Vitamin C Prevents Offspring DNA Methylation Changes Associated with Maternal Smoking in Pregnancy

Abstract

Rationale: Infants whose mothers smoked during pregnancy demonstrate lifelong decreases in pulmonary function. DNA methylation changes associated with maternal smoking during pregnancy have been described in placenta and cord blood at delivery, in fetal lung, and in buccal epithelium and blood during childhood. We demonstrated in a randomized clinical trial ( ClinicalTrials.gov identifier, NCT00632476) that vitamin C supplementation to pregnant smokers can lessen the impact of maternal smoking on offspring pulmonary function and decrease the incidence of wheeze at 1 year of age.

Objectives: To determine whether vitamin C supplementation reduces changes in offspring methylation in response to maternal smoking and whether methylation at specific CpGs is also associated with respiratory outcomes.

Methods: Targeted bisulfite sequencing was performed with a subset of placentas, cord blood samples, and buccal samples collected during the NCT00632476 trial followed by independent validation of selected cord blood differentially methylated regions, using bisulfite amplicon sequencing.

Measurements and main results: The majority (69.03%) of CpGs with at least 10% methylation difference between placebo and nonsmoker groups were restored (by at least 50%) toward nonsmoker levels with vitamin C treatment. A significant proportion of restored CpGs were associated with phenotypic outcome with greater enrichment among hypomethylated CpGs.

Conclusions: We identified a pattern of normalization in DNA methylation by vitamin C supplementation across multiple loci. The consistency of this pattern across tissues and time suggests a systemic and persistent effect on offspring DNA methylation. Further work is necessary to determine how genome-wide changes in DNA methylation may mediate or reflect persistent effects of maternal smoking on lung function.

Keywords: ascorbic acid; asthma; epigenetics; nicotine; prenatal exposure.

Figures

Figure 1.

DNA methylation changes associated with maternal smoking at targeted loci are blunted by vitamin C supplementation. Heatmaps were generated with CpGs differentially methylated between nonsmokers and placebo groups by pairwise t test (nominal P ≤ 0.05). Raw methylation was log2 scaled and mean centered for each CpG to normalize distribution before statistical testing (see the color keys). Samples from patients born to vitamin C–supplemented smokers (green, V1–Vn) cluster closer to the nonsmoking group (blue, N1–Nn) than to the placebo group (red, P1–Pn) in (A) placenta, (B) cord blood, and (C) buccal epithelium.

Figure 2.

Vitamin C treatment reverses the majority of significant DNA methylation changes associated with maternal smoking at targeted loci. Points represent mean methylation for nonsmokers (N), placebo-treated smokers (P), and vitamin C–supplemented smokers (V) at each CpG with significant change in methylation (≥10%; nominal P ≤ 0.05) between the placebo and nonsmoker groups. CpGs are clustered by the direction of change with maternal smoking (HYPER/HYPO) and lines represent the direction of change at each CpG in nonsmokers versus placebo and placebo versus vitamin C mean methylation.

Figure 3.

Select cord blood differentially methylated regions (DMRs) associated with maternal smoking: (A) Runt related transcription factor 1 (RUNX1); (B) growth factor independent 1 transcriptional repressor (GFI1); (C) aryl-hydrocarbon receptor repressor (AHRR); and (D) bone morphogenetic protein 4 (BMP4). DMRs were defined with the comb-p package to identify enriched regions of spatially related pairwise P values between nonsmokers and placebo samples. Plots on the left demonstrate the overall trend in methylation changes across the DMR by showing the mean methylation for nonsmokers (N), placebo (P), and vitamin C (V) at each false discovery rate–significant CpG. On the right, DMRs are plotted by treatment group with spatial orientation to the genome, with base position on the x-axis and methylation (mean ± SEM) on the y-axis.

Figure 4.

PR domain–containing 8 (PRDM8) is hypomethylated across multiple cord blood differentially methylated regions (DMRs) and restored by vitamin C (VitC). PRDM8 CpGs identified by meta-analysis as hypomethylated with maternal smoking overlapped with four regions identified in the NCT00632476 cohort with significantly increased methylation in vitamin C–supplemented smokers relative to placebo-treated smokers. The DMR plots (A–D) show the mean ± SEM methylation for each group in cord blood relative to chromosomal position. (A) chr4_81110074_PRDM8; (B) chr4_81111140_PRDM8; (C) chr4_81118138_PRDM8; (D) chr4_81122567_PRDM8.