Mitochondrial DNA methylation in placental tissue: a proof of concept study by means of prenatal environmental stressors

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 ENVIR<i>ON</i>AGE 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 (<i>D-loop</i> and <i>LDLR2</i>) by bisulphite pyrosequencing. Additionally, we measured DNA methylation on nuclear genes involved in mitochondrial maintenance (<i>PINK1, DNA2</i>, and <i>POLG1</i>) and assessed mtDNA content using qPCR. Absolute <i>D-loop</i> 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 <i>D-loop</i> 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 <i>D-loop</i> methylation). Most notably, our data demonstrates relevant levels of mtDNA methylation in placenta tissue, with significant associations between prenatal exposure to environmental stressors and <i>D-loop</i> methylation.

既往研究已证实，产前暴露于环境应激源与线粒体DNA（mitochondrial DNA, mtDNA）甲基化存在关联，但近期多项研究对甲基化评估的准确性及其生物学相关性提出了质疑。本研究针对胎盘组织的mtDNA甲基化展开探究，同时考量了核污染、亚硫酸氢盐转化及PCR偏倚等方法学层面的问题。本研究从ENVIRONAGE出生队列中选取了三组研究对象（每组n=20）：妊娠期间吸烟的母亲组（日均吸烟13.2支）、空气污染物高暴露组（PM₂.₅：16.0±1.4 μg/m³，黑碳：1.8±0.3 μg/m³）以及空气污染物低暴露的对照组（非吸烟母亲，PM₂.₅：10.6±1.7 μg/m³，黑碳：0.9±0.1 μg/m³）。本研究通过亚硫酸氢盐焦磷酸测序技术，对位移环控制区（D-loop与LDLR2）两个区域的DNA甲基化水平进行定量检测。此外，我们还检测了参与线粒体维持功能的核基因（PINK1、DNA2及POLG1）的DNA甲基化水平，并通过qPCR评估了mtDNA含量。相较于对照组，重度吸烟母亲组与空气污染物高暴露母亲组的D-loop绝对甲基化水平均显著升高，分别提升0.36%（95%置信区间（confidence interval, CI）：0.06%~0.66%）与0.47%（95%置信区间（confidence interval, CI）：0.20%~0.73%）。本研究结果的相关性得到进一步验证：D-loop甲基化水平与胎盘mtDNA含量呈显著负相关（r=-0.40，p=0.002），且与新生儿出生体重存在关联——当D-loop甲基化水平升高四分位数间距（interquartile range, IQR）时，出生体重降低106.98 g（95%置信区间（confidence interval, CI）：-209.60 g~-4.36 g）。尤为重要的是，本研究数据证实胎盘组织中存在具有生物学意义的mtDNA甲基化水平，且产前暴露于环境应激源与D-loop甲基化之间存在显著关联。