Methotrexate (MTX) is used in patients with malignant and autoimmune diseases. This drug is primarily excreted unchanged in the urine, and its net excretion occurs via active secretory and reabsorptive processes. We characterized the interaction of MTX with human organic-anion transporting polypeptide transporter (OATP) 1A2, which is expressed in tissues important for MTX disposition and toxicity, such as the intestine, kidney, liver, and endothelial cells of the blood-brain barrier. In Xenopus laevis oocytes expressing OATP1A2, the uptake of the model substrate, estrone-3-sulfate (ES), was enhanced 30-fold compared with uninjected oocytes. MTX uptake in oocytes expressing OATP1A2 was saturable (K m ϭ 457 Ϯ 118 M; V max ϭ 17.5 Ϯ 4.9 pmol/oocyte/60 min) and sensitive to extracellular pH. That is, acidic pHs stimulated MTX uptake by as much as 7-fold. Seven novel protein-altering variants were identified in 270 ethnically diverse DNA samples. Four protein-altering variants in OATP1A2 exhibited altered transport of ES and/or MTX. The common variant, protein reference sequence (p.) Ile13Thr, was hyperfunctional for ES and MTX and showed a 2-fold increase in the V max for ES. The common variant, p. Glu172Asp, exhibited reduced maximal transport capacity for ES and MTX. p. Arg168Cys was hypofunctional, and p. Asn277DEL was nonfunctional. Because of its expression on the apical membrane of the distal tubule and in tissues relevant to MTX disposition and toxicity, these findings suggest that OATP1A2 may play a role in active tubular reabsorption of MTX and in MTX-induced toxicities. Furthermore, genetic variation in OATP1A2 may contribute to variation in MTX disposition and response.

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The human concentrative nucleoside transporter, CNT3 (SLC28A3), plays an important role in mediating the cellular entry of a broad array of physiological nucleosides and synthetic anticancer nucleoside analog drugs. As a first step toward understanding the genetic basis for interindividual differences in the disposition and response to antileukemic nucleoside analogs, we examined the genetic and functional diversity of CNT3. In all, 56 variable sites in the exons and flanking intronic region of SLC28A3 were identified in a collection of 270 DNA samples from US populations (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). Of the 16 coding region variants, 12 had not been previously reported. Also, 10 resulted in amino-acid changes and three of these had total allele frequencies of Z1%. Nucleotide diversity (p) at nonsynonymous and synonymous sites was estimated to be 1.81 Â 10 4 and 18.13 Â 10 4 , respectively, suggesting that SLC28A3 is under negative selection. All nonsynonymous variants, constructed by site-directed mutagenesis and expressed in Xenopus laevis oocytes, transported purine and pyrimidine model substrates, except for c. 1099G4A (p. Gly367Arg). This rare variant alters an evolutionarily conserved site in the putative substrate recognition domain of CNT3. The presence of three additional evolutionarily conserved glycine residues in the vicinity of p. Gly367Arg that are also conserved in human paralogs suggest that these glycine residues are critical in the function of the concentrative nucleoside transporter family. The genetic analysis and functional characterization of CNT3 variants suggest that this transporter does not tolerate nonsynonymous changes and is important for human fitness.

Although considerable progress has been made toward characterizing human DNA sequence variation, there remains a deficiency in information on human phenotypic variation at the single-gene level. We systematically analyzed the function of all protein-altering variants of eleven membrane transporters in heterologous expression systems. Coding-region variants were identified by screening DNA from a large sample (n = 247-276) of ethnically diverse subjects. In total, we functionally analyzed 88 protein-altering variants. Fourteen percent of the polymorphic variants (defined as variants with allele frequencies Ն1% in at least one major ethnic group) had no activity or significantly reduced function. Decreased function variants had significantly lower allele frequencies and were more likely to alter evolutionarily conserved amino acid residues. However, variants at evolutionarily conserved positions with approximately normal activity in cellular assays were also at significantly lower allele frequencies, suggesting that some variants with apparently normal activity in biochemical assays may influence occult functions or quantitative degrees of function that are important in human fitness but not measured in these assays. For example, eight (14%) of the 58 variants for which we had measured the transport of at least two substrates showed substrate-specific defects in transport. These variants and the reduced function variants provide plausible candidates for disease susceptibility or variation in clinical drug response.Since the completion of the human genome project, considerable progress has been made in characterizing the nature and degree of human DNA sequence variation (Cargill et al. 1999;Halushka et al. 1999;Patil et al. 2001;Stephens et al. 2001;Leabman et al. 2003). Numerous single nucleotide polymorphisms in all regions of the human genome have been identified, including coding and noncoding regions of genes and large intergenic regions. However, there remains a lack of information connecting genetic polymorphisms to human phenotypic variation. As a consequence, current attempts to predict the functional significance of genetic variants from sequence data alone (from interspecies sequence comparisons or degree of chemical change for amino acid substitutions, as examples) (Sunyaev et al. 2000(Sunyaev et al. , 2001(Sunyaev et al. , 2003Chasman and Adams 2001;Fay et al. 2001;Muller et al. 2001;Henikoff 2001, 2003;Wang and Moult 2001;Ramensky et al. 2002) are lacking in support from empirical data. Furthermore, we have little information on the fraction of natural variants with altered or potentially deleterious function that are harbored in healthy populations. In order to understand variation in normal human physiology and responses to the environment (including, for example, drug-response phenotypes), it is important to understand the range of phenotypic variation that is associated with genetic variation in human populations.Analysis of causal mutations in Mendelian diseases has demonstrated that the majority...