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

Objective: To construct a pyrosequencing method applicable to the methylation of genes related to environmental chemical pollutant exposure and apply it to detect the gene methylation modification of peripheral blood cell DNA of coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs). Methods: The methylation level of the Ras association domain family 1A (RASSF1A) gene in the peripheral blood of workers occupationally exposed to PAHs was detected by bisulfite sequencing. Fragments with relatively concentrated methylation sites that showed an obvious response to the exposure factor, that is, with significant statistical differences between the two groups of people, were selected as methylation-sensitive regions. A pyrosequencing method was constructed for this region and verified in coke oven workers. Results: The constructed pyrosequencing method was characterized by being faster, simpler, more accurate and more economical, and could effectively distinguish workers occupationally exposed to PAHs from control workers. Among them, the methylation modification level of RASSF1A in coke oven-exposed workers increased by 87.15% (9.32% ± 3.82% in the exposed group and 4.98% ± 2.28% in the control workers, P < 0.01). Conclusion: Selecting pyrosequencing fragments based on the results of bisulfite sequencing of people exposed to exogenous chemicals has good chemical specificity and accuracy and can be used for the detection of DNA methylation in large samples of occupationally exposed people.

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。