Transcriptomic variation of pharmacogenes in multiple human tissues and lymphoblastoid cell lines.

Variation in the expression level and activity of genes involved in drug disposition and action ('pharmacogenes') can affect drug response and toxicity, especially when in tissues of pharmacological importance. Previous studies have relied primarily on microarrays to understand gene expression differences, or have focused on a single tissue or small number of samples. The goal of this study was to use RNA-sequencing (RNA-seq) to determine the expression levels and alternative splicing of 389 Pharmacogenomics Research Network pharmacogenes across four tissues (liver, kidney, heart and adipose) and lymphoblastoid cell lines, which are used widely in pharmacogenomics studies. Analysis of RNA-seq data from 139 different individuals across the 5 tissues (20-45 individuals per tissue type) revealed substantial variation in both expression levels and splicing across samples and tissue types. Comparison with GTEx data yielded a consistent picture. This in-depth exploration also revealed 183 splicing events in pharmacogenes that were previously not annotated. Overall, this study serves as a rich resource for the research community to inform biomarker and drug discovery and use.

Genetic and epigenetic regulation of the organic cation transporter 3, SLC22A3.

Human organic cation transporter 3 (OCT3 and SLC22A3) mediates the uptake of many important endogenous amines and basic drugs in a variety of tissues. OCT3 is identified as one of the important risk loci for prostate cancer, and is markedly underexpressed in aggressive prostate cancers. The goal of this study was to identify genetic and epigenetic factors in the promoter region that influence the expression level of OCT3. Haplotypes that contained the common variants, g.-81G>delGA (rs60515630) (minor allele frequency 11.5% in African American) and g.-2G>A (rs555754) (minor allele frequency>30% in all ethnic groups) showed significant increases in luciferase reporter activities and exhibited stronger transcription factor-binding affinity than the haplotypes that contained the major alleles. Consistent with the reporter assays, OCT3 messenger RNA expression levels were significantly higher in Asian (P<0.001) and Caucasian (P<0.05) liver samples from individuals who were homozygous for g.-2A/A in comparison with those homozygous for the g.-2G/G allele. Studies revealed that the methylation level in the basal promoter region of OCT3 was associated with OCT3 expression level and tumorigenesis capability in various prostate cancer cell lines. The methylation level of the OCT3 promoter was higher in 62% of prostate tumor samples compared with matched normal samples. Our studies demonstrate that genetic polymorphisms in the proximal promoter region of OCT3 alter the transcription rate of the gene and may be associated with altered expression levels of OCT3 in human liver. Aberrant methylation contributes to the reduced expression of OCT3 in prostate cancer.

Screening a large reference sample to identify very low frequency sequence variants: comparisons between two genes.

Most human sequence variation is in the form of single-nucleotide polymorphisms (SNPs). It has been proposed that coding-region SNPs (cSNPs) be used for direct association studies to determine the genetic basis of complex traits. The success of such studies depends on the frequency of disease-associated alleles, and their distribution in different ethnic populations. If disease-associated alleles are frequent in most populations, then direct genotyping of candidate variants could show robust associations in manageable study samples. This approach is less feasible if the genetic risk from a given candidate gene is due to many infrequent alleles. Previous studies of several genes demonstrated that most variants are relatively infrequent (<0.05). These surveys genotyped small samples (n<75) and thus had limited ability to identify rare alleles. Here we evaluate the prevalence and distribution of such rare alleles by genotyping an ethnically diverse reference sample that is more than six times larger than those used in previous studies (n=450). We screened for variants in the complete coding sequence and intron-exon junctions of two candidate genes for neuropsychiatric phenotypes: SLC6A4, encoding the serotonin transporter; and SLC18A2, encoding the vesicular monoamine transporter. Both genes have unique roles in neuronal transmission, and variants in either gene might be associated with neurobehavioral phenotypes.

Discovery of regulatory elements in human ATP-binding cassette transporters through expression quantitative trait mapping.

