Predisposition to disease and the response to drugs are highly variable in human populations,41-42
with expression level differences in various genes contributing to interindividual differences in disease risk and drug response43-44
. We assessed the influence of ethnicity, gender, and local genetic variation on the expression levels of ABC transporters in lymphoblastoid cell lines, and used the detected expression quantitative trait loci (eQTL) to guide experimental assays of putative regulatory genomic regions.
For 19 of the 49 ABC genes in the human genome, ethnicity was significantly associated with expression levels. Since ABC transporters are known to affect the disposition of many drugs, metabolites and endogenous ligands,1-2
such population-dependent expression can ultimately lead to differential susceptibility to disease or outcome of drug therapy among populations. For example, we observed that the expression level of the hepatic bile salt efflux pump (ABCB11/BSEP), which when mutated is associated with familial, drug-induced and pregnancy-induced intrahepatic cholestasis,13, 45-46
was significantly lower in the Asian than in the European and African samples. Though our data cannot be directly extrapolated to expression level differences in the liver, if such population differences in hepatic expression levels of ABCB11 occurred, our data would suggest that Asians may be more susceptible to intrahepatic cholestasis mediated by BSEP. Intriguingly, the incidence of xxx is higher in Asians than in European women, and the possible connection with population dependent BSEP expression thus warrants further studies.
The largest difference among populations was observed for ABCG1, with expression levels on average 1.8 fold higher in the Asian compared to the Caucasian samples. Notably, in contrast to the several cases of statistically significant population dependent expression, ABCG1 was the only ABC transporter for which gender was significantly associated with expression. ABCG1 mediates the efflux of cellular cholesterol to high density lipoprotein (HDL),5, 47
and the higher average expression levels in women, and in the Asian population, suggests that ABCG1 expression might contribute to the relatively higher HDL plasma levels observed in these populations.48-49
Again, some caution in extrapolation of our data in lymphoblastoid cell lines to liver must be exercised.
Previous studies have indicated that genetic differences account for a substantial part of observed gene expression differences among populations.50-51
This was also observed here for some of our genes, with population differences in allele frequencies generally well correlated with population average expression. Particularly, the SNP rs2888327 was only observed in the CEU population, which had a higher average expression of the proximally located ABCA12 gene than the ASI and YRI populations (). On the whole, however, the contribution of population and gender was small compared to the total ABC gene expression variation, consistent with other studies of population effects on gene expression,50-52
and also with the observation that 85-95% of human genetic variation is due to within-population variation.53-54
With current advances in sequencing techniques, access to near-complete personal genomes is not far from reality. However, for these large-scale datasets to be truly useful, much work is needed in functionally annotating the human genome. So far, our knowledge of non-coding regions is greatly lagging behind that of protein coding regions. Especially little is known about the extent and function of distantly located regulatory elements such as enhancers and silencers. Evolutionary constraints on non-coding sequences have been successfully used to identify regions with regulatory function.17-19
However, not all evolutionarily conserved regions have detectable functions in in vitro and in vivo enhancer assays.18-19, 55
Furthermore, many regulatory elements are likely located in regions not detectable using comparative genomics.56-57
Alternatively, DNAse I hypersensitivity,37-38
binding of enhancer-associated proteins such as the acetyltransferase and transcriptional coactivator p300,40, 55
and epigenetic modifications such as histone methylation and acetylation39-40
have been used to predict the genome-wide locations of regulatory regions. While highly promising, additional experimental validation of predicted regulatory regions is needed to determine the predictive value of these methods.
Here, we used a complementary approach to identify putative regulatory regions, through the association of genetic polymorphism with expression levels of ABC transporters. We identified 24 tag SNPs that were significantly associated with ABC transporter expression levels in lymphoblastoid cell lines. These were in linkage disequilibrium with a total of 104 interrogated SNPs with a minor allele frequency >5% in at least one population. After prioritizing the initial list of possible eQTLs based on association strength, genomic location, predicted alteration of transcription factor binding sites and species conservation, 16 regions within a ±50 000 bp range surrounding 7 ABC transporters were selected for experimental validation. This eQTL-centered approach led to the identification of five genomic regions that resulted in at least fourfold increased or decreased reporter activity, and six additional regions with at least twofold altered activities. Allele-specific regulatory function was shown for five of the regions. Genetic variation in these regions can thus contribute to differential expression among individuals, and can ultimately affect drug response or susceptibility to disease.
Previous analyses of expression quantitative traits in lymphoblastoid cell lines20, 22
have focused on global properties of regulatory polymorphisms, and have not included experimental verification of identified associations. In our gene family focused effort, 5 to 11 of 16 tested associations were experimentally replicated depending on the reporter assay cutoff used. However, five regions did not alter reporter activity. While significant signals in the reporter assay suggest causative regulatory effects of the examined regions, interpreting the lack of signal is less straight-forward. It could be a consequence of false positives in the association analysis, but could also result from linkage disequilibrium between the examined region and the actual causative site. In addition, combinatorial regulation of gene expression in vivo is difficult to replicate in an artificial system that does not include the larger genomic context.
We recently used a combination of comparative genomics and transcription factor binding site predictions to select 50 putative regulatory regions in important hepatic drug transporters from the ABC and SLC families.58
The enhancer activities of these regions were assayed in mice using hydrodynamic tail vein injection, which results in a highly localized expression in the liver. Using this technique, 12/50 (24%) of the prioritized regions showed experimental activity in vivo. Notably, however, none of the regions with experimental regulatory impact in the present study were detectably conserved among placental mammals, suggesting that eQTL mapping provides additional information compared to comparative genomics approaches.
The present work thus shows the general utility of expression level quantitative trait loci in defining genomic regions with regulatory potential, and, specifically, demonstrates transcriptional regulation of human ABC transporters by several hitherto uncharacterized genomic regions. Genetic and epigenetic variation in the identified regions can thus lead to altered transport of drugs and their metabolites, and of endogenous solutes such as cholesterol, fatty acids and peptide fragments. Ultimately, this can cause inter-individual variation in the pharmacological response to drugs and in important physiological processes, including cellular lipid metabolism, lysosomal protein degradation and in antigen presentation on the cell surface.