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Susan J Johns

Publications and source records attributed to Susan J Johns.

At least 19 recordsLinked to original sources
PubMed2006

Interaction of methotrexate with organic-anion transporting polypeptide 1A2 and its genetic variants.

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 (Km = 457 +/- 118 microM; Vmax = 17.5 +/- 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.

Alleles↗
PubMed2006

The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse.

Gene trapping is a method of generating murine embryonic stem (ES) cell lines containing insertional mutations in known and novel genes. A number of international groups have used this approach to create sizeable public cell line repositories available to the scientific community for the generation of mutant mouse strains. The major gene trapping groups worldwide have recently joined together to centralize access to all publicly available gene trap lines by developing a user-oriented Website for the International Gene Trap Consortium (IGTC). This collaboration provides an impressive public informatics resource comprising approximately 45 000 well-characterized ES cell lines which currently represent approximately 40% of known mouse genes, all freely available for the creation of knockout mice on a non-collaborative basis. To standardize annotation and provide high confidence data for gene trap lines, a rigorous identification and annotation pipeline has been developed combining genomic localization and transcript alignment of gene trap sequence tags to identify trapped loci. This information is stored in a new bioinformatics database accessible through the IGTC Website interface. The IGTC Website (www.genetrap.org) allows users to browse and search the database for trapped genes, BLAST sequences against gene trap sequence tags, and view trapped genes within biological pathways. In addition, IGTC data have been integrated into major genome browsers and bioinformatics sites to provide users with outside portals for viewing this data. The development of the IGTC Website marks a major advance by providing the research community with the data and tools necessary to effectively use public gene trap resources for the large-scale characterization of mammalian gene function.

Animals↗
PubMed2005

The human organic anion transporter 3 (OAT3; SLC22A8): genetic variation and functional genomics.

The human organic anion transporter, OAT3 (SLC22A8), plays a critical role in renal drug elimination, by mediating the entry of a wide variety of organic anions, including a number of commonly used pharmaceuticals, into the renal proximal tubular cells. To understand the nature and extent of genetic variation in OAT3, and to determine whether such variation affects its function, we identified OAT3 variants in a large, ethnically diverse sample population and studied their transport activities in cellular assays. We identified a total of 10 distinct coding-region variants, which altered the encoded amino acid sequence, in DNA samples from 270 individuals (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). The overall prevalence of these OAT3 variants was relatively low among the screened population, with only three variants having allele frequencies of >1% in a particular ethnic group. Clones of each variant were created by site-directed mutagenesis, expressed in HEK-293 cells, and tested for function using the model substrates, estrone sulfate (ES) and cimetidine (CIM). The results revealed a high degree of functional heterogeneity among OAT3 variants, with three variants (p. Arg149Ser, p. Gln239Stop, and p. Ile260Arg) that resulted in complete loss of function, and several others with significantly reduced function. One of the more common variants (p. Ile305Phe), found in 3.5% of Asian-Americans, appeared to have altered substrate specificity. This variant exhibited a reduced ability to transport ES, but a preserved ability to transport CIM. These data suggest that genetic variation in OAT3 may contribute to variation in the disposition of drugs.

Alleles↗
PubMed2005

Functional characterization and haplotype analysis of polymorphisms in the human equilibrative nucleoside transporter, ENT2.

The equilibrative nucleoside transporter 2 (ENT2; SLC29A2) is a bidirectional transporter that is involved in the disposition of naturally occurring nucleosides as well as a variety of anticancer and antiviral nucleoside analogs. The goal of the current study was to evaluate the function of genetic variants in ENT2 in cellular assays and to determine the haplotype structure of the coding and flanking intronic region of the gene. As part of a large study focused on genetic variation in membrane transporters (Leabman et al., 2003), DNA samples from ethnically diverse populations (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans, and 7 Pacific Islanders) were screened for variants in membrane transporters, including SLC29A2. Fourteen polymorphic sites in SLC29A2 were found, including 11 in the coding region. Five protein-altering variants were identified: three nonsynonymous variants, and two deletions. Each of the protein-altering variants was found at a very low frequency, occurring only once in the sample population. The nonsynonymous variants and the deletions were constructed via site-directed mutagenesis and were subsequently characterized in Xenopus laevis oocytes. All variants were able to take up inosine with the exception of ENT2-Delta845-846, which resulted in a frameshift mutation that prematurely truncated the protein. ENT2 showed very infrequent variation compared with most other transporter proteins studied, and it was found that five haplotypes were sufficient to describe the entire sample set. The low overall genetic diversity in SLC29A2 makes it unlikely that variation in the coding region contributes significantly to clinically observed differences in drug response.