ATP-binding cassette (ABC) membrane transporters determine the disposition of many drugs, metabolites and endogenous compounds. Coding region variation in ABC transporters is the cause of many genetic disorders, but much less is known about the genetic basis and functional outcome of ABC transporter expression level variation. We used genotype and mRNA transcript level data from human lymphoblastoid cell lines to assess population and gender differences in ABC transporter expression, and to guide the discovery of genomic regions involved in transcriptional regulation. Nineteen of 49 ABC genes were differentially expressed between individuals of African, Asian and European descent, suggesting an important influence of race on expression level of ABC transporters. Twenty-four significant associations were found between transporter transcript levels and proximally located genetic variants. Several of the associations were experimentally validated in reporter assays. Through influencing ABC expression levels, these single-nucleotide polymorphisms may affect disease susceptibility and response to drugs.

Genotype-dependent effects of inhibitors of the organic cation transporter, OCT1: predictions of metformin interactions.

Common genetic variants of the liver-specific human organic cation transporter 1 (OCT1; SLC22A1) have reduced transport capacity for substrates such as the antidiabetic drug metformin. The effect of the reduced OCT1 function on drug interactions associated with OCT1 has not been investigated and was, therefore, the focus of the study presented here. HEK293 cells expressing human OCT1-reference or the variants R61C, V408M, M420del and G465R were first used to study the kinetics and inhibition pattern of the OCT1 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). In the second part OCT1-mediated (14)C-metformin uptake was studied in the presence of drugs administered concomitantly with metformin. Transport studies using ASP(+) showed that the function of the variants decreased in the following order: OCT1-reference=V408M=M420del >R61C >>G465R. Variants M420del and R61C were more sensitive to drug inhibition, with IC(50) values up to 23 times lower than those of the OCT1-reference. Uptake studies using (14)C-metformin were in qualitative agreement with those using ASP(+), with the exception that a larger reduction in transport capacity was observed for M420del. Concomitantly administered drugs, such as verapamil and amitriptyline, revealed potential drug-drug interactions at clinical plasma concentrations of metformin for OCT1-M420del.

PBPK Modeling of the Effect of Reduced Kidney Function on the Pharmacokinetics of Drugs Excreted Renally by Organic Anion Transporters.

Altered pharmacokinetics (PK) in subjects with chronic kidney disease (CKD) may lead to dosing adjustment of certain drugs in subjects with CKD. It can be valuable to quantitatively predict PK in CKD for the management of drug dosing in these subjects. We developed physiologically based pharmacokinetic (PBPK) models of seven renally eliminated drugs: adefovir, avibactam, entecavir, famotidine, ganciclovir, oseltamivir carboxylate, and sitagliptin. These drugs are all substrates of renal organic anion transporters (OATs). Drug models verified using PK data from healthy subjects (HS) were coupled with physiological models representing CKD that incorporated prior knowledge of effects of CKD on hepatic and renal elimination. The models reasonably described clinically observed PK changes in subjects with CKD (compared to subjects with normal renal function), with predicted AUC changes within 50% of the observed changes. PBPK models can be used to prospectively predict PK of renally eliminated OAT substrates in subjects with CKD.

The pharmacogenetics research network: from SNP discovery to clinical drug response.

The NIH Pharmacogenetics Research Network (PGRN) is a collaborative group of investigators with a wide range of research interests, but all attempting to correlate drug response with genetic variation. Several research groups concentrate on drugs used to treat specific medical disorders (asthma, depression, cardiovascular disease, addiction of nicotine, and cancer), whereas others are focused on specific groups of proteins that interact with drugs (membrane transporters and phase II drug-metabolizing enzymes). The diverse scientific information is stored and annotated in a publicly accessible knowledge base, the Pharmacogenetics and Pharmacogenomics Knowledge base (PharmGKB). This report highlights selected achievements and scientific approaches as well as hypotheses about future directions of each of the groups within the PGRN. Seven major topics are included: informatics (PharmGKB), cardiovascular, pulmonary, addiction, cancer, transport, and metabolism.

Stereoselective renal clearance of pindolol in humans.

