PAH, Epigenetic and Asthma Figure 4. Methylation status of a region in the ACSL3 59CGI analyzed by bisufite genomic sequencing and methylation specific PCR (MSPCR). A) Schematic diagram of the CG content (%) in the 59 flanking region of the ACSL3 gene. A CGI of 1058 bp was located at the 59 end of ACSL3 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 ACSL3 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 ACSL3 59-CGI of all samples was further analyzed by MS-PCR using the same sets of primers. Results are shown in Table 4 and Table 5. High PAH ($ the cohort median of 2.3 ng/m3), Low PAH (,the cohort median). doi:10.1371/journal.pone.0004488.g004 were quantified by SYBR Green-based real time RT-PCR with normalization to b-actin as previously described [14,16]. Primers specific for quantification of transcripts of the selected genes are listed in Table 8. Three separate real time RT-PCRs were performed on each FPT sample to obtain a mean value. The 2- DDCt method was used to calculate the relative expression level of transcripts normalized to b-actin. The mean value from a universal reference RNA (Clontech, CA) was arbitrarily assigned an abundance value of 1.00 for each gene. Mean values from all other samples were normalized against this sample. Statistical methods The Kendall coefficient of concordance (tau) was calculated to measure the strength of the relationship between the percent of methylation of a gene’s promoter CGI in UCWBC DNA samples and its transcript expression level in matched FPT RNA samples. Ninety-five percent confidence intervals and 2-tailed p-values were obtained to determine the expected range and significance of observed tau values. Chi-square tests were performed to investigate possible differences in the percents of African Americans, males, and methylated subjects between categories of PAH exposure, (,2.41, $2.41). Odds ratios and 95% confidence intervals of high PAH exposure for each of the three characteristics were calculated and tested for statistical significance. The cutpoint of 2.41 was determined from an ROC curve analysis following logistic regression of methylation status on continuously measured PAH exposure. This value separated methylated and unmethylated subjects according to ‘high/low’ PAH exposure. We considered MSPCR to be methylated above 2.41 and unmethylated below 2.41. Figure 5. Receiver operating characteristics (ROC) plot for 56 children on PAH values and methylation status of ACSL3 analyzed by MSPCR. Point labels are values of PAH of each children. Statistical software, sas, was used for ROC plot and R was used for determination of cutoff value. The cutoff value corresponds to the desired sensitivity and specificity (or 1-specificity). doi:10.1371/journal.pone.0004488.g005 PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4488