Animals↗
PubMed2005

Genetic analysis and functional characterization of polymorphisms in the human concentrative nucleoside transporter, CNT2.

The concentrative nucleoside transporter CNT2 (SPNT1; SLC28A2) plays a role in the absorption and disposition of naturally occurring nucleosides, as well as nucleoside analog drugs. The aim of the present study was to characterize genetic variation in SLC28A2, the gene encoding CNT2, and to functionally analyse non-synonymous variants of CNT2, as a first step towards understanding whether genetic variation in this nucleoside transporter contributes to variation in response to nucleoside analogs. As part of a larger study, DNA samples from an ethnically diverse population (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans and seven Pacific Islanders) were screened and 10 coding region variants of CNT2 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. Six non-synonymous variants were identified, and all were able to transport guanosine. The four common variants (>1% in the sample population) were further characterized with the anti-viral nucleoside analog drug ribavirin. No differences were observed among the four common variants in the uptake kinetics of 3H-ribavirin (Km in microM: 35.6+/-9.27 for CNT2-reference, 40.7+/-6.47 for CNT2-P22L, 31.2+/-15.8 for CNT2-S75R, 26.7+/-6.13 for CNT2-S245T and 49.9+/-14.6 for CNT2-F355S). The variant CNT2-F355S exhibited a change in specificity for the naturally occurring nucleosides, inosine and uridine. All non-synonymous variants of CNT2 took up guanosine, and the four variants examined showed no significant difference in ribavirin kinetics. However, CNT2-F355S (3% allele frequency in the African-American sample) was found to alter specificity for naturally occurring nucleosides, which may have implications for nucleoside homeostasis.

Alleles↗
PubMed2005

Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1).

OBJECTIVES: The organic anion transporter, OAT1 (SLC22A6), plays a role in the renal elimination of many drugs and environmental toxins. The goal of this study was to identify and functionally characterize OAT1 variants as a first step towards understanding whether genetic variation in OAT1 may contribute to interindividual differences in renal elimination of xenobiotics. METHODS: As part of a larger study, 276 DNA samples from an ethnically diverse population were screened and 12 coding region variants of OAT1 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. A small family-based clinical study was conducted to determine the renal elimination of a model OAT1 substrate, adefovir (an antiviral agent) in human subjects who possessed a non-functional variant, OAT1-R454Q. RESULTS: Six non-synonymous variants were identified; two (OAT1-R50 H and OAT1-R293W) were present at > or = 1% in at least one ethnic population. These two variants exhibited normal uptake of p-aminohippurate, ochratoxin A and methotrexate assayed in X. laevis oocytes. One variant, OAT1-R454Q, was non-functional with respect to the above substrates. In the clinical study, there was no significant decrease in the renal secretory clearance of adefovir in family members heterozygous for OAT1-454Q in comparison to those with the reference transporter, OAT1-454R. CONCLUSIONS: These data indicate that the coding region of OAT1 has low genetic and functional diversity and suggest that coding region variants of OAT1 may not contribute substantially to interindividual differences in renal elimination of xenobiotics.

Adenine↗
PubMed2004

Functional and genetic diversity in the concentrative nucleoside transporter, CNT1, in human populations.

