Schematic of the workflow applied to identify regulatory regions and SNPs in ABC genes. The number of SNPs assessed in each analysis step is shown in the shaded panel to the right. A: Gene expression data for ABC genes were downloaded and extracted from the Wellcome Trust Sanger Institute GeneVar database. Expression levels for all 210 unrelated individuals were used to determine population differences in ABC gene expression. B: Genotype data was downloaded from the HapMap database, and all SNPs within 50 kb of each ABC gene was used in the further analyses. C: Tag SNPs were determined for each of the CEU, YRI and ASI (JPT+CHB) populations separately. D: Associations between ABC expression levels and SNPs were determined in each population. E: Tag SNPs significantly associated with expression levels, together with all HapMap SNPs in linkage disequilibrium (r² ≥ 0.8) with them, were prioritized for experimental follow-up based on individual association significance, SNP location, species conservation, and predicted altered transcription factor binding. F: The ~500 bp regions surrounding each selected SNPs were cloned into a luciferase reporter vector (pGL4.23[luc2/minP] vector). G: Luciferase activity was assessed in five different cell lines (ACHN, HEK293T, HCT-116, HepG2 and Huh-7) for each reporter construct. Constructs resulting in decreased luciferase activity compared to the negative control were subsequently cloned into an alternative vector carrying a strong SV40 promoter (pGL3-Promoter vector). H: Allele-specific effects on luciferase activity was determined for all significant regulatory regions.

ABC transporter genes exhibiting significant population differences in expression levels in lymphoblastoid cell lines. ANOVA was used to determine differential expression between the CEU, YRI and ASI populations. The plot shows ABC genes with significant differences after correcting for multiple tests (Bonferroni corrected p < 0.001). Bottom panels show log₂ normalized expression values for each population. Top panels show relative differences compared to the overall mean across all populations, in original untransformed units.

SNPs that significantly associate with expression levels of ABC transporters in lymphoblastoid cell lines from three populations. A: The most significant association across all populations is shown for each ABC gene with a significant local SNP association. B: Population differences in ABCA12 expression (see Fig. 3) can be attributed to a private SNP in the CEU population. Genotypes are shown for the coding strand.

Association of a SNP in ABCD1 with expression levels in lymphoblastoid cell lines from the YRI population, and with luciferase reporter activity. A: The SNP rs17091297 is correlated with ABCD1 mRNA levels in the YRI population (Bonferroni-corrected p < 0.05). B: The major allele results in decreased luciferase activity compared to the control, when inserted upstream of a strong SV40 promoter. Luciferase activity is shown relative to the background promoter activity. Values are shown as mean ± S. E. M. from four separate experiments, each performed in triplicate.

Comparisons of expression of ABC transporter genes in lymphoblastoid cell lines among four populations. The correlations of normalized probe intensities are shown across all pairs of populations. Probe intensities are shown as filled circles, and the standard error of the mean as grey ellipses. For most probes, the standard errors were smaller than the size of the plot symbols.

Genome-wide distribution of loci associated with ABC gene expression. The uncorrected significance of each SNP’s association with expression levels in lymphoblastoid cell lines in the CEU, YRI and ASI populations is shown as a function of relative genomic position, divided per chromosome. The relative locations of human ABC genes are shown in the top panel.

Regulation of luciferase reporter activity by genomic regions surrounding SNPs in or near ABC transporter genes. A: Putative regulatory elements were cloned into the weak promoter luciferase vector pGL4.23[luc2/minP]. B: Regions resulting in negative regulation in the weak promoter assay were assayed in an alternative vector containing the strong SV40 promoter (pGL3[Promoter]). Luciferase activity is shown relative to that of a vector only containing the promoter and the luciferase gene. The activity of each region was assessed in multiple cell lines (from left to right, for each construct: ACHN, HCT-116, HEK293T, HepG2 and Huh-7; the latter was only used in the weak promoter assay). Red, yellow, light blue, and dark blue bars: luciferase activity >4×, >2×, <0.5×, and <0.25× background promoter activity, respectively. Values are shown as mean ± S. E. M. from three to five separate experiments, each performed in triplicate.