Reproducibility and intraindividual variation over days in buccal cell DNA methylation of two asthma genes, interferon γ (IFNγ) and inducible nitric oxide synthase (iNOS)

The biological mechanisms responsible for the development of asthma symptoms in children following acute exposure to air pollution and other triggers are complex. These include the induction of oxidative stress pathways and formation of excessive reactive oxygen species in the airways [1-5]. Also, exposure to diesel and other combustion products may upregulate proallergic T helper (Th) 2 immune mechanisms [1,6-9]. Epigenetic regulation of gene expression associated with airway inflammation and allergic immune responses following exposure to air pollutants has been proposed as a key molecular step linking environmental exposures with altered asthma gene expression and asthma symptoms [10-14].To date, clinical research on epigenetic changes in asthma and other complex diseases has been limited, especially in children [11,12]. One cross-sectional study by White and colleagues observed promoter demethylation of the allergy counter-regulatory and Th1 cytokine interferon γ (IFNγ) gene in association with in vitro differentiation of CD4+ neonatal T cells [15]. Another study by Kwon and colleagues found phytohemagglutinin (PHA) and dust mite allergen stimulation of CD4+ T lymphocytes induced small increases in the degree of demethylation in several CpG loci of the Th2 interleukin (IL)-4 promoter (CpG-80, CpG+5) in adult asthmatic subjects, when compared to the control group [16]. The changes in DNA methylation at the IFNγ promoter were less consistent. Recently, Breton and colleagues sampled children living in Southern California in one of the first large cohort studies analyzing DNA methylation of asthma genes in buccal cells [17]. They hypothesized that buccal cell DNA methylation levels in two genes important to the production of proinflammatory nitric oxide, namely arginase (ARG) and inducible nitric oxide synthases (iNOS), would be associated inversely with fractional exhaled nitric oxide (FeNO) levels measured concurrently at one timepoint. They found that methylation