GENE ASSOCIATION STUDIES OF SCHIZOPHRENIA & TARDIVE DYSKINESIA

By Clement Zai

A thesis submitted in conformity with the requirements For the degree of Doctor of Philosophy Institute of Medical Science University of Toronto

Copyright by Clement C. Zai (2008)

Clement Zai Thesis Title: Gene Association Studies of Schizophrenia and Tardive Dyskinesia Degree: Doctor of Philosophy Year of Convocation: June, 2008 Name: Clement C. H. Zai Department: Institute of Medical Science, University of Toronto ABSTRACT Schizophrenia (SCZ) is a severe neuropsychiatric disorder with a genetic component. Most candidate gene association studies have given mixed results. We investigated the GABAA receptor 2 subunit gene GABRG2, the dopamine receptor gene DRD3, and the Brain-derived neurotrophic factor gene BDNF that is required for D3 expression by genotyping polymorphisms spanning and surrounding these genes for association with SCZ, as well as suicidal behaviour. We also examined the BDNF, DRD3, as well as the dopamine receptor gene DRD2 and Protein Kinase B gene AKT1 for association with Tardive Dyskinesia (TD), a potentially irreversible motor side effect of long-term antipsychotic medication. Our analysis included single-marker tests, haplotype tests, and gene-gene interactions. We found a haplotype in the 5 region of GABRG2 to be associated with SCZ in both families and matched case-control samples. We also found two synonymous DRD2 polymorphisms, rs6275 (C939T) and rs6277 (C957T), and their haplotypes, as well as a polymorphism 5 of DRD3, rs905568, to be associated with TD. Further, we reviewed two putative functional DRD2 polymorphisms, -141C Ins/Del and TaqIA, in TD and found TaqIA 3 of the gene to be associated with TD in a meta-analysis. Lastly, we found a significant interaction between AKT1 rs3730358 and DRD2 C939T in TD. Though replication studies are required, these results contribute to the future development of genetic tests to assess

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Clement Zai for the risks of SCZ and TD, leading to better outcome for patients suffering from these debilitating conditions.

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Clement Zai ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. James L. Kennedy, and my co-supervisor, Dr. Albert H. C. Wong, for their guidance, teaching, and insightful comments on my projects as well as advice on my future career path. I would like to also thank members of my supervisory committee, Dr. Paul Fletcher and Dr. John Vincent, and other members of my examination committee, Dr. Jose Nobrega and Dr. Oksana Suchowerski. I would like to acknowledge the graduate coordinators and administrative staff at the Institute of Medical Science. Many thanks to members of the Kennedy lab and Wong lab: Nicole King and Mawahib Semeralul for their help in study designs; Mary Smirniw, Sharah Mar, and Andrea Smart for their administrative assistance; past and present research analysts (Joanne Brathwaite, Natalie Bulgin, Sahar Ehtesham, Olga Likhodi, Laura Miler, Sajid Shaikh, David Sibony, Tricia Sicard, Maria Tampakeras, Subi Tharmalingam, Joseph Trakalo, Gregory Wong, Pamela Zuker) for their technical support so that my experiments could run smoothly; my past and present student colleagues (Dr. Paul Arnold, Poonam Batra, Renan deSouza, Dr. Marc Fadel, Laura Feldcamp, Nipa Haque, Dr. Daniela Hlousek, Rudi Hwang, Dr. Timothy Klempan, Dr. Wiplove Lamba, Frankie Lee, Dr. Livia Martucci, Anjali Rastogi, Dr. Anil Srivastava, Dr. John Strauss) for making my doctoral experience stimulating and fun; post-doctoral fellows and visiting scientists (Drs. Vincenzo De Luca, Yuko Hirata, Mirko Manchia, Daniel Mller, Xingqun Ni, Claudia Rothe, Marco Romano-Silva, Arun Tiwari) for their valuable input throughout my research projects. I would further like to thank the collaborators (Drs. Gary Remington, John Roder, Tatiana Lipina, Bernard Le Foll, Herbert Meltzer, Jeffrey Lieberman, Steven Potkin) for their contributions in the studies and manuscripts, and of course, the patients without whom my doctoral studies would not have been possible.

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Clement Zai I would like to thank my many relatives and family friends who have provided encouragements throughout the years. Lastly, I would like to thank my parents, brother and sister, who have always believed in me even when I was in doubt, and have supported me unconditionally through it all. To them I will dedicate this work.

Clement Zai CONTENTS CHP SECTION TITLE PAGE

ABSTRACT .. ii ACKNOWLEDGEMENTS .. iv

TABLE OF CONTENTS .. vi List of Abbreviations List of Figures ... List of Tables 1 INTRODUCTION 1.1 SCHIZOPHRENIA 1.1.1 1.1.2 1.1.3 1.1.4 1.1.5 Diagnostic Criteria and Epidemiology .. 1 Molecular Genetics Studies ... 2 Candidate Pathways and Genes Suicidal Behaviour in SCZ ... 7 20 x xii xiv 1

Pharmacogenetics .. 21

1.2 TARDIVE DYSKINESIA 1.2.1 1.2.2 Diagnostic Criteria and Epidemiology .. 23 Candidate Pathways and Genes 26

1.3 DOPAMINE 1.3.1 1.3.2 1.3.3 Dopamine neurotransmission 32 The Dopamine DRD2 Gene .. The Dopamine DRD3 Gene .. 33 37

1.4 GABA

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Clement Zai 1.4.1 1.4.2 GABA neurotransmission . The GABRG2 Gene ... 39 39

1.5 METHODOLOGIES GENETIC ASSOCIATION STUDIES 1.5.1 1.5.2 Case-control association studies ... Transmission-Disequilibrium Tests and Family-Based Association Test 41 41 43 45

1.6 RATIONALE 2 ORIGINAL RESEARCH ARTICLE: The -aminobutryic acid type A receptor 2 subunit gene is associated with Schizophrenia and Suicidal Behaviour (manuscript to be submitted) 2.1 Abstract . 2.2 Introduction ... 2.3 Patients and Methods 2.4 Results ... 2.5 Discussion . 3 ORIGINAL RESEARCH ARTICLE: Association study of BDNF and DRD3 genes in Schizophrenia (manuscript to be submitted) 3.1 Abstract . 3.2 Introduction ... 3.3 Patients and Methods

46 47 51 53 55 61

62 63 69

3.4 Results ... 72 3.5 Discussion . 4 ORIGINAL RESEARCH ARTICLE: 74 86-87

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Clement Zai Genetic study of BDNF, DRD3, and their interaction in Tardive Dyskinesia (manuscript to be submitted) 4.1 Abstract . 4.2 Introduction ... 4.3 Patients and Methods 88 89 94

4.4 Results ... 97 4.5 Discussion . 5 ORIGINAL RESEARCH ARTICLE: Association study of Tardive Dyskinesia and twelve DRD2 polymorphisms in Schizophrenia Patients (published in International Journal of Neuropsychopharmacology) 5.1 Abstract . 5.2 Introduction ... 5.3 Patients and Methods 113 114 118 100 111112

5.4 Results ... 121 5.5 Discussion . 6 ORIGINAL RESEARCH PUBLICATION: Meta-Analysis of Two Dopamine D2 receptor gene Polymorphisms with Tardive Dyskinesia in Schizophrenia Patients (published in Molecular Psychiatry) 6.1 Introduction ... 6.2 Patients and Methods 135 136 124 134

6.3 Results and Discussion .. 138

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Clement Zai 7 ORIGINAL RESEARCH ARTICLE Genetic study of eight AKT1 gene polymorphisms and their interaction with DRD2 gene polymorphisms in Tardive Dyskinesia (manuscript to be submitted) 7.1 Abstract . 7.2 Introduction ... 7.3 Patients and Methods 142 143 146 140141

7.4 Results ... 148 7.5 Discussion . 8 DISCUSSION 8.1 Summary of Findings and Implications 8.2 Limitations and Considerations 8.2.1 Sample Characteristics and Power . 8.2.2 Multiple Testing . 8.3 Future Directions 8.3.1 Gene-gene Interactions ... 173 8.3.2 Gene-environment Interactions .. 173 8.3.3 Whole Genome Association ... 176 8.4 Concluding Remarks . 177 9 REFERENCES .. 179 168 170 161 151

