环境化学污染物暴露相关基因甲基化焦磷酸测序方法的构建和应用

Construction of an environmental pollution-responsive gene-specific pyrosequencing assay

目的:构建适用于环境化学污染物暴露相关基因甲基化的焦磷酸测序方法,应用于检测多环芳烃暴露的焦炉工人外周血细胞DNA的基因甲基化修饰。方法:采用亚硫酸氢盐修饰后测序检测多环芳烃职业暴露工人外周血中RAS相关结构域家族基因1A(RASSF1A)的甲基化水平,选择对暴露因素响应明显,即在两组人群中存在显著统计学差异的甲基化位点较集中的片段作为甲基化敏感区域。针对该区域构建焦磷酸测序方法,并在焦炉工人中进行验证。结果:所构建的焦磷酸测序方法具有更快速、简便、准确及经济的特点,能有效区分多环芳烃职业暴露工人与对照工人,其中,焦炉暴露工人RASSF1A甲基化修饰水平升高了87.15%(暴露组为9.32%±3.82%,对照工人为4.98%±2.28%,P<0.01)。结论:基于外源性化学物暴露人群的亚硫酸氢盐修饰后测序结果选取焦磷酸测序片段具有良好的化学物特异性和准确度,可用于职业暴露人群大样本DNA甲基化检测。

OBJECTIVE：To establish a pollutant-responsive gene-specific pyrosequencing assay for quantitative measurement of DNA methylation in peripheral blood lymphocytes of coke-oven workers.METHODS：We firstly analyzed DNA methylation levels of RAS-associated domain family 1 （RASSF1A） in 69 coke-oven workers and 46 controls using bisulfate sequencing method. The hyper-methylated hotspots in the exposure group were recognized as the target region for design of specific primers of pyrosequencing. As a pilot study,we examined the methylation status ofRASSF1A in 11 paired coke-oven workers and controls.RESULTS：Pyrosequencing was the optimized assay for DNA methylation detection. Compared to BSP assay,pyrosequencing exhibited advantages in efficiency,simplicity,accuracy and economical performances. We were able to distinguish occupational exposed workers from controls effectively using this method. The methylation level ofRASSF1A in coke-oven workers was increased by 87.15% compared to that in the controls （9.32%±3.82% and 4.98%±2.28%,respectively,P〈0.01）.CONCLUSION：Compared to the BSP assay, gene-specific pyrosequencing method exhibited strength in specificity and accuracy of methylation detection of environmental pollutant-responsive gene in PAHs-exposed workers.