遗传易感性的基因背景。.二、探讨了VKH综合征、Behcet病、急性前葡萄膜炎（AAU）、强直性脊柱炎（AS）伴发AAU等多种葡萄膜炎遗传背景的异同，发现FCGR3A、LACC1、CEBPB-PTPN1、ADO-EGR2和RIPK2等的基因多态性与Behcet病相关，ANTXR2、ERAP1/2、RUNX3、miR-143、miR-146a、miR-9-3和miR-205等基因的多态性与AS伴发AAU相关，部分揭示了这些葡萄膜炎临床表现的异质性。.三、探讨了VKH综合征全基因组的甲基化水平以及甲基化与基因多态的关系，发现9个非MHC区和4个MHC区的CpG位点在两组间存在着显著的甲基化水平差异，利用其中8个CpG位点作为VKH综合征的早期诊断，AUC可以达到84.95%，95%CI为79.49%-90.41%。.四、探讨了相关遗传变异与环境因素等相互作用参与葡萄膜炎发病的生物学机制，我们对VKH综合征患者、Behcet病患者、正常对照者的肠道微生物进行16S rRNA测序、宏基因组分析，并利用试验性自身免疫性葡萄膜炎小鼠模型（EAU）进行Behcet病患者粪便移植，发现嗜胆菌属和副杆菌属及副普氏菌等机会致病菌在Behcet病患者肠道微生物的比例显著高于正常人，而产丁酸盐的梭菌属和产甲烷菌属显著低于正常人，将BD患者粪便移植于EAU小鼠动物模型，所诱导EAU的炎症反应程度显著加重。