Table_11_Maternal Smoking During Pregnancy Induces Persistent Epigenetic Changes Into Adolescence, Independent of Postnatal Smoke Exposure and Is Associated With Cardiometabolic Risk.xlsb

Background: Several studies have shown effects of current and maternal smoking during pregnancy on DNA methylation of CpG sites in newborns and later in life. Here, we hypothesized that there are long-term and persistent epigenetic effects following maternal smoking during pregnancy on adolescent offspring DNA methylation, independent of paternal and postnatal smoke exposure. Furthermore, we explored the association between DNA methylation and cardiometabolic risk factors at 17 years of age.
Materials and Methods: DNA methylation was measured using the Illumina HumanMethylation450K BeadChip in whole blood from 995 participants attending the 17-year follow-up of the Raine Study. Linear mixed effects models were used to identify differential methylated CpGs, adjusting for parental smoking during pregnancy, and paternal, passive, and adolescent smoke exposure. Additional models examined the association between DNA methylation and paternal, adolescent, and passive smoking over the life course. Offspring CpGs identified were analyzed against cardiometabolic risk factors (blood pressure, triacylglycerols (TG), high-density lipoproteins cholesterol (HDL-C), and body mass index).
Results: We identified 23 CpGs (genome-wide p level: 1.06 × 10−7) that were associated with maternal smoking during pregnancy, including associated genes AHRR (cancer development), FTO (obesity), CNTNAP2 (developmental processes), CYP1A1 (detoxification), MYO1G (cell signalling), and FRMD4A (nicotine dependence). A sensitivity analysis showed a dose-dependent relationship between maternal smoking and offspring methylation. These results changed little following adjustment for paternal, passive, or offspring smoking, and there were no CpGs identified that associated with these variables. Two of the 23 identified CpGs [cg00253568 (FTO) and cg00213123 (CYP1A1)] were associated with either TG (male and female), diastolic blood pressure (female only), or HDL-C (male only), after Bonferroni correction.
Discussion: This study demonstrates a critical timing of cigarette smoke exposure over the life course for establishing persistent changes in DNA methylation into adolescence in a dose-dependent manner. There were significant associations between offspring CpG methylation and adolescent cardiovascular risk factors, namely, TG, HDL-C, and diastolic blood pressure. Future studies on current smoking habits and DNA methylation should consider the importance of maternal smoking during pregnancy and explore how the persistent DNA methylation effects of in utero smoke exposure increase cardiometabolic risk.

### 背景
已有多项研究证实，当前吸烟及孕期母亲吸烟会对新生儿及日后生命进程中CpG位点（CpG site）的DNA甲基化（DNA methylation）产生影响。本研究假设，孕期母亲吸烟会对子代青少年的DNA甲基化产生长期且持久的表观遗传（epigenetic）效应，且该效应不受父亲吸烟及产后吸烟暴露的影响。此外，本研究还探究了17岁时DNA甲基化与心脏代谢风险因子之间的关联。

### 材料与方法
本研究针对Raine研究（Raine Study）的995名参与17年随访的受试者，采集其全血样本，通过Illumina人类甲基化450K微珠芯片（Illumina HumanMethylation450K BeadChip）检测DNA甲基化水平。采用线性混合效应模型筛选差异甲基化CpG位点（differentially methylated CpGs），校正孕期父母吸烟、父亲吸烟、被动吸烟及青少年时期吸烟暴露等混杂因素。额外构建模型，探究生命进程中父亲吸烟、青少年时期吸烟及被动吸烟与DNA甲基化之间的关联。对筛选出的子代CpG位点，分析其与心脏代谢风险因子——包括血压、三酰甘油（triacylglycerols, TG）、高密度脂蛋白胆固醇（high-density lipoproteins cholesterol, HDL-C）及体质量指数——之间的关联。

### 结果
本研究共筛选出23个与孕期母亲吸烟相关的CpG位点（全基因组显著性P值：1.06 × 10^−7），涉及的基因包括AHRR（参与癌症发生）、FTO（与肥胖相关）、CNTNAP2（参与发育过程）、CYP1A1（参与解毒过程）、MYO1G（参与细胞信号转导）及FRMD4A（与尼古丁依赖相关）。敏感性分析显示，母亲吸烟与子代甲基化水平之间存在剂量-反应关系。在校正父亲吸烟、被动吸烟或子代自身吸烟后，上述结果未发生显著变化；且未筛选出与这些吸烟暴露因素相关的CpG位点。经邦费罗尼校正（Bonferroni correction）后，23个位点中的2个[cg00253568（关联FTO基因）及cg00213123（关联CYP1A1基因）]分别与三酰甘油（男女均相关）、舒张压（仅女性相关）或高密度脂蛋白胆固醇（仅男性相关）存在关联。

### 讨论
本研究证实，生命进程中香烟烟雾暴露存在关键窗口期，可通过剂量依赖的方式使DNA甲基化产生持久改变，并持续至青少年时期。子代CpG位点甲基化水平与青少年时期心脏代谢风险因子（即三酰甘油、高密度脂蛋白胆固醇及舒张压）之间存在显著关联。未来针对当前吸烟习惯与DNA甲基化的研究，应考虑孕期母亲吸烟的重要性，并探究宫内烟雾暴露所导致的持久性DNA甲基化改变如何增加心脏代谢疾病风险。