Demographic and Exposure Characteristics of the Cohorts.

<p>*significant difference between the two cohorts at alpha  = 0.05 level, using t-test.</p><p>** significant difference between the two cohorts at alpha  = 0.05 level, using chi-square test.</p

Population characteristics.

<p>Note: ^*Among N = 606 samples with available data.</p

Methylation status of a region in the <i>ACSL3</i> 5′CGI analyzed by bisufite genomic sequencing and methylation specific PCR (MSPCR).

<p>A) Schematic diagram of the CG content (%) in the 5′ flanking region of the <i>ACSL3</i> gene. A CGI of 1058 bp was located at the 5′ end of <i>ACSL3</i> including transcription start site (TSS), untranslated region (UTR) exon1 and intron 1. Individual CG sites are marked as red vertical lines in the genomic DNA sequence. The PCR-amplified regions were indicated by lines and methylation status of this region were determined by BSPCR and MSPCR. B) The diagram represents the methylation status of MSPCR-amplified regions (R1–R3) of 20 samples with high or low PAH exposure with or without asthma assayed by bisulfite genomic sequencing. 6 clones were sequenced from each sample. The PAH level of each sample is shown. Open circle: unmethylated CG; closed circle: methylated CG. MSPCR-primers were then specially designed on particular regions (R1–R3). Overall Met% of <i>ACSL3</i> promoter is shown. Statistical difference was accepted at p<0.05* when compared with the group of low PAH without asthma. C) Representative results from MSPCR analyses on Region 1 (R1) of samples from the low and high PAH group. M: methylated; and U: unmethylated. The methylation status of <i>ACSL3</i> 5′-CGI of all samples was further analyzed by MS-PCR using the same sets of primers. Results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004488#pone-0004488-t004" target="_blank">Table 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004488#pone-0004488-t005" target="_blank">Table 5</a>. High PAH (≥ the cohort median of 2.3 ng/m3), Low PAH (</p

Number (%) of African Americans, Males, and Methylated Subjects by PAH Exposure Categories (<2.41, > = 2.41)^a.

<p>Note: ^*Odds Ratios, 95% Confidence Intervals (OR, 95%CI) measuring the odds of high PAH exposure in African Amercians (Compared to Dominicans), males (Compared to Females), and subjects with methylated ACSL3 5′CGI (compared to those with unmehtylated CGI) assayed by MSPCR (N = 56).</p>a<p>The PAH cutpoint value 2.41 was obtained from an ROC curve where we considered that methylation status could be determined by PAH exposure level. We considered ACSL3 5′CGI to be methylated (determined by MSPCR) above 2.41 and unmethylated below 2.41. The value of the cutpoint determined the sensitivity (i.e. number of correctly classified unmethylated subjects or true positives), the specificity (i.e. number of correctly classified unmethylated subjects or true negatives), and the number of false positives and false negatives. The median of samples with PAH exposure <2.41 (N = 30) was 3.58 where that of samples with PAH exposure > = 2.41 was 1.55.</p>b<p>p<0.001. Odds Ratio(OR) = 13.8, which may also be interpreted as the odds of ACSL3 5′CGI methylated when PAH = or >2.41 (compared to <2.41).</p

Concordance of degree of methylation of 5′CGI(s) in UCWBC samples and gene expression levels in FPT samples.

<p>Note: % of methylated cytosine in the CGI(s) (regions mark as BSPCR in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004488#pone-0004488-g002" target="_blank">Fig 2</a>) 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.</p>*<p>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.</p

The workflow chart outlines how to discover the methylated genes in a step-by-step manner.

<p>Initially, ten umbilical cord white blood cell (UCWBC) samples from children with maternal (prenatal) PAH exposure above and ten with prenatal exposure below the cohort median were selected irrespective of gender and ethnicity. DNA was isolated from the sample was dichotomized according to the median of prenatal PAH exposure obtained from 48-hour personal air monitoring of the mothers during pregnancy (the cohort median is 2.3 ng/m3). Candidates with differentially methylation status between the samples from the two groups were discovered by Methylation Sensitive Restriction Fingerprinting (MSRF) following by subcloning and sequencing. Candidates were further identified by <i>in silico</i> analysis from databases from NCBI and UCSF genomic centre. Candidates which contained CpG island (CGI; CG rich region with >60% GC content and obs/exp ratio >0.6) on its 5′ flanking region were chosen for bisulfite sequencing to confirm the dependency of the methylation status of the CGI in a candidate gene on maternal PAH exposure. Six genes confirmed to have the latter relation were subjected to gene expression analysis in the 14 matched fetal placental tissues (FPT). The <i>ACSL3</i> was found to have the highest concordance between degree of CGI methylation and level of gene expression in reverse manner. It was therefore selected for the final analyses for correlation between methylation status of its CGI and transplacental PAH exposure and/or parent reported asthma symptoms of childhood asthma up to age 5 in a case-control study comprised of 56 participants. For this part of the study, Methylation specific-PCR (MSPCR) protocol was optimized to analyze the methylation status of R1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004488#pone-0004488-g004" target="_blank">Figure 4A</a> was used as the high throughput method to analyze this larger sample set.</p

Differentially methylated candidate genes with 5′CpG Islands identified with MSRF.

<p>Note: Sequences were identified based on BLAT (UCSC genome center database) and RefSeq (NCBI database) search.</p><p>Abbreviation: NA, not available.</p

Total maternal PAH exposure levels in the study sample as compared to the full cohort.

<p>Note: ^*There are 127 participants in the full cohort for whom we are missing prenatal PAH. No significant differences by ttest (means) or kruskal-wallis (medians). p25, p50 and p75 denote means at the 25, 50 and 75 percentile.</p

Characteristics of Subjects (Number, % Column Total) by ACSL3 5′CGI methylation Status and Asthma Status Assayed by MSPCR (N = 56).

<p>Note: ^ap = 0.03. OR = 3.9 measuring the odds of asthma given ACSL3 5′-CGI was methylated (M) verus unmethylated (U).</p>b<p>p<0.001 testing the equality of median PAH exposure levels between subjects with methylated and unmethylated ACSL3 5′CGI in their UCWBCs.</p

Real-time PCR analysis of gene expressions of <i>ACSL3</i> in non-small cell lung cancer H1299 cell line in response to (A) 5-aza-deoxycytidine (5AZA-dC) and (B) benzo[a]pyrene (BaP) and bisulfite genomic sequencing analysis of <i>ACSL3</i> promoter methylation status in response to BaP (C).

<p>A: Cells were treated with 0.5 and 1.0 µM 5-AZA-dC with DMSO as control every 2 days for a total of 8 days. B: Cells were treated with 0.01, 0.1 and 1.0 nM BaP with DMSO as control every 2 days for a total of 4 days. RNA was isolated, reverse transcribed and underwent real-time PCR. The 2-ΔΔCt method was used to calculate the relative expression level of transcripts normalized to <i>β-actin</i>. *Statistically significant differences between exposed and control was accepted at p<0.05. C: Diagram represents methylation status of <i>ACSL3</i> promoter of H1299 cells exposed to BaP assayed by bisulfite genomic sequencing. Cells were treated with 0.01, 0.1 and 1.0 nM BaP with DMSO as control every 2 days for a total of 4 days. Open circle: unmethylated CG; closed circle: methylated CG. Putative transcription factor binding sites such as Sp1, AP2, GCF, c-fos and junB are shown in scale with the particular CG sites on the promoter.</p