线粒体DNA变异介导ROS调控核DNA甲基化在类风湿关节炎发生机制中的研究

Rheumatoid arthritis, a progressive autoimmune disease, is characterized with synovial lining hyperplasia, pannus formation and destruction of cartilage and bone. Epigenetics study demonstrate that RA patients appear to be genome-wide hypomethylation phenomenon, while the mechanism has still not been elucidated. Family genetic is one of the major factor pathogenesis of RA. Previous studies demonstrate that RA families comply with maternal inheritance. It has reported that excess ROS can change the status of genomic DNA methylation. We raise the hypothesis that mtDNA mutation-mediated ROS production can regulate inflammatory cytokines methylation status, thereby affecting the expression of inflammatory cytokines. Upon previous study, we draft to RA-related variants through retrospective clinical trial, evaluate the diagnostic value of familial RA in multi-pedigrees and validate the relationship between the high risk mtDNA and RA in a prospective cohort study. We use genetically mitochondrial cell lines and mouse models to analyze the impact of mtDNA mutation on mitochondrial biochemical function, and detect the differences distribution of genome-wide methylation and expression levels of immune-related factors, investigate the potential pathway of mtDNA mutation aggravate arthritis. RA provide a basis for seeking new therapeutic targets and the design of more effective treatment strategies. In this study, we aimed to find out the familial RA-associated mtDNA variants and its role in the development of RA occurrence, then provide the basis for seeking new diagnostic biomarkers for familial rheumatoid arthritis.

类风湿关节炎（RA）是一种以滑膜组织炎性增生、血管翳形成、关节软骨及骨质破坏为特征的进行性自身免疫性疾病。表观遗传学显示RA患者存在全基因组低甲基化现象，其机制一直尚未阐明。家族遗传是RA发病的主要因素之一，申请者前期发现RA家系呈母系遗传特征，文献报道ROS可改变核DNA甲基化状态，因此我们提出mtDNA变异可介导ROS调控核DNA甲基化状态进而影响炎症因子表达的假说。本项目拟采用回顾性临床试验筛选出RA相关mtDNA易感位点，家系分析明确家族性RA高风险mtDNA位点，前瞻性队列研究验证该位点与RA的相关性。同时利用转线粒体细胞系和小鼠模型分析mtDNA变异对线粒体生化功能的影响，检测全基因组甲基化分布差异及免疫相关因子表达水平，深入探讨mtDNA变异加重关节炎症的潜在作用途径。旨在发现家族性RA相关mtDNA变异及其在RA发生中的作用，为寻求家族性RA新的分子靶标提供依据。

氧自由基（reactive oxygen species，ROS）作为线粒体氧化磷酸化的副产物在类风湿关节炎（rheumatoid arthritis，RA）的发生发展中发挥重要作用。近期，研究报道了4个核基因编码线粒体相关蛋白上存在大量低频变异，其累积效应与RA关节侵蚀性相关。已有文献报道RA患者滑膜组织中存在高浓度的ROS和大量损伤的mtDNA。但是，外周血mtDNA变异与RA的关系目前尚不明确。我们通过二代测序技术对年龄、性别匹配的RA和健康人群外周血中mtDNA全基因组进行测序，绘制了RA患者mtDNA变异图谱。研究证实了RA患者外周血中存在大量mtDNA变异，通过SKAT、SKAT-O等基因负荷检验明确RA患者mtDNA变异主要富集在MT-ATP6和MT-ND5上。此外，发现了4个RA相关的低频、异质性体细胞变异，未发现RA显著相关的胚系变异。同时分析了mtDNA遗传背景与中国RA人群的关系，揭示了mtDNA单倍群F1及其亚群F1a’c和F1a可能是RA的保护性因素。本研究有助于深入探讨mtDNA变异介导的ROS与RA发病机制的关系，将为RA的诊断、疾病监测和治疗提供实验基础。