DNA methylation in airway and parenchymal fibroblasts from individuals with and wthout COPD. DNA methylation in airway and parenchymal fibroblasts from individuals with and wthout COPD

Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease of the lungs that is currently the fourth leading cause of death worldwide. Genetic factors account for only a small amount of COPD risk, but epigenetic mechanisms including DNA methylation, have the potential to mediate the interactions between an individual?s genetics and environmental exposure. DNA methylation is highly cell type specific and individual cell type studies of DNA methylation in COPD are sparse. Fibroblasts are present within the airway and parenchyma of the lung and contribute to the aberrant deposition of extracellular matrix in COPD. No assessment or comparison of genome-wide DNA methylation profiles in airway and parenchymal fibroblasts from individuals with and without COPD has been undertaken. These data provide valuable insight into the molecular mechanisms contributing to COPD and the differing pathologies of small airways disease and emphysema in COPD. Methods: Genome-wide DNA methylation was evaluated at over 485,000 CpG sites using the Illumina Infinium HumanMethylation450 BeadChip array in airway (non-COPD n=8, COPD n=7) and parenchymal fibroblasts (non-COPD n=18, COPD n=28) isolated from individuals with and without COPD. Targeted gene expression was assessed by qPCR in matched RNA samples. Results: Differentially methylated DNA regions were identified between cells isolated from individuals with and without COPD in both airway and parenchymal fibroblasts. Only in parenchymal fibroblasts was differential DNA methylation associated with differential gene expression. A second analysis of differential DNA methylation variability identified 359 individual differentially variable CpG sites in parenchymal fibroblasts. No differentially variable CpG sites were identified in airway fibroblasts. Five differentially variable methylated CpG sites, associated with three genes were subsequently assessed for gene expression differences. Two genes (OAT and GRIK2) displayed significantly increased gene expression in cells isolated from individuals with COPD. Conclusions: Differential and variable DNA methylation was associated with COPD status in parenchymal fibroblasts but not airway fibroblasts. Aberrant DNA methylation was associated with altered gene expression imparting biological function to DNA methylation changes. Changes in DNA methylation are therefore implicated in the molecular mechanisms underlying COPD pathogenesis and may represent novel therapeutic targets. Overall design: Genome-wide DNA methylation was evaluated at over 485,000 CpG sites using the Illumina Infinium HumanMethylation450 BeadChip array in airway (non-COPD n=8, COPD n=7) and parenchymal fibroblasts (non-COPD n=18, COPD n=28) isolated from individuals with and without COPD.

背景：慢性阻塞性肺疾病（Chronic obstructive pulmonary disease, COPD）是一类异质性肺部疾病，目前位列全球第四大死亡病因。遗传因素仅能解释小部分COPD发病风险，而包括DNA甲基化（DNA methylation）在内的表观遗传机制，可介导个体遗传因素与环境暴露之间的相互作用。DNA甲基化具有高度的细胞类型特异性，但目前针对COPD中DNA甲基化的单细胞类型研究仍较为匮乏。成纤维细胞存在于肺部气道与肺实质中，可促进COPD患者体内细胞外基质的异常沉积。目前尚无针对伴与不伴COPD个体的气道及肺实质成纤维细胞全基因组DNA甲基化谱进行评估或比较的研究。本数据集可为阐明COPD的分子发病机制，以及COPD中小气道疾病与肺气肿的不同病理特征提供宝贵的研究视角。
方法：本研究采用Illumina Infinium HumanMethylation450 BeadChip芯片，对从伴或不伴COPD个体中分离得到的气道成纤维细胞（非COPD组n=8，COPD组n=7）与肺实质成纤维细胞（非COPD组n=18，COPD组n=28）中超过485,000个CpG位点（CpG site）开展全基因组DNA甲基化检测。通过qPCR对匹配的RNA样本进行靶向基因表达分析。
结果：在气道与肺实质成纤维细胞中，均鉴定出了伴与不伴COPD个体来源细胞之间的差异甲基化DNA区域。仅在肺实质成纤维细胞中，差异DNA甲基化与差异基因表达存在显著关联。针对差异DNA甲基化变异性的二次分析，在肺实质成纤维细胞中鉴定出359个独立的差异可变CpG位点，而在气道成纤维细胞中未发现任何差异可变CpG位点。后续针对与3个基因相关的5个差异可变甲基化CpG位点开展基因表达差异评估，结果显示OAT与GRIK2这两个基因在COPD个体来源的细胞中呈现显著升高的表达水平。
结论：差异甲基化与可变DNA甲基化在肺实质成纤维细胞中与COPD患病状态相关，而在气道成纤维细胞中无此关联。异常DNA甲基化与基因表达改变相关，赋予了DNA甲基化变化生物学功能。因此，DNA甲基化改变参与了COPD发病的分子机制，或可成为新型治疗靶点。
整体实验设计：本研究采用Illumina Infinium HumanMethylation450 BeadChip芯片，对从伴或不伴COPD个体中分离得到的气道成纤维细胞（非COPD组n=8，COPD组n=7）与肺实质成纤维细胞（非COPD组n=18，COPD组n=28）中超过485,000个CpG位点开展全基因组DNA甲基化检测。