DNA methylation mediates the effect of maternal smoking during pregnancy on birthweight of the offspring

Int J Epidemiol. 2015 Aug;44(4):1224-37. doi: 10.1093/ije/dyv048. Epub 2015 Apr 10.


Background: We examined whether the effect of maternal smoking during pregnancy on birthweight of the offspring was mediated by smoking-induced changes to DNA methylation in cord blood.

Methods: First, we used cord blood of 129 Dutch children exposed to maternal smoking vs 126 unexposed to maternal and paternal smoking (53% male) participating in the GECKO Drenthe birth cohort. DNA methylation was measured using the Illumina HumanMethylation450 Beadchip. We performed an epigenome-wide association study for the association between maternal smoking and methylation followed by a mediation analysis of the top signals [false-discovery rate (FDR) < 0.05]. We adjusted both analyses for maternal age, education, pre-pregnancy BMI, offspring's sex, gestational age and white blood cell composition. Secondly, in 175 exposed and 1248 unexposed newborns from two independent birth cohorts, we replicated and meta-analysed results of eight cytosine-phosphate-guanine (CpG) sites in the GFI1 gene, which showed the most robust mediation. Finally, we performed functional network and enrichment analysis.

Results: We found 35 differentially methylated CpGs (FDR < 0.05) in newborns exposed vs unexposed to smoking, of which 23 survived Bonferroni correction (P < 1 × 10(-7)). These 23 CpGs mapped to eight genes: AHRR, GFI1, MYO1G, CYP1A1, NEUROG1, CNTNAP2, FRMD4A and LRP5. We observed partial confirmation as three of the eight CpGs in GFI1 replicated. These CpGs partly mediated the effect of maternal smoking on birthweight (Sobel P < 0.05) in meta-analysis of GECKO and the two replication cohorts. Differential methylation of these three GFI1 CpGs explained 12-19% of the 202 g lower birthweight in smoking mothers. Functional enrichment analysis pointed towards activation of cell-mediated immunity.

Conclusions: Maternal smoking during pregnancy was associated with cord blood methylation differences. We observed a potentially mediating role of methylation in the association between maternal smoking during pregnancy and birthweight of the offspring. Functional network analysis suggested a role in activating the immune system.

Keywords: ALSPAC; DOHaD; Epigenetic epidemiology; GECKO; Generation R; epigenome-wide association study; fetal programming.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Birth Weight / genetics*
  • Cohort Studies
  • DNA Methylation*
  • DNA-Binding Proteins / genetics*
  • Epigenesis, Genetic
  • Female
  • Fetal Blood
  • Genome-Wide Association Study
  • Humans
  • Infant, Newborn
  • Linear Models
  • Male
  • Maternal Exposure / adverse effects*
  • Pregnancy
  • Prenatal Exposure Delayed Effects / genetics*
  • Risk Factors
  • Smoking / adverse effects*
  • Transcription Factors / genetics*


  • DNA-Binding Proteins
  • GFI1 protein, human
  • Transcription Factors