ABSTRACT

While previous studies have demonstrated that prenatal exposure to environmental stressors is associated with mitochondrial DNA (mtDNA) methylation, more recent investigations are questioning the accuracy of the methylation assessment and its biological relevance. In this study, we investigated placental mtDNA methylation while accounting for methodological issues such as nuclear contamination, bisulphite conversion, and PCR bias. From the ENVIRONAGE birth cohort, we selected three groups of participants (n = 20/group). One group with mothers who smoked during pregnancy (average 13.2 cig/day), one group with high air pollutant exposure (PM_2.5: 16.0 ± 1.4 µg/m³, black carbon: 1.8 ± 0.3 µg/m³) and one control group (non-smokers, PM_2.5: 10.6 ± 1.7 µg/m³, black carbon: 0.9 ± 0.1 µg/m³) with low air pollutant exposure. DNA methylation levels were quantified in two regions of the displacement loop control region (D-loop and LDLR2) by bisulphite pyrosequencing. Additionally, we measured DNA methylation on nuclear genes involved in mitochondrial maintenance (PINK1, DNA2, and POLG1) and assessed mtDNA content using qPCR. Absolute D-loop methylation levels were higher for mothers that smoked extensively (+0.36%, 95% CI: 0.06% to 0.66%), and for mothers that were highly exposed to air pollutants (+0.47%, 95% CI: 0.20% to 0.73%). The relevance of our findings is further supported, as D-loop methylation levels were correlated with placental mtDNA content (r = −0.40, p = 0.002) and associated with birth weight (−106.98 g, 95% CI: −209.60 g to −4.36 g for an IQR increase in D-loop methylation). Most notably, our data demonstrates relevant levels of mtDNA methylation in placenta tissue, with significant associations between prenatal exposure to environmental stressors and D-loop methylation.

摘要

虽然先前的研究表明，产前暴露于环境压力源与线粒体 DNA (mtDNA) 甲基化有关，但最近的研究质疑甲基化评估的准确性及其生物学相关性。在这项研究中，我们研究了胎盘 mtDNA 甲基化，同时考虑了诸如核污染、亚硫酸氢盐转化和 PCR 偏差等方法学问题。从 ENVIR ON AGE 出生队列中，我们选择了三组参与者（n = 20/组）。一组母亲在怀孕期间吸烟（平均 13.2 支/天），一组高空气污染物暴露（PM _2.5：16.0 ± 1.4 µg/m ³，黑碳：1.8 ± 0.3 µg/m ³）和一个对照组（非吸烟者，下午_2.5：10.6 ± 1.7 µg/m ³，黑碳：0.9 ± 0.1 µg/m ³ )，空气污染物暴露量低。通过亚硫酸氢盐焦磷酸测序在置换环控制区的两个区域（D-环和LDLR2）中量化 DNA 甲基化水平。此外，我们测量了参与线粒体维持的核基因（PINK1、DNA2和POLG1）的 DNA 甲基化，并使用 qPCR 评估了 mtDNA 含量。绝对D 环大量吸烟的母亲（+0.36%，95% CI：0.06% 至 0.66%）和高度暴露于空气污染物的母亲（+0.47%，95% CI：0.20% 至 0.73%）的甲基化水平更高. 我们的研究结果的相关性得到进一步支持，因为D 环甲基化水平与胎盘 mtDNA 含量相关（r = -0.40，p = 0.002）并与出生体重相关（-106.98 g，95% CI：-209.60 g 至 - D-环甲基化IQR 增加 4.36 g ）。最值得注意的是，我们的数据证明了胎盘组织中 mtDNA 甲基化的相关水平，产前暴露于环境压力源与D 环甲基化之间存在显着关联。