Altered glutamate neurotransmission is implicated in the etiology of schizophrenia (SCZ) and the pharmacogenetics of response to clozapine (CLZ), which is the drug of choice for treatment-resistant SCZ. Response to antipsychotic therapy is highly variable, although twin studies suggest a genetic component. We investigated the association of 10 glutamate system gene variants with CLZ response using standard genotyping procedures. GRM2 (rs4067 and rs2518461), SLC1A2 (rs4354668, rs4534557, and rs2901534), SLC6A9 (rs12037805, rs1978195, and rs16831558), GRIA1 (rs2195450), and GAD1 (rs3749034) were typed in 163 European SCZ/schizoaffective disorder patients deemed resistant or intolerant to previous pharmacotherapy. Response was assessed following 6 months of CLZ monotherapy using change in Brief Psychiatric Rating Scale (BPRS) scores. Categorical and continuous response variables were analyzed using χ2 tests and analysis of covariance, respectively. We report no significant associations following correction for multiple testing. Prior to correction, nominally significant associations were observed for SLC6A9, SLC1A2, GRM2, and GRIA1. Most notably, CC homozygotes of rs16831558 located in the glycine transporter 1 gene (SLC6A9) exhibited an allele dose-dependent improvement in positive symptoms compared to T allele carriers (puncorrected = 0.008, pcorrected = 0.08). To clarify the role of SLC6A9 in clinical response to antipsychotic medication, and CLZ in particular, this finding warrants further investigation in larger well-characterized samples.

Keywords:
Clozapine, Pharmacogenetics, Schizophrenia, Glutamate, Glycine transporter 1 (SLC6A9)
1.
Shan D, Yates S, Roberts RC, McCullumsmith RE: Update on the neurobiology of schizophrenia: a role for extracellular microdomains. Minerva Psichiatr 2012;53:233-249.
2.
Luby ED, Cohen BD, Rosenbaum G, Gottlieb JS, Kelley R: Study of a new schizophrenomimetic drug; sernyl. AMA Arch Neurol Psychiatry 1959;81:363-369.
3.
Luisada PV: The phencyclidine psychosis: phenomenology and treatment. NIDA Res Monogr 1978;21:241-253.
4.
Javitt DC, Zukin SR: Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry 1991;148:1301-1308.
5.
Lahti AC, Weiler MA, Tamara-Michaelidis BA, Parwani A, Tamminga CA: Effects of ketamine in normal and schizophrenic volunteers. Neuropsychopharmacology 2001;25:455-467.
6.
Moghaddam B, Javitt D: From revolution to evolution: the glutamate hypothesis of schizophrenia and its implication for treatment. Neuropsychopharmacology 2012;37:4-15.
7.
Kane J, Honigfeld G, Singer J, Meltzer H: Clozapine for the treatment-resistant schizophrenic. A double-blind comparison with chlorpromazine. Arch Gen Psychiatry 1988;45:789-796.
8.
Borgio JG, Bressan RA, Barbosa Neto JB, et al: Refractory schizophrenia: a neglected clinical problem. Rev Bras Psiquiatr 2007;29:292-293.
9.
Elkis H, Meltzer HY: Refractory schizophrenia. Rev Bras Psiquiatr 2007;29(suppl 2):S41-S47.
10.
Solanki RK, Singh P, Munshi D: Current perspectives in the treatment of resistant schizophrenia. Indian J Psychiatry 2009;51:254-260.
11.
Vojvoda D, Grimmell K, Sernyak M, Mazure CM: Monozygotic twins concordant for response to clozapine. Lancet 1996;347:61.
12.
Horáček J, Libiger C, Höschl K, Borzova I, Hendrychová J: Clozapine-induced concordant agranulocytosis in monozygotic twins. Int J Psychiatry Clin Pract 2001;5:71-73.
13.
Mata I, Madoz V, Arranz MJ, Sham P, Murray RM: Olanzapine: concordant response in monozygotic twins with schizophrenia. Br J Psychiatry 2001;178:86.
14.
Hoyer C, Kranaster L, Leweke FM, Klosterkötter J, Meyer-Lindenberg A, Koethe D: Familial differential treatment response in schizophrenia - lessons from a case of three affected siblings. Pharmacopsychiatry 2010;43:196-197.
