Table 1. Differentially methylated candidate genes with 59CpG Islands identified with MSRF. Clone Pr1 Pr2 Hyper- methylation Chr Band A1 A2 A3 A4 A5 A6 A7 A8 A9 B1 B2 B3 B4 D1 D2 D4 D5 D6 D7 D8 D9 E1 E2 E3 E4 F1 F3 F4 G1 G2 D10 20 20 20 20 20 20 20 20 20 22 22 22 22 22 22 22 22 22 22 22 22 22 7 7 7 7 10 10 10 22 22 21 21 21 21 21 21 21 21 21 23 23 23 23 23 23 23 23 23 23 23 23 23 11 11 11 11 11 11 11 11 11 High PAH 8q24.11 High PAH 6p25.3 Low PAH High PAH High PAH High PAH High PAH High PAH High PAH High PAH High PAH High PAH High PAH Low PAH Low PAH Low PAH Low PAH High PAH High PAH Low PAH Low PAH Low PAH High PAH High PAH High PAH High PAH High PAH High PAH Low PAH Low PAH High PAH 16q13 8q24.3 6p25.1 1q41 19p13.3 10q26.3 11q12.1 8p23.1 1q32.2 2p24.2 3q24 1q41 2p24.2 1q32.1 15q22.2 1q41 19p13.2 2q36.1 11q25 1p32.3 14q21.1 4q21.22 16p13.11 16q24.2 Xq13.1 11q23.1 10p14 13q32.3 16q23.1 Gene homology NIK and IKKbetta-binding protein cDNA clone IMAGE:4825327 cDNA clone FLJ14311 Protein in DNA double-strand break repair, RAD21 Dual specificity phosphatase 22, DUSP22 Beta-defensin 107, DEFB107 mRNA for KIAA0463 protein, partial cds WIAF-4002-STS Human THudson EST STS cDNA Vacuolar protein sorting-associated protein 13C, VPS13C Acyl-CoA synthetase long-chain family member 3, ACSL3 cDNA clone DKFZp686H1949 Clone BNGH42007037, similar to zinc finger protein GLI1 CTAGE-5B protein mRNA, complete cds, alternatively spliced Junctophilin 3, JPH3 Ribosomal protein S4, X-linked Scm-like with four mbt domains 2, SFMBT2 Small inducible cytokine A17 precursor, CCL17 WW domain-containing oxidoreductase isoform , WWOX Location INTRON15 59 END INTRON2 59 END INTRON1 39 END INTRON4 INTRON1 EXON7 INTRON1 INTRON1 INTRON1 INTRON3 INTRON8 INTRON2 CGI at 59 end ,1 kb ,1 kb PAH, Epigenetic and Asthma Gene Ontology NF-kappaB-inducing kinase acitivity Double strand break repair Stress kinase involved in MAPK pathway Defense response Trans-Golgi to endosome transport Integral to membrane Translation Chemokine activity INTRON1 ,1 kb Fatty acid metabolism INTRON1 ,2 kb Gene transcription INTRON1 ,1 kb Gene transcription Stearoyl-CoA desaturase 5, SCD5 INTRON1 ,1 kb Fatty acid metabolism Note: Sequences were identified based on BLAT (UCSC genome center database) and RefSeq (NCBI database) search. Abbreviation: NA, not available. doi:10.1371/journal.pone.0004488.t001 analyses of samples from children with prenatal PAH exposure above (high PAH) or below (low PAH) the cohort median are illustrated in Figure 4C. Thus, MSPCR of R1 was chosen as the high throughput method for investigating the methylation status of the ACSL3 59-CGI in the larger sample comprised of 56 children. Receiver Operating Curve (ROC) analysis of data from 56 subjects identified the maternal PAH exposure cutpoint value that best distinguished methylated versus unmethylated ACSL3 59CGI status determined by MSPCR to be 2.41 ng/m3 (Figure 5). The Area Under ROC curve (AUC) value was 0.82 suggesting excellent separability of the subjects with methylated or unmethylated ACSL3 59CGI. Using the ‘‘optimal’’ cutpoint value, methylation status was predicted with 75% sensitivity, 82% specificity. Table 4 shows that 30 of the 56 children had a methylated ACSL3 59-CGI; 81% of these 30 children were born to mothers with PAH exposure equal or exceeding 2.41 ng/m3, the cutpoint value determined by the ROC analysis. Thus, methylation was positively and significantly associated with maternal PAH PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4488