Smoking and blood DNA methylation: an epigenome-wide association study and assessment of reversibility

We conducted a genome-wide association study of blood DNA methylation and smoking, attempted replication of previously discovered associations, and assessed the reversibility of smoking-associated methylation changes. DNA methylation was measured in baseline peripheral blood samples for 5,044 participants in the Melbourne Collaborative Cohort Study. For 1,032 participants, these measures were repeated using blood samples collected at follow-up, a median of 11 years later. A cross-sectional analysis of the association between smoking and DNA methylation and a longitudinal analysis of changes in smoking status and changes in DNA methylation were conducted. We used our cross-sectional analysis to replicate previously reported associations for current (N = 3,327) and former (N = 172) smoking. A comprehensive smoking index accounting for the biological half-life of smoking compounds and several aspects of smoking history was constructed to assess the reversibility of smoking-induced methylation changes. This measure of lifetime exposure to smoking allowed us to detect more associations than comparing current with never smokers. We identified 4,496 cross-sectional associations at P −7, including 3,296 annotated to 1,326 genes that were not previously implicated in smoking-associated DNA methylation changes at this significance threshold. We replicated the majority of previously reported associations (P −7) for current and former smokers. In our data, we observed for former smokers a substantial degree of return to the methylation levels of never smokers, compared with current smokers (median: 74%, IQR = 63-86%), corresponding to small values (median: 2.75, IQR = 1.5–5.25) for the half-life parameter of the comprehensive smoking index. Longitudinal analyses identified 368 sites at which methylation changed upon smoking cessation. Our study demonstrates the usefulness of the comprehensive smoking index to detect associations between smoking and DNA methylation at CpGs across the genome, replicates the vast majority of previously reported associations, and quantifies the reversibility of smoking-induced methylation changes.本研究针对血液DNA甲基化与吸烟行为开展全基因组关联分析（genome-wide association study），尝试对既往已发现的关联进行重复验证，并评估吸烟相关甲基化改变的可逆性。研究对象为墨尔本协作队列研究（Melbourne Collaborative Cohort Study）的5044名参与者，我们对其基线外周血样本开展了DNA甲基化检测。其中1032名参与者的甲基化检测结果通过随访时采集的血液样本完成了重复测定，两次采样的中位间隔时长为11年。我们分别开展了吸烟与DNA甲基化关联的横断面分析（cross-sectional analysis），以及吸烟状态变化与DNA甲基化变化的纵向分析（longitudinal analysis）。我们利用本次横断面分析结果，对既往报道的当前吸烟者（样本量N=3327）与既往吸烟者（样本量N=172）相关关联进行了重复验证。我们构建了综合吸烟指数（comprehensive smoking index），该指数纳入了吸烟相关化合物的生物学半衰期以及吸烟史的多个维度，用于评估吸烟诱导的甲基化改变的可逆性。相较于仅对比当前吸烟者与从未吸烟者的分析方式，这一终身吸烟暴露量指标能够帮助我们检测到更多的关联信号。我们共鉴定出4496个符合P < 10^−7显著性阈值的横断面关联位点，其中3296个位点被注释到1326个基因，且在该阈值下，这些基因此前未被发现与吸烟相关DNA甲基化改变存在关联。我们对既往报道的绝大多数当前吸烟者与既往吸烟者相关关联完成了重复验证（P < 10^−7）。在本研究数据中，与当前吸烟者相比，既往吸烟者的甲基化水平已显著向从未吸烟者回归（中位恢复比例为74%，四分位间距为63%~86%），这一结果对应综合吸烟指数的半衰期参数取值较小（中位值为2.75，四分位间距为1.5~5.25）。纵向分析共鉴定出368个在戒烟后甲基化水平发生改变的CpG位点。本研究证实了综合吸烟指数可用于检测全基因组范围内吸烟与CpG位点DNA甲基化之间的关联，重复验证了绝大多数既往报道的关联，并量化了吸烟诱导的甲基化改变的可逆性。