In this study, pindolol, a beta-adrenoceptor blocking agent marketed as a racemic mixture, was used as a model compound to investigate stereoselective renal clearance of organic cations in human beings. Six normal subjects received an oral dose of 20 mg racemic pindolol. Heart rate and blood pressure were measured throughout the study. A stereospecific high performance liquid chromatographic procedure was used to quantitate the concentrations of d- and l-pindolol in plasma and urine. Renal clearance and other pharmacokinetic parameters of both enantiomers were calculated and compared. The renal clearance of l-pindolol was greater than that of d-pindolol in all subjects. The renal clearance (mean +/- SD) was 240 +/- 55 ml/min for l-pindolol and 200 +/- 51 ml/min for d-pindolol (P less than 0.01). Since stereoselective binding to plasma proteins was not observed, differences in renal clearance between d- and l-pindolol were caused by either stereoselective renal transport, or stereoselective renal metabolism. The area under the plasma concentration-time curve, the amount of drug excreted, and the half-life of l-pindolol were greater than those of d-pindolol, which suggests that pindolol was also eliminated stereoselectively by nonrenal routes. The slopes of the resting heart rate vs. the plasma concentration of l-pindolol were significantly less than zero and were significantly correlated to the pretreatment heart rate, which supports the hypothesis that intrinsic sympathetic tone largely determines the effect of pindolol on the resting heart rate. The observation that pindolol is eliminated stereoselectively by the kidney may have clinical implications for other racemic drugs that are renally eliminated.

A Molecular Basis for Innovation in Drug Excipients.

Excipients are ubiquitous in drug formulation, ensuring that active ingredient drugs are properly released on dosing, retain their properties over time, and are palatable, among other roles. Despite their crucial roles, surprisingly little is known about their systemic availability and activities on molecular targets. Here we review key excipient properties, introduce a public-accessible database that enumerates and categorizes them, and sketch a strategy for exploring their possible direct actions on molecular targets.

New Pharmacogenomics Research Network: An Open Community Catalyzing Research and Translation in Precision Medicine.

The goal of pharmacogenomics research is to discover genetic polymorphisms that underlie variation in drug response. Increasingly, pharmacogenomics research involves large numbers of patients and the application of new technologies and methodologies to enable discovery. The Pharmacogenomics Research Network (PGRN) has become a community-driven network of investigators spanning scientific and clinical disciplines. Here, we highlight the activities and types of resources that enable PGRN members to enhance and drive basic and translational research in pharmacogenomics.

Variants in Pharmacokinetic Transporters and Glycemic Response to Metformin: A Metgen Meta-Analysis.

Therapeutic response to metformin, a first-line drug for type 2 diabetes (T2D), is highly variable, in part likely due to genetic factors. To date, metformin pharmacogenetic studies have mainly focused on the impact of variants in metformin transporter genes, with inconsistent results. To clarify the significance of these variants in glycemic response to metformin in T2D, we performed a large-scale meta-analysis across the cohorts of the Metformin Genetics Consortium (MetGen). Nine candidate polymorphisms in five transporter genes (organic cation transporter [OCT]1, OCT2, multidrug and toxin extrusion transporter [MATE]1, MATE2-K, and OCTN1) were analyzed in up to 7,968 individuals. None of the variants showed a significant effect on metformin response in the primary analysis, or in the exploratory secondary analyses, when patients were stratified according to possible confounding genotypes or prescribed a daily dose of metformin. Our results suggest that candidate transporter gene variants have little contribution to variability in glycemic response to metformin in T2D.

Genomewide Association Studies in Pharmacogenomics: Meeting Report of the NIH Pharmacogenomics Research Network-RIKEN (PGRN-RIKEN) Collaboration.

Genomewide association studies (GWAS) have resulted in the identification of many heritable genetic factors that underlie risk for human disease or variation in physiologic traits. In contrast, there are fewer GWAS of drug response phenotypes, despite extensive unexplained interindividual variability. To address this urgent need, the NIH Pharmacogenomics Research Network (PGRN) and the Center for Integrative Medical Sciences (IMS) at RIKEN support a collaboration, PGRN-RIKEN, with the goal of accelerating GWAS of drug response phenotypes.

