de Andres María C, Perez-Pampin Eva, Calaza Manuel, Santaclara Francisco J, Ortea Ignacio, Gomez-Reino Juan J, Gonzalez Antonio
Laboratorio de Investigacion 10 and Rheumatology Unit, Instituto de Investigación Sanitaria-Hospital Clínico Universitario de Santiago, Travesia de Choupana, s/n, 15706, Santiago de Compostela, Spain.
Department of Medicine, University of Santiago de Compostela, Rúa de San Francisco, s/n, 15782, Santiago de Compostela, Spain.
Arthritis Res Ther. 2015 Aug 29;17(1):233. doi: 10.1186/s13075-015-0748-5.
DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results.
Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR).
Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression.
Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.
DNA甲基化是一种调节基因表达的表观遗传机制，在类风湿关节炎（RA）患者血液中的研究尚不充分，因为此前仅对确诊RA患者的T细胞和外周血单个核细胞（PBMC）进行过研究，且结果相互矛盾。
从19例早期RA患者和17例健康对照中分离出五种主要血细胞亚群：T细胞、B细胞、NK细胞、单核细胞和多形核白细胞。患者样本在开始使用甲氨蝶呤（MTX）治疗前及治疗1个月后采集。分析包括采用高效液相色谱-电喷雾电离-串联质谱-选择反应监测（HPLC-ESI-MS/MS-SRM）进行DNA甲基化检测，以及通过定量聚合酶链反应（qPCR）检测七种甲基化特异性酶的表达水平。
未使用改善病情抗风湿药（DMARD）的早期RA患者在T细胞和单核细胞中表现出整体DNA低甲基化，同时维持性DNA甲基转移酶DNA甲基转移酶1（DNMT1）的表达较低，B细胞中该酶表达也降低。此外，在单核细胞中发现参与去甲基化的双加氧酶10-11易位蛋白1（TET1）、TET2和TET3的表达显著增加，在T细胞中TET2表达增加。B细胞中DNMT3A的表达也有适度降低，T细胞和B细胞中生长停滞和DNA损伤诱导蛋白45A（GADD45A）的表达也降低。MTX治疗可逆转T细胞和单核细胞中的低甲基化，使其与对照组无异，并增加B细胞中的整体甲基化。此外，DNMT1和DNMT3A表达降低的趋势有所逆转。
我们的结果证实了RA患者存在整体DNA低甲基化，且在某些血细胞亚群中具有特异性，同时甲氨蝶呤治疗可使其逆转。这些变化伴随着甲基化相关酶水平的平行变化，提示在这一水平上存在调控的可能性。
