Associations between Maternal Tobacco Smoke Exposure and the Cord Blood CD4+CD4+<math> <mrow> <mtext>CD</mtext> <msup> <mn>4</mn> <mo>+</mo> </msup> </mrow> </math> DNA Methylome

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+CD4+$\text{CD}{4}^{+}$ DNA methylome. Methods: The methylomes of 20 Hispanic white newborns (n=10n=10$n=10$ exposed to any maternal tobacco smoke in pregnancy; n=10n=10$n=10$ unexposed) from the Maternal and Child Health Study (MACHS) were profiled by whole-genome bisulfite sequencing (median coverage: 6.5×6.5×$6.5\times$). 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(pF⁢D⁢R)<0.05$\left(p_{FDR}\right)<0.05$]. From these CpGs, RADMeth identified 557 differentially methylated regions (DMRs) that were overrepresented (p<0.05p<0.05$p<0.05$) in important regulatory regions, including enhancers. Of nine DMRs that could be queried in a reduced representation bisulfite sequencing (RRBS) study of adult CD4+CD4+$\text{CD}{4}^{+}$ cells (n=9n=9$n=9$ smokers; n=10n=10$n=10$ nonsmokers), four replicated (p<0.05p<0.05$p<0.05$). Additionally, a CpG in the promoter of SLC7A8 (percent methylation difference: −9.4%−9.4%$-9.4\%$ comparing exposed to unexposed) replicated (p<0.05p<0.05$p<0.05$) in an EPIC (Illumina) array study of cord blood CD4+CD4+$\text{CD}{4}^{+}$ cells (n=14n=14$n=14$ exposed to sustained maternal tobacco smoke; n=16n=16$n=16$ unexposed) and in a study of adult CD4+CD4+$\text{CD}{4}^{+}$ cells across two platforms (EPIC: n=9n=9$n=9$ smokers; n=11n=11$n=11$ nonsmokers; 450K: n=59n=59$n=59$ smokers; n=72n=72$n=72$ nonsmokers). Conclusions: Maternal tobacco smoke exposure in pregnancy is associated with cord blood CD4+CD4+$\text{CD}{4}^{+}$ 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+CD4+$\text{CD}{4}^{+}$ cells. https://doi.org/10.1289/EHP3398.