Metabolomic and Genome-wide Association Studies Reveal Potential Endogenous Biomarkers for OATP1B1.

Transporter-mediated drug-drug interactions (DDIs) are a major cause of drug toxicities. Using published genome-wide association studies (GWAS) of the human metabolome, we identified 20 metabolites associated with genetic variants in organic anion transporter, OATP1B1 (P < 5 × 10-8 ). Of these, 12 metabolites were significantly higher in plasma samples from volunteers dosed with the OATP1B1 inhibitor, cyclosporine (CSA) vs. placebo (q-value < 0.2). Conjugated bile acids and fatty acid dicarboxylates were among the metabolites discovered using both GWAS and CSA administration. In vitro studies confirmed tetradecanedioate (TDA) and hexadecanedioate (HDA) were novel substrates of OATP1B1 as well as OAT1 and OAT3. This study highlights the use of multiple datasets for the discovery of endogenous metabolites that represent potential in vivo biomarkers for transporter-mediated DDIs. Future studies are needed to determine whether these metabolites can serve as qualified biomarkers for organic anion transporters. Quantitative relationships between metabolite levels and modulation of transporters should be established.

A Longitudinal HbA1c Model Elucidates Genes Linked to Disease Progression on Metformin.

One-third of type-2 diabetic patients respond poorly to metformin. Despite extensive research, the impact of genetic and nongenetic factors on long-term outcome is unknown. In this study we combine nonlinear mixed effect modeling with computational genetic methodologies to identify predictors of long-term response. In all, 1,056 patients contributed their genetic, demographic, and long-term HbA1c data. The top nine variants (of 12,000 variants in 267 candidate genes) accounted for approximately one-third of the variability in the disease progression parameter. Average serum creatinine level, age, and weight were determinants of symptomatic response; however, explaining negligible variability. Two single nucleotide polymorphisms (SNPs) in CSMD1 gene (rs2617102, rs2954625) and one SNP in a pharmacologically relevant SLC22A2 gene (rs316009) influenced disease progression, with minor alleles leading to less and more favorable outcomes, respectively. Overall, our study highlights the influence of genetic factors on long-term HbA1c response and provides a computational model, which when validated, may be used to individualize treatment.

Substrate selectivity, potential sensitivity and stoichiometry of Na(+)-nucleoside transport in brush border membrane vesicles from human kidney.

Recently, we demonstrated the presence of a Na(+)-nucleoside cotransport mechanism that transports both purine and pyrimidine nucleosides in human renal brush-border membrane vesicles (BBMV) (Gutierrez et al. (1992) Biochim. Biophys. Acta 1105, 1-9). The objective of this study was to further elucidate the characteristics of this cotransport system in terms of electrical potential sensitivity, stoichiometry and substrate selectivity with respect to nucleoside analogs. In BBMV from human kidney, Na(+)-thymidine uptake was stimulated by an inside negative potential difference created by K+ and valinomycin. A hyperbolic relationship between initial rate of uridine uptake and Na+ concentration was obtained suggesting a Na(+)-nucleoside coupling stoichiometry of 1:1. Our previous study had demonstrated that the pyrimidines, thymidine, cytidine, and uridine and the purines, adenosine, 2'-deoxyadenosine, and guanosine, but not inosine and formycin B, were substrates of this system. To further define the substrate selectivity of the transporter, the interaction of the drugs, 2-chloroadenosine (2-ClAdo), 5-fluorouridine (5-FUrd) and 5-iodo-2'-deoxyuridine (5-IdUrd), nucleoside analogs that are modified on the base moiety was studied. The three compounds inhibited Na(+)-thymidine uptake in the vesicles via a competitive mechanism. The IC50 values for 2-ClAdo, 5-FUrd and 5-IdUrd were 75, 49, and 16 microM, respectively. In addition, 5-IdUrd trans-stimulated the initial uptake of thymidine into the vesicles suggesting that the two compounds share the same transporter. Collectively, these data suggest that Na(+)-nucleoside transport in the human renal brush-border membrane is an electrogenic process and that the kidney may play a role in the disposition and targeting of clinically important nucleoside analogs.

