Our first objective was to assess the reproducibility of buccal DNA collection and quantification of DNA methylation of asthma genes among a cohort of young children. We found that replicate and field duplicate samples correlated strongly. These results suggest our field and laboratory procedures (including bisulfite conversion, pyrosequencing) are robust, and that collection and measure of buccal cell DNA methylation in cohort studies can have great utility. Also they allow us to start answering fundamental questions about the stability of DNA methylation in buccal cells over time, questions that have significant implications for the understanding of environmental epigenetic regulation in children.
Upon confirming the feasibility and reproducibility of these measures, our next step was to assess acute differences in levels of buccal cell DNA methylation of select sites on two asthma genes tested days apart. We found low within-subject correlations for both IFNγ (r = 0.56 and 0.23 for CpG-186, CpG-54, respectively) and iNOS (r = 0.20 and 0.19 for CpG-359, CpG-352, respectively) upon repeat testing over a 4 to 7 day period. Combined with the high level of reproducibility previously demonstrated, these data suggest that methylation levels can change acutely in both genes. These findings are novel in that research measuring short-term changes in methylation has been limited, with only a few examples to date [23, 26]. For example, Baccarelli and colleagues tested blood DNA methylation levels in long interspersed nucleotide elements (LINE 1) and Alu element (Alu) as surrogates of global methylation levels after acute exposure to black carbon/soot. They found an association between ambient black carbon levels and LINE 1, but not Alu, demethylation suggesting that global epigenetic regulation may occur in association with measures of recent air pollution exposure. As a second example, Tarantini and colleagues, in addition to assessing acute changes in global methylation (LINE 1, Alu), assessed PBMC DNA methylation levels in the proinflammatory iNOS gene over a 3-day period. Interestingly, they found an association between concentrations of PM10 particles and iNOS demethylation, implicating this regulator of airway inflammation as a gene whose expression may depend in part on alteration of DNA methylation levels.
We reasoned that genes expressed in buccal cells, like those in peripheral blood mononuclear cells, also could undergo acute changes in DNA methylation, presumably following recent changes in environmental triggers. Inducible NOS was a main focus of this investigation because of Tarantini's and colleagues' reports, and because of its reported expression in the buccal mucosa . Interestingly, methylation levels and interquartile ranges observed in this study of inner city asthmatic children were strikingly similar to the report by Breton and colleagues in a Southern California cohort, suggesting that some of our new results may be generalizable to other cohorts.
The second main focus, namely susceptibility of buccal cells to undergo DNA methylation in the promoter region of IFNγ, was in response to considerable previous work that suggests methylation of IFNγ is critical to its gene expression [15, 28]. This body of work also includes our previous findings in mice that CD4+ T cells undergo increases in IFNγ DNA methylation in multiple CpG sites following exposures to diesel exhaust particles. In these experiments, methylation levels were measured once after 3 weeks of diesel exposure. The hypermethylation that occurred over this period was suspected to induce silencing of the IFNγ gene and downregulation of the production of proallergic IgE antibodies, as indicated by the observed inverse correlation between IFNγ methylation levels and IgE . Indeed the CpG sites studied here (CpG-186 and CpG-54) are conserved in mice . One of the few studies of human cells to date, conducted by Gonsky and colleagues, looked at the same loci in the promoter of IFNγ (CpG-186 and CpG-54) in lamina propria T cells and peripheral blood T cells . Their group found that a 5% reduction in methylation of CpG-54 in the promoter region of IFNγ was associated with a threefold increase in IFNγ gene expression. While our study did not link the changes in IFNγ promoter methylation in buccal cells over time with downstream biological events as the Liu et al. and Gonsky et al. studies did, it does for the first time show that the time course for changes in DNA methylation can be as short as several days in a pediatric cohort.
We acknowledge several limitations to the study. First, a limited number of asthma genes, and CpG sites per gene, were studied. Examination of additional CpG sites and asthma genes may help elucidate the time course of epigenetic change of other genes important to airway inflammation. To date it still needs to be ascertained how methylation levels across multiple CpG sites may impact gene transcription differentially, though early evidence suggests that particular sites, such as in proximal gene promoters such as IFNγ CpG-54, may be critical . Alternately, other evidence suggests that CpG methylation in the intron could affect elongation and thereby gene transcription [30, 31]. Also, the sample size was small, though sufficient to evaluate the quality of the reproduced data. The data display ranges of methylation that may be shown to be biologically meaningful in future studies. The buccal cell collection does not test for cell specific effects nor necessarily represent what occurs in respiratory epithelium. The magnitude of changes may vary across tissues. Moreover, in the absence of corresponding gene expression data, it may be difficult to know whether their epigenetic changes led to downstream molecular events. Repeat findings in other cohorts would be helpful to validate these results.