Jason M. Gow

Very important pharmacogene summary: ABCB1 (MDR1, P-glycoprotein).

ABCB1 descriptionABCB1 (MDR1) is one of many ubiquitous adenosine triphosphate (ATP)-binding cassette (ABC) genes present in all kingdoms of life that is responsible for cellular homeostasis [1–3]. ABC genes encode transporter and channel proteins possessing multiple membrane-spanning domains that f

Substrate-Dependent Effects of Human ABCB1 Coding Polymorphisms

One of the many obstacles to effective drug treatment is the efflux transporter P-glycoprotein (P-gp), which can restrict the plasma and intracellular concentrations of numerous xenobiotics. Variable drug response to P-gp substrates suggests that genetic differences in ABCB1 may affect P-gp transport. The current study examined how ABCB1 variants alter the P-gp-mediated transport of probe substrates in vitro. Nonsynonymous ABCB1 variants and haplotypes with an allele frequency ≥2% were transiently expressed in HEK293T cells, and the transport of calcein acetoxymethyl ester and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY-FL)-paclitaxel was measured in the absence or presence of the P-gp inhibitor cyclosporin A. The A893S, A893T, and V1251I variants and the N21D/1236C>T/A893S/3435C>T haplotype altered intracellular accumulation compared with reference P-gp in a substrate-dependent manner. It is interesting that certain variants showed altered sensitivity to cyclosporin A inhibition that was also substrate-specific. These functional data demonstrate that nonsynonymous polymorphisms in ABCB1 may selectively alter P-gp transport and drug-drug interactions in a substrate- and inhibitor-dependent manner.

Genetic Variation in the Proximal Promoter of ABC and SLC Superfamilies: Liver and Kidney Specific Expression and Promoter Activity Predict Variation

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 (π) 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.

FUNCTIONAL CHARACTERIZATION OF ABCC2 PROMOTER POLYMORPHISMS AND ALLELE SPECIFIC EXPRESSION

Multidrug resistance protein 2 (MRP2, ABCC2) is an efflux membrane transporter highly expressed in liver, kidney and intestine with important physiological and pharmacological roles. The goal of this study was to investigate the functional significance of promoter region polymorphisms in ABCC2 and potential allele-specific expression. Twelve polymorphisms in the 1.6 kb region upstream of the translation start site were identified by resequencing 247 DNA samples from ethnically diverse individuals. Luciferase reporter gene assays showed that ABCC2 −24C>T both alone and as part of a common haplotype (−24C>T/−1019A>G/−1549G>A) increased promoter function 35% compared with the reference sequence (P<0.0001). No other common variants or haplotypes affected ABCC2 promoter activity. Allele-specific expression was also investigated as a mechanism to explain reported associations of the synonymous ABCC2 3972C>T variant with pharmacokinetic phenotypes. In Caucasian liver samples (n=41) heterozygous for the 3972C>T polymorphism, the 3972C allele was preferentially transcribed relative to the 3972T allele (P<0.0001). This allelic imbalance was particularly apparent in samples with haplotypes containing two or three promoter/untranslated region variants (−1549G>A, −1019A>G and −24C>T). The observed allelic imbalance was not associated with hepatic or renal ABCC2 mRNA expression. Additional mechanisms will need to be explored to account for the interindividual variation in ABCC2 expression and MRP2 function.

The Effects of ABCB1 3′-Untranslated Region Variants on mRNA Stability

Genetic variation in ABCB1, encoding P-glycoprotein (P-gp), is a potential cause of interindividual variation in drug response. Numerous studies have focused on the effects of coding region variants on P-gp expression and function, whereas few noncoding region variants have been investigated. The 3′-untranslated region (UTR) regulates mRNA levels or stability via RNA-protein interactions with mRNA degradation machinery. mRNA stability is a key regulatory step controlling ABCB1 mRNA expression that ultimately affects P-gp levels and function. We hypothesized that ABCB1 3′-UTR polymorphisms alter mRNA stability by disrupting RNA-protein interactions. An ethnically diverse panel of DNA samples was sequenced to identify 3′-UTR polymorphisms and determine allele frequencies. The three most common variants, along with reference ABCB1, were stably expressed in cells in order to measure mRNA half-life. The calculated half-life for ABCB1 reference in HEK293 cells was 9.4 ± 1.3 h and was similar to that estimated for the 3′-UTR variants. Endogenous ABCB1 mRNA decay was similar in lymphoblastoid cell lines carrying 3′-UTR variant and reference alleles. Although the examined ABCB1 3′-UTR variants have no effect on ABCB1 mRNA stability, these data represent one of the first attempts to determine the influence of genetic variation in UTRs on ABCB1 mRNA levels.