Further characterization of the sodium-dependent nucleoside transporter (N3) in choroid plexus from rabbit.

The Na+/nucleoside cotransporter in rabbit choroid plexus differs from Na+/nucleoside cotransporters in other tissues in terms of substrate selectivity and stoichiometry. The overall goal of this study was to further characterize the kinetics of this system (N3). Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP and treated with valinomycin and K+. Na+/thymidine uptake at 30 s in the presence of an inside negative potential difference was significantly greater than in the absence of a potential difference. Na+/thymidine uptake was not significantly affected by replacing chloride with either thiocyanate or sulfate. The Km of Na+/guanosine uptake was 149, 85.2 and 30.5 microM in the presence of a 25, 50 and 100 mM Na+ gradient, respectively, whereas the Vmax was unaffected, suggesting that Na+ binds first to the cotransporter, then, the nucleoside. Therapeutically relevant base-modified nucleoside analogs, 5-fluorouridine, 2-chloroadenosine and 5-iododeoxyuridine, significantly inhibited Na+/thymidine uptake with IC50 values (mean +/- S.E.) of 12.0 +/- 2.3, 21.3 +/- 2.2 and 24.4 +/- 2.1 microM, respectively, whereas nucleoside analogs structurally modified on the ribose ring, 3'-azidothymidine, dideoxyinosine and dideoxycytidine (100 microM) did not. These studies suggest that Na+/nucleoside cotransport in the choroid plexus is electrogenic and is not dependent on chloride. This cotransporter, which is present in choroid plexus but not in renal brush-border membrane vesicles from rabbit, may play a role in the disposition of clinically relevant base-modified nucleoside analogs into and out of the brain.

Na+-dependent gamma-aminobutyric acid (GABA) transport in the choroid plexus of rabbit.

The goal of this study was to examine the mechanisms of transport of gamma-aminobutyric acid (GABA) in the choroid plexus. Choroid plexus slices from the rabbit were depleted of ATP with 2,4-dinitrophenol. GABA accumulated in the choroid plexus slices in a concentrative manner in the presence of an inwardly-directed Na+ gradient. Uptake occurred in the presence of Cl-; replacement of Cl- with gluconate abolished uptake. SCN-, NO3- or Br- were able to support uptake in the absence of Cl- to a significant extent (80, 68 and 61% of control, respectively). GABA uptake was saturable (Km of 37 +/- 8.5 microM, Vmax of 409 +/- 43 nmol/g/min). Na+-driven GABA uptake was inhibited by beta-alanine (IC50 = 22.9 microM) and hypotaurine (IC50 = 21.9 microM) but less potently by nipecotic acid (IC50 = 244 microM) and hydroxy-nipecotic acid (IC50 = 284 microM). Betaine, L-(2,4)-diaminobutyric acid, guvacine and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol were weak inhibitors (IC50 > 500 microM). GABA inhibited Na+-driven uptake of taurine (IC50 = 230 microM); taurine, however, did not inhibit GABA uptake (IC50 > 1 mM). RT-PCR, using degenerate primers for cloned GABA transporters, did not result in the amplification of a band from rat choroid plexus RNA. The location of the choroid plexus in the ventricles of the brain, and its role in the secretion of the cerebrospinal fluid, suggest a role for the choroid plexus Na+-GABA transporter in the disposition of GABA in the brain.

Characterization of a sodium-dependent taurine transporter in rabbit choroid plexus.

