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）已将尼古丁依赖与吸烟行为相关基因，与心血管、肺部及恶性疾病的发病风险升高联系起来。本研究使用人类甲基化450K芯片（Human Methylation 450 K array），对464名个体（22名当前吸烟者与263名既往吸烟者）的外周血DNA开展表观全基因组关联研究（epigenome wide association study）。在包含356名双胞胎（41名当前吸烟者与104名既往吸烟者）的独立样本中完成验证后，我们在15个不同的基因座中鉴定出30个探针，所有探针在联合分析中均达到全基因组显著性水平（P < 5×10⁻⁸）。除探针cg17024919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。我们验证了全部9个已知基因座，并在GPR15邻近的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基因座中发现了6个全新的基因座。大多数与吸烟相关的先导探针（15个中的13个）与开放染色质区域（FAIRE与DNaseI超敏位点）或/和组蛋白H3赖氨酸27乙酰化（H3K27Ac）峰（ENCODE数据集（ENCODE data set））重叠，而这些区域正是调控元件的标志性区域。吸烟对DNA甲基化的影响在戒烟超过3个月后可部分逆转。我们首次报道了单核苷酸多态性（Single Nucleotide Polymorphism，SNP）rs2697768与吸烟对DNA甲基化位点cg03329539之间存在具有统计学意义的交互作用。我们的研究证据表明，cg03329539的甲基化SNP（metSNP）可调控位于该甲基化位点下游约95kb处的CHRND基因的表达。本研究结果提示，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持续性健康风险。