Differential methylation analysis in human whole blood DNA from healthy smokers and non-smokers

To better characterize smoking–associated methylation changes in whole blood, we used Illumina HumanMethylation450 BeadChip to assess DNA samples from current (SM, n=172) and never smokers (NS, n=81). 253 individual study participants consisting of 172 smokers and 81 non-smokers enrolled between 1993 and 1995 as healthy volunteers from the general public in Durham and Chapel Hill, North Carolina . These subjects were part of a community-based sample comprised of 294 healthy unrelated blacks and whites and have been described in several studies (Jones et al. 1993; Bell et al. 1995; Li et al. 2000; Lunn et al. 1999). All non-smokers were self-reported as not having smoked >100 cigarettes in their lifetime. Smokers reported their average daily cigarette consumption for the past 3 months. The analysis of samples was carried out under an approved human subject protocol (NIEHS 86-E-0037). Nucleated DNA from whole blood was collected after sucrose-induced osmotic lysis of cells followed by phenol-chloroform extraction and DNA was stored in Tris-EDTA buffer at -20°C. The Human Methylation 450 BeadChip (Illumina) was used to measure methylation by the NCI Center for Genome Research. Specifically, 500 ng of DNA was bisulfite converted using the EZ-DNA Methylation kit (Zymo Research), hybridized to HumanMethylation450 BeadChip arrays and then scanned with an iScan microarray scanner (Illumina) following the manufacturer’s protocols. The ChAMP pipeline was used to extract and Normalize data from iDat files (Morris et al. 2014).

为了更好地表征全血中与吸烟相关的甲基化变化，我们使用Illumina HumanMethylation450 BeadChip（Illumina人类甲基化450K芯片）对当前吸烟者（SM，n=172）和从未吸烟者（NS，n=81）的DNA样本进行检测。
本研究共纳入253名个体受试者，其中172名吸烟者与81名非吸烟者，于1993至1995年间从北卡罗来纳州达勒姆和教堂山的普通人群中招募为健康志愿者。这些受试者属于一项包含294名健康无亲缘关系黑人和白人的基于社区的研究队列，此前已有多项研究对该队列进行过报道（Jones等，1993；Bell等，1995；Li等，2000；Lunn等，1999）。
所有非吸烟者均自述终身吸烟量未超过100支；吸烟者则自述了过去3个月的日均吸烟量。
样本分析流程已通过人类受试者研究方案审批（NIEHS 86-E-0037）。
全血经蔗糖诱导细胞渗透性裂解后，采用酚-氯仿法提取有核细胞DNA，随后将DNA保存于Tris-EDTA缓冲液中，置于-20℃低温冰箱储存。
本研究由美国国家癌症研究所基因组研究中心（NCI Center for Genome Research）使用HumanMethylation 450 BeadChip（Illumina人类甲基化450K芯片）完成甲基化水平检测。具体实验流程如下：取500 ng DNA样本，使用EZ-DNA Methylation试剂盒（Zymo Research）进行亚硫酸氢盐转化，随后按照制造商操作规程将样本杂交至HumanMethylation450 BeadChip芯片阵列，最后使用iScan微阵列扫描仪（Illumina）完成扫描。
研究采用ChAMP流程（Morris等，2014）从iDat文件中提取数据并完成标准化处理。