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Clement Zai List of Abbreviations 6-OHDA AC5 AIMS AKT1 ANKK1 AP BDNF cAMP CATIE COMT CSF CYP DA DAOA/G72 DARPP32 DISC1 DRD2 DRD3 DRD4 DRD5 DSM DTNBP1 FBAT GABA GABRA3 GABRB2 GABRG2 GSK3 GSTM1/P1/T1 GPX1 HPL HTR2A HTR2C HTR6 LI MAO MAPK MDR1 NMDA NOS1/3 NQO1 NR2B NRG1 OR 6-hydroxydopamine Adenylate cyclase 5 Abnormal Involuntary Movement Scale Protein kinase B gene ankyrin repeat and kinase domain containing 1 Antipsychotic Brain-derived neurotrophic factor gene 3-5-cyclic adenosine momophosphate Clinical Antipsychotic Trials of Intervention Effectiveness Catechol-O-methyltransferase gene Cerebrospinal fluid Cytochrome P450 genes dopamine D-amino acid oxidase activator gene Dopamine and cAMP-regulated phosphoprotein gene (aka PPP1R1B) Disrupted in Schizophrenia 1 gene Dopamine D2 receptor gene Dopamine D3 receptor gene Dopamine D4 receptor gene Dopamine D5 receptor gene Diagnostic and Statistical Manual of Mental Disorders Dysbindin (Dystrobrevin-binding protein) gene Family-based association Test -amino-butyric-acid GABAA 3 subunit gene GABAA 2 subunit gene GABAA 2 subunit gene Glycogen synthase kinase 3 Glutathione S-transferase //1 gene Glutathione peroxidase 1 gene Haloperidol Serotonin 5HT2A receptor gene Serotonin 5HT2C receptor gene Serotonin 5HT6 receptor gene Latent inhibition Monoamine oxidase mitogen-activated protein kinase (aka ERK) P-glycoprotein (Multidrug Resistance), aka ABCB1 N-methyl-D-aspartate Nitric oxide synthase 1(neuronal)/3(endothelial) gene NAD(P)H:Quinone acceptor oxidoreductase type 1 gene NMDA receptor 2B subunit gene Neuregulin gene Odds ratio

Clement Zai PET PKC PPI PRODH RGS4 SCZ SLC6A3 SLC6A4 SNP SOD SPECT TD TDT TPH1/2 Positron emission tomography protein kinase C Prepulse inhibition Proline dehydrogenase gene Regulator of G-protein signalling gene Schizophrenia Dopamine transporter DAT1 gene Serotonin transporter 5HTT gene Single nucleotide polymorphism Superoxide dismutase Single photon emission computed tomography Tardive dyskinesia Transmission disequilibrium Test Tryptophan hydroxylase 1/2 genes

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Clement Zai List of Figures 1 2 3a 3b 4a 4b 5 Candidate genes of Schizophrenia Linkage disequilibrium plot among the five GABRG2 gene polymorphisms used Schematic diagram of the DRD3 gene with its exons and introns Schematic diagram of the BDNF gene with its exons and introns Linkage disequilibrium plot among the 10 DRD3 gene polymorphisms used Linkage disequilibrium plot among the six BDNF gene polymorphisms used p-values from analyses of two-marker interactions between BDNF and DRD3 polymorphisms in relation to schizophrenia diagnosis given by HELIXTREE program 6 p-values from analyses of two-marker interactions between BDNF and DRD3 polymorphisms in association with the history of suicide attempt(s) given by HELIXTREE program 7a Linkage disequilibrium plot among the three ZNF80 and 10 DRD3 gene polymorphisms used 7b 8 Linkage disequilibrium plot among the six BDNF gene polymorphisms used p-values from analyses of two-marker interactions between BDNF and DRD3 polymorphisms in association to AIMS given by HELIXTREE Program 9 10 11 12 13 Schematic diagram of the DRD2 gene with its exons and introns Linkage disequilibrium plot among the 12 DRD2 gene polymorphisms used Schematic diagram of the AKT1 gene with its exons and introns Linkage disequilibrium plot for the eight AKT1 gene polymorphisms used p-values from analyses of two-marker interactions between DRD2 and AKT1 129 132 154 158 159 108 110 107 85 18-19 59 76 76 80 81 84

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Clement Zai polymorphisms in association to AIMS given by HELIXTREE Program 14 Interaction between DRD2_rs6275 (C939T) and AKT1_rs3730358 in AIMS 160

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Clement Zai List of Tables 1 2 3 Candidate Genes of Schizophrenia Candidate Gene Studies of Tardive Dyskinesia Genetic analysis of GABRG2 markers and schizophrenia using paired casecontrol samples 4 Family-based association test using FBAT for GABRG2 polymorphisms and haplotypes 5 Results considering suicidal behaviour in schizophrenia patients and GABRG2 polymorphisms 6a Genetic analysis of DRD3 markers and schizophrenia using paired case-control samples 6b Genetic analysis of BDNF markers and schizophrenia using paired case-control samples 7 Family-based association test using FBAT for DRD3 and BDNF singlenucleotide polymorphisms and HBAT for two-marker haplotypes 8 Results considering suicidal behaviour in schizophrenia patients with BDNF and DRD3 polymorphisms 9 ABI assays-on-demand and assays-by-design with information on their corresponding BDNF and DRD3 polymorphisms used 10a Statistical analyses on demographics as well as total AIMS scores and TD diagnoses with DRD3 genotypes 10b Statistical analyses on demographics as well as total AIMS scores and TD diagnoses with BDNF and ZNF80 genotypes 105 104 103 82-83 79 78 77 60 58 17 31 57

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Clement Zai 11 Results from 2 tests of allele frequencies of each of the 19 polymorphisms versus tardive dyskinesia diagnoses for our Caucasian and African-American samples 12 p-values from analyses of two-marker haplotypes across ZNF80, DRD3, and BDNF genes in association to tardive dyskinesia and AIMS using COCA-PHASE and QT-PHASE respectively 13 Statistical analysis on demographics as well as total AIMS scores and tardive dyskinesia diagnoses with genotypes of the 12 polymorphisms in DRD2 14 Results from 2 test of allele frequencies of each of the 12 DRD2 polymorphisms versus tardive dyskinesia diagnoses for both Caucasian and African-American populations 15 Global p-values from analyses of DRD2 two-marker haplotypes in association to tardive dyskinesia and AIMS using COCA-PHASE and QT-PHASE respectively 16 17 Summary for meta-analysis of DRD2 Taq1A and 141C Ins/Del polymorphisms Assays-on-Demand with information on their corresponding AKT1 polymorphisms used in the present study 18 Statistical analyses on demographics as well as total AIMS scores and tardive dyskinesia occurrence with each of the eight AKT1 polymorphisms 19 Results from 2 tests of allele frequencies of each of the eight AKT1 polymorphisms versus tardive dyskinesia occurrence for both Caucasian and African-American populations 157 156 139 155 133 131 130 109 106

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Clement Zai CHAPTER 1

I. Introduction

INTRODUCTION

1.1 SCHIZOPHRENIA 1.1.1 Diagnostic criteria and Epidemiology Schizophrenia (SCZ) is a severe debilitating neuropsychiatric disorder. It is generally characterized by positive and negative symptoms. Positive symptoms refer to hallucinations, delusions and thought disorder. Hallucinations in schizophrenia are typically auditory and are of voices talking with or about the patient. Delusions are often paranoid and can include false beliefs of persecution, grandiosity, external control, and special powers. Thought disorder is usually manifest in disorganized speech that can be disjointed and erratic, and disorganized behaviours. Negative symptoms include poverty of speech (alogia), greatly diminished motivation (avolition), and a lack of emotion. The diagnostic criteria require that these signs and symptoms be present for at least six months, and are especially prominent for a significant portion of a one-month time period unless successfully treated (APA, 2000). They often cause significant disturbances at work and/or in interpersonal relationships. The lifetime risk of schizophrenia is estimated to be nearly 1% for the general population, regardless of ethnicity, and to be approximately equal between the sexes. However, a meta-analysis of non-overlapping samples from 38 published studies on gender and SCZ found males to be at 40% increased risk compared to females (Odds Ratio, OR=1.42; 95% CI: 1.30-1.56) (Aleman et al, 2003). The reported age of onset varies from study to study, ranging from 25 to 35 years (Hafner and an der Heiden, 1997). There appears to be an earlier age of onset in males compared to females, with

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I. Introduction

our sample reporting a difference of 2.1 years (Renou et al, 2007). Being born or living in cities appears to increase the risk of SCZ two folds compared to living in rural areas (Pedersen et al, 2001; Marcelis et al, 1998). The authors cautioned that the increased SCZ rate in urban areas could be partially due to easier access to medical care in urban areas, thus artificially increasing the observed SCZ rates in these areas. There was an estimated 234,300 SCZ patients in Canada in 2004, with healthcare costs estimated at over $2 billion CAD. Combined with lost productivity due to unemployment, morbidity, and mortality due to SCZ, the total cost estimate of SCZ was $6.85 billion CAD (Goeree et al, 2005). It is important to note that these costs did not take into account emotional and monetary costs for relatives of individuals with SCZ, as well as the chronic social effects of SCZ.

