PAH, Epigenetic and Asthma Figure 1. The workflow chart outlines how to discover the methylated genes in a step-by-step manner. 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 in silico 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 59 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 ACSL3 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 Figure 4A was used as the high throughput method to analyze this larger sample set. doi:10.1371/journal.pone.0004488.g001 investigation on the larger sample of 56 children, the following quality control experiments were performed. First, bisulfite sequencing analyses of an amplicon encompassing the entire predicted ACSL3 59-CGI (Figure 4A, BSPCR marks the amplicon) were performed on the 20 UWBC DNA samples; five from each of the high or low PAH groups with or without asthma. Bisulfite sequencing data (Figure 4B) showed that hypermethylation of this CGI was found to be strongly associated with maternal exposure to a PAH level higher than the cohort median of 2.3 ng/m3. Asthma status did not seem to affect methylation status of ACSL3 promoter. Second, based on bisulfite sequencing data, we designed and optimized three methylation-sensitive-PCR (MSPCR) protocols [27,28] to amplify three regions (R1, R2 and R3) within the CGI that exhibited the greatest differential methylation changes between samples from the ‘‘high PAH’’ and the ‘‘low PAH’’ groups regardless of asthma status (Figure 4A, R1–3 are marked). These three regions were closely flanking the transcriptional start site (TSS). We further validated that the methylation status of R1 exhibited the strongest correlation with bisulfite sequencing data from analyses of the entire CGI. Representative data from MSPCR PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4488