Using comparative genomics, we identified noncoding ECRs in the vicinity of nine liver membrane transporters (SLC22A1, SLCO1B1, SLCO1B3, ABCC2, ABCB11, SLC22A7, SLCO2B1, SLC10A1, and SLC47A1) that have well-characterized drug interactions. These sequences were then ranked for the prevalence of previously characterized liver TFBSs, and the top 50 sequences were assayed for liver enhancer activity using the mouse hydrodynamic tail vein injection technique. The initial screening resulted in twelve ECRs that exhibited luciferase activity at or above the cutoff value (1.8-fold). This value could be more stringent in future screenings because we saw that the majority of ECRs with activity between 1.8 and 2.0-fold decreased in reporter gene activity when followed up and subsequently lost statistical significance.
Five sequences that exhibited significant enhancer activity were further characterized for sequence variation in an ethnically diverse cohort. These were in the vicinity of ABCB11
, and SLC47A1
. We found eleven common variants within these five ECRs that we assayed for differential enhancer expression. Of these, three showed significant enhancer activity differences as compared with their reference allele. These variants showed significant minor allele frequency differences between the ethnic groups, suggesting that enhancer variants can also contribute to ethnic-specific differences in drug response, similar to what has been observed for coding SNPs. We performed a more extensive analysis of one enhancer, ECR35. This enhancer is located within an intron of SLCO1A2
, a transporter that interacts with a variety of statins, anticancer and antibacterial drugs.30
We identified four different common haplotypes within this ECR, two of which led to significantly reduced enhancer expression levels.
Using human liver tissue samples, we were able to show that ECR35*3 by itself led to reduced SLCO1A2 mRNA levels. However, this SNP in combination with ECR35*1 did not cause a significant reduction of SLCO1A2 mRNA levels despite demonstrating a trend to reduced expression. This suggests that ECR35*1 may mitigate the ability of ECR35*3 to reduce expression of SLCO1A2. Analysis of ECR35 variants for the six liver-specific TFBSs used to rank the ECRs found no changes in binding sites for either ECR35*1 or ECR35*3.
We observed a correlation for the ECR35 variants between the enhancer assay results and SLCO1A2
mRNA expression levels. Our studies were limited due to our small human liver tissue sample size (n
= 88) and the large variation in SLCO1A2
mRNA expression levels (22.8 ± 24.9). Additional studies would have to be done to explicitly link ECR35 SNP*3 as the causative SNP that leads to reduced SLCO1A2
expression. We also found another variant that exhibited increased enhancer activity in another enhancer, ECR32, which is near SLC10A1
, an uptake transporter for bile salts and cyclosporin A.3
Performing drug-associated studies with this variant would also be of interest because the ECR32*1 variant led to increased reporter gene activity relative to the reference allele.
With ECR35 variants leading to significantly reduced reporter gene and mRNA expression levels, we analyzed their potential effects on OATP1A2 and its associated drug interactions. Coding mutations in OATP1A2 have been shown to affect the transport of MTX32
and other substances,33
and the SLCO1B1–SLCO1A2
genomic region has been found to be associated with reduced MTX clearance and gastrointestinal toxicity.31
Potentially, the reduced MTX clearance could be due to lower SLCO1A2
mRNA expression, attributable to the ECR35 variants. Although we did not observe ECR35 SNPs to be in strong LD with the MTX GWAS-associated SNPs in the SLCO1B1
genomic region, we did observe haplotype ECR35*1*3 to be associated with increased MTX clearance. This haplotype led to slightly reduced reporter gene expression in the enhancer assay but did not meet the criteria for significance (). However, in this population, SNP*1 was found to be in strong LD (D′ = 0.991; r2
= 0.939) with the nonsynonymous SLCO1A2
rs10841795 SNP (T38C; Ile13Thr), which was previously shown to lead to increased MTX uptake when expressed in Xenopus laevis
In the kidney, the predominant organ involved in MTX elimination, OATP1A2 is localized to the apical domain of distal nephrons.33
Higher MTX uptake, as observed in association with rs10841795, should lead to increased reabsorption and lower MTX clearance, which is contrary to our observations. The differences between our in vivo
observations and these functional studies might be due to the assay having been carried out in vitro
and/or the differences in pH concentrations in the kidney. The pH of the distal tubule is variable, and MTX transport by the reference OATP1A2 allele has been shown to be highly dependent on pH.32
Further studies need to be performed to determine the actual in vivo functional outcomes of rs10841795 and SNP*1, considering their effect on OATP1A2 in the kidney, liver, intestine, and brain. As for ECR35*3, all of our assays were carried out on liver tissues; therefore, its functional role in the kidney—the major organ associated with MTX clearance—is unknown. It would be interesting to investigate whether ECR35 is important for SLCO1A2 expression in the kidney and whether the enhancer affects interindividual drug interactions of other OATP1A2- transported drugs in general.
This study is the first to link nucleotide variations in enhancers with drug-associated genes, providing further evidence that regulatory sequences could be important determinants of differential drug response and ADEs. With the advancement of sequencing technologies, individual genome sequences will soon be available at an affordable price to the general public. The ability to obtain the genetic blueprint of an individual will greatly advance the quality of pharmacological treatment by tailoring drugs to fit that blueprint and ultimately reduce ADEs and improve drug efficacy overall. However, the major hurdle will be the development of high-throughput functional assays that can rapidly interpret the pharmacological nature of the nucleotide change(s) in a given individual, both in coding and in regulatory sequences.