Functional hot spots in human ATP-binding cassette transporter nucleotide binding domains

The human ATP‐binding cassette (ABC) transporter superfamily consists of 48 integral membrane proteins that couple the action of ATP binding and hydrolysis to the transport of diverse substrates across cellular membranes. Defects in 18 transporters have been implicated in human disease. In hundreds of cases, disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs). The functional impact of the majority of ABC transporter nsSNPs has yet to be experimentally characterized. Here, we combine experimental mutational studies with sequence and structural analysis to describe the impact of nsSNPs in human ABC transporters. First, the disease associations of 39 nsSNPs in 10 transporters were rationalized by identifying two conserved loops and a small α‐helical region that may be involved in interdomain communication necessary for transport of substrates. Second, an approach to discriminate between disease‐associated and neutral nsSNPs was developed and tailored to this superfamily. Finally, the functional impact of 40 unannotated nsSNPs in seven ABC transporters identified in 247 ethnically diverse individuals studied by the Pharmacogenetics of Membrane Transporters consortium was predicted. Three predictions were experimentally tested using human embryonic kidney epithelial (HEK) 293 cells stably transfected with the reference multidrug resistance transporter 4 and its variants to examine functional differences in transport of the antiviral drug, tenofovir. The experimental results confirmed two predictions. Our analysis provides a structural and evolutionary framework for rationalizing and predicting the functional effects of nsSNPs in this clinically important membrane transporter superfamily.

Response to “Predictable difficulty or difficulty to predict”

Using our automated bioinformatics tool, we recently classified 40 point mutations in ABC transporters as “neutral” or “disease-associated,” followed by experimentally testing three of the predictions.1 Scanning the scientific literature, Varadi et al. found support for nine of our predictions, whereas suggesting that 10 were incorrect. However, the interpretation of the literature by Varadi et al. is itself not unambiguous, as illustrated by at least 6 of the 10 examples of presumed misprediction. The first five examples are likely pleiotropic mutations, with a different impact on a different function: Mutation Q141K in the breast cancer resistance protein BCRP does not significantly influence the disposition of HIV drug lamivudine,2 thus not supporting the unequivocal “disease-associated” classification by Varadi et al. Mutation P269S in BCRP is associated with a 40% decrease in vesicular uptake of [(3)H]estrone-3-sulfate and [(3)H]methotrexate compared to the wild-type form,3 which is inconsistent with the unambiguous “neutral” annotation. Mutation V444A in the bile salt export pump BSEP does not change taurocholate transport function in vitro;4 homozygous V444A mutations were prevalent in both progressive familial intrahepatic cholestasis patients and in noncholestatic patients;5 and this variant has also been observed in other healthy populations.6 Thus, a clearcut classification as “disease-associated” is not justified. Mutant V1251I of ABCB1 showed decreased transport of BODIPY-FL-paclitaxel, but increased transport of calcein-AM in an assay using transfected HEK293T cells,7 arguing against an unequivocal “disease-associated” classification. Mutation S1141T in ABCB1 showed either normal or increased transport in the yeast assay,8 and increased function in transfected HEK293T cells.7 Therefore, this mutation should not be classified simply as “disease-associated.” The final, sixth case is an example of the lack of evidence for the classification by Varadi et al.: Mutation A1291T in ABCC6 occurs simultaneously with a nonsense mutation (Y1069X) in the analyzed form of the transporter.9 Therefore, we do not yet know whether the nonsense mutation or the A1291T mutation (or both) is causative of the disease state, contrary to a definitive “disease-associated” classification. If one accepts the qualifications above, 5 of the 40 predictions by our automated tool are invalidated, including one by our experiments1 and four by the scientific literature cited by Varadi et al.; and 11 mutations are confirmed, including two by our experiments1 and nine by the literature cited by Varadi et al. The corresponding accuracy level (69%) is in fact comparable to that estimated by the random forest test (Fig. 4 in Ref.1). In conclusion, we agree with Varadi et al. that there are inherent difficulties in predicting the functional impact of point mutations. Moreover, as we illustrate above, there are also inherent difficulties in characterizing the functional impact of mutations by experiment, and in interpreting the corresponding scientific literature. Classification of mutations into only two classes (such as our “neutral” and “disease-associated” classes) is a serious limitation, given the complexities of protein localization, degradation, interactions with other small molecules and macromolecules, as well as function in general, resulting in the pleiotropy of some mutations.7 Nevertheless, the difficulty of the problem does not mean we should not address it as long as the progress is properly qualified, as we believe we did in our publication.