15.
Bleehen T: Clozapine: Literature Review. Basel, Sandoz Pharma, 1993.
16.
Potkin SG, Bera R, Gulasekaram B, Costa J, Hayes S, Jin Y, Richmond G, Carreon D, Sitanggan K, Gerber G, Telford J, Plon L, Plon H, Park L, Chang Y-J, Oldroyd J, Cooper TB: Plasma clozapine concentrations predict clinical response in treatment-resistant schizophrenia. J Clin Psychiatry 1994;55(suppl B): 133-136.
17.
Davis JM, Chen N, Glick ID: A meta-analysis of the efficacy of second-generation antipsychotics. Arch Gen Psychiatry 2003;60:553-564.
18.
Lieberman JA, Safferman AZ, Pollack S, Szymanski S, Johns C, Howard A, Kronig M, Bookstein P, Kane JM: Clinical effects of clozapine in chronic schizophrenia: response to treatment and predictors of outcome. Am J Psychiatry 1994;151:1744-1752.
19.
Müller DJ, De Luca V, Kennedy JL: Overview: towards individualized treatment in schizophrenia. Drug Dev Res 2003;60:75-94.
20.
Gunduz-Bruce H, McMeniman M, Robinson DG, Woerner MG, Kane JM, Schooler NR, Lieberman JA: Duration of untreated psychosis and time to treatment response for delusions and hallucinations. Am J Psychiatry 2005;162:1966-1969.
21.
Perkins DO, Gu H, Boteva K, Lieberman JA: Relationship between duration of untreated psychosis and outcome in first-episode schizophrenia: a critical review and meta-analysis. Am J Psychiatry 2005;162:1785-1804.
22.
Kohlrausch FB: Pharmacogenetics in schizophrenia: a review of clozapine studies. Rev Bras Psiquiatr 2013;35:305-317.
23.
Potkin SG, Jin Y, Bunney BG, Costa J, Gulasekaram B: Effect of clozapine and adjunctive high-dose glycine in treatment-resistant schizophrenia. Am J Psychiatry 1999;156:145-147.
24.
Heresco-Levy U: Glutamatergic neurotransmission modulation and the mechanisms of antipsychotic atypicality. Prog Neuropsychopharmacol Biol Psychiatry 2003;27:1113-1123.
25.
Malhotra AK, Adler CM, Kennison SD, Elman I, Pickar D, Breier A: Clozapine blunts N-methyl-D-aspartate antagonist-induced psychosis: a study with ketamine. Biol Psychiatry 1997;42:664-668.
26.
Daly DA, Moghaddam B: Actions of clozapine and haloperidol on the extracellular levels of excitatory amino acids in the prefrontal cortex and striatum of conscious rats. Neurosci Lett 1993;152:61-64.
27.
Yamamoto BK, Cooperman MA: Differential effects of chronic antipsychotic drug treatment on extracellular glutamate and dopamine concentrations. J Neurosci 1994;14:4159-4166.
28.
Banerjee SP, Zuck LG, Yablonsky-Alter E, Lidsky TI: Glutamate agonist activity: implications for antipsychotic drug action and schizophrenia. Neuroreport 1995;6:2500-2504.
29.
Arvanov VL, Liang X, Schwartz J, Grossman S, Wang RY: Clozapine and haloperidol modulate N-methyl-D-aspartate- and non-N-methyl-D-aspartate receptor-mediated neurotransmission in rat prefrontal cortical neurons in vitro. J Pharmacol Exp Ther 1997;283:226-234.
30.
Kubota T, Jibiki I, Enokido F, Nakagawa H, Watanabe K: Effects of MK-801 on clozapine-induced potentiation of excitatory synaptic responses in the perforant path-dentate gyrus pathway in chronically prepared rabbits. Eur J Pharmacol 2000;395:37-42.
31.
Ninan I, Jardemark KE, Wang RY: Differential effects of atypical and typical antipsychotic drugs on N-methyl-D-aspartate and electrically evoked responses in the pyramidal cells of the rat medial prefrontal cortex. Synapse 2003;48:66-79.
32.
