Smoking-Associated Site-Specific Differential Methylation in Buccal Mucosa in the COPDGene Study

Abstract

DNA methylation is a complex, tissue-specific phenomenon that can reflect both endogenous factors and exogenous exposures. Buccal brushings represent an easily accessible source of DNA, which may be an appropriate surrogate tissue in the study of environmental exposures and chronic respiratory diseases. Buccal brushings were obtained from a subset of current and former smokers from the COPDGene study. Genome-wide DNA methylation data were obtained in the discovery cohort (n = 82) using the Illumina HumanMethylation450K array. Empirical Bayes methods were used to test for differential methylation by current smoking status at 468,219 autosomal CpG sites using linear models adjusted for age, sex, and race. Pyrosequencing was performed in a nonoverlapping replication cohort (n = 130). Current smokers were significantly younger than former smokers in both the discovery and replication cohorts. Seven CpG sites were associated with current smoking at a false discovery rate less than 0.05 in the discovery cohort. Six of the seven significant sites were pyrosequenced in the replication cohort; five CpG sites, including sites annotated to CYP1B1 and PARVA, were replicated. Correlations between cumulative smoke exposure and time since smoking cessation were observed in a subset of the significantly associated CpG sites. A significant correlation between reduced lung function and increased radiographic emphysema with methylation at cg02162897 (CYP1B1) was observed among female subjects. Site-specific methylation of DNA isolated from buccal mucosa is associated with exposure to cigarette smoke, and may provide insights into the mechanisms underlying differential susceptibility toward the development of smoking-related chronic respiratory diseases.

Keywords: DNA methylation; buccal mucosa; smoking.

Figure 1.

Volcano plot of methylation by current smoking status in Infinium II analysis. The difference in mean methylation for each CpG site is plotted on the x axis, whereas the log-transformed P value is plotted on the y axis. Each point represents an individual CpG site. The dotted red line denotes the threshold for significance at a false discovery rate less than 0.05, whereas the blue dashed line corresponds to an unadjusted P value less than 0.001.

Figure 2.

Correlation between buccal DNA methylation and time since smoking cessation in former smokers (n = 52) in the discovery cohort. Time since smoking cessation (in years) is plotted on the x axis, whereas percent methylation is plotted on the y axis for (A) cg09853702 (Pearson’s r = −0.57; P = 9.4 × 10⁻⁶), (B) cg02162897 (Pearson’s r = 0.58; P = 6.2 × 10⁻⁶), and (C) cg21371809 (Pearson’s r = −0.30; P = 0.03). Males are represented by triangles, whereas females are represented by circles.