1.1.2 Molecular Genetic Studies Family, twin, and adoption studies have provided evidence for a genetic component to SCZ risk. Overall, there is a ten-fold increase in the risk of schizophrenia for a first-degree relative of a SCZ patient compared to an average global prevalence of 0.7-0.8% (Mowry, 2000; Saha et al, 2005). The concordance rate (presence of the same trait in both members of a twin pair), between monozygotic twins (sharing ~100% DNA sequence identity) is 45-75%, and that between dizygotic twins (sharing ~50% sequence identity) is lower at 4-15% (Kendler, 1983; McGuffin et al, 1984; Farmer et al, 1987; Onstad et al, 1991; Cannon et al, 1998). While the monozygotic and dizygotic twin concordance rates are well above the expected global average incidence rate of 1%, the monozygotic concordance rate of well below 100% suggests that genetic factors are insufficient in totally explaining the etiology of SCZ (LaBuda et al, 1993). From these twin studies, the heritability of SCZ could be estimated at ~60-70%. 2

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I. Introduction

Adoption studies have been useful in separating the relative contribution of familial environment from genetic factors in SCZ risk (Wahlberg et al, 1997). In a study of 361 Finnish families, adopted children whose biological mothers had schizophrenia have over a four-fold increase in the rate of SCZ compared to adopted children of non-SCZ biological mothers (Tienari, 1991). A similar study in Denmark showed a ten-fold increase in SCZ rate (Kety et al, 1994). The results from adoption studies should be reviewed with caution, as the adopted children were exposed to prenatal and often perinatal environment with their biological mothers. Kety and coworkers (1994) attempted to resolve this issue by comparing the prevalence rate of SCZ among paternal half-siblings of SCZ and non-SCZ individuals. They found higher rate of SCZ among paternal half-siblings of SCZ (13%) compared to those of non-SCZ (2%) (Kety et al, 1994). The lifetime risk of schizophrenia of an individual with both parents suffering from SCZ is ~46%, a rate that is much lower than if SCZ is a dominant trait (75%), or if SCZ is a recessive trait (100%) (Tsuang et al, 1982; Faraone et al, 1985; Mortensen et al, 1998). Therefore, it is likely that SCZ is a complex disorder with multiple genetic and environmental components each contributing a small risk (Tsuang et al, 2001). To begin unravelling the genetic contribution of schizophrenia, familial syndromes with schizophrenia-like phenotypes were investigated. Chromosomal aberrations have been reported in families with schizophrenia and other psychiatric disorders. Inversion at 4(q13;q25) was detected in a multigenerational Hong Kong family with multiple SCZ probands (Mensah et al, 2007). An extra copy of a portion of the 5q chromosomal region was reported in an extended family (Bassett et al, 1988), followed by the report of a SCZ patient with an interstitial deletion at 5q21-23.1 (Bennett et al, 1997). Other chromosomal abnormalities have also been reported, including single families with a balanced translocation between chromosomal regions 1p22 and 7q22 (t(1;7)(p22;q22)) (Gordon et al, 1994), t(2;18)(p11.2;p11.2) (Maziade et al, 1993), 3

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I. Introduction

t(6;11)(q14.2;q25) (Holland and Gosden, 1990), t(4; 13)(p16.1;q21.31) (Itokawa et al, 2004), an inversion on chromosome 4 (Palmour et al, 1994), or partial trisomy at 5p (Malaspina et al, 1992). 22q11 deletion occurs in velocardiofacial (DiGeorge) syndrome patients of which 18% have psychotic symptoms, a rate that is much higher than the overall prevalence of 1%; conversely, at least 2% of schizophrenia patients were reported to have 22q11 deletions compared to the 0.025% prevalence rate of 22q11 deletion syndrome in the general population (Karayiorgou et al, 1995; Murphy, 2002). A balanced (1;11)(q42.1;q14.3) translocation was found in a large Scottish family with high frequency of psychiatric disorders including schizophrenia (Millar et al, 2000; Blackwood et al, 2001). In each study, however, the chromosomal aberration does not completely co-segregate with the SCZ phenotype; thus these chromosomal abnormalities alone are not sufficient to cause SCZ. Since the first linkage study done by Sherrington et al (1988), over 35 genome scans have been conducted to search for linkage between genetic markers throughout the human genome and the hypothetical SCZ locus. Sherrington and coworkers analyzed seven families with 39 SCZ or schizophreniform disorder patients and found the long arm of chromosome 5 (5q) to be linked to SCZ (Sherrington et al, 1988). However, investigations in independent samples soon after did not yield significant results in the same region (Kennedy et al, 1988; St Clair et al, 1989; McGuffin et al, 1990; Aschauer et al, 1990). To date, many chromosomal regions have been linked to SCZ, with repeated positive linkage findings in chromosomal regions 1q21-42 (Brzustowicz et al, 2000; Gurling et al, 2001; Hovatta et al, 1999; Ekelund et al, 2000; Blackwood et al, 2001), 5q21-q33 (Schwab et al, 1997; Camp et al, 2001; Gurling et al, 2001; Straub et al, 1997; 2002a; DeLisi et al, 2002; Paunio et al, 2001; Devlin et al, 2002; Sklar et al, 2004), 6p24-p22 (Moises et al, 1995; SCLG, 1996; Straub et al, 2002a; Schwab et al, 1995; 2000; Fallin et al, 2003), 6q21-q25 (Cao et al, 1997; Kaufmann et al, 1998; Martinez et al, 1999, 4

Clement Zai

I. Introduction

Levinson et al, 2000; Lindholm et al, 2001), 8p22-p21 (SCLG, 1996; Blouin et al, 1998; Kaufmann et al, 1998; Brzustowicz et al, 1999; Gurling et al, 2001; Garver et al, 2001; Straub et al, 2002a; Stefansson et al, 2002), each having been cited in at least five studies. The mixed results could be due to variability in sample characteristics such as ethnicity, as well as diagnostic procedure and criteria. In addition, the presumed mode of inheritance and penetrance are important. Evidence of linkage is traditionally provided by the odds of observing the cosegregation of a genetic marker and SCZ by chance. A genetic marker is considered linked to SCZ if the observed degree of cosegregation could occur only once for every 1000 or more cases; that is, if the logarithm of the odds is more than 3. If the mode of inheritance is nonMendelian, then linkage findings will be difficult to interpret. The lack of strong replication in linkage studies could also be the result of small effect size for each susceptibility region that can only be detected with very large sample sizes. Badner and Gershon (2002) attempted to resolve the inconsistencies by performing a meta-analysis of 18 genome scans using multiple-scan probability (MSP) and found three chromosomal regions showing significant linkage to SCZ: 8p, 13q, and 22q. Lewis et al (2003) performed a meta-analysis of 20 previously published genome scans. The multi-centre study group ranked linkage scores of 30cM bins across the genome for each study, and computed the average rank for each 30cM bin across all 20 studies. Using a permutation test, they found significant linkage at 2q, as well as a number of nominally significant regions including 5q, 3p, 11q, 6p, 1q, 22q, 8p, 20q, 14p, 16q, 18q, 10p, 15q, 6q, and 17q (Lewis et al, 2003). Additional linkage support was more recently provided for 4q33-q35.1 (Vawter et al, 2006), 5q31-q35 (Sklar et al, 2004), 6p22 (Maziade et al, 2005), 6q23 (Lerer et al, 2003), 8p23.3-q12 (Suarez et al, 2006), 13q13 (Maziade et al, 2005), 15q26 (Vazza et al, 2007), and 18q21 (Maziade et al, 2005).

Clement Zai

I. Introduction

Another approach in elucidating the genetic basis of SCZ is to study the candidate genes. DNA variants, polymorphisms, in HTR2A at 13q14-q21, DRD2 at 11q23, DRD3 at 3q13.3, COMT at 22q11.21, DTNBP1 at 6p22.3, NRG1 at 8p12, RGS4 at 1q23.3, DISC1 at 1q42.1, NR2B at 12p12, DAOA/G72 at 13q33.2-q34, BDNF at 11p13, PRODH at 22q11.21, AKT1 at 14q32.32 genes have been examined more than once for possible association in SCZ families and casecontrol samples. The roles of some of these candidate pathways and genes in SCZ are discussed below (Figure 1; Table 1). Mice with mutant or deficient expression of some of these candidate genes have been generated and tested for SCZ related phenotypes, prepulse inhibition (PPI) and latent inhibition (LI). PPI of the acoustic startle reflex refers to a paradigm that measures sensorimotor gating where a weak prepulse stimulus reduces the startle reflex to a startle-eliciting pulse stimulus that follows shortly after (Hoffman and Searle, 1965). PPI has been demonstrated in a variety of animal species (van den Buuse et al, 2005). PPI deficit has been consistently reported in schizophrenia patients (Braff et al, 1992; Ludewig et al, 2003). LI is commonly considered as a form of salience (or attentional) learning, reflecting the ability to ignore stimuli that do not previously predict any significant outcomes. Hence, LI deficit may indicate a vulnerability to distraction by irrelevant stimuli. Baruch et al (1988) initially reported LI deficiency in schizophrenia patients, followed by Gray et al (1992) (Williams et al., 1998). LI can be disrupted by amphetamine treatment and rescued by antipsychotics in animals (Solomon and Staton, 1982; Weiner et al., 1984; Feldon and Weiner, 1992; Moser et al., 2000; Weiner, 2003; Meyer et al., 2004) and human subjects (Gray et al., 1992b; Kumari et al., 1999).

