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. 2009 Feb 16;4(2):e4488. doi: 10.1371/journal.pone.0004488

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Perera et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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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 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 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.