Maternal smoking during pregnancy is associated with mitochondrial DNA methylation

Abstract

Maternal smoking during pregnancy (MSDP) has detrimental effects on fetal development and on the health of the offspring into adulthood. Energy homeostasis through ATP production via the mitochondria (mt) plays a key role during pregnancy. This study aimed to determine if MSDP resulted in differences in DNA methylation to the placental mitochondrial chromosome at the transcription and replication control region, the D-Loop, and if these differences were also present in an alternate neonatal tissue (foreskin) in an independent birth cohort. We investigated mtDNA methylation by bisulfite-pyrosequencing in two sections of the D-Loop control region and in long interspersed nuclear element-1 (LINE-1) genomic sequences in placenta from 96 mother-newborn pairs that were enrolled in a Rhode Island birth cohort along with foreskin samples from 62 infants from a Kentucky birth cohort. In both placenta and foreskin, mtDNA methylation in the light chain D-Loop region 1 was positively associated with MSDP in placenta (difference+2.73%) (P=0.001) and foreskin (difference+1.22%) (P=0.08). Additionally, in foreskin, a second segment of the D-Loop-heavy chain region 1 showed a small but significant change in methylation with MSDP (+0.4%, P=0.04). No methylation changes were noted in either tissue at the LINE-1 repetitive element. We identified a similar pattern of epigenetic effect to mitochondria arising in cells from different primordial lineages and in different populations, associated with MSDP. These robust and consistent results build evidence that MSDP may impact mt D-Loop methylation, as one mechanism through which this exposure affects newborn health.

Keywords: DNA methylation; maternal smoking; mitochondria; placenta; pregnancy.

Figure 1

Map of mitochondrial genome and sequences on the MT chromosome used for methylation analysis. The outer circle (green) represents the heavy chain MT chromosome and the inner circle (purple) being the MT light chain ( A ). The 16,569 base chromosome is numbered at various segments for orientation. Coding genes are labeled on the H & L chains and the 12S and 16S ribosomal RNA genes. tRNAs are represented by thick black bars. The D-Loop control region is contained within bases 576–16,024. Yellow rectangle outlines the section of the mitochondrial chromosome within which the methylation assays were designed. The specific MT-chromosomal sequences and CpG sites analyzed in D-Loop are detailed ( B )

Figure 2

Mitochondrial and global methylation associated with maternal smoking during pregnancy in placenta . Median methylation % was measured for each pyrosequencing assay from placenta of mothers comparing NS ( n  = 37), 1T smokers ( n  = 23) and FT pregnancy smokers ( n  = 36). Mitochondrial LDLR1 showed significantly increased methylation in full pregnancy smokers ( P = 0.001, Kruskal-Wallis, NS to FT P = 0.001, Dunn’s post hoc) ( A ). HDLR1 ( B ) showed no differences in methylation among the smoking groups ( P  = 0.66). Horizontal lines represent the median. Global methylation measured via LINE-1 (C) showed no differences between the groups ( P = 0.87). Horizontal lines represent the mean (LINE-1)

Figure 3

Mitochondrial and global methylation associated with maternal smoking during pregnancy in foreskin. Median methylation % was measured for each pyrosequencing assay from foreskin of infants of the Kentucky birth cohort. Mitochondrial LDLR1 showed a trend of increased methylation in FT pregnancy smokers ( n = 21) vs. NS ( n = 41) ( P = 0.08, Mann–Whitney) (A). Mitochondrial HDLR1 showed significantly increased methylation in FT pregnancy smokers ( n = 20) vs. NS ( n = 38) ( P = 0.04, Mann–Whitney) (B) (four individual samples failed pyrosequencing assay for HDLR1). Horizontal lines represent the median. Global methylation measured via LINE-1 (C) showed no differences between the groups ( P = 0.58, t -test), (FT n = 13, NS n = 21). Horizontal lines represent the mean (LINE-1)