Associations between Maternal Tobacco Smoke Exposure and the Cord Blood CD4+ DNA Methylome

Abstract

Background: Maternal tobacco smoke exposure has been associated with altered DNA methylation. However, previous studies largely used methylation arrays, which cover a small fraction of CpGs, and focused on whole cord blood. Objectives: The current study examined the impact of in utero exposure to maternal tobacco smoke on the cord blood CD4+ DNA methylome. Methods: The methylomes of 20 Hispanic white newborns (n=10 exposed to any maternal tobacco smoke in pregnancy; n=10 unexposed) from the Maternal and Child Health Study (MACHS) were profiled by whole-genome bisulfite sequencing (median coverage: 6.5×). Statistical analyses were conducted using the Regression Analysis of Differential Methylation (RADMeth) program because it performs well on low-coverage data (minimizes false positives and negatives). Results: We found that 10,381 CpGs were differentially methylated by tobacco smoke exposure [neighbor-adjusted p-values that are additionally corrected for multiple testing based on the Benjamini-Hochberg method for controlling the false discovery rate (FDR) (pFDR)<0.05]. From these CpGs, RADMeth identified 557 differentially methylated regions (DMRs) that were overrepresented (p<0.05) in important regulatory regions, including enhancers. Of nine DMRs that could be queried in a reduced representation bisulfite sequencing (RRBS) study of adult CD4+ cells (n=9 smokers; n=10 nonsmokers), four replicated (p<0.05). Additionally, a CpG in the promoter of SLC7A8 (percent methylation difference: −9.4% comparing exposed to unexposed) replicated (p<0.05) in an EPIC (Illumina) array study of cord blood CD4+ cells (n=14 exposed to sustained maternal tobacco smoke; n=16 unexposed) and in a study of adult CD4+ cells across two platforms (EPIC: n=9 smokers; n=11 nonsmokers; 450K: n=59 smokers; n=72 nonsmokers). Conclusions: Maternal tobacco smoke exposure in pregnancy is associated with cord blood CD4+ DNA methylation in key regulatory regions, including enhancers. While we used a method that performs well on low-coverage data, we cannot exclude the possibility that some results may be false positives. However, we identified a differentially methylated CpG in amino acid transporter SLC7A8 that is highly reproducible, which may be sensitive to cigarette smoke in both cord blood and adult CD4+ cells. https://doi.org/10.1289/EHP3398