Clement Zai 1.1.3 Candidate Pathways and Genes The Serotonin HTR2A Gene

I. Introduction

The serotonin hypothesis of SCZ arose from the pharmacological evidence that Lysergic acid diethylamide (LSD), an indoleamine that resembles serotonin, produces psychotic/hallucinogenic symptoms. The affinity and agonistic effects of hallucinogenic agents to the 5HT2A receptor was correlated to their hallucinogenic potential (Glennon RA et al, 1984; Vollenweider et al, 1997). The serotonin 2A receptor (HTR2A) gene is located at 13q14-q21, a SCZ susceptibility region (Blouin et al, 1998; Brzustowicz et al, 1999; Maziade et al, 2005; Badner and Gershon, 2002). The exon 1 synonymous T102C polymorphism (Ser34Ser; Warren et al, 1993) has been examined in SCZ samples. The C allele has been associated with a 20% decrease in 5HT2A receptor levels in the temporal cortex (Polesskaya and Sokolov, 2002). A meta-analysis of 31 case-control studies showed a significant association between the C allele and CC genotype and SCZ in European Caucasians but not in East Asians (Abdolmaleky et al, 2004). The odds ratio for the C allele in European Caucasians was 1.2 (CI: 1.1-1.3), and that for the CC genotype in European Caucasians was 1.5 (CI: 1.1-2.0). This allele distribution could account for the decreased messenger RNA (mRNA) expression in post-mortem brain samples of SCZ patients compared to healthy controls (Polesskaya and Sokolov, 2002). The serotonin system may influence SCZ development by its effect on glutamatergic neurotransmission (Aghajanian and Marek, 1997). Studying the behavioural phenotypic effects of HTR2A gene ablation will uncover its possible role in SCZ. The Regulator of G-protein Signalling 4 RGS4 Gene Regulator of G-protein Signalling 4 (RGS4), on 1q23, was identified as a SCZ candidate gene in a microarray study where RGS4 levels were found decreased (Mirnics et al, 2001). RGS4

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I. Introduction

was later found genetically associated with SCZ (Chowdari et al, 2002; Chen et al, 2004; Morris et al, 2004). However, other studies (Sobell et al, 2005), including recent meta-analyses (Talkowski et al, 2006a; Guo et al, 2006; Li et al, 2006a) did not support these findings. RGS4 is expressed throughout much of the central nervous system (Erdely et al, 2004), with a role in the negative regulation of G-protein signalling from receptors for neurotransmitters including dopamine (Taymans et al, 2003) and glutamate (De Blasi et al, 2001). Mice with the RGS domain within the endogenous Rgs4 deleted by Cre-mediated recombination exhibit intact PPI and similar locomotor activity compared to wildtype mice (Grillet et al, 2005), suggesting that the role of RGS4 in SCZ may be modest. The N-methyl-D-aspartate (NMDA) receptor 2 subunit NR2B Gene The glutamate hypothesis of SCZ arises from observations that altered glutamate system components are seen in SCZ and that glutamatergic antagonists such as phencyclidine (PCP) and ketamine produce psychotic symptoms. Examples of this type of evidence include reduced glutamate in the CSF of SCZ patients (Kim et al, 1980), and the effectiveness of D-cycloserine, which regulates glutamate receptor function, in treating negative symptoms of SCZ when combined with standard antipsychotics (Goff et al, 1995; Evins et al, 2002). Glutamate has also been shown to modulate dopamine function (Floresco et al, 1998; Floresco et al, 2001). More specifically, N-methyl-D-aspartate (NMDA) receptor activation in the prefrontal cortex and striatum enhances presynaptic dopamine release (Matsumoto et al, 2003). NR2B at chromosomal region 12p12, which codes for the ionotropic NMDA glutamate receptor 2 subunit (GRIN2B), has been investigated in SCZ. Although post-mortem NR2B mRNA levels were not significantly different between SCZ and controls, two studies found association of NR2B polymorphisms with SCZ, and association with the T-200C polymorphism in the 5 region of the gene was recently

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I. Introduction

replicated (Martucci et al, 2006), and strengthened in an independent meta-analysis of six studies (Li et al, 2007a). Testing Nr2b-mutant mice for SCZ-related phenotypes (eg PPI, LI) may uncover the mechanism underlying this association. The D-amino acid oxidase activator G72 (DAOA) Gene D-amino acid oxidase activator (G72, or DAOA) is located at 13q22-q34, another SCZ susceptibility region (Itokawa et al, 2004; Maziade et al, 2005). DAOA activates D-amino acid oxidase (DAAO), which metabolizes D-serine, an agonist of the glutamatergic NMDA receptor. Its expression is increased in post-mortem prefrontal cortical brain regions of SCZ patients (Korostishevsky et al, 2004). Chumakov et al (2002) found DAOA to be associated with SCZ in two Caucasian samples. The association between the G72 and SCZ was replicated in eight samples out of nine that were reported (Shinkai et al, 2007), and confirmed in a recent metaanalysis by Detera-Wadleigh and McMahon (2006). Since Daoa is not present in rodents, studies of its function are carried out through transgenic mice that ectopically express human DAOA. More work is needed to elucidate the function and functional variants of this gene. The Neuregulin 1 NRG1 Gene Neuregulin 1, coded by NRG1 on 8p22-p11, is another strong candidate of SCZ because of repeated linkage findings of the chromosomal region being linked to SCZ (SCLG, 1996; Blouin et al, 1998; Kaufman et al, 1998; Brzustowicz et al, 1999; Gurling et al, 2001; Garver et al, 2001; Straub et al, 2002a; Stefansson et al, 2002). Neuregulin 1 is a growth and differentiation factor that binds ErbB receptor tyrosine kinases, and is involved in the formation and remodelling of the synapse, as well as neurotransmitter function (Falls, 2003). NRG1 has been shown to upregulate NMDAR 2C subunit expression (Ozaki et al, 1997). It has also been reported to increase and decrease GABAA receptor subunit expression, depending on cell type

Clement Zai

I. Introduction

(Rieff et al, 1999; Okada and Corfas, 2004). mRNA for one isoform of NRG1 was increased in SCZ post-mortem brains (Hashimoto et al, 2004). Stefansson et al (2002) originally reported an association between NRG1 and SCZ in an Icelandic and Scottish sample. Since then, the genetic association between NRG1 and SCZ has been replicated in a majority of studies, with a recent meta-analysis showing significant association of two promoter microsatellites in an East Asian population, and four adjacent single-nucleotide polymorphisms in a Caucasian sample (Li et al, 2006a), highlighting the different linkage disequilibrium structures between the two ethnic groups. Phenotypes of mice with only one functional copy of Nrg1 (+/-) resemble those of ErbB4+/- mice, suggesting that the behavioural effects of NRG1 deficiency are transduced through ErbB4. Both mouse lines were hyperactive and had PPI deficit (Stefansson et al, 2002; Hong et al, 2008). Additional behavioural tests indicated Nrg1+/- to have LI deficit (Rimer et al, 2005) and delay in habituation to novel environment (OTuathaigh et al, 2006). The Dystrobrevin-binding protein, or Dysbindin DTNBP1 Gene The DTNBP1 gene, which codes for dysbindin, resides on 6p24-p21, a region repeatedly found to be linked to SCZ from genome scans (Moises et al, 1995; Straub et al, 2002a; Schwab et al, 1995; Fallin et al, 2003; Lerer et al, 2003; Maziade et al, 2005). It binds -dystrobrevin, a member of the dystrophin protein complex located at the synapse. The complex may be involved in glutamate release (Numakawa et al, 2004), as well as recruiting GABAA receptor to the postsynaptic density (Knuesel et al, 1999). Dysbindin protein (Talbot et al, 2004) and mRNA (Weickert et al, 2004) levels were significantly lower in the prefrontal cortex of SCZ patients compared to matched controls. Straub et al (2002b) found several single-nucleotide polymorphisms (SNPs) and their haplotypes to be associated with SCZ in an Irish family sample. A recent meta-analysis of all published genetic association papers on nine single-nucleotide

