DNA methylome signature in rheumatoid arthritis. Homo sapiens

Objectives: Epigenetics can influence disease susceptibility and severity. While DNA methylation of individual genes has been explored in autoimmunity, no unbiased systematic analyses have been reported. Therefore, a genome-wide evaluation of DNA methylation loci in fibroblast-like synoviocytes (FLS) isolated from the site of disease in rheumatoid arthritis (RA) was performed. Methods: Genomic DNA was isolated from six RA and five osteoarthritis (OA) FLS lines and evaluated using the Illumina HumanMethylation450 chip. Cluster analysis of data was performed and corrected using Benjamini–Hochberg adjustment for multiple comparisons. Methylation was confirmed by pyrosequencing and gene expression was determined by qPCR. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes. Results: RA and control FLS segregated based on DNA methylation, with 1859 differentially methylated loci. Hypomethylated loci were identified in key genes relevant to RA, such as CHI3L1, CASP1, STAT3, MAP3K5, MEFV and WISP3. Hypermethylation was also observed, including TGFBR2 and FOXO1. Hypomethylation of individual genes was associated with increased gene expression. Grouped analysis identified 207 hypermethylated or hypomethylated genes with multiple differentially methylated loci, including COL1A1, MEFV and TNF. Hypomethylation was increased in multiple pathways related to cell migration, including focal adhesion, cell adhesion, transendothelial migration and extracellular matrix interactions. Confirmatory studies with OA and normal FLS also demonstrated segregation of RA from control FLS based on methylation pattern. Conclusions: Differentially methylated genes could alter FLS gene expression and contribute to the pathogenesis of RA. DNA methylation of critical genes suggests that RA FLS are imprinted and implicate epigenetic contributions to inflammatory arthritis. Overall design: Fibroblast-like synoviocyte cell-lines from osteoarthritis (OA) and rheumatoid arthritis (RA) patients.

研究目的：表观遗传学（Epigenetics）可影响疾病易感性与疾病严重程度。尽管自身免疫性疾病中单个基因的DNA甲基化已得到探索，但目前尚无无偏倚的系统性分析报道。为此，本研究对从类风湿关节炎（rheumatoid arthritis, RA）病变部位分离的成纤维样滑膜细胞（fibroblast-like synoviocytes, FLS）中的DNA甲基化位点开展全基因组水平评估。
研究方法：从6例RA患者与5例骨关节炎（osteoarthritis, OA）患者的FLS株中提取基因组DNA，采用Illumina HumanMethylation450芯片进行检测。对数据进行聚类分析，并通过Benjamini-Hochberg校正法对多重比较进行校正。采用焦磷酸测序验证甲基化水平，通过实时定量聚合酶链反应（quantitative PCR, qPCR）检测基因表达水平。采用京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）进行通路富集分析。
研究结果：RA与对照FLS的DNA甲基化模式存在显著分离，共鉴定出1859个差异甲基化位点。在与RA相关的关键基因中鉴定出低甲基化位点，包括CHI3L1、CASP1、STAT3、MAP3K5、MEFV及WISP3；同时也观察到高甲基化位点，涉及TGFBR2与FOXO1。单个基因的低甲基化与基因表达上调相关。分组分析共鉴定出207个携带多个差异甲基化位点的高/低甲基化基因，包括COL1A1、MEFV及TNF。细胞迁移相关通路的低甲基化水平显著升高，包括黏着斑、细胞黏附、跨内皮迁移及细胞外基质相互作用通路。针对OA与正常FLS的验证实验同样证实，RA与对照FLS可基于甲基化模式实现分离。
研究结论：差异甲基化基因可改变FLS的基因表达，进而参与RA的发病机制。关键基因的DNA甲基化状态提示RA FLS存在表观遗传印记，表明表观遗传调控在炎症性关节炎的发病中发挥重要作用。
实验整体设计：取自骨关节炎与类风湿关节炎患者的成纤维样滑膜细胞株。