宫内重金属暴露及相关基因甲基化与非综合征室间隔缺损的病例对照研究

A case-control study will be used to investigate the relationship between intrauterine exposures to selected heavy metals, DNA methylation changes of related genes, and the risk of nonsyndromic ventricular septal defects (NsVSD). The main contents are as follows：1)Envrionmental exposure study: the placental lead (Pb), mercury (Hg) and cadmium (Cd) of 200 NsVSD cases and 200 controls will be assessed, and the assocition of heavy metal exposure with NsVSD will be analyzed, and then to decide which one is the major pollutant. 2)DNA methylation study: According to different concentration levels of major heavy metal in control groups, 40 control infants will be selected and their genomic methylation of cord blood DNA and placenta DNA will be examined by NimbleGen CpG Promoter Assay to screen out 5 candidate loci for each type of tissue that are prone to methylation changes with prenatal heavy metal exposures. In addtion, about ten target regions including LINE-1/Alu repetitive elements, CpG islands in genes implicated in fetal heart development and one-carbon-related metabolisms,will be selected as candidate loci for DNA methylation analysis. Using the Sequenom MassArray Methylation platform, DNA methylation of these CpG sites in cord blood and placenta tissues will be detected for all cases and controls, and the relationship between loci-specific methylation and NsVSD will be evaluated, thus loci related to NsVSD risk will be identified for the next analysis.3) Using DNA samples extracted from cord blood, placenta and heart tissues of 20 aborted fetus without heart defects, the correlation of NsVSD-related loci-specific methylation between three different tissue DNA specimens will be assessed, then the applicability of methylation changes in cord blood and placenta tissues as surrogate marks in human CHD studies will be assessed. It is noteworthy that this part of research depends on the availability of tissue samples. Data from above studies will be incorporated into logistic regression models to explore the independent and combined effects of intrauterine Pb,Hg and Cd exposures, DNA methylation alterations on CHD risk, and possible mechanisms.

课题采用病例-对照研究探索宫内重金属暴露、DNA甲基化和非综合征室间隔缺损(NsVSD)的关系。主要内容1）环境暴露研究:检测200对病例和对照胎盘组织铅汞镉含量,关联分析鉴别主要污染物；2)DNA甲基化研究:根据主要污染物水平选取40例对照，NimbleGen CpG Promoter甲基化芯片检测脐血/胎盘DNA，筛选暴露敏感甲基化位点各5个；同时从LINE-1/Alu序列、心脏发育和一碳代谢基因中选约10个目标CpG区域；Sequenom MassArray Methylation技术检测全部病例和对照的胎盘及脐血DNA目标区域甲基化水平，确定NsVSD相关联的位点；3）视标本收集情况，拟用20例无畸形引产胎儿的胎盘/脐血和心脏组织DNA分析NsVSD风险位点甲基化水平的相关性，评价脐血/胎盘甲基化标记在人群CHD研究中的适用性。Logistic回归探讨暴露、DNA甲基化对畸形风险的影响及可能机制。

背景：采用病例-对照设计，探讨宫内铅汞镉砷暴露、DNA甲基化和NsVSD 的关系。.方法：基于出生队列流行病学数据和生物标本开展研究，石墨炉原子吸收光谱法检测胎盘组织重金属干重含量，Illumina 850K甲基化芯片检测胎盘和脐血DNA。非条件Logistic回归分析暴露、甲基化和疾病的关系。.结果：1）重金属暴露研究：373例对照和288例先天性心脏病（CHD，含171例NsVSD）中，病例组胎盘干重铅汞镉含量高于对照组。铅含量每增加105ng/g，CHD和NsVSD的发病风险增加，OR为1.08(95%CI：1.01-1.16和1.09(95%CI：1.01-1.17）；汞含量每增加65ng/g CHD 和NsVSD 风险增加，OR为1.06（95%CI：1.01-1.10）和1.05（95%CI：1.00-1.11）；砷含量每增加88ng/g，CHD风险增加（OR=1.20，95%CI：1.00-1.44）。2）DNA甲基化研究：基于49例对照胎盘DNA、60例对照和45例NsVSD脐血DNA全基因组甲基化谱数据，铅暴露相关差异甲基化位点（DMP）和区域（DMR）在胎盘和脐血DNA中有差别；多个DMP和DMR或与暴露、NsVSD有关；验证了5个DMP（FUS基因cg01699425、FDX1 cg09762563、EPHA5 cg10554702、UPK3B cg22995176和UBB cg27351675）的甲基化水平；调整混杂因素后，Logistic分析发现NsVSD与cg01699425（OR=1.41，95%CI:1.12-1.45）、cg22995176（OR=1.20,95%CI：1.03-1.39）和cg27351675（OR=2.21，95%CI：1.50-3.26）的高甲基化相关联（为0.01甲基化水平/β值对应的OR）；cg09762563（OR=0.88,95%CI：0.78-1.00）和cg10554702（OR=0.86,95%CI：0.76-0.98）高甲基化则可能为保护因素。.结论：胎儿宫内暴露于铅、汞等重金属元素是NsVSD的危险因素，暴露相关的差异甲基化位点也与疾病发生风险相关联。