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I. Introduction

polymorphisms in DTNBP1 showed modest association between five of these SNPs and SCZ (Li et al, 2007b). Li et al (2003) reported a non-sense mutation in DTNBP1 in Hermansky-Pudlak syndrome type 7, an albinism marked by bleeding problems and lung fibrosis. Preliminary behavioural tests on mice with spontaneous Dtnbp1 mutations revealed intact PPI, but additional SCZ-related behavioural tests need to be conducted. Other components of the dystrophin protein complex should be investigated for genetic association with SCZ in the future. The Disrupted-in-Schizophrenia 1 DISC1 Gene The Disrupted-in-Schizophrenia 1 gene (DISC1) was discovered because the gene is truncated by a balanced t(1;11) translocation in a multigenerational Scottish family with multiple members affected by SCZ and other psychiatric disorders (Blackwood et al, 2001). An exon-12 frameshift mutation was also discovered in an American family affected by SCZ (Sachs et al, 2005). Additional support for a role of DISC1 in SCZ came from linkage studies that point to chromosomal region 1q42 to be a SCZ susceptibility region for the general population (Brzustowicz et al, 2000; Gurling et al, 2001; Hovatta et al, 1999; Ekelund et al, 2000; Blackwood et al, 2001). The nonsynonymous rs821616 (Ser704Cys) polymorphism was associated with SCZ in a Caucasian sample (Callicott et al, 2005). The results were partially replicated by Qu et al (2007), albeit with a different risk allele in an East Asian sample. Other reports did not find association between DISC1 polymorphisms and SCZ (Devon et al, 2001; Hennah et al, 2003; Thomson et al, 2005; Zhang et al, 2006). A meta-analysis of rs821616 in six studies did not find a significant association with SCZ (Rastogi et al, in preparation). DISC1 expression appeared similar between SCZ and healthy controls in the Stanley postmortem brain sample, but Sawamura et al (2005) found nuclear DISC1 mRNA to be enriched in the SCZ group. Lipska et al (2006) also reported no significant difference in DISC1 expression between post-mortem brain samples of SCZ patients and controls. They found, however, that the levels of 11

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I. Introduction

DISC1-binding proteins FEZ1, LIS1, and NUDEL were significantly lower in SCZ (Lipska et al, 2006). These protein levels were also associated with the risk allele of rs821597, a polymorphism close to rs831616 (Lipska et al, 2006). Using numerous yeast-two-hybrid experiments with DISC1 and strong DISC1-binding proteins, Camargo and coworkers (2007) constructed a DISC1 interactome consisting of 127 proteins involved in microtubule function, neurodevelopment, cAMP catabolism, among others, and some interacting proteins overlap with the interactome of dysbindin (Camargo et al, 2007). Several research groups have generated different mutant or transgenic mouse lines that express various mutant DISC1 proteins (Clapcote et al, 2007; Li et al, 2007c; Pletnikov et al, 2008; Hikida et al, 2007); these mice were shown to have SCZ-related phenotypes, including deficits in PPI and LI. The Catechol-O-methyltransferase COMT Gene Catechol-O-methyltransferase (COMT) is linked to a SCZ susceptibility region at 22q11 (Karajiorgou et al, 1995; Lewis et al, 2003). Deletion of this chromosomal region is associated with velocardiofacial syndrome, where approximately 25% of its sufferers exhibit psychotic symptoms (Murphy et al, 1999). COMT metabolizes dopamine to homovanillic acid (HVA) in brain regions including the prefrontal cortex. Its role in dopamine metabolism suggests it may play a part in the dopamine hypothesis of SCZ (Egan et al, 2001; Joober et al, 2002; Tunbridge et al, 2006). The dopamine hypothesis of SCZ has endured for decades because of two key observations. All antipsychotics block D2 DA receptors to some extent (Carlsson and Lindqvist, 1963; Creese et al, 1976; Seeman et al, 1976; Kapur and Mamo, 2003). Amphetamine, the dopamine agonist that inhibits dopamine reuptake, induces psychotic features in nonschizophrenia individuals. Amphetamine also exacerbates psychotic symptoms in SCZ patients (Casey et al, 1961; Curran et al, 2004). However, it was not until 1996 that a link between 12

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I. Introduction

dopamine and SCZ was provided in SCZ patients (Laruelle et al, 1996). Striatal dopamine release in response to amphetamine was enhanced in acute SCZ patients. In addition, there was a positive correlation between the release of dopamine and positive symptom severity as well as response to dopamine antagonists (Breier et al, 1997; Abi-Dargham et al, 1998; Laruelle et al, 1999). Experimental models also support the dopamine hypothesis of SCZ. Increased dopamine sensitivity has been found in rodents that have undergone neonatal hippocampal lesions, chronic administration of various antipsychotics or psychotogenic agents, genetic deletion of dopamine system genes such as DRD1, or birth by Caesarean sections (Seeman et al, 2005). These manipulations all resulted in increased D2 DA receptor levels given by autoradiography and competition assays (Seeman et al, 2005). Using positron emission topography on SCZ patients, Meyer-Lindenberg et al (2002) observed blunted task-induced increase in regional cerebral blood flow in the prefrontal cortex that correlated with increased striatal dopamine uptake, suggesting that hypofunction in the prefrontal cortex may be related to dopamine hyperactivity in the subcortical striatal region (Meyer-Lindenberg et al, 2002). Altered HVA levels have been found in SCZ (Davidson and Davis, 1988; Green et al, 1993a, b). The results are difficult to compare due to possible effects of antipsychotic treatment (Sedvall and Wode-Helgodt, 1980). HVA levels also correlated with the severity of positive symptoms (Maas et al, 1997) and treatment response (Pickar et al, 1990). They were inversely correlated to negative symptoms (Lindstrom, 1985). Association studies of the functional COMT Val158Met polymorphism and SCZ have yielded mixed results, with some studies indicating increased risk with the low-activity Met allele and others indicating increased risk with the high-activity Val allele. Some studies reported no significant association. A meta-analysis of case-control studies did not find Val158Met to be 13

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I. Introduction

associated with SCZ (Munafo et al, 2005). Comt-deficient mice exhibit intact PPI and similar open-field activity levels to wildtype mice (Gogos et al, 1998). However, a meta-analysis of family studies found the Val allele to be significantly associated with SCZ (Glatt et al, 2003). Val158Met has been associated with performance on the Wisconsin Card Sort Test and the Nback Task, both cognitive examinations being dependent on dorsolateral prefrontal cortical activity (Weinberger et al, 1986; Berman et al, 1995; Callicott et al, 2000). More specifically, the high-activity Val allele carriers performed significantly worse than Met allele carriers (Joober et al, 2002; Egan et al, 2001). SCZ patients scored significantly worse in the Wisconsin Card Sort Test than healthy controls (Egan et al, 2001). Overall, the association of COMT Val158Met with SCZ and performance on cognitive tests, together with cognitive deficit reported in SCZ patients, suggest that COMT is associated with cognitive deficit in SCZ. Further studies into other SCZrelated phenotypes in rodents and humans are warranted. The Proline dehydrogenase PRODH Gene Similar to COMT, Proline dehydrogenase (PRODH) is also located at 22q11. PRODH may regulate glutamate release (Renick et al, 1999). PRODH is widely expressed in the brain. It is localized within the mitochondria where it converts proline to D-1-pyrroline-5-carboxylate, which is a precursor for glutamate and GABA. Liu et al (2002) detected an initial association between PRODH and schizophrenia in three independent samples. While later genetic studies yielded mixed results and the meta-analysis did not find a significant association (Li et al, 2006b), mice expressing a truncated form of PRODH exhibit significant deficit in PPI (Gogos et al, 1999). Revisiting PRODH with additional polymorphisms is warranted. The -aminobutyric acid 2 subunit GABRB2 Gene

14

Clement Zai

I. Introduction

The GABRB2 gene, which codes for the -aminobutyric acid A receptor 2 subunit, is localized on chromosomal region 5q33.2, a SCZ susceptibility region (Schwab et al, 1997; Camp et al, 2001; Gurling et al, 2001; Straub et al, 2002a; DeLisi et al, 2002; Paunio et al, 2001; Devlin et al, 2002; Sklar et al, 2004; Lewis et al, 2003). It has been a target of SCZ genetic studies because of the GABA hypothesis of SCZ. The GABA hypothesis of SCZ stemmed from pathological data. GABA neuron density was decreased in SCZ (Reynolds et al, 2001; Cotter et al, 2002). Picrotoxin, a GABAA receptor antagonist, disrupted PPI in rats (Japha et al, 1999). GAD67, an enzyme that synthesizes GABA, is decreased (Akbarian et al, 1995), while GAT1, a GABA transporter, is increased (Sundman-Eriksson et al, 2002) in SCZ patients. An earlier study reported no significantly different mRNA levels of GABAA receptor subunits between SCZ patients and controls using in-situ hybridization (Akbarion et al, 1995). Recently, mice with genetic ablation of Gabra3 were generated. These mice exhibited over 50% reduction in PPI, suggesting that GABRA3 may be a candidate gene for SCZ and other related disorders (Yee et al, 2005). GABA may also influence the development of SCZ through its interaction with the dopamine system. Early reports of the possible interaction between the GABA and dopamine systems came from rodent studies. Pycock and Horton (1979) found that dopamine-induced hyperactivity in rats was suppressed by GABA uptake inhibition or GABA agonists. Dopamine axons are colocalized with GABA neurons in the prefrontal cortex (Benes et al, 1993), where GABA may inhibit the dopamine release (Dewey et al, 1992). Dopamine agonists, on the other hand, have been shown to inhibit pallidal GABA release (Floran et al, 1997). Genetically, Lo et al (2004) reported an initial association of six intronic polymorphisms in GABRB2 with SCZ. The findings were partially replicated in some (Petryshen et al, 2005; Liu et al, 2005a; Yu et al, 2006; Lo et al, 2007; Zhao et al, 2007), but not other studies (Ambrosio et

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I. Introduction

al, 2005; Ikeda et al, 2005; Jamra et al, 2007). The most consistent findings appeared to be centred on two adjacent polymorphisms (rs1816071 and rs1816072) in intron 8 (Liu et al, 2005a; Yu et al, 2006; Lo et al, 2007). These two polymorphisms were associated with changes in GABRB2 mRNA levels in post-mortem brain tissues, where overall GABRB2 mRNA levels were reduced in SCZ patients (Zhao et al, 2006). Animal behavioural studies will help uncover the involvement of its gene product in SCZ development.

