Maternal Smoking during Pregnancy and DNA-Methylation in Children at Age 5.5 Years: Epigenome-Wide-Analysis in the European Childhood Obesity Project (CHOP)-Study

Abstract

Mounting evidence links prenatal exposure to maternal tobacco smoking with disruption of DNA methylation (DNAm) profile in the blood of infants. However, data on the postnatal stability of such DNAm signatures in childhood, as assessed by Epigenome Wide Association Studies (EWAS), are scarce. Objectives of this study were to investigate DNAm signatures associated with in utero tobacco smoke exposure beyond the 12th week of gestation in whole blood of children at age 5.5 years, to replicate previous findings in young European and American children and to assess their biological role by exploring databases and enrichment analysis. DNA methylation was measured in blood of 366 children of the multicentre European Childhood Obesity Project Study using the Illumina Infinium HM450 Beadchip (HM450K). An EWAS was conducted using linear regression of methylation values at each CpG site against in utero smoke exposure, adjusted for study characteristics, biological and technical effects. Methylation levels at five HM450K probes in MYO1G (cg12803068, cg22132788, cg19089201), CNTNAP2 (cg25949550), and FRMD4A (cg11813497) showed differential methylation that reached epigenome-wide significance according to the false-discovery-rate (FDR) criteria (q-value<0.05). Whereas cg25949550 showed decreased methylation (-2% DNAm ß-value), increased methylation was observed for the other probes (9%: cg12803068; 5%: cg22132788; 4%: cg19089201 and 4%: cg11813497) in exposed relative to non-exposed subjects. This study thus replicates previous findings in children ages 3 to 5, 7 and 17 and confirms the postnatal stability of MYO1G, CNTNAP2 and FRMD4A differential methylation. The role of this differential methylation in mediating childhood phenotypes, previously associated with maternal smoking, requires further investigation.

Fig 1

Volcano plot (A) and QQ-plot (B) of 433 313 differentially methylated CpG sites for 366 children at age 5.5 exposed vs. not-exposed to maternal smoking beyond week 12 of gestation. Figures are based on linear regression of M-values on exposure variable and adjustment factors.

Fig 2

Regional association of CpG methylation at MYO1G (A), CNTNAP2 (B) and FRMD4A (C) loci with prenatal smoking status. The direction of differential methylation is marked with arrows (↑ increase or ↓ decrease). Regional association plots are shown using the gene map (UCSC genome browser, hg19) with a graph of–log₁₀ p-values on the y-axis, the nucleotide position on the x-axis, and the position of selected Illumina Infinium HumanMethylation 450 BeadChip probes. Abbreviations: CGI: CpG island; TSS: transcription start site; FDR: false discovery rate p-value.