Doug Stryke

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.

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.

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 (πNS) 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 conserved/evolutionarily 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 conserved/evolutionarily 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.

A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012–14): a retrospective cohort study

BACKGROUND Enterovirus D68 was implicated in a widespread outbreak of severe respiratory illness across the USA in 2014 and has also been reported sporadically in patients with acute flaccid myelitis. We aimed to investigate the association between enterovirus D68 infection and acute flaccid myelitis during the 2014 enterovirus D68 respiratory outbreak in the USA. METHODS Patients with acute flaccid myelitis who presented to two hospitals in Colorado and California, USA, between Nov 24, 2013, and Oct 11, 2014, were included in the study. Additional cases identified from Jan 1, 2012, to Oct 4, 2014, via statewide surveillance were provided by the California Department of Public Health. We investigated the cause of these cases by metagenomic next-generation sequencing, viral genome recovery, and enterovirus D68 phylogenetic analysis. We compared patients with acute flaccid myelitis who were positive for enterovirus D68 with those with acute flaccid myelitis but negative for enterovirus D68 using the two-tailed Fishers exact test, two-sample unpaired t test, and Mann-Whitney U test. FINDINGS 48 patients were included: 25 with acute flaccid myelitis, two with enterovirus-associated encephalitis, five with enterovirus-D68-associated upper respiratory illness, and 16 with aseptic meningitis or encephalitis who tested positive for enterovirus. Enterovirus D68 was detected in respiratory secretions from seven (64%) of 11 patients comprising two temporally and geographically linked acute flaccid myelitis clusters at the height of the 2014 outbreak, and from 12 (48%) of 25 patients with acute flaccid myelitis overall. Phylogenetic analysis revealed that all enterovirus D68 sequences associated with acute flaccid myelitis grouped into a clade B1 strain that emerged in 2010. Of six coding polymorphisms in the clade B1 enterovirus D68 polyprotein, five were present in neuropathogenic poliovirus or enterovirus D70, or both. One child with acute flaccid myelitis and a sibling with only upper respiratory illness were both infected by identical enterovirus D68 strains. Enterovirus D68 viraemia was identified in a child experiencing acute neurological progression of his paralytic illness. Deep metagenomic sequencing of cerebrospinal fluid from 14 patients with acute flaccid myelitis did not reveal evidence of an alternative infectious cause to enterovirus D68. INTERPRETATION These findings strengthen the putative association between enterovirus D68 and acute flaccid myelitis and the contention that acute flaccid myelitis is a rare yet severe clinical manifestation of enterovirus D68 infection in susceptible hosts. FUNDING National Institutes of Health, University of California, Abbott Laboratories, and the Centers for Disease Control and Prevention.

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 ∼45 000 well-characterized ES cell lines which currently represent ∼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 () 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.

Role of Organic Cation Transporter 3 (SLC22A3) and Its Missense Variants in the Pharmacologic Action of Metformin

Objectives The goals of this study were to determine the role of organic cation transporter 3 (OCT3) in the pharmacological action of metformin and to identify and functionally characterize genetic variants of OCT3 with respect to the uptake of metformin and monoamines. Methods For pharmacological studies, we evaluated metformin-induced activation of AMP-activated protein kinase, a molecular target of metformin. We used quantitative PCR and immunostaining to localize the transporter and isotopic uptake studies in cells transfected with OCT3 and its nonsynonymous genetic variants for functional analyses. Results Quantitative PCR and immunostaining showed that OCT3 was expressed high on the plasma membrane of skeletal muscle and liver, target tissues for metformin action. Both the OCT inhibitor, cimetidine, and OCT3-specific short hairpin RNA significantly reduced the activating effect of metformin on AMP-activated protein kinase. To identify genetic variants in OCT3, we used recent data from the 1000 Genomes and the Pharmacogenomics of Membrane Transporters projects. Six novel missense variants were identified. In functional assays, using various monoamines and metformin, three variants, T44M (c.131C>T), T400I (c.1199C>T) and V423F (c.1267G>T) showed altered substrate specificity. Notably, in cells expressing T400I and V423F, the uptakes of metformin and catecholamines were significantly reduced, but the uptakes of metformin, 1-methyl-4-phenylpyridinium and histamine by T44M were significantly increased more than 50%. Structural modeling suggested that these two variants may be located in the pore lining (T400) or proximal (V423) membrane-spanning helixes. Conclusion Our study suggests that OCT3 plays a role in the therapeutic action of metformin and that genetic variants of OCT3 may modulate metformin and catecholamine action.