The concentrative nucleoside transporter, CNT1 (SLC28A1), mediates the cellular uptake of naturally occurring pyrimidine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs. As a first step toward understanding whether genetic variation in CNT1 contributes to variation in the uptake and disposition of clinically used nucleoside analogs, we determined the haplotype structure and functionally analyzed all coding region variants of CNT1 identified in ethnically diverse populations (100 African Americans, 100 European Americans, 30 Asians, 10 Mexican Americans, and 7 Pacific Islanders) (Leabman et al., 2003). A total of 58 coding region haplotypes were identified using PHASE analysis, 44 of which contained at least one amino acid variant. More than half of the coding region haplotypes were population-specific. Using site-directed mutagenesis, 15 protein-altering CNT1 variants, including one amino acid insertion and one base pair (bp) deletion, were constructed and expressed in Xenopus laevis oocytes. All variant transporters took up [3H]thymidine with the exception of CNT1-Ser546Pro, a rare variant, and CNT1-1153del, a single bp deletion found at a frequency of 3% in the African American population. The bp deletion results in a frame-shift followed by a stop-codon. The anticancer nucleoside analog gemcitabine had a reduced affinity for CNT1-Val189Ile (a common CNT1 variant found at a frequency of 26%) compared with reference CNT1 (IC50=13.8 +/- 0.60 microM for CNT1-reference and 23.3 +/- 1.5 microM for CNT1-Val189Ile, p<0.05). These data suggest that common genetic variants of CNT1 may contribute to variation in systemic and intracellular levels of anti-cancer nucleoside analogs.

Amino Acid Sequence↗
PubMed2003

Natural variation in human membrane transporter genes reveals evolutionary and functional constraints.

Membrane transporters maintain cellular and organismal homeostasis by importing nutrients and exporting toxic compounds. Transporters also play a crucial role in drug response, serving as drug targets and setting drug levels. As part of a pharmacogenetics project, we screened exons and flanking intronic regions for variation in a set of 24 membrane transporter genes (96 kb; 57% coding) in 247 DNA samples from ethnically diverse populations. We identified 680 single nucleotide polymorphisms (SNPs), of which 175 were synonymous and 155 caused amino acid changes, and 29 small insertions and deletions. Amino acid diversity (pi(NS)) in transmembrane domains (TMDs) was significantly lower than in loop domains, suggesting that TMDs have special functional constraints. This difference was especially striking in the ATP-binding cassette superfamily and did not parallel evolutionary conservation: there was little variation in the TMDs, even in evolutionarily unconserved residues. We used allele frequency distribution to evaluate different scoring systems (Grantham, blosum62, SIFT, and evolutionarily conservedevolutionarily unconserved) for their ability to predict which SNPs affect function. Our underlying assumption was that alleles that are functionally deleterious will be selected against and thus under represented at high frequencies and over represented at low frequencies. We found that evolutionary conservation of orthologous sequences, as assessed by evolutionarily conservedevolutionarily unconserved and SIFT, was the best predictor of allele frequency distribution and hence of function. European Americans had an excess of high frequency alleles in comparison to African Americans, consistent with a historic bottleneck. In addition, African Americans exhibited a much higher frequency of population specific medium-frequency alleles than did European Americans.

DNA↗
PubMed2003

Evolutionary conservation predicts function of variants of the human organic cation transporter, OCT1.

The organic cation transporter, OCT1, is a major hepatic transporter that mediates the uptake of many organic cations from the blood into the liver where the compounds may be metabolized or secreted into the bile. Because OCT1 interacts with a variety of structurally diverse organic cations, including clinically used drugs as well as toxic substances (e.g., N-methylpyridinium, MPP(+)), it is an important determinant of systemic exposure to many xenobiotics. To understand the genetic basis of extensive interindividual differences in xenobiotic disposition, we functionally characterized 15 protein-altering variants of the human liver organic cation transporter, OCT1, in Xenopus oocytes. All variants that reduced or eliminated function (OCT1-R61C, OCT1-P341L, OCT1-G220V, OCT1-G401S, and OCT1-G465R) altered evolutionarily conserved amino acid residues. In general, variants with decreased function had amino acid substitutions that resulted in more radical chemical changes (higher Grantham values) and were less evolutionarily favorable (lower blosum62 values) than variants that maintained function. A variant with increased function (OCT1-S14F) changed an amino acid residue such that the human protein matched the consensus of the OCT1 mammalian orthologs. Our results indicate that changes at evolutionarily conserved positions of OCT1 are strong predictors of decreased function and suggest that a combination of evolutionary conservation and chemical change might be a stronger predictor of function.

Alleles↗
PubMed2003

BayGenomics: a resource of insertional mutations in mouse embryonic stem cells.

