Table 3. Concordance of degree of methylation of 59CGI(s) in UCWBC samples and gene expression levels in FPT samples. Gene Name ACSL3 RAD21 DUSP22 SCD5 SFMBT2 WWOX tau* 20.45 20.30 20.31 20.11 20.23 +0.22 Confidence Interval [20.76, 20.15] [20.70, +0.09] [20.66, +0.05] [20.49, +0.26] [20.57, +0.10] [20.11, +0.55] p-value ,0.01 0.13 0.09 0.55 0.18 0.20 Note: % of methylated cytosine in the CGI(s) (regions mark as BSPCR in Fig 2) was determined by bisulfite sequencing; levels of gene expression was measured as steady state transcript levels in FPT samples matched to UCWBC samples (n = 14) quantified by real time RT-PCR. *The Kendall tau coefficient was calculated between % of methylation and gene expression; negative sign indicates inverse relationship. The larger the negative value means greater negative correlation and statistical significance was determined by a 2-tailed test with p,0.05. doi:10.1371/journal.pone.0004488.t003 exposure level. The odds ratio (OR) of having a methylated ACSL3 59CGI in UCWBC DNA was 13.8, 95% CI = [3.8, 50.2] 3 (p,0.001), given the maternal PAH exposure was $2.41 ng/m , 3 compared to those exposed to ,2.41 ng/m . ACSL3 59CGI methylation status was not affected by gender or ethnicity. Relationship between ACSL3 59 CGI methylation and asthma classification Table 5 shows the percentage of subjects with methylated or unmethylated ACSL3 59 CGI according to asthma classification. Methylation of the ACSL3 59CGI was positively and significantly associated with asthma classification. The odds ratio (OR) relating methylation status and asthma classification among all 56 subjects was 3.9, 95% CI = [1.1, 14.3] (p = 0.03). Eleven of 15 children classified as asthmatic (73%) had UCWBC DNA with a methylated ACSL3 59-CGI. Table 5 further shows that the median (minimum, maximum) values of maternal PAH exposure differed significantly by methylation status of the ACSL3 59-CGI: methylation = 3.39 ng/m3 (1.11, 34.5) and unmethyla- tion = 1.70 ng/m3 (0.49, 3.33), respectively (p,0.001). All mater- nal PAH exposure values except the maximum (34.5 ng/m3) were 3 below 7.7 ng/m . When the latter analysis was repeated excluding the extreme high PAH value, the p-value was unchanged. Discussion In this exploratory study, we have identified ACSL3 as a candidate biomarker/gene whose 59-CGI methylation status appears to be related to transplacental PAH exposure and further associated with PAH-associated asthma. Thus, ACSL3 may be the first potential surrogate endpoint for environmentally related childhood asthma. If confirmed, such a biomarker holds promise in assessing PAH exposure and as a clinically relevant predictor for asthma risk in children born to mothers exposed to air pollutants such as traffic-related combustion emissions. We used a set of stringent criteria for the identification, selection and validation of candidates, which resulted in choosing ACSL3 the final candidate for the 56-sample study. We first used an unbiased method, MSRF, to identify both hypo- and hyper- methylated sequences in the high PAH group compared to the low PAH group. In silico analyses then revealed that 19 out of 31 sequences identified were homologous to known genes and 6 of PAH, Epigenetic and Asthma these aligned to a CGI(s) located in the 59 flanking region of a known gene (59-CGI). These 6 genes therefore were chosen as putative candidates for further investigation. By comparing the degree of methylation of their 59CGI in UCWBC DNA samples and levels of gene expression in FPT RNA samples we identified ACSL3 to have the highest inverse concordance in vivo. We then employed a lung cancer cell line (H1299) to demonstrate that the demethylation of ACSL3 59-CGI by 5-aza-dC treatment induced enhancement of gene expression in H1299. Conversely, the treatment of H1299 with low doses of BaP, a PAH, was associated with dose-dependent hypermethylaiton of ACSL3 5-CGI and gene silencing. Collectively, these data strongly support methylation of the 59-CGI of ACSL3 in gene regulation. They also provide evidence that PAH could directly induce ACSL3 promoter methylation. This well though out, step-wise approach allowed us to identify a candidate to be tested in the 56-sample study to determine whether methylation of the ACSL3 59CGI could be a stand alone biomarker for PAH exposure and/or PAH-associated childhood asthma. Measuring human exposure to chemicals such as PAHs and determining their relationship to disease outcomes is a challenge for environmental health, especially when children are the study subjects. Although some studies have found a positive correlation between PAH-induced DNA adducts and mutagenesis or cancer development [29] others have failed to establish clear cause-effect relations between PAH adducts and the induction of these or other disease outcomes [30], suggesting that other mechanisms may be involved. Moreover, some of the methods for measuring genotoxic mechanisms such as adducts are complex and low-throughput. Therefore, it is of significance that, in this investigative study, we have developed a relatively simple, high throughput assay based on methylation of a sequence in the promoter of the ACSL3 gene that appears to distinguish children born to mothers with high airborne PAH exposure from those who were not (OR = 13.8; p,0.001) with high sensitivity (75%) and specificity (85%). The Area Under ROC curve (AUC) value for this assay was 0.82 suggesting excellent separability of the subjects with the methyl- ated or unmethylated ACSL3sequence. This optimized MSPCR protocol allows for a rapid determination of ACSL3 59-CGI methylation status in large sample sets, which is a normal requirement in population studies. It also has the added advantage to be developed into a minimally invasive test since only a small amount of DNA from UCWBC is required for the assay. If validated, such an assay is expected to have a high utility in the assessment of PAH exposure in future population-based studies. Multiple epidemiological studies have suggested that asthma risk is determined by prenatal and/or early life exposure to pollutants and allergens that modify later life lung function and T helper cell allergic phenotype [11]. Epigenetic modifications of regulatory genes crucial to the development of asthma-related pathophysiol- ogy have been put forth as a possible mechanistic basis of this disease [11]. Thus, asthma may be caused by epigenetic changes such as DNA methylation, post-translational modifications of histones, dysregulation of non-coding RNAs, and chromatin alterations. At present, the best studied mechanism is DNA methylation-mediated disruption of gene regulation. In this regard, experimental data indicating that DNA methylation of genes critical to the differentiation of T helper cells could skew their response towards or away from an allergic phenotype lend support to this theory [31–34]. Additionally, we recently demonstrated that inhaled diesel exposure (a source of PAHs) and intranasal Aspergillus fumigatus induced specific DNA methyl- ation changes in the promoters of interferon-c and interleukin-4 in T helper cells, respectively, and altered circulating levels of IgE in PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4488