Julie R. Field

Systematic comparison of phenome-wide association study of electronic medical record data and genome-wide association study data

Candidate gene and genome-wide association studies (GWAS) have identified genetic variants that modulate risk for human disease; many of these associations require further study to replicate the results. Here we report the first large-scale application of the phenome-wide association study (PheWAS) paradigm within electronic medical records (EMRs), an unbiased approach to replication and discovery that interrogates relationships between targeted genotypes and multiple phenotypes. We scanned for associations between 3,144 single-nucleotide polymorphisms (previously implicated by GWAS as mediators of human traits) and 1,358 EMR-derived phenotypes in 13,835 individuals of European ancestry. This PheWAS replicated 66% (51/77) of sufficiently powered prior GWAS associations and revealed 63 potentially pleiotropic associations with P < 4.6 × 10−6 (false discovery rate < 0.1); the strongest of these novel associations were replicated in an independent cohort (n = 7,406). These findings validate PheWAS as a tool to allow unbiased interrogation across multiple phenotypes in EMR-based cohorts and to enhance analysis of the genomic basis of human disease.

Tyr-95 and Ile-172 in Transmembrane Segments 1 and 3 of Human Serotonin Transporters Interact to Establish High Affinity Recognition of Antidepressants

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [125I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition (∼10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.

The IGNITE network: a model for genomic medicine implementation and research

BackgroundPatients, clinicians, researchers and payers are seeking to understand the value of using genomic information (as reflected by genotyping, sequencing, family history or other data) to inform clinical decision-making. However, challenges exist to widespread clinical implementation of genomic medicine, a prerequisite for developing evidence of its real-world utility.MethodsTo address these challenges, the National Institutes of Health-funded IGNITE (Implementing GeNomics In pracTicE; www.ignite-genomics.org) Network, comprised of six projects and a coordinating center, was established in 2013 to support the development, investigation and dissemination of genomic medicine practice models that seamlessly integrate genomic data into the electronic health record and that deploy tools for point of care decision making. IGNITE site projects are aligned in their purpose of testing these models, but individual projects vary in scope and design, including exploring genetic markers for disease risk prediction and prevention, developing tools for using family history data, incorporating pharmacogenomic data into clinical care, refining disease diagnosis using sequence-based mutation discovery, and creating novel educational approaches.ResultsThis paper describes the IGNITE Network and member projects, including network structure, collaborative initiatives, clinical decision support strategies, methods for return of genomic test results, and educational initiatives for patients and providers. Clinical and outcomes data from individual sites and network-wide projects are anticipated to begin being published over the next few years.ConclusionsThe IGNITE Network is an innovative series of projects and pilot demonstrations aiming to enhance translation of validated actionable genomic information into clinical settings and develop and use measures of outcome in response to genome-based clinical interventions using a pragmatic framework to provide early data and proofs of concept on the utility of these interventions. Through these efforts and collaboration with other stakeholders, IGNITE is poised to have a significant impact on the acceleration of genomic information into medical practice.

Targeting glutamate synapses in schizophrenia

Although early clinical observations implicated dopamine dysfunction in the neuropathology of schizophrenia, accumulating evidence suggests that multiple neurotransmitter pathways are dysregulated. The psychotomimetic actions of NMDA receptor antagonists point to an imbalance of glutamatergic signaling. Encouragingly, numerous preclinical and clinical studies have elucidated several potential targets for increasing NMDA receptor function and equilibrating glutamatergic tone, including the metabotropic glutamate receptors 2, 3 and 5, the muscarinic acetylcholine receptors M(1) and M(4), and the glycine transporter GlyT1. Highly specific allosteric and orthosteric ligands have been developed that modify the activity of these novel target proteins, and in this review we summarize both the glutamatergic mechanisms and the novel compounds that are increasing the promise for a multifaceted pharmacological approach to treat schizophrenia.

Electronic health record design and implementation for pharmacogenomics: a local perspective

Purpose:The design of electronic health records to translate genomic medicine into clinical care is crucial to successful introduction of new genomic services, yet there are few published guides to implementation.Methods:The design, implemented features, and evolution of a locally developed electronic health record that supports a large pharmacogenomics program at a tertiary-care academic medical center was tracked over a 4-year development period.Results:Developers and program staff created electronic health record mechanisms for ordering a pharmacogenomics panel in advance of clinical need (preemptive genotyping) and in response to a specific drug indication. Genetic data from panel-based genotyping were sequestered from the electronic health record until drug–gene interactions met evidentiary standards and deemed clinically actionable. A service to translate genotype to predicted drug-response phenotype populated a summary of drug–gene interactions, triggered inpatient and outpatient clinical decision support, updated laboratory records, and created gene results within online personal health records.Conclusion:The design of a locally developed electronic health record supporting pharmacogenomics has generalizable utility. The challenge of representing genomic data in a comprehensible and clinically actionable format is discussed along with reflection on the scalability of the model to larger sets of genomic data.Genet Med 15 10, 833–841.Genetics in Medicine (2013); 15 10, 833–841. doi:10.1038/gim.2013.109