Kargieman L, Santana N, Mengod G, Celada P, Artigas F: Antipsychotic drugs reverse the disruption in prefrontal cortex function produced by NMDA receptor blockade with phencyclidine. Proc Natl Acad Sci USA 2007;104:14843-14848.
33.
Evins AE, Amico ET, Shih V, Goff DC: Clozapine treatment increases serum glutamate and aspartate compared to conventional neuroleptics. J Neural Transm 1997;104:761-766.
34.
Fitzgerald LW, Deutch AY, Gasic G, Heinemann SF, Nestler EJ: Regulation of cortical and subcortical glutamate receptor subunit expression by antipsychotic drugs. J Neurosci 1995;15(pt 2):2453-2461.
35.
Spurney CF, Baca SM, Murray AM, Jaskiw GE, Kleinman JE, Hyde TM: Differential effects of haloperidol and clozapine on ionotropic glutamate receptors in rats. Synapse 1999;34:266-276.
36.
Puckett C, Gomez CM, Korenberg JR, Tung H, Meier TJ, Chen XN, Hood L: Molecular cloning and chromosomal localization of one of the human glutamate receptor genes. Proc Natl Acad Sci USA 1991;88:7557-7561.
37.
Fribourg M, Moreno JL, Holloway T, Provasi D, Baki L, Mahajan R, Park G, Adney SK, Hatcher C, Eltit JM, Ruta JD, Albizu L, Li Z, Umali A, Shim J, Fabiato A, MacKerell AD Jr, Brezina V, Sealfon SC, Filizola M, González-Maeso J, Logothetis DE: Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs. Cell 2011;147:1011-1023.
38.
Joo A, Shibata H, Ninomiya H, Kawasaki H, Tashiro N, Fukumaki Y: Structure and polymorphisms of the human metabotropic glutamate receptor type 2 gene (GRM2): analysis of association with schizophrenia. Mol Psychiatry 2001;6:186-192.
39.
Ichise T, Kano M, Hashimoto K, Yanagihara D, Nakao K, Shigemoto R, Katsuki M, Aiba A: mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination. Science 2000;288:1832-1835.
40.
Santos SD, Carvalho AL, Caldeira MV, Duarte CB: Regulation of AMPA receptors and synaptic plasticity. Neuroscience 2009;158:105-125.
41.
Melone M, Vitellaro-Zuccarello L, Vallejo-Illarramendi A, Pérez-Samartin A, Matute C, Cozzi A, Pellegrini-Giampietro DE, Rothstein JD, Conti F: The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine. Mol Psychiatry 2001;6:380-386.
42.
Li X, Francke U: Assignment of the gene SLC1A2 coding for the human glutamate transporter EAAT2 to human chromosome 11 bands p13-p12. Cytogenet Cell Genet 1995;71:212-213.
43.
Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, Welty DF: Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 1996;16:675-686.
44.
Demjaha A, Egerton A, Murray RM, Kapur S, Howes OD, Stone JM, McGuire PK: Antipsychotic treatment resistance in schizophrenia associated with elevated glutamate levels but normal dopamine function. Biol Psychiatry 2014;75:e11-e13.
45.
Javitt DC, Duncan L, Balla A, Sershen H: Inhibition of system A-mediated glycine transport in cortical synaptosomes by therapeutic concentrations of clozapine: implications for mechanisms of action. Mol Psychiatry 2005;10:275-287.
46.
Kim KM, Kingsmore SF, Han H, Yang-Feng TL, Godinot N, Seldin MF, Caron MG, Giros B: Cloning of the human glycine transporter type 1: molecular and pharmacological characterization of novel isoform variants and chromosomal localization of the gene in the human and mouse genomes. Mol Pharmacol 1994;45:608-617.
47.
Jones EM, Fernald A, Bell GI, Le Beau MM: Assignment of SLC6A9 to human chromosome band 1p33 by in situ hybridization. Cytogenet Cell Genet 1995;71:211.
48.
Dong E, Nelson M, Grayson DR, Costa E, Guidotti A: Clozapine and sulpiride but not haloperidol or olanzapine activate brain DNA demethylation. Proc Natl Acad Sci USA 2008;105:13614-13619.
49.
Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, Tobin AJ: Two genes encode distinct glutamate decarboxylases. Neuron 1991;7:91-100.
50.
Bu DF, Erlander MG, Hitz BC, Tillakaratne NJ, Kaufman DL, Wagner-McPherson CB, Evans GA, Tobin AJ: Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene. Proc Natl Acad Sci USA 1992;89:2115-2119.
51.
Taylor SF, Tso IF: GABA abnormalities in schizophrenia: a methodological review of in vivo studies. Schizophr Res 2015;167:84-90.
52.
American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, revised (DSM-III-R). Washington, American Psychiatric Association, 1987.
53.
American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders (DSM-V). Washington, American Psychiatric Association, 1994.
54.
World Medical Association: WMA Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects. 2013. http://www.wma.net/en/30publications/10policies/b3/index.html.
55.
Ward LD, Kellis M: HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants. Nucleic Acids Res 2012;40:D930-D934.
56.
Boyle AP, Hong EL, Hariharan M, Cheng Y, Schaub MA, Kasowski M, Karczewski KJ, Park J, Hitz BC, Weng S, Cherry JM, Snyder M: Annotation of functional variation in personal genomes using RegulomeDB. Genome Res 2012;22:1790-1797.
57.
Xu Z, Taylor JA: SNPinfo: integrating GWAS and candidate gene information into functional SNP selection for genetic association studies. Nucleic Acids Res 2009;37:W600-W605.
58.
Lahiri DK, Nurnberger JI Jr: A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991;19:5444.
59.
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC: PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007;81:559-575.
60.
Barrett JC, Fry B, Maller J, Daly MJ: Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263-265.
61.
Dudbridge F: Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data. Hum Hered 2008;66:87-98.
62.
Gauderman WJ, Morrison JM: QUANTO: a computer program for power and sample size calculations for genetic-epidemiology studies. 2006. http://biostats.usc.edu/software.
63.
Nyholt DR: A simple correction for multiple testing for single-nucleotide polymorphisms in linkage disequilibrium with each other. Am J Hum Genet 2004;74:765-769.
64.
Thierry-Mieg D, Thierry-Mieg J: AceView: a comprehensive cDNA-supported gene and transcripts annotation. Genome Biol 2006;7(suppl 1):S12.1-14.
65.
Harvey RJ, Yee BK: Glycine transporters as novel therapeutic targets in schizophrenia, alcohol dependence and pain. Nat Rev Drug Discov 2013;12:866-885.
66.
Smith KE, Borden LA, Hartig PR, Branchek T, Weinshank RL: Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors. Neuron 1992;8:927-935.
67.
Kuryatov A, Laube B, Betz H, Kuhse J: Mutational analysis of the glycine-binding site of the NMDA receptor: structural similarity with bacterial amino acid-binding proteins. Neuron 1994;12:1291-1300.
68.
Laube B, Hirai H, Sturgess M, Betz H, Kuhse J: Molecular determinants of agonist discrimination by NMDA receptor subunits: analysis of the glutamate binding site on the NR2B subunit. Neuron 1997;18:493-503.
69.
Javitt DC: Glutamate as a therapeutic target in psychiatric disorders. Mol Psychiatry 2004;9:984-997.
70.
Singh SP, Singh V: Meta-analysis of the efficacy of adjunctive NMDA receptor modulators in chronic schizophrenia. CNS Drugs 2011;25:859-885.
71.
Lane HY, Huang CL, Wu PL, Liu YC, Chang YC, Lin PY, Chen PW, Tsai G: Glycine transporter I inhibitor, N-methylglycine (sarcosine), added to clozapine for the treatment of schizophrenia. Biol Psychiatry 2006;60:645-649.
72.
Schwieler L, Linderholm KR, Nilsson-Todd LK, Erhardt S, Engberg G: Clozapine interacts with the glycine site of the NMDA receptor: electrophysiological studies of dopamine neurons in the rat ventral tegmental area. Life Sci 2008;83:170-175.
73.
Schwieler L, Erhardt S: Inhibitory action of clozapine on rat ventral tegmental area dopamine neurons following increased levels of endogenous kynurenic acid. Neuropsychopharmacology 2003;28:1770-1777.
74.
