Genetic variation in the proximal promoter of ABC and SLC superfamilies: liver and kidney specific expression and promoter activity predict variation

Abstract

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp) and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

Figure 1. Ethnic breakdown of proximal promoter SNPs.

The number of SNPs present in each ethnic group are represented by shaded bars: African Americans (black), European Americans (dark grey), Mexican Americans (light grey), and Asian Americans (white). SNPs are categorized based on the minor allele frequency (MAF) occurring in each corresponding ethnic group.

Figure 2. Location of nucleotide diversity in transporter promoters relative to the transcription start site.

Nucleotide diversity was estimated using the mutation parameter (θ) and the average heterozygosity per site parameter (π), in a 100 bp sliding window. The diversity parameters are plotted as functions of the center of the sliding window. Blue line: African Americans; red line: European Americans; gold line: Mexican Americans; green line: Asian Americans; and black line: combined population.

Figure 3. Nucleotide diversity in transporters expressed in human liver and/or kidney.

Transporters were included in the analysis if expressed in the top one-third of all assessed probes in mRNA microarray experiments, or if significantly expressed in real time PCR experiments (See Materials and Methods). A list of the transporters is provided as Table S5. Nucleotide diversity is shown for the proximal promoter (black bars) and the 5′ flanking region (open bars).

Figure 4

A. Reporter activity of proximal promoter constructs in human cell lines. The proximal promoters were amplified from human genomic DNA and cloned into pGL-4 gene expression vectors. Luciferase activity was measured 24 h after transfection of a panel of human cell lines. Promoter activity is expressed as log₂ [(1+ Firefly luciferase/Renilla luciferase)/Average of the negative control]. Promoters were classified as having high, intermediate or low activity if the average relative activity across multiple cell lines was above 4, between 2 and 4, or below 2, respectively. B. Nucleotide diversity in high, intermediate and low activity promoters.