PAH, Epigenetic and Asthma Figure 2. Schematic diagram of CG content (%) in the 59 flanking region of A) ACSL3; B) DUSP22; C) RAD21; D) SCD5; E) SFMBT2 and F) WWOX. The CGI(s) (shaded in blue in the genomic DNA sequence) were identified in silico based on a CG content .60%, with an observed/expected ratio of 0.6 according to instructions from MethPrimer. TSS: Transcription start site. The nested PCR-amplified region was indicated by a line with double arrow heads and methylation status of this region was determined by bisulfite genomic sequencing. doi:10.1371/journal.pone.0004488.g002 Table 2. Percentage of promoter methylation (Met%) in UCWBC DNA samples and relative expression ratio (RER) in FPT RNA samples of all six candiate genes*. Sample Low PAH #813 #826 #926 #954 #1066 #1090 #755 High PAH #784 #831 #876 #901 #946 #1079 #1107 PAH level 1.71 1.80 1.50 1.15 1.22 1.47 0.85 PAH level 34.48 2.41 2.65 2.82 2.84 3.66 2.76 ACSL3 Overall Met% 42 45 48 35 67 45 45 Overall Met% 85 81 94 95 95 88 93 RAD 21 Overall RER Met% 0.25 46 0.48 56 3.07 35 0.26 36 0.43 45 1.88 40 0.03 35 RER Overall Met% 0.15 80 0.34 90 0.13 85 0.14 85 0.04 90 0.22 78 0.16 78 DUSP22 Overall Met% RER 2.22 52 0.58 42 6.36 35 0.66 36 0.79 36 0.56 36 1.19 44 RER Overall Met% 1.33 80 2.18 85 759.55 85 0.80 85 0.54 84 2.28 90 0.34 78 RER 26.72 9.31 6984.8 12.70 2.85 148.16 13.45 RER 30.25 5.15 192.40 10.72 4.23 12.95 2.47 SCD5 Overall Met% 25 33 33 33 60 48 33 Overall Met% 68 68 86 85 82 82 75 SFMBT2 Overall RER Met% 0.46 44 0.71 36 1.17 68 0.00 55 57.70 36 0.09 36 1.41 44 RER Overall Met% 1.41 78 0.95 68 2.48 68 0.19 75 0.05 85 0.94 85 0.17 78 WWOX Overall Met% RER 204.08 85 3.06 85 1438.5 86 79.09 78 9308.0 78 62.25 75 173.87 85 RER Overall Met% 51.68 33 0.29 51 342.03 35 15.07 35 160.54 40 25.11 40 99.13 42 RER 0.87 0.74 0.25 0.04 0.01 3.95 0.13 RER 0.48 0.16 0.13 0.97 0.03 0.62 0.27 Note: *Overall Met% is the percentage of methylated cytosine in the CGI(s) (regions mark as BSPCR in Fig 2) was determined by bisulfite sequencing; levels of gene expression normalized to b-actin level as a ratio using values of transcripts in FPT samples matched to UCWBC samples (n = 14) determined by real time RT-PCR. doi:10.1371/journal.pone.0004488.t002 PLoS ONE | www.plosone.org 5 February 2009 | Volume 4 | Issue 2 | e4488