基因组特异等位基因表达研究砷暴露毒性效应易感性个体差异

Arsenic,a commen enviromental toxicant, can lead to a multitude of toxic effects including induction of cancer. The mechanism underlying the genentic and epigenetic variation associated with interindividual sensitivity and diversified toxicity in the response to arsenic exposure is unclear. Genome-wide association study and gene expression quantitative trait loci mapping are the most widely used contemporary approaches to connect genetic variation to phenotypic diversity. However, the requirement of large-scale population samples is expensive and time consuming. Moreover, the existance of environmental confounding factors limits their application to study gene-environment interactions.In this project, we plan to use allele-specific expression analysis, a method with the advantage of a within-sample control - the other allele - to sensitively and specifically investigate the effects of genetic and epigenetic differences on cis -regulatory element in diploid genomes,in conbination with allele-specific DNA-protein interactions assay ,bioinformatic and statistic analysis, using 45 unrelated EBV-transformed lymphoblastoid cell lines （ Han Chinese individuals collected from Beijing ）provided by Hapmap Project, to explore the mechanism underlying the interindividual diversity of toxicological effects of arsenic exposure. The function of candidate SNPs will be further validated by allele-specific gene espression in cell lines derived from another 150 (50 families ) Han Chinese individuals collected from the south) and freshly isolated lymphocytes from healthy population. The results derived from this project will provide a basis and strategy for the establishment of individual-based risk evaluation and preventive policy for the environmental exposure of arsenic and other environmental toxicants.

砷是一种常见环境毒物，引起癌症和其他各种疾病，个体间砷毒性效应敏感性差异与遗传、表观遗传和环境因素密切相关，但机制不明确。全基因组关联和表达数量性状位点分析已广泛运用在遗传性状和疾病关系的研究，但是大样本、费时耗钱以及大量环境因素的混杂干扰，限制了其在遗传性状与环境暴露有关疾病关系的研究。鉴于基因组特异等位基因表达分析以细胞内等位基因表达自身相互对照，结果直观,混杂因素少,本项目以该方法为基础,结合能反映细胞内DNA原位转录调控的功能学实验,以及生物信息学和生物统计分析,利用HapMap收集并完成基因组基因分型的人群来源小样本类淋巴母细胞系（中国汉族）为砷暴露模型, 研究砷毒性效应敏感性个体差异的遗传和表观遗传因素,并在另外中国南方汉族家系来源的淋巴细胞系以及健康人群新鲜分离淋巴细胞中验证结果。研究方法和结果将为制定基于个体遗传差异的砷和其他环境毒物的人群风险评价体系和预防策略提供依据。

砷对人体健康影响为科学研究证明。低剂量暴露后，个体效应差异有文献报道，原因并不明确，尤其个体遗传特性在砷暴露致病过程中的机制不明确。个体特定DNA序列在外界环境作用下，通过特定基因调控机制，转录特定RNA，表达各种数量的蛋白质，决定细胞的代谢。个体基因SNP差异, miRNA和IncRNA 表达差异，都最终作用细胞调控基因表达，产生不同代谢反应。本研究目标是发现个体间在低剂量砷暴露后，基因表达的差异，推断相关基因的可能调控机制.1)孕期砷暴露(10ug/L 和50ug/L)对仔代海马神经元各种mRNA、miRNA和lncRNA表达的影响，为人群研究基因调控差异奠定基础。按差异倍数（FC）>2或<0.5及组间P值<0.05筛选出差异表达mRNA，以及差异miRNA和IncRNA预测的靶基因进行GO和KEGG Pathway分析.发现仔鼠海马神经元细胞砷暴露引起上调基因基本为嗅觉受体相关以及甲基转移酶，下调基因为具有肿瘤坏死因子受体、细胞生长因子结合蛋白，功能线粒体核糖蛋白、神经营养蛋白、组蛋白等。主要干扰了嗅觉传导、神经配体交互、不饱和脂肪酸代谢等代谢通路，特定miRNA变化可能调控参与β诱导转化生长因子、线粒体相关的凋亡诱导因子、金属相关的转运蛋白等功能，l特定ncRNA表达可能调控了嗅觉受体相关基因。研究提示孕期砷暴露（即使低剂量）可引起后代海马神经系统发育的改变，发现了砷暴露影响的特定mRNA，miRNA和IncRNA。结果发表因故未完成。.2）研究正常和职业重金属暴露人群淋巴细胞体外砷暴露后的基因表达差异。正常和血液砷含量高人员淋巴细胞在体外砷染毒后，等位基因的变化差异。推断可能的DNA调控机制。连续健康体检，推断基因表达与健康的联系。由于健康个体，血浆游离DNA主要来源于正常细胞的细胞凋亡， 血浆游离DNA被认为是与一些调控蛋白结合，故没有降解而存在血液中，测定血浆游离DNA的序列推断基因调控的位点。课题完成了血液样本收集，部分样本血清砷浓度监测，EBV 病毒转染淋巴细胞和血浆游离DNA提取测序。结果发表因故未完成。