Epigenome-450K-wide methylation signatures of active cigarette smoking: The Young Finns Study

Abstract

Smoking as a major risk factor for morbidity affects numerous regulatory systems of the human body including DNA methylation. Most of the previous studies with genome-wide methylation data are based on conventional association analysis and earliest threshold-based gene set analysis that lacks sensitivity to be able to reveal all the relevant effects of smoking. The aim of the present study was to investigate the impact of active smoking on DNA methylation at three biological levels: 5'-C-phosphate-G-3' (CpG) sites, genes and functionally related genes (gene sets). Gene set analysis was done with mGSZ, a modern threshold-free method previously developed by us that utilizes all the genes in the experiment and their differential methylation scores. Application of such method in DNA methylation study is novel. Epigenome-wide methylation levels were profiled from Young Finns Study (YFS) participants' whole blood from 2011 follow-up using Illumina Infinium HumanMethylation450 BeadChips. We identified three novel smoking related CpG sites and replicated 57 of the previously identified ones. We found that smoking is associated with hypomethylation in shore (genomic regions 0-2 kilobases from CpG island). We identified smoking related methylation changes in 13 gene sets with false discovery rate (FDR) ≤ 0.05, among which is olfactory receptor activity, the flagship novel finding of the present study. Overall, we extended the current knowledge by identifying: (i) three novel smoking related CpG sites, (ii) similar effects as aging on average methylation in shore, and (iii) a novel finding that olfactory receptor activity pathway responds to tobacco smoke and toxin exposure through epigenetic mechanisms.

Keywords: Metylation; Tobacco smoke; epigenomics.

Figure 1. Manhattan plot and quantile-quantile (Q-Q) plot of epigenome-wide association analysis of smoking habit

(A) Manhattan plot showing the P-values of genome-wide CpG sites. X-axis represents position of the CpG sites on each chromosome. Y-axis represents negative log10 of the P-values for the association. The dotted line indicates false discovery rate (FDR)-corrected significance threshold and the solid horizontal line represents Bonferroni-corrected significance threshold (experiment-wide significance). The annotations for the three novel CpG sites and corresponding genes (if annotation is known) are shown. (B) Quantile-quantile plot showing genomic inflation factor (lambda = 1.11) of the epigenome-wide association study. The genomic inflation factor (ratio of the median of the empirically observed distribution of the test statistic to the expected median) represents the extent of inflation and false positive rate in the results.

Figure 2. Box plots showing differences in average methylation of all CpG sites belonging to genes of the analyzed gene sets in smokers and non-smokers

X-axis represents smoking habit (active smokers and non-smokers). Y-axis represents mean methylation β-values of CpG sites belonging to genes of the analyzed gene sets.

Figure 3. Effect of smoking on average methylation level in different genomic regions in our genome

Box plots showing smoking-related differences in average methylation levels in different genomic regions. X-axis represents smoking habit (active smokers and non-smokers). Y-axis represents average methylation β values of CpG sites belonging to different genomic regions. Abbreviations and definitions for gene regions: TSS200, 0–200 bases upstream of the transcriptional start site; TSS1500, 200–1500 bases upstream of the TSS; 5′UTR, within the 5′ untranslated region, between the TSS and the ATG start site; Body, between the ATG and stop codon irrespective of the presence of introns, exons, TSS, or promoters; 3′UTR, between the stop codon and poly A signal. Definitions for CpG islands: Shores, 0–2 kb from CpG island; Shelves, 2–4 kb from CpG island.