Fig. 2

Visualization and verification of differentially methylated probes. a Volcano plot showing mean methylation differences between FASD and control (x axis) versus log transformed p-values (y axis). 1661 CpG sites with an FDR <0.05 were considered significantly differently methylated between FASD and control, but 1003 of these were ethnically confounded, resulting in the final 658 probes shown in blue. b Heatmap of top 50 most significant up- (top) and down-methylated (bottom) probes in control (left, gray) versus FASD cases (right, blue). The percent methylation values (ranging from 0 to 1) are adjusted for the covariates from the regression model and then centered, scaled, and trimmed, resulting in a standardized DNA methylation level ranging from −2 to +2 (black–white scale). The mean percent methylation value (beta) for each probe (red–blue scale) is the mean methylation value, after adjustment for covariates, for all samples. c Verification with pyrosequencing in both FASD (blue) and control (gray) samples. The top panel displays DNA methylation levels measured by the 450K array, the bottom panel, the levels for the same CpG sites measured with pyrosequencing. These CpGs were located in the gene body of SHANK3 (cg10793758), NOS1AP (cg02858267), CACNA1A (cg24800175), and SNED1 (cg19075225), or in the 3′ UTR of NOS1AP (cg12486795). Those found in NOS1AP were located in a CpG island, while those in SHANK3 and CACNA1A were located in a north shelf or shore, respectively. The CpG associated with SNED1 was not located near any CpG island