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I. Introduction

Table 1. Some Schizophrenia candidate genes and their association with schizophrenia according to chromosomal location, genetic association, biology/animal models, expression alterations, and meta-analysis results. The number of + indicates the strength of association. ND not determined (modified from Ross et al, 2006). Gene AKT1 COMT DAOA(G72) DISC1 DRD2 DRD3 DTNBP1 GABRB2 HTR2A NR2B NRG1 PRODH RGS4 Locus 14q22-32 22q11 13q32-34 1q42 11q23 3q13.3 6p22 5q34 13q14-21 12p12 8p12-21 22q11 22q11 Linkage + ++++ +++ ++++ ++ ++ ++++ ++++ +++ + ++++ ++++ ++++ Association Biology ++ ++ ++++ ++ +++++ ++ +++++ ++++ ++++ ++++ +++++ ++ ++ ++ +++ +++ ++++ ++++ +++ ++ ++ +++ ++ +++ ++ ++ Expression Metaanalysis ++ ND + + ND +++ + + ++++ +++ +++ ++ ++ +++ ++ + ++ +++ ND +++ + +++ +++ + ++ +

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I. Introduction

18

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I. Introduction

Figure 1. Some SCZ candidate genes and their possible interactions in the central nervous system. Dysbindin (DTNBP1), part of the dystrophin protein complex, is involved in neuronal survival via AKT1, as well as glutamate and dopamine release (Numakawa et al, 2004). Neuregulin (NRG1), via its receptor ErbB4, increases the expression of glutmatergic NMDA and GABAA receptor subunits (Ozaki et al, 1997; Okada and Corfas, 2004). The dopamine D1 receptor is involved in the expression of Brain-derived Neurotrophic Factor (BDNF), which in turn through its receptor tyrosine kinase TrkB, is required for the expression of the dopamine D3 receptor (DRD3) (Guillin et al, 2001). AKT1 increases the expression of GABAA receptors and phosphorylates GABAA 2 subunit (GABRB2) (Wang et al, 2003). Proline dehydrogenase (PRODH) regulates glutamate release (Renick et al, 1999). Regulator of G-protein Signaling 4 (RGS4) regulates dopamine (Taymans et al, 2003) and glutamate receptor (Aghajanian and Marek, 1997) signaling. The serotonin 2A receptor (HTR2A) inhibits the release of dopamine (Fink and Gothert, 2007). G72 (D-amino acid oxidase activator) upregulates glutamatergic NMDA receptor activity by increasing the synthesis of D-serine (Panatier et al, 2006).

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Clement Zai 1.1.3 Suicidal Behaviour in Schizophrenia

I. Introduction

An estimated 12% of SCZ deaths are due to suicide (Brown, 1997). It is the single largest cause of excess mortality in SCZ compared to the general population (Sartorius et al, 1986). Suicide attempts in general tend to occur more often within families (Johnson et al, 1998; Brent et al, 2002). Twin studies showed greater concordance among monozygotic twins than dizygotic twins (Roy and Segal, 2001; Statham et al, 1998). A recent review of 32 twin studies estimated the heritability of suicidal behaviour to be 30-55% (Voracek and Loibl, 2007). The serotonergic system has been examined for genetic associations with suicidal behaviour, but a meta-analysis of HTR2A did not reveal a major effect of genetic variation on suicidal behaviour in SCZ patients (Li et al, 2006c). The Tryptophan hydroxylase genes TPH1 and TPH2 have been associated with suicide (Paik et al, 2000; Zhang et al, 2007), but the results were not replicated by other investigators (Viana et al, 2006; De Luca et al, 2005a, 2006a; Mann et al, 2008). Variation in the serotonin transporter gene (SLC6A4) intron 2 VNTR is associated with suicide (DeLuca et al, 2006b; Correa et al, 2004; Bayle et al, 2003) in a majority of studies (except Chong et al, 2000a), with the short low-activity allele being associated with history of suicide attempt. More comprehensive genotyping efforts, together with molecular studies may help clarify the role of serotonin system genes in suicidal behaviour. Only a few genes in other neurotransmitter systems have been explored for association with suicidal behaviour. Persson and coworkers (1997) tested the gene coding tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of monoamines dopamine and noradrenaline, and did not find a tetranucleotide repeat to be associated with suicide attempts in Swedish mixed psychiatric patients. The gene coding for the dopamine-metabolizing COMT has also been investigated in suicidal behaviour, with some reports finding the low-activity Met allele to be associated (Nolan et al, 2000; Ono et al, 2004), and others not finding an association (Russ et al, 2000; Liou et al, 2001; De Luca et al, 2005b). 20

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I. Introduction

The 141C deletion allele in the promoter region of the DRD2 gene was found to be associated with suicdality in a sample of alcoholics (Johann et al, 2005). The exon 3 variable number tandem repeat polymorphism within the DRD4 gene coding for the D4 DA receptor was not found to be associated with suicidal behaviour (Persson et al, 1999; Zalsman et al, 2004). BacaGarcia et al (2004) did not find an association between the GABRA3 gene coding for the GABAA receptor 3 subunit, while Hong CJ et al (2003) did not find an association between the brainderived neurotrophic factor gene BDNF and suicidal behaviour. More detailed examinations of genes in the dopamine and GABA systems in addition to those in the serotonin system are required.

1.1.4 Pharmacogenetics of SCZ There is currently no cure for schizophrenia. It is treated with antipsychotic medications. Efficacy and side effects remain major concerns. Antipsychotics (APs) used to treat SCZ symptoms are originally categorized into two groups, typical and atypical APs that differ in their ability to induce catalepsy in rodents. Some clinicans define typical APs, including chlorpromazine, haloperidol, and perphenazine, as having more specific dopamine D2 DA receptor antagonism. They define atypical APs, including olanzapine, risperidone, and quetiapine, as having a different pharmacological profile from typical APs (Geddes et al, 2000; Meltzer, 1989). Yet others differentiate between them by defining typical APs as being effective in treating positive symptoms of SCZ but with a high propensity for developing unfavourable motor side effects such as tardive dyskinesia (TD), and atypical APs as being effective in treating both positive and negative symptoms of SCZ but with a higher chance of developing metabolic adverse effects such as weight gain (Meltzer, 2004; Nasrallah, 2008). Recently, the Clinical

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I. Introduction

Antipsychotic Trials of Intervention Effectiveness (CATIE) study was carried out to determine and compare the efficacy and side effects of several atypical (second-generation) antipsychotics (olanzapine, quetiapine, risperidone, ziprasidone) and the typical (or first-generation) antipsychotic perphenazine (Lieberman et al, 2005). Results from the 18-month long CATIE trial found that the rate of discontinuation (mostly due to inefficacy of treatment) was similar among the APs, with the exception of olanzapine being most efficacious. On the other hand, the rate of discontinuation due to weight gain is highest in the olanzapine group, while the rate of discontinuation due to extrapyramidal side effects was highest in the perphenazine group. Overall, the rate of discontinuation due to any intolerable adverse effects was not significantly different among all antipsychotic groups (Lieberman et al, 2005). This landmark multi-centre study encourages the development of predictive tests for clinical response and side effects of APs. Meta-analyses have indicated the role of HTR2A T102C and H452Y in clozapine response (Arranz et al, 1998a), and HTR2C in clozapine-induced weight gain (De Luca et al, 2007). The most studied area in pharmacogenetics of SCZ is Tardive Dyskinesia (TD). Because both typical and atypical antipsychotics have similar efficacy in relieving schizophrenia symptoms (Lieberman et al, 2005), predicting which patients are vulnerable to TD is a high priority for psychiatrists in treatment selection.

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Clement Zai 1.2 TARDIVE DYSKINESIA (TD) 1.2.1 Diagnostic Criteria and Epidemiology

I. Introduction

Tardive Dyskinesia (TD) was first mentioned by Faurbye (1964), with tardive emphasizing delayed onset of the impairment of voluntary movements, with the DSM-IV diagnosis requiring generally at least three months of antipsychotic exposure. It is a potentially irreversible movement disorder caused by long-term antipsychotic exposure, characterized by involuntary athetoid (slow), choreiform (fast), and/or rhythmic (stereotypic) movements affecting mostly orofacial muscles, with more severe cases involving the trunk and limbs. Obvious manifestations of TD include tongue protrusions, grimaces, lip smacking, puckering, and pursing. Prevalence data is difficult to interpret due to heterogeneous study populations and different TD assessment methodologies. Kane and Smith (1982) reviewed 56 studies from 1959 to 1979 and found TD occurrence to range from 0.5 to 65%. Yassa and Jeste (1992) reported a prevalence of 24% in 39187 patients pooled from 76 studies. Using the Schooler and Kane (1982) criteria for TD occurrence, Woerner et al (1991) reported that 23.4% of neuroleptictreated mixed population had TD. Muscettola et al (1993) reported a prevalence of 19.1% in 1651 patients with various psychiatric disorders using the Schooler and Kane criteria. Despite the widely variable results, there appears to be a common trend toward higher prevalence of TD in elderly subjects. Age has been the most consistently reported risk factor for TD, beginning with a report by Smith and Baldessarini (1980) of a linear correlation between age and TD severity and prevalence. Fenton (2000) reviewed 14 studies and found a positive correlation between TD occurrence and age in schizophrenia patients, from 12% in patients at or below age 30 years, to 42% in patients at least 60 years of age. Two prospective studies reported cumulative incidence of TD over 3 to 6 years of antipsychotic exposure (Jeste et al, 1995; Kane et al, 1995). Kane et al (1995) found the incidence increasing from 5% after 1 year to 26% after 23

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I. Introduction

6 years in 850 adults with a mean age of 29 years. Jeste et al (1995), on the other hand, reported the incidence to be 26% after 1 year and to increase to 60% after 3 years in a cohort of 266 patients with a higher mean age of 65 years. There have been mixed results in the studies of relationship between sex and TD. Some studies report higher risk in females (Smith and Dunn, 1979; Musettatola et al, 1993); others report higher risk in males (van Os et al, 1999) or no difference in risk between the sexes (Caligiuri et al, 1997). Yassa and Jeste (1992) conducted a review of 75 studies on sex differences in TD. Using six studies with data grouped according to age, they found no difference in risk between the two sexes in lower age groups but an increased risk in females over 50 years of age compared to males in the same age group (Yassa and Jeste, 1992). This agedependent risk may affect results in other studies that do not control for both sex and age, such as a meta-analysis reporting a higher TD incidence in female subjects (Smith and Dunn, 1979). Chong et al (2002) assessed TD in 537 East Asian SCZ inpatients and found the TD rate to be 29-40%, which is comparable to that in Caucasian SCZ patients. African Americans appear to be more likely to develop TD after adjusting for dose and duration of antipsychotic treatment (Morgenstern and Glazer, 1993; Glazer et al, 1993). Swartz et al (1997) reviewed literature on ethnicity and TD and concluded that a mixture of genetic and environmental factors including diet, alcohol, tobacco, substance use, as well as types and dosages of medication contribute to TD development. Since TD is a drug-induced movement disorder, the dose and choice of medication may affect risk for developing TD. The dose of antipsychotics affects the risk of TD at the lower dose range, as the target neurotransmitter receptor may be saturated at the lower doses (Baldessarini, 1988). Conventional neuroleptics like haloperidol and chlorpromazine were shown to be more likely to induce TD than atypical drugs, however clozapine appears to be unique in having 24

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extremely low risk for TD. Newer antipsychotics like risperidone, olanzapine, aripiprazole, and quetiapine have been reported to be associated with TD as well (Buzan, 1996; Silberbauer, 1998; Ananth and Kenan, 1999; Herran and Vazquez-Barquero, 1999; Ghelber and Belmaker, 1999; Karama and Lal, 2004; Gharabawi et al, 2006; Maytal et al, 2006; Bhanji and Margolese, 2004; Bressan et al, 2004; Rizos et al, 2007). However, the quality of most studies was hindered by mixed medical/medication history, short study duration, poly-pharmacy, and changes in medication over time (Tarsy and Baldessarini, 2006). Several reviews and meta-analyses have been conducted after the year 2000, with most of them pointing to similar overall tolerability between conventional and atypical antipsychotics (Geddes et al, 2000; Wahlbeck et al, 2001). Conventional neuroleptics have less favourable motor side effect profile than atypical antipsychotics only at high doses (Geddes et al, 2000). At lower doses or in combination with the prophylactic benztropine, conventional neuroleptics have similar extrapyramidal side effect profiles to atypical antipsychotics (Geddes et al, 2000; Rosenheck et al, 2003). Recently, the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) study was carried out to determine and compare the efficacy and side effects of several atypical antipsychotics (olanzapine, quetiapine, risperidone, ziprasidone) and the typical antipsychotic perphenazine (Lieberman et al, 2005). Results from the 18-month long CATIE trial found that while the rate of discontinuation due to extrapyramidal side effects was highest in the perphenazine group, the rate of discontinuation due to any intolerable adverse effect was not significantly different between perphenazine and the atypical antipsychotics (Lieberman et al, 2005). Tobacco, alcohol, and substance use have long been associated with TD. Smokers appeared to be more likely to develop TD (Yassa et al, 1987; Binder et al, 1987), as are alcoholics (van Os et al, 1997; Dixon et al, 1992; Olivera et al, 1990). One study attributed the 25

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I. Introduction

three-fold increased TD risk in alcohol abusers to neurotoxic effects or intermittent neuroleptic exposure due to poor compliance (Duke et al, 1994). The motor side effects of TD interfere with normal voluntary movements, and causes discrimination by others, thus these side effects greatly reduce treatment compliance and worsen outcome. Predicting which patients will likely develop TD remains an important issue in treatment prescription.

1.2.2 Candidate Pathways and Genes for TD Genetics is a prominent factor in determining the risk of TD, as suggested by increased incidence of TD in families reported by several family studies (Yassa and Ananth, 1981; Youssef et al, 1989; Mller et al, 2001). Genes that influence the pharmacokinetics, pharmacodynamics, and oxidative stress associated with antipsychotics (APs) have been considered for TD risk (see Table 2). Pharmacokinetic factors influence the level of APs at site of action; that is, their neurotransmitter targets. They include absorption from site of administration, distribution, and metabolism. CYP2D6 and CYP1A2 genes have been the primary focus for pharmacokinetics studies of AP treatment. The results related to TD have been promising. CYP2D6 is a noninducible enzyme that metabolizes around 30% of commonly prescribed drugs, including many typical and atypical antipsychotics (Shimada et al, 1994). It has received much attention in TD genetics studies because it is highly polymorphic (Patsopoulos et al, 2005). A recent metaanalysis of CYP2D6 studies observed an odds ratio of 1.64 for poor metabolizing genotypes with TD (Patsopoulos et al, 2005). Basile and coworkers (2000) found an association between the CYP1A2 CC734 genotype and the AIMS score (p=0.008), with partial replication by an independent group (Tiwari et al, 2005a). Other CYP genes also deserve attention in TD genetic studies. Tiwari and coworkers (2005b) found a trend for association with CYP3A4, which 26

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I. Introduction

eliminates approximately half of commonly prescribed APs. CYP17 converts Dehydroepiandrosterone (DHEA) to pregnenolone, both of which have neuroprotective effects (Waters et al, 1997; Maurice et al, 2000), and pregnenolone increases dopamine release from rat nucleus accumbens (Barrot et al, 1999). Segman and coworkers (2002) did not find an association between CYP17 and TD, unless it was combined with the DRD3 Gly9 allele. de Leon (2005) found that the low-expression alleles of CYP3A5 were over-represented in TD compared to non-TD, and that the gene coding for Multidrug resistance (MDR1), a drug transporter across membranes, was not associated with TD. With regards to pharmacodynamics, dopamine system genes have been popular candidates since all APs target the dopamine system to some extent. Although the non-human primate TD model showed irreversible decreases in dopamine turnover in the caudate and substantia nigra (Shannak and Hornykiewicz, 1980), independent researchers have not found TDassociated alterations in D1 or D2 DA receptor levels in the vacuous chewing movement model (Knable et al, 1994) and in post-mortem human tissues (Crow et al, 1982). More recently, brainimaging studies have provided stronger evidence supporting the involvement of D2 occupancy in antipsychotic-induced motor side effects (Kapur et al, 2000; Nordstrom et al, 1993). From PET scans of SCZ patients using [11C]-raclopride as the substrate, Kapur et al (2000) demonstrated that high D2 occupancy (>80%) by haloperidol was associated with extrapyramidal side effects. More long-term, follow-up studies with additional subjects are required to determine the effect of dopamine receptor occupancy in TD emergence and severity. The DRD2 gene has received much attention in light of the observation that D2 DA receptor is the target of all antipsychotics. Chen and coworkers (1997a) found an initial association of Taq1A with TD. The association could not be replicated in a number of later studies (Hori et al, 2001a; Chong et al, 2003a; Segman et al, 2003) until recently, when several 27

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I. Introduction

laboratories found haplotypes containing Taq1A to be associated with TD (Srivastava et al, 2006; Liou et al, 2006a). Other DRD2 polymorphisms have been investigated, but no significant association has been reported. In a number of studies, the sample sizes might have limited the statistical power to detect an association between DRD2 and TD (Lattuada et al, 2005; Inada et al, 1997; Segman et al, 2003), especially if the polymorphisms that were used have low minor allele frequencies (Ser311Cys, for example), and the quantitative AIMS scores were not used in the analyses. More thorough examination of DRD2 gene using both qualitative and quantitative analyses are required. Badri et al (1996) was the first to describe an association between the D3 receptor gene (DRD3) Ser9Gly polymorphism and TD (Basile et al., 1999). The findings were corroborated by Steen et al (1997) and two subsequent meta-analyses (Lerer et al., 2002; Bakker et al., 2006). Odds ratios for the Gly allele of 1.3 and 1.1 suggest a consistent but minor role in TD. Only Srivastava and coworkers tested polymorphisms other than Ser9Gly in TD, and they found no significant association with DRD3 in their North Indian sample (Srivastava et al, 2006). The exon 3 48 base-pair variable number tandem repeat polymorphism in the D4-coding DRD4 gene was found to be associated with TD in one report (Lattuada et al, 2005), but the finding was not replicated in two other studies (Srivastava et al, 2006; Segman et al, 2003). Other dopamine system genes, namely COMT (Srivastava et al, 2006; Lai et al, 2005; Matsumoto et al, 2004a; Herken et al, 2003), monoamine oxidase MAO (Matsumoto et al, 2004a), and dopamine transporter SLC6A3 (Srivastava et al, 2006; Segman et al, 2003; Inada et al, 1997) genes, have not been associated with TD. However, their role in TD cannot be dismissed without thorough examination with additional polymorphisms spanning each gene. The serotonergic system is also a target of many APs and serotonin may inhibit dopamine neurotransmission (Kapur and Remington, 1996). Therefore, genes in this system have also been 28

Clement Zai

I. Introduction

studied in TD. An association was initially reported for HTR2A C102 and TD (Segman et al., 2001; Tan et al., 2001). Although two research groups were unable to replicate the findings (Basile et al., 2001; Deshpande et al, 2005), a meta-analysis later supported this finding, especially in older patients (Lerer et al., 2005). The S23 allele in the X-linked 2C receptor gene (HTR2C) was found to be associated with TD (Segman et al, 2000), but the finding was not replicated by Deshpande et al (2005). Zhang et al (2002a) found an association with another polymorphism, G-697C. Other serotonergic system genes, serotonin 6 receptor (HTR6) (Segman et al, 2003; Ohmori et al, 2002), serotonin transporter (SLC6A4) (Segman et al, 2003; Chong et al, 2000; Herken et la, 2003), and tryptophan hydroxylase (TPH1) (Segman et al, 2003), were not found to be associated with TD. Oxidative stress has also been implicated in the pathophysiology of TD. The brain is vulnerable to oxidative stress for several reasons. It uses a great deal of energy. It has large amounts of polyunsaturated fatty acids that are substrates for lipid peroxidation cascades. Further, certain brain regions, the basal ganglia in particular, contain large amounts of transition metals, some of which are involved in the formation of hydroxyl radicals via superoxide dismutase (SOD). The basal ganglia is also rich in dopamine, a neurotransmitter that can autooxidize to produce dopamine quinones free radicals, or be metabolized by monoamine oxidase (MAO), with hydrogen peroxide as a byproduct. Acute haloperidol also increased murine brain oxidized-to-reduced glutathione ratio; the increase could be blocked by the inhibition of monoamine oxidase enzyme by deprenyl (Cohen et al, 1989). Brain superoxide dismutase and catalase levels were significantly reduced accompanied by increased membrane lipid oxidation products after long-term haloperidol administration in rats (Pillai et al, 2007). Thus, haloperidol increases oxidative stress at least partly through increased dopamine metabolism. Sagara (1998) also found increased levels of oxidized glutathione in neuronal cultures, but found that oxidative 29

Clement Zai

I. Introduction

stress originated from the mitochondria and not dopamine metabolism. Vitamin E and melatonin, both with antioxidant activity, have both been tested in TD. A meta-analysis of the effect of Vitamin E on TD found 28.3% of Vitamin-E treated patients to have decreased AIMS scores by at least one third compared to 4.6% of placebo-treated patients (Barak et al, 1998). A double-blind placebo controlled trial found melatonin to decrease average AIMS scores by 2.45 compared to a reduction of 0.77 in the placebo group (Shamir et al, 2001). Genetic studies have suggested that the manganese superoxide dismutase MnSOD Ala9 allele may be protective against TD (Hori et al., 2000; Galecki et al, 2006), though Zhang and coworkers (2002b) could not replicate the finding. The nitric-oxide synthase NOS1 gene was not associated with TD (Shinkai et al., 2002; Wang et al, 2004). The association found between TD and the NADPH quinone oxidoreductase gene NQO1 T750 (Pae et al. 2004) was not replicated by Hori and coworkers (2006), though Liou et al (2005) found a trend for higher average AIMS scores in T750 carriers than in C750 carriers, which is in agreement with the Pae et al paper. The NOS3 (Liou et al, 2006b) and glutathione S-transferase GSTM1 (Lattuada et al, 2005) findings require replication, while the GPX1 (Shinkai et al, 2006), GSTT1 (de Leon et al, 2005), GSTP1 (Shinkai et al, 2005), and APOE (Halford et al, 2006) genes need to be examined more thoroughly. Although there have been several positive results, conclusions regarding the effects of these candidate genes (DRD3, DRD2, HTR2A, CYP2D6, CYP1A2) cannot be drawn yet. It is necessary for these findings to be replicated. In addition a thorough investigation across these genes needs to be performed (see Table 2).

30

Clement Zai

I. Introduction

Table 2. Tardive Dyskinesia candidate genes and their association with tardive dyskinesia. Polymorphisms investigated, number of studies with positive (+) and negative (-) findings, and meta-analysis (meta) results. ? Insufficient information.
Gene CYP1A2 Polymorphisms rs762551 (C163A), rs2470890 (C1545T), *1F, rs2472304, rs3743484, rs762551 (C734A), rs2069514 (G-2964A) *1-*15, *17-*19, *25, *26, *31, *36, *41, *45, duplication rs2740574 (*1B) *3, *6 rs743572 (promoter-T/C) rs1045642? (C3435T), G2677AT rs686, rs4532 (DdeI), rs13306309 (T229A), A-48G, rs5330 (S50R), rs5331 (A199S) rs1800497 (TaqIA), rs1799732 (-141C Ins/Del), rs1799978 (A-241G), rs1079597 (TaqIB), rs1800498 (TaqID), rs6275 (NcoI), rs1801028 (S311C) rs6280 (Ser9Gly), rs324036, rs1503670, rs905568 rs1800955 (C-521T), exon3-VNTR, promoter-120bp-repeat 3-VNTR, rs27072 (G2319A) rs4680 (Val158Met), rs11544669? (L112L), rs3838146? (3UTR-C Ins/Del) Promoter-30bp-repeat rs1799836 (intron13A/G) rs6313 (T102C), rs6314 (H452Y), rs6311 (A-1438G) rs6318 (C23S) rs1805054 (C267T) LPR rs1800532 (A218C) rs4880 (A9V, A16V) rs2682826? Intron4-27bp-repeat, rs1799983 (D298E), rs2070744? (T-786C) rs1800566 (C750T, P187S, C609T) rs1050450 (P197L, P200L) Gene deletion Gene deletion rs1695 (I105V) (+) 3 7 0 1 0 0 0 4 (-) 4 9 1 0 1 1 2 8 Meta Comments ND Boke, 2007; Tiwari, 2007; Schulze, 2001; Chong, 2003b; Matsumoto, 2004b; sig in smokers (Basile, 2000) (+) Patsopooulos, 2005: LOF risk ND ND ND ND ND ND Tiwari, 2005 De Leon, 2005 Segman, 2002 De Leon, 2005 Srivastava, 2006 Chen, 1997a; Inada, 1997; Hori, 2001; Kaiser, 2002; Chong, 2003a; Lattuada, 2005; de Leon, 2005; Srivastava, 2006; Liou, 2006a Lerer, 2002; Bakker, 2006: Gly9 risk Segman, 2003; Lattuada, 2005; Srivastava, 2006; Lee, 2007 Inada, 1997; Segman, 2003; Srivastava, 2006 Lai, 2005; Matsumoto, 2004a; Herken, 2003; Srivastava, 2006 Matsumoto, 2004a Matsumoto, 2004a Lerer, 2005: C102 risk Zhang, 2002a; Segman, 2000 Segman, 2003; Ohmori, 2002 Segman, 2003; Chong, 2000b; Herken, 2003 Segman, 2003 Zhang, 2002b; Hori, 2000 Wang, 2004; Shinkai, 2002 Liou, 2006b Pae, 2004a; Liou, 2005; Hori, 2006 Shinkai, 2006 Pae, 2004b; de Leon, 2005 De Leon, 2005 Shinkai, 2005

CYP2D6 CYP3A4 CYP3A5 CYP17 MDR1 DRD1

DRD2

DRD3 DRD4 DAT1 COMT MAOA MAOB HTR2A HTR2C HTR6