Effects of smoking on genome-wide DNA methylation profiles: A study of discordant and concordant monozygotic twin pairs

Abstract

Background: Smoking-associated DNA methylation levels identified through epigenome-wide association studies (EWASs) are generally ascribed to smoking-reactive mechanisms, but the contribution of a shared genetic predisposition to smoking and DNA methylation levels is typically not accounted for.

Methods: We exploited a strong within-family design, that is, the discordant monozygotic twin design, to study reactiveness of DNA methylation in blood cells to smoking and reversibility of methylation patterns upon quitting smoking. Illumina HumanMethylation450 BeadChip data were available for 769 monozygotic twin pairs (mean age = 36 years, range = 18-78, 70% female), including pairs discordant or concordant for current or former smoking.

Results: In pairs discordant for current smoking, 13 differentially methylated CpGs were found between current smoking twins and their genetically identical co-twin who never smoked. Top sites include multiple CpGs in CACNA1D and GNG12, which encode subunits of a calcium voltage-gated channel and G protein, respectively. These proteins interact with the nicotinic acetylcholine receptor, suggesting that methylation levels at these CpGs might be reactive to nicotine exposure. All 13 CpGs have been previously associated with smoking in unrelated individuals and data from monozygotic pairs discordant for former smoking indicated that methylation patterns are to a large extent reversible upon smoking cessation. We further showed that differences in smoking level exposure for monozygotic twins who are both current smokers but differ in the number of cigarettes they smoke are reflected in their DNA methylation profiles.

Conclusions: In conclusion, by analysing data from monozygotic twins, we robustly demonstrate that DNA methylation level in human blood cells is reactive to cigarette smoking.

Funding: We acknowledge funding from the National Institute on Drug Abuse grant DA049867, the Netherlands Organization for Scientific Research (NWO): Biobanking and Biomolecular Research Infrastructure (BBMRI-NL, NWO 184.033.111) and the BBRMI-NL-financed BIOS Consortium (NWO 184.021.007), NWO Large Scale infrastructures X-Omics (184.034.019), Genotype/phenotype database for behaviour genetic and genetic epidemiological studies (ZonMw Middelgroot 911-09-032); Netherlands Twin Registry Repository: researching the interplay between genome and environment (NWO-Groot 480-15-001/674); the Avera Institute, Sioux Falls (USA), and the National Institutes of Health (NIH R01 HD042157-01A1, MH081802, Grand Opportunity grants 1RC2 MH089951 and 1RC2 MH089995); epigenetic data were generated at the Human Genomics Facility (HuGe-F) at ErasmusMC Rotterdam. Cotinine assaying was sponsored by the Neuroscience Campus Amsterdam. DIB acknowledges the Royal Netherlands Academy of Science Professor Award (PAH/6635).

Keywords: DNA methylation; discordant twins; epidemiology; epigenetics; genetics; genomics; global health; human; identical twins; twin study.

Figure 1.. DNA methylation analysis in smoking discordant and smoking concordant monozygotic twin pairs.

Blood DNA methylation profiles (Illumina 450k array) from six groups of monozygotic twin pairs were analysed.

Figure 2.. Study flowchart.

Figure 3.. Top differentially methylated loci identified in monozygotic twin pairs discordant for current smoking.

(a) Manhattan plot of the epigenome-wide association study (EWAS) in 53 smoking discordant monozygotic twin pairs (current versus never). The red horizontal line denotes the epigenome-wide significance threshold (Bonferroni correction) and 13 CpGs with significant differences are highlighted. (b) Mean within-pair differences in monozygotic twin pairs at the 13 CpGs that were epigenome-wide significant in smoking discordant monozygotic pairs. Mean within-pair differences of the residuals obtained after correction of methylation β-values for covariates are shown for 53 monozygotic pairs discordant for current/never smoking, 66 monozygotic pairs discordant for current/former smoking, 72 monozygotic pairs discordant for former/never smoking, 83 concordant current smoking monozygotic pairs, 88 concordant former smoking monozygotic pairs, and 406 concordant never smoking monozygotic pairs. (c) QQ-plot showing p-values from the EWAS in 53 smoking discordant monozygotic twin pairs (current versus never). P-values for CpGs located nearby significant SNPs from the genome-wide association study (GWAS) of smoking initiation are plotted in blue and all other genome-wide CpGs are plotted in orange.

Figure 3—figure supplement 1.. DNA methylation levels in current/never smoking discordant monozygotic twin pairs.

Figures show the DNA methylation β-values (unadjusted for any covariates) in 53 discordant monozygotic twin pairs for the 13 significant CpGs.

Figure 4.. DNA methylation differences in smoking discordant and smoking concordant pairs.

(a) Distributions of the mean absolute within-pair differences in discordant and concordant pairs at the top 1000 CpGs with the lowest p-value from the epigenome-wide association study (EWAS) in discordant monozygotic pairs (current versus never smokers). (b) Scatterplot of cigarettes smoked per day in 80 concordant current smoking monozygotic pairs with complete data. (c) Scatterplot of within-pair differences in cigarettes smoked per day versus DNA methylation at cg05575921 (AHRR) in 80 concordant current smoking monozygotic pairs with complete data. (d) Scatterplot of within-pair differences in DNA methylation at cg05575921 (AHRR) versus time since quitting smoking (years) in 63 pairs discordant for former smoking.