Cigarette smoking reduces DNA methylation levels at multiple genomic loci but the effect is partially reversible upon cessation

Smoking is a major risk factor in many diseases. Genome wide association studies have linked genes for nicotine dependence and smoking behavior to increased risk of cardiovascular, pulmonary, and malignant diseases. We conducted an epigenome wide association study in peripheral-blood DNA in 464 individuals (22 current smokers and 263 ex-smokers), using the Human Methylation 450 K array. Upon replication in an independent sample of 356 twins (41 current and 104 ex-smokers), we identified 30 probes in 15 distinct loci, all of which reached genome-wide significance in the combined analysis <i>P</i> &lt; 5 × 10⁻⁸. All but one probe (cg17024919) remained significant after adjusting for blood cell counts. We replicated all 9 known loci and found an independent signal at <i>CPOX</i> near <i>GPR15.</i> In addition, we found 6 new loci at <i>PRSS23</i>, <i>AVPR1B</i>, <i>PSEN2</i>, <i>LINC00299</i>, <i>RPS6KA2,</i> and <i>KIAA0087.</i> Most of the lead probes (13 out of 15) associated with cigarette smoking, overlapped regions of open chromatin (FAIRE and DNaseI hypersensitive sites) or / and H3K27Ac peaks (ENCODE data set), which mark regulatory elements. The effect of smoking on DNA methylation was partially reversible upon smoking cessation for longer than 3 months. We report the first statistically significant interaction between a SNP (rs2697768) and cigarette smoking on DNA methylation (cg03329539). We provide evidence that the metSNP for cg03329539 regulates expression of the <i>CHRND</i> gene located circa 95 Kb downstream of the methylation site. Our findings suggest the existence of dynamic, reversible site-specific methylation changes in response to cigarette smoking , which may contribute to the extended health risks associated with cigarette smoking.

吸烟是多种疾病的重要危险因素。全基因组关联研究（Genome Wide Association Study）已证实，尼古丁依赖与吸烟行为相关的基因，与心血管、肺部及恶性疾病的发病风险升高存在显著关联。本研究采用人类甲基化450K芯片平台，对464名受试者（22名当前吸烟者、263名既往吸烟者）的外周血DNA开展表观基因组全关联研究。在包含356名双生子的独立验证队列（41名当前吸烟者、104名既往吸烟者）中完成验证后，我们在15个独立基因座中鉴定出30个探针位点，所有位点在合并分析中均达到全基因组显著性阈值（P < 5×10⁻⁸）。除cg17024919这一探针外，其余所有探针在校正血细胞计数后仍保持统计学显著性。本研究重复验证了全部9个已知基因座，并在GPR15基因邻近区域的CPOX基因位点发现了一个独立信号。此外，我们在PRSS23、AVPR1B、PSEN2、LINC00299、RPS6KA2及KIAA0087基因区域中发现了6个全新的基因座。与吸烟相关的15个核心探针中，有13个探针的靶向区域与开放染色质（FAIRE与DNaseI超敏位点）或/和标记调控元件的H3K27Ac峰（ENCODE数据集）存在重叠。吸烟对DNA甲基化的调控效应在戒烟超过3个月后可部分逆转。本研究首次报道了单核苷酸多态性（Single Nucleotide Polymorphism，SNP）rs2697768与吸烟行为在cg03329539位点的DNA甲基化水平上存在具有统计学显著性的交互作用。我们的研究证实，cg03329539位点的甲基化相关单核苷酸多态性（metSNP）可调控位于该甲基化位点下游约95kb处的CHRND基因的表达。本研究结果表明，吸烟可诱导产生动态、可逆的位点特异性DNA甲基化改变，这或可参与介导吸烟相关的长期健康风险。