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

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). Overall design: 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).

为更精准地表征全血中与吸烟相关的甲基化修饰变化，我们采用Illumina HumanMethylation450 BeadChip（Illumina人类甲基化450K芯片）对当前吸烟者（SM，n=172）与从未吸烟者（NS，n=81）的DNA样本开展检测。实验整体设计：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年至1995年间从美国北卡罗来纳州达勒姆与教堂山的普通人群中招募为健康志愿者。该队列属于一项基于社区的健康样本，包含294名无亲缘关系的黑人和白人健康受试者，相关队列信息已在多项研究中被报道（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个月内的日均香烟消耗量。样本分析工作遵循经批准的人类受试者研究方案（NIEHS 86-E-0037）开展。全血有核DNA通过蔗糖诱导细胞渗透裂解后收集，经酚-氯仿抽提获得，随后将DNA保存于Tris-EDTA（三羟甲基氨基甲烷-乙二胺四乙酸）缓冲液中，置于-20℃低温保存。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）使用人类甲基化450K芯片（Illumina）完成甲基化水平检测。具体实验流程如下：取500 ng基因组DNA，使用EZ-DNA甲基化试剂盒（Zymo Research）进行亚硫酸氢盐转化，将转化后的样本杂交至HumanMethylation450 BeadChip芯片阵列，随后按照制造商的操作规范，使用iScan微阵列扫描仪（Illumina）完成芯片扫描。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。