Tobacco-smoking-related differential DNA methylation: 27K discovery and replication

Abstract

Tobacco smoking is responsible for substantial morbidity and mortality worldwide, in particular through cardiovascular, pulmonary, and malignant pathology. CpG methylation might plausibly play a role in a variety of smoking-related phenomena, as suggested by candidate gene promoter or global methylation studies. Arrays allowing hypothesis-free searches on a scale resembling genome-wide studies of SNPs have become available only very recently. Methylation extents in peripheral-blood DNA were assessed at 27,578 sites in more than 14,000 gene promoter regions in 177 current smokers, former smokers, and those who had never smoked, with the use of the Illumina HumanMethylation 27K BeadChip. This revealed a single locus, cg03636183, located in F2RL3, with genome-wide significance for lower methylation in smokers (p = 2.68 × 10(-31)). This was similarly significant in 316 independent replication samples analyzed by mass spectrometry and Sequenom EpiTyper (p = 6.33 × 10(-34)). Our results, which were based on a rigorous replication approach, show that the gene coding for a potential drug target of cardiovascular importance features altered methylation patterns in smokers. To date, this gene had not attracted attention in the literature on smoking.

Figure 1

Mixed Linear Regression p Values of the Sex-Adjusted Association of Smoking Status with Methylation Intensities at 27,578 CpG Sites –log₁₀(P) versus –log₁₀(rank) plot.

Figure 2

Distribution of Methylation Intensities by Smoking Status at the CpG Site cg03636183 Located in F2RL3 (A) Histograms of “beta values” obtained in the 27K genome-wide screening (discovery measurements). (B) cg03636183 methylation extent as determined by the Sequenom MassArray, validating the discovery measurements in the same samples. (C) cg03636183 methylation measured by the Sequenom MassArray in a nonoverlapping replication sample set.

Figure 3

Example Results of the Sequenom MassArray Analysis of cg03636183 Methylation Extents The graphs show the detection intensity (y axis) of amplicon fragments after base-specific cleavage. The latter results in fragments of different masses (x axis) for originally methylated and nonmethylated CpG sites. The proportion of methylated CpG at this locus can thus be determined by the ratio of detection intensities at masses corresponding to fragments originating from methylated and nonmethylated CpG sites (blue and red vertical lines, respectively). The graphs originating from samples with 17%, 59%, and 98% methylation at cg03636183 (from left to right) show clearly distinguishable peaks.