The BayGenomics gene-trap resource (http://baygenomics.ucsf.edu) provides researchers with access to thousands of mouse embryonic stem (ES) cell lines harboring characterized insertional mutations in both known and novel genes. Each cell line contains an insertional mutation in a specific gene. The identity of the gene that has been interrupted can be determined from a DNA sequence tag. Approximately 75% of our cell lines contain insertional mutations in known mouse genes or genes that share strong sequence similarities with genes that have been identified in other organisms. These cell lines readily transmit the mutation to the germline of mice and many mutant lines of mice have already been generated from this resource. BayGenomics provides facile access to our entire database, including sequence tags for each mutant ES cell line, through the World Wide Web. Investigators can browse our resource, search for specific entries, download any portion of our database and BLAST sequences of interest against our entire set of cell line sequence tags. They can then obtain the mutant ES cell line for the purpose of generating knockout mice.

Animals↗
PubMed2003

Functional characterization in yeast of genetic variants in the human equilibrative nucleoside transporter, ENT1.

The human equilibrative nucleoside transporter, ENT1, appears to play a critical role in the disposition of nucleosides and nucleoside analogs used clinically as anti-viral and anti-cancer drugs. Recently, we identified variants of ENT1 in an ethnically diverse DNA sample set from 247 individuals, focusing primarily on the coding region. The goal of the present study was to analyse the haplotype structure and functionally characterize the variants of ENT1. We observed that a single haplotype, ENT1*1, accounted for 91.3% of the 494 chromosomes. Functional analysis in Saccharomyces cerevisiae revealed no differences in the kinetics of uptake of nucleosides and nucleoside analogs by the two non-synonymous variant transporters, ENT1-I216T and ENT1-E391K, and the reference ENT1. These results, together with the observation that there are few haplotypes of ENT1, indicate that coding region variants of ENT1 do not contribute to inter-individual differences in response to nucleoside analog drugs.

Amino Acid Sequence↗
PubMed2003

Sequence diversity and haplotype structure in the human ABCB1 (MDR1, multidrug resistance transporter) gene.

OBJECTIVES: There is increasing evidence that polymorphism of the ABCB1 (MDR1) gene contributes to interindividual variability in bioavailability and tissue distribution of P-glycoprotein substrates. The aim of the present study was to (1) identify and describe novel variants in the ABCB1 gene, (2) understand the extent of variation in ABCB1 at the population level, (3) analyze how variation in ABCB1 is structured in haplotypes, and (4) functionally characterize the effect of the most common amino acid change in P-glycoprotein. METHODS AND RESULTS: Forty-eight variant sites, including 30 novel variants and 13 coding for amino acid changes, were identified in a collection of 247 ethnically diverse DNA samples. These variants comprised 64 statistically inferred haplotypes, 33 of which accounted for 92% of chromosomes analyzed. The two most common haplotypes, ABCB1*1 and ABCB1*13, differed at six sites (three intronic, two synonymous, and one non-synonymous) and were present in 36% of all chromosomes. Significant population substructure was detected at both the nucleotide and haplotype level. Linkage disequilibrium was significant across the entire ABCB1 gene, especially between the variant sites found in ABCB1*13, and recombination was inferred. The Ala893Ser change found in the common ABCB1*13 haplotype did not affect P-glycoprotein function. CONCLUSION: This study represents a comprehensive analysis of ABCB1 nucleotide diversity and haplotype structure in different populations and illustrates the importance of haplotype considerations in characterizing the functional consequences of ABCB1 polymorphisms.

Base Sequence↗
PubMed2003

SNP analysis and presentation in the Pharmacogenetics of Membrane Transporters Project.

The multidisciplinary UCSF Pharmacogenetics of Membrane Transporters project seeks to systematically identify sequence variants in transporters and to determine the functional significance of these variants through evaluation of relevant cellular and clinical phenotypes. The project is structured around four interacting cores: genomics, cellular phenotyping, clinical phenotyping, and bioinformatics. The bioinformatics core is responsible for collecting, storing, and analyzing the information obtained by the other cores and for presenting the results, in particular, for the genomic data. Most of this process is automated using locally developed software written in Python, an open source language well suited for rapid, modular development that meets requirements that are themselves constantly evolving. Here we present the details of transforming ABI trace file data into useful information for project investigators and a description of the types of data analysis and display that we have developed.

Amino Acid Sequence↗