CRESTAR: CRESTAR: development of pharmacogenetic biomarkers for schizophrenia. 2011. http://www.crestar-project.eu/.
75.
Meltzer HY, Okayli G: Reduction of suicidality during clozapine treatment of neuroleptic-resistant schizophrenia: impact on risk-benefit assessment. Am J Psychiatry 1995;152:183-190.
76.
Kinon BJ, Liu-Seifert H, Adams DH, Citrome L: Differential rates of treatment discontinuation in clinical trials as a measure of treatment effectiveness for olanzapine and comparator atypical antipsychotics for schizophrenia. J Clin Psychopharmacol 2006;26:632-637.
77.
Tsunoka T, Kishi T, Ikeda M, Kitajima T, Yamanouchi Y, Kinoshita Y, Okochi T, Okumura T, Inada T, Ozaki N, Iwata N: Association analysis of group II metabotropic glutamate receptor genes (GRM2 and GRM3) with mood disorders and fluvoxamine response in a Japanese population. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:875-879.
78.
Tsunoka T, Kishi T, Kitajima T, Okochi T, Okumura T, Yamanouchi Y, Kinoshita Y, Kawashima K, Naitoh H, Inada T, Ujike H, Yamada M, Uchimura N, Sora I, Iyo M, Ozaki N, Iwata N: Association analysis of GRM2 and HTR2A with methamphetamine-induced psychosis and schizophrenia in the Japanese population. Prog Neuropsychopharmacol Biol Psychiatry 2010;34:639-644.
79.
Dallaspezia S, Poletti S, Lorenzi C, Pirovano A, Colombo C, Benedetti F: Influence of an interaction between lithium salts and a functional polymorphism in SLC1A2 on the history of illness in bipolar disorder. Mol Diagn Ther 2012;16:303-309.
80.
Ohnuma T, Augood SJ, Arai H, McKenna PJ, Emson PC: Expression of the human excitatory amino acid transporter 2 and metabotropic glutamate receptors 3 and 5 in the prefrontal cortex from normal individuals and patients with schizophrenia. Brain Res Mol Brain Res 1998;56:207-217.
81.
Mallolas J, Hurtado O, Castellanos M, Blanco M, Sobrino T, Serena J, Vivancos J, Castillo J, Lizasoain I, Moro MA, Dávalos A: A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke. J Exp Med 2006;203:711-717.
82.
Deng X, Shibata H, Ninomiya H, Tashiro N, Iwata N, Ozaki N, Fukumaki Y: Association study of polymorphisms in the excitatory amino acid transporter 2 gene (SLC1A2) with schizophrenia. BMC Psychiatry 2004;4:21.
83.
Nagai Y, Ohnuma T, Karibe J, Shibata N, Maeshima H, Baba H, Hatano T, Hanzawa R, Arai H: No genetic association between the SLC1A2 gene and Japanese patients with schizophrenia. Neurosci Lett 2009;463:223-227.
84.
Addington AM, Gornick M, Duckworth J, Sporn A, Gogtay N, Bobb A, Greenstein D, Lenane M, Gochman P, Baker N, Balkissoon R, Vakkalanka RK, Weinberger DR, Rapoport JL, Straub RE: GAD1 (2q31.1), which encodes glutamic acid decarboxylase (GAD67), is associated with childhood-onset schizophrenia and cortical gray matter volume loss. Mol Psychiatry 2005;10:581-588.
85.
Lundorf MD, Buttenschøn HN, Foldager L, Blackwood DH, Muir WJ, Murray V, Pelosi AJ, Kruse TA, Ewald H, Mors O: Mutational screening and association study of glutamate decarboxylase 1 as a candidate susceptibility gene for bipolar affective disorder and schizophrenia. Am J Med Genet B Neuropsychiatr Genet 2005;135B:94-101.
86.
Straub RE, Lipska BK, Egan MF, Goldberg TE, Callicott JH, Mayhew MB, Vakkalanka RK, Kolachana BS, Kleinman JE, Weinberger DR: Allelic variation in GAD1 (GAD67) is associated with schizophrenia and influences cortical function and gene expression. Mol Psychiatry 2007;12:854-869.
© 2016 S. Karger AG, Basel
2016
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.