Validation of differential DNA methylation in newborns exposed to tobacco smoke during gestation using bisulfite pyrosequencing

Maternal exposure to tobacco smoke during pregnancy has been associated with many negative child health outcomes. Tobacco smoke exposure alters DNA methylation in the developing embryo/fetus and may be a mechanism that increases risk of later life disease. Previous studies have identified CpG sites in umbilical cord blood that are associated with in utero tobacco smoke exposure. We sought to validate findings for CpG sites within several of the top hit genes, AHRR , CYP1A1 , and GFI1, using targeted quantitative bisulfite pyrosequencing. Comparing results from cord blood specimens of tobacco smoke-exposed to unexposed newborns, we confirmed significance at all previously identified CpG sites tested, including one in AHRR (p=0.007), three in CYP1A1 (p<0.0001), and one in GFI1 (p=0.008). These assays also captured novel differentially methylated CpGs located near the identified sites that were not included in the prior array-based studies (p value range, 0.02 to <0.0001). These results validate the prior findings and provide a simplified and more economical approach to analysis of CpG sites for expanded use as biomarkers of in utero tobacco smoke exposure.