X chromosome-wide analysis identifies DNA methylation sites influenced by cigarette smoking

Background: Tobacco smoking is a major cause of chronic disease worldwide. Smoking may induce cellular and molecular changes including epigenetic modification, with both short-term and long-term modification patterns that may contribute to phenotypic expression of diseases. Recent epigenome-wide association studies (EWAS) have identified dozens of smoking-related DNA methylation (DNAm) sites. However, the X chromosomal DNAm sites have been largely overlooked due to a lack of an analytical framework for dealing with the sex-dimorphic distribution. To identify novel smoking-related DNAm sites on the X chromosome, we examined the modality of each X chromosomal DNAm site and conducted a sex-specific association study of cigarette smoking. Results: We used a discovery sample of 139 middle-age twins, and three replication samples of 78 twins, 464 and 333 unrelated individuals including 47, 17, 22, and 89 current smokers, respectively. After correction for multiple testing, the top smoking-related DNAm sites in BCOR and TSC22D3 were significantly hypermethylated and hypomethylated, respectively, among current smokers. These smoking-associated sites were replicated with meta-analysis p-values of 9.17 x 10(-12) and 1.61 x 10(-9). For both sites, the smoking effects on methylation levels were larger in males than that in females. Conclusions: Our findings highlight the importance of investigating X chromosome methylation patterns and their associations with environmental exposures and disease phenotypes and demonstrate a robust statistical methodology for such study. Existing EWAS of human diseases should incorporate the X chromosomal sites to complete a comprehensive epigenome-wide scan.