The Structure–Function Linkage Database

The Structure–Function Linkage Database (SFLD, http://sfld.rbvi.ucsf.edu/) is a manually curated classification resource describing structure–function relationships for functionally diverse enzyme superfamilies. Members of such superfamilies are diverse in their overall reactions yet share a common ancestor and some conserved active site features associated with conserved functional attributes such as a partial reaction. Thus, despite their different functions, members of these superfamilies ‘look alike’, making them easy to misannotate. To address this complexity and enable rational transfer of functional features to unknowns only for those members for which we have sufficient functional information, we subdivide superfamily members into subgroups using sequence information, and lastly into families, sets of enzymes known to catalyze the same reaction using the same mechanistic strategy. Browsing and searching options in the SFLD provide access to all of these levels. The SFLD offers manually curated as well as automatically classified superfamily sets, both accompanied by search and download options for all hierarchical levels. Additional information includes multiple sequence alignments, tab-separated files of functional and other attributes, and sequence similarity networks. The latter provide a new and intuitively powerful way to visualize functional trends mapped to the context of sequence similarity.

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 ≥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.

The Human Multidrug Resistance Protein 4 (MRP4, ABCC4) : Functional Analysis of a Highly Polymorphic Gene

ABCC4 encodes multidrug resistance protein 4 (MRP4), a member of the ATP-binding cassette family of membrane transporters involved in the efflux of endogenous and xenobiotic molecules. The aims of this study were to identify single nucleotide polymorphisms of ABCC4 and to functionally characterize selected nonsynonymous variants. Resequencing was performed in a large ethnically diverse population. Ten nonsynonymous variants were selected for analysis of transport function based on allele frequencies and evolutionary conservation. The reference and variant MRP4 cDNAs were constructed by site-directed mutagenesis and transiently transfected into human embryonic kidney cells (HEK 293T). The function of MRP4 variants was compared by measuring the intracellular accumulation of two antiviral agents, azidothymidine (AZT) and adefovir (PMEA). A total of 98 variants were identified in the coding and flanking intronic regions of ABCC4. Of these, 43 variants are in the coding region, and 22 are nonsynonymous. In a functional screen of ten variants, there was no evidence for a complete loss of function allele. However, two variants (G187W and G487E) showed a significantly reduced function compared to reference with both substrates, as evidenced by higher intracellular accumulation of AZT and PMEA compared to the reference MRP4 (43 and 69% increase in accumulation for G187W compared with the reference MRP4, with AZT and PMEA, respectively). The G187W variant also showed decreased expression following transient transfection of HEK 293T cells. Further studies are required to assess the clinical significance of this altered function and expression and to evaluate substrate specificity of this functional change.

Genetic variants in multidrug and toxic compound extrusion-1, hMATE1, alter transport function

hMATE1 (human multidrug and toxin compound extrusion-1; encoded by SLC47A1) is thought to have an important function in the renal and hepatic elimination of drugs, endogenous compounds and environmental toxins. The goals of this study were to identify genetic variants of hMATE1 and to determine their effects on hMATE1 transport function. We identified four synonymous and six nonsynonymous, coding region variants in DNA samples from 272 individuals (68 Caucasians, 68 African Americans, 68 Asian Americans and 68 Mexican Americans). The overall prevalence of hMATE1 nonsynonymous variants was relatively low with three singleton variants and three variants having allele frequencies ⩾2% in a specific ethnic group. The nonsynonymous hMATE1 variants were constructed and stably transfected into HEK-293 cells. Uptake studies using four known hMATE1 substrates (paraquat, metformin, tetraethylammonium and oxaliplatin) were performed in cells transfected with hMATE1 reference or variants. We found that two singleton variants, G64D and V480M, produced a complete loss of function for all four tested substrates whereas three polymorphic variants (allele frequencies ⩾2%), L125F, V338I and C497S, significantly altered the transport function in a substrate-dependent manner. Confocal microscopy studies were consistent with functional studies suggesting that the altered function of the variants was due to altered localization to the plasma membrane. These data suggest that nonsynonymous variants in hMATE1 may alter drug disposition and ultimately affect clinical drug response.