Biobanks and Electronic Medical Records: Enabling Cost-Effective Research

Linking of data from electronic medical records to biological specimens enables cost-effective and rapid genomic analyses. The use of electronic medical record data linked to biological specimens in health care settings is expected to enable cost-effective and rapid genomic analyses. Here, we present a model that highlights potential advantages for genomic discovery and describe the operational infrastructure that facilitated multiple simultaneous discovery efforts.

Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

To identify potential determinants of substrate selectivity in serotonin (5‐HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuTAa) structure reported by Yamashita et al. (Nature 2005;437:215–223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuTAa is only 17%, it increases to above 50% in the first shell of the putative 5‐HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to critically assess and validate their predictive power: A single 5‐HT binding mode was identified that retains the amine placement observed in the LeuTAa structure, matches site‐directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5‐HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5‐HT deep in the binding pocket of the SERT with the 5‐position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 Å of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected. Proteins 2009.

Discovery, synthesis, and structure-activity relationship development of a series of N-4-(2,5-dioxopyrrolidin-1-yl)phenylpicolinamides (VU0400195, ML182): characterization of a novel positive allosteric modulator of the metabotropic glutamate receptor 4 (mGlu(4)) with oral efficacy in an antiparkinsonian animal model.

There is an increasing amount of literature data showing the positive effects on preclinical antiparkinsonian rodent models with selective positive allosteric modulators of metabotropic glutamate receptor 4 (mGlu(4)). However, most of the data generated utilize compounds that have not been optimized for druglike properties, and as a consequence, they exhibit poor pharmacokinetic properties and thus do not cross the blood-brain barrier. Herein, we report on a series of N-4-(2,5-dioxopyrrolidin-1-yl)phenylpicolinamides with improved PK properties with excellent potency and selectivity as well as improved brain exposure in rodents. Finally, ML182 was shown to be orally active in the haloperidol induced catalepsy model, a well-established antiparkinsonian model.

Dopamine Signaling Architecture in Caenorhabditis elegans

1. Aims: In this review, we highlight the identification and analysis of molecules orchestrating dopamine (DA) signaling in the nematode Caenorhabditis elegans, focusing on recent characterizations of DA transporters and receptors.2. Methods: We illustrate the isolation and characterization of molecules important for C. elegans DA synthesis, packaging, reuptake and signaling and examine how mutations in these proteins are being exploited through in vitro and in vivo paradigms to yield novel insights of protein structure, DA signaling pathways and DA-supported behaviors.3. Results: DA signaling in the worm, as in man, arises by synaptic and nonsynaptic release from a small number of cells that exert modulatory control over a larger network underlying C. elegans behavior.4. Conclusions: The C. elegans model system offers unique opportunities to elucidate ill-defined pathways that support DA release, inactivation, and signaling in addition to clarifying mechanisms of DA-mediated behavioral plasticity. Further use of the model offers prospects for the identification of novel genes and proteins whose study may yield benefits for DA-supported neural disorders in man.

Transgenic elimination of high-affinity antidepressant and cocaine sensitivity in the presynaptic serotonin transporter

Serotonin [i.e., 5-hydroxytryptamine (5-HT)]–targeted antidepressants are in wide use for the treatment of mood disorders, although many patients do not show a response or experience unpleasant side effects. Psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (i.e., “ecstasy”), also impact 5-HT signaling. To help dissect the contribution of 5-HT signaling to the actions of these and other agents, we developed transgenic mice in which high-affinity recognition of multiple antidepressants and cocaine is eliminated. Our animals possess a modified copy of the 5-HT transporter (i.e., SERT, slc6a4) that bears a single amino acid substitution, I172M, proximal to the 5-HT binding site. Although the M172 substitution does not impact the recognition of 5-HT, this mutation disrupts high-affinity binding of many competitive antagonists in transfected cells. Here, we demonstrate that, in M172 knock-in mice, basal SERT protein levels, 5-HT transport rates, and 5-HT levels are normal. However, SERT M172 mice display a substantial loss of sensitivity to the selective 5-HT reuptake inhibitors fluoxetine and citalopram, as well as to cocaine. Through a series of biochemical, electrophysiological, and behavioral assays, we demonstrate the unique properties of this model and establish directly that SERT is the sole protein responsible for selective 5-HT reuptake inhibitor-mediated alterations in 5-HT clearance, in 5-HT1A autoreceptor modulation of raphe neuron firing, and in behaviors used to predict the utility of antidepressants.