原发性胆汁性肝硬化易感基因RANK/RANKL致病位点的探索和功能研究

Primary biliary cirrhosis (PBC) is the most common autoimmune liver disease with high heritability. Although GWAS has identified multiple susceptibility variants associated with PBC, the real causal variants and their functions remain unknown. Substantial work is warranted to dissect the functional consequences of genetic susceptible variants. Our preliminary studies have identified several PBC associated variants adjacent to RANK/RANKL in UK population. Moreover, we found that RANK/RANKL had higher expression level in liver tissues in PBC patients compared to healthy controls, AIH, PSC and HBV patients. Therefore, we attempt to combine molecular genetics, bioinformatics and functional studies, to explore how the genetic variants in RANK/RANKL contribute to the pathogenesis of PBC. Several issues will be addressed in this study: fine mapping of the genetic variants in RANK/RANKL with Chinese population and UK population; in-vivo and in-vitro studies to understand the molecular mechanisms of the candidate variants; relationship between genetic variants and the clinical manifestations and treatment response in PBC patients. Our study will shed new light on elucidating the molecular mechanisms of PBC as well as personal medicine.

原发性胆汁性肝硬化（PBC）是最常见的自身免疫性肝病，表现出很强的遗传易感性。尽管全基因组关联分析(GWAS)已识别了多个PBC易感位点，但真正的致病基因和功能位点仍有待发掘。我们分析WTCCC PBC数据发现，RANK/RANKL基因区域的遗传变异显著影响PBC易感性。进一步的预实验研究结果表明，RANK/RANKL在PBC患者肝内表达上调且呈疾病特异性。本项目拟利用我们已建立的PBC临床标本库，结合英国人群数据，采用精细定位发掘RANK/RANKL基因调控区域的PBC易感位点；利用荧光素酶报告基因、染色质免疫共沉淀等技术研究遗传变异对目标基因表达的调控机制；进一步在细胞水平及小鼠模型中研究致病位点参与PBC发病的免疫学机制；建立预测模型，探讨遗传变异与临床特征和药物应答等之间的关系。本研究有望发现PBC新的致病功能位点，完善PBC发病的分子机制，为疾病个性化诊疗提供理论依据。

原发性胆汁性胆管炎（原名原发性胆汁性肝硬化，简称PBC）是一种重要的自身免疫性肝病。遗传因素在PBC发病机制中发挥重要作用。首先，我们研究了候选易感基因RANK/RANKL参与疾病发生发展的分子遗传学及免疫学机制。基于大样本中国人群的病例对照分析，我们发现RANK/RANKL基因并不显著影响疾病的易感性。然而，RANK/RANKL通过调控肝内免疫稳态影响疾病的进展。PBC患者肝内RANK/RANKL信号通路明显活化，主要表达于肝内汇管区炎症细胞，且与疾病的严重程度呈正相关；RANK/RANKL可通过调控肝内免疫细胞的功能，导致肝内局部免疫微环境的紊乱。RANKL可作用于髓系源性抑制细胞（MDSCs）表面的RANK，激活下游NF-kB通路，从而促使MDSCs扩增与活化，进而促进肝内免疫稳态失衡和纤维化进展。因此，RANKL诱导的MDSCs信号途径可作为自身免疫性肝病未来免疫治疗的靶点。其次，通过全基因组关联分析，我们发现了多个新的PBC易感基因。其中，ARID3A与RANK/RANKL都参与调控B细胞及髓系细胞的发育与成熟。结合精细定位和功能注释，初步确定ARID3A基因调控区的遗传变异（位于基因组增强子区）是PBC潜在的致病功能位点。我们通过荧光素酶报告基因、表达数量性状、染色质免疫共沉淀、染色质构象捕获等方法确定该遗传易感位点通过与转录因子NR2C2结合，调控ARID3A的基因表达。免疫组化实验也发现ARID3A在肝内表达水平与PBC患者肝内炎症和纤维化程度正相关。我们的研究发现了PBC新的易感基因和功能位点，完善了PBC发生发展的机制，为探索新的治疗策略提供依据。