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

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.

KEYWORDS:

• Mitochondria
• mtDNA
• methylation
• placenta
• air pollution
• smoking
• prenatal
• DOHaD
• markers
• birthweight

Acknowledgments

The authors thank the participating women and neonates, as well as the staff of the maternity ward, midwives, and the staff of the clinical laboratory of East-Limburg Hospital in Genk. This research is supported by the Flemish Scientific Fund N1518119 and G082317N. Bram Janssen and Dries Martens are postdoctoral fellows funded by the Research Foundation Flanders (FWO) (12W3218N and 12X9620N respectively).

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Research Foundation Flanders [G082317N]; Research Foundation Flanders [N1518119].