Taurine, a beta-amino acid, plays an important role as a neuromodulator and is necessary for the normal development of the brain. Since de novo synthesis of taurine in the brain is minimal and in vivo studies suggest that taurine does not cross the blood-brain barrier, we examined whether the choroid plexus, the blood-cerebrospinal fluid barrier, plays a role in taurine transport in the central nervous system. The uptake of [3H]taurine into ATP-depleted choroid plexus from rabbit was substantially greater in the presence of an inwardly directed Na+ gradient, whereas in the absence of a Na+ gradient taurine accumulation was negligible. A transient inside-negative potential gradient enhanced the Na(+)-driven uptake of taurine into the tissue slices, suggesting that the transport process is electrogenic. Na(+)-driven taurine uptake was saturable with an estimated Vmax of 111 +/- 20.2 nmol/g per 15 min and a Km of 99.8 +/- 29.9 microM. The estimated coupling ratio of Na+ and taurine was 1.80 +/- 0.122. Na(+)-dependent taurine uptake was significantly inhibited by beta-amino acids, but not by alpha-amino acids, indicating that the transporter is selective for beta-amino acids. Na(+)-dependent taurine uptake showed some selectivity for anions: the accumulation was comparable in the presence of Cl-, Br- and thiocyanate whereas I-, SO4(2-) and gluconate did not stimulate the uptake significantly. Collectively, our results demonstrate that taurine is transported in the choroid plexus via a Na(+)-dependent, saturable and apparently beta-amino acid selective mechanism. This process may be functionally relevant to taurine homeostasis in the brain.

Molecular cloning and functional expression of a rabbit renal organic cation transporter.

A cDNA encoding an organic cation transporter (rbOCT1) was isolated from rabbit kidney. The cDNA encodes a 554 amino acid protein that is highly homologous to other mammalian organic cation transporters. rbOCT1 mediated 3H-1-methyl-4-phenylpyridinium (3H-MPP+) transport in Xenopus laevis oocytes was saturable, sensitive to membrane potential, and inhibited by various organic cations. rbOCT1 mRNA transcripts are expressed in the kidney, liver, and intestine.

Nucleoside transport in brush border membrane vesicles from human kidney.

The goal of this study was to elucidate the mechanisms of nucleoside transport in the brush border membrane of the human kidney. [3H]Uridine was transported into brush border membrane vesicles (BBMV) from human kidney via Na(+)-independent and Na(+)-dependent processes. The Na(+)-dependent transport was saturable (Km = 4.76 +/- 0.39 microM; Vmax = 6.42 +/- 0.17 pmol/mg proteins per s) and was trans-stimulated by unlabeled uridine. Structural analogs of uridine (100 microM), 2'-deoxyuridine (2-dU) and dideoxyuridine (ddU), significantly inhibited Na(+)-uridine uptake into BBMV. Previous studies have suggested that Na(+)-nucleoside co-transport occurs via two major systems (Vijayalakshmi et al. (1988) J. Biol. Chem. 263, 19419-19423). One system (cit) is generally pyrimidine-selective; thymidine serves as a model substrate. The other system (cif) is generally purine-selective; formycin B serves as a model substrate. Uridine and adenosine are substrates of both systems. Thymidine and cytidine (100 microM), but not formycin B (100 microM) inhibited Na(+)-uridine uptake. In addition, [3H]thymidine exhibited an Na(+)-driven overshoot phenomenon whereas [3H]formycin B did not. Na(+)-thymidine uptake was inhibited by (100 microM) adenosine, uridine, guanosine, but not by formycin B and inosine. Further studies demonstrated that guanosine trans-stimulated thymidine uptake suggesting that guanosine and thymidine share a common transporter in the human renal BBMV. A different pattern was identified in BBMV from the rabbit kidney where both [3H]thymidine and [3H]formycin B as well as [3H]uridine exhibited a transient Na(+)-driven overshoot phenomenon. Collectively, these data suggest that in rabbit renal BBMV both cif and cit systems are present whereas in human renal BBMV, there appears to be a single concentrative Na(+)-nucleoside cotransport system that interacts with uridine, cytidine, thymidine, adenosine and guanosine but not with formycin B and inosine. The system is similar to the previously described cit system except that guanosine is also a substrate.