Genetic contribution to variation in DNA methylation at maternal smoking-sensitive loci in exposed neonates

Abstract

Epigenome-wide DNA methylation association studies have identified highly replicable genomic loci sensitive to maternal smoking during gestation. The role of inter-individual genetic variation in influencing DNA methylation, leading to the possibility of confounding or bias of such associations, has not been assessed. We investigated whether the DNA methylation levels at the top 10 CpG sites previously associated with exposure to maternal smoking during gestation were associated with individual genetic variation at the genome-wide level. Genome-wide association tests between DNA methylation at the top 10 candidate CpG and genome-wide SNPs were performed in 736 case and control participants of the California Childhood Leukemia Study. Three of the strongest maternal-smoking sensitive CpG sites in newborns were significantly associated with SNPs located proximal to each gene: cg18146737 in the GFI1 gene with rs141819830 (P D 8.2£10¡44 ), cg05575921 in the AHRR gene with rs148405299 (P D 5.3£10 ¡10), and cg12803068 in the MYO1G gene with rs61087368 (P D 1.3£10 ¡18). For the GFI1 CpG cg18146737, the underlying genetic variation at rs141819830 confounded the association between maternal smoking and DNA methylation in our data (the regression coefficient changed from ¡0.02 [P D 0.139] to ¡0.03 [P D 0.015] after including the genotype). Our results suggest that further studies using DNA methylation at cg18146737, cg05575921, or cg12803068 that aim to assess exposure to maternal smoking during gestation should include genotype at the corresponding SNP. New methods are required for adequate and routine inclusion of genotypic influence on DNA methylation in epigenome-wide association studies to control for potential confounding.

Correlates of Prenatal and Early-Life Tobacco Smoke Exposure and Frequency of Common Gene Deletions in Childhood Acute Lymphoblastic Leukemia

“…Germline ( i.e. non-tumor) DNA was extracted from neonatal dried bloodspots and bisulfite-treated, before being assayed using Illumina HumanMethylation450 Beadchip arrays (450K arrays), with data normalized to remove batch and plate-position effect as previously described (24). Genotype data were also available at SNP rs148405299, a recently identified methyl-quantitative trait locus (methyl-QTL) for methylation at the AHRR CpG cg05575921 (24).…”

“…non-tumor) DNA was extracted from neonatal dried bloodspots and bisulfite-treated, before being assayed using Illumina HumanMethylation450 Beadchip arrays (450K arrays), with data normalized to remove batch and plate-position effect as previously described (24). Genotype data were also available at SNP rs148405299, a recently identified methyl-quantitative trait locus (methyl-QTL) for methylation at the AHRR CpG cg05575921 (24). Decreased methylation at cg05575921 is strongly associated with in utero exposure to maternal smoking (24, 25), and not paternal or maternal preconception smoking or mother’s exposure to secondhand smoke (26).…”

“…Genotype data were also available at SNP rs148405299, a recently identified methyl-quantitative trait locus (methyl-QTL) for methylation at the AHRR CpG cg05575921 (24). Decreased methylation at cg05575921 is strongly associated with in utero exposure to maternal smoking (24, 25), and not paternal or maternal preconception smoking or mother’s exposure to secondhand smoke (26). …”

“…This model was adjusted for gestational age, sex, race/ethnicity, estimated cell-mixture, methylation array batch number and genotype at the methyl-QTL SNP rs148405299, as previously described (24). Multivariable Poisson regression analysis was carried out to test the association between DNA methylation at cg05575921 (in beta-values) and total number of deletions in each case (as the outcome), adjusted for the same variables listed above.…”

Population epigenetics

“…However, even this paradigm of epigenetic association may be undermined by what appear to be substantial effects of DNA sequence variation and cell subtype effects for the informative loci [30–32], raising the possibility that these DNA methylation changes are substantially due to allelic variants for these meQTLs segregating non-randomly into the smoker and non-smoker groups, and blood cell subtypes being altered as a response to cigarette smoking.…”