MHC区域非编码插入缺失片段（InDels）对类风湿关节炎发病的作用及其调控机制研究

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic joint inflammation leading to bone erosions and joint destruction. Genetic factors play important role in RA pathogenesis, many of them remain unidentified. Human leukocyte antigens (HLA) genes within major histocompatibility complex (MHC) region, were the first and still remain to be the best described genetic risk factors contributing to RA. However, due to the high linkage disequilibrium in MHC region, it is difficult to define the causal gene(s) or additional independent risks using the conventional HLA genotyping or chip-based methodology. Recently our group has performed a deep sequencing for entire MHC region and further Sanger sequencing for validation. By the targeted sequencing and bioinformatics analysis, we found a number of novel DNA insertion-deletion fragments (Indels) were strongly associated with ACPA-positive RA, and majority of the Indels are non-coding variants. From ENCODE, RegulomeDB, and rVarBase databases, there are abundant transcription factor binding and classic epigenetic signatures for an enhancer functions in this region, suggesting that they are active enhancers. In addition, several Indels displayed eQTLs effects from Blood eQTL databases. To further verify and functional characterize these genetic variants, varied molecular biology experiments in vitro will be carried out. To further understand the molecular mechanisms of these Indels contributed to RA susceptibility and pathogenesis, we will generate conditional knock-in or knock -out mice specific for the Indels with significant functional properties, and study the impact of these Indels in experimental arthritis models. We expect that the combination of functional study with MHC deep sequencing will lead to a better understanding in the role of non-coding DNA variants in RA pathogenesis.

类风湿关节炎（RA）是以慢性关节炎症和骨破坏为主要特征的自身免疫性疾病，具有明显遗传倾向。MHC是迄今最为公认的RA遗传相关区域。本课题组近期通过对MHC全区域深度测序和进一步独立验证，新发现多个核苷酸插入或缺失片段(insertions-deletions, Indels)与ACPA阳性RA发病显著相关，其中绝大多数Indels为非编码DNA序列。通过系统生物信息学分析预测和功能注释，提示这些RA发病相关Indels所在区域包含多个转录因子结合域；并具有增强子序列所特有的表观遗传学特征；数个Indels具有潜在eQTL作用。为进一步证实这些Indels调控功能，课题组将通过一系列经典和现代分子生物技术，研究其转录调控功能并鉴定其关键靶基因。最后，我们将选择数个功能最显著的Indels构建条件性敲除或敲入鼠, 进一步在关节炎模型定向研究RA高风险Indels的致病作用和分子机制。

类风湿关节炎（RA）是以慢性关节炎症和骨破坏为主要特征的自身免疫性疾病，具有明显遗传倾向。MHC是迄今最为公认的RA遗传相关区域。课题组前期通过对MHC区域深度测序，除证实HLA-DRB1共享表位为RA高风险遗传因素外，还新发现了其他重要的ACPA阳性RA独立危险因素，包括多个核苷酸插入或缺失片段(Indels)。通过系统生物信息学分析预测，提示这些RA发病相关Indels所在区域包含多个转录因子结合域；并具有增强子序列所特有的表观遗传学特征。在此研究基础上，课题组拟进一步探讨：1）Indels所在非编码DNA区域的关键调控元件；2）鉴定和验证调控元件（增强子）的关键靶基因；3）采用条件性敲除/敲入鼠和实验性关节炎模型，进一步明确RA高风险Indels在关节炎症中的作用及分子机制。通过四年的研究，课题组进一步发现：1）数个H3K27ac和H3K4me1富集区域，提示这些高风险Indels所在区域的增强子活性程度明显增强；2）采用pGL3荧光素酶报告载体系统，进一步验证了高活性度的Indels相关增强子；3）采用CRISPR/Cas9编辑系统，鉴定了Indels相关增强子可能调控的关键靶基因。.截止目前，受本项目资助，已发表SCI论文6篇（课题负责人均为通讯或共同通讯作者），其中5篇为JCR分区Q1区文章。包括1篇发表于风湿免疫学领域权威期刊《Annals of the Rheumatic Diseases》（IF: 27.973，Q1）；另外4篇分别发表在风湿免疫学一流期刊《Rheumatology（Oxford）》（IF: 7.046，Q1），以及《Frontiers in Immunology》（IF: 8.786，Q1, 共2篇）和《Frontiers in Genetics》（IF: 4.772，Q1）；此外，在投论文1篇。课题负责人作为第一发明人，获得国家发明专利1项。共获得省部级成果奖或科技奖3项：包括《教育部高等学校科学研究优秀成果奖》1项（一等奖，第四完成人）、《北京医学科技奖》1项（一等奖，第五完成人）、《中华医学科技奖》1项（二等奖，第五完成人）。课题负责人作为导师，培养博士研究生1名，硕士研究生2名；作为副导师，协助培养博士研究生1名。参加国际学术交流3次，美国风湿病学会年会壁报展示1次，国际狼疮大会壁报展示1次。

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