中国汉族人Behcet病致病基因的鉴定及功能研究

Beh?et disease is a blinding ophthalmopathy common seen in China. So far there is no effective mean of prevention and treatment for this disease, because its pathogenic mechanism remains unclear. This present project is aimed at identifying the common variants or rare variants lead to Beh?et's disease and exploring the genetic basis mechanism for Beh?et disease. Based on our previous GWAS identified three Beh?et's disease associated loci-1p32.3, 3p21, 21q22.3, and reported two loci-1q31, 1p31.3 in the literature, we will determine the minimum haplotype block by the regional fine mapping and haplotype analysis were performed to, and then re-sequence the determined region, finally identify the causal common variant. Based on three Beh?et's disease pedigrees family collected by us, we will perform exome sequencing to look for rare variants for the disease, and then further confirm the identified variants by sporadic of patients and normal controls. The function studies in vitro was used to detect the effect of disease-causing mutations on gene mRNA expression, protein level, protein structure by quantitative PCR, Western blot and so on. We then constructed mutant and wild-type plasmid to detect the effect of disease-related mutations on Th17/Treg cell percentage and cytokine secretion. Transgenosis rat model was used to examine the genetic variants biological function and its mechanism. Through this research, we may identify the disease-causal mutations of Beh?et's disease, elucidate its biological functions, and provide the new targets for individualized prevention and treatment of Beh?et's disease.

Beh?et病是我国常见的致盲眼病，迄今尚无有效的防治手段，原因在于其致病机制尚不明确。本项目将从常见变异和/或罕见变异导致Beh?et病两个方面探讨Beh?et病的遗传学基础及其作用机制，基于我们GWAS鉴定的3个疾病相关区域-1p32.3，3p21，21q22.3和文献报道的2个区域-1q31, 1p31.3，本项目将对这5个区域的GWAS数据结合千人基因组数据，确定最小单体型块，通过重测序鉴定致病变异；基于我们收集到的3个疾病家系，利用外显子组重测序寻找疾病罕见变异，散发患者及对照进一步明确致病变异。通过体内、外功能检测致病变异对mRNA表达、蛋白水平、蛋白结构等的影响，检测相关变异对Th17/Treg比例及炎性细胞因子分泌的影响；通过致病基因诱导鼠疾病模型，进而研究其生物学功能和机制。通过此研究将可能鉴定Beh?et病致病变异，并阐明其生物学功能，将为此病的个体化防治提供新的靶点。

我们课题组主要围绕葡萄膜炎的遗传学发病机制进行研究，获得了以下成果：.1.利用Affymetrix人全基因组SNP芯片对149例中国汉族Behcet病患者和951例正常对照进行了全基因组关联研究，发现新的Behcet病易感基因STAT4 (P=3.36×10-7-6.20×10-9)，并证实STAT4基因变异通过影响Th17细胞因子IL-17的产生参与疾病的发生。研究结果发表在Arthritis Rheum （IF= 8.955）并获Nature Reviews Rheumatology亮点推荐。.2.对补体拷贝数变异与葡萄膜炎进行了关联研究及功能研究，发现Behcet病患者补体4血清水平显著升高。大于2拷贝C4A在Behcet病患者中显著增加(P=1.65×10-7,OR=2.84)。逻辑回归分析证实C4与Behcet病的相关性完全独立于HLA-B51。功能学研究发现高拷贝数C4A显著增加C4A表达，并增加炎症因子IL-6的产生。研究结果发表在Arthritis Rheum（IF= 8.955）杂志上。.3. 我们还发现了IL23R-C1orf141、EGR2-ADO-ZNF365在内的2个中国汉族人VKH综合征新易感区。通过功能学研究，我们发现IL23R SNPs并未引起IL23R蛋白结构/活性的变化，而可能通过影响IL23R的转录和表达的改变，进而影响IL-23/IL-17通路的激活，最终参与VKH综合征的发生。该结果申请人以第一作者发表在Nature Genetics杂志（IF：31.616）。为进一步阐明IL23R信号通路的作用，我们还对IL23R通路相关分子进行了研究，发现Rorc、IL17F、IL23A的另一种遗传变异形式，拷贝数变异，通过改变基因自身的转录水平，进而引起下游炎症因子的过度分泌，最终导致Behcet病的发生或发展（Hou S et al. Ophthalmology，IF：6.75）。.综上，本课题共发表SCI论文14篇，总IF值98.356分，平均IF值7.03分（全世界眼科62份SCI杂志平均IF值为2.04）。研究执行阶段，课题负责人获得国家优秀青年基金、国家重点研发计划、重庆市杰出青年基金、重庆市青年拔尖人才计划、重庆市基础研究重大项目等项目，入选国家百千万人才工程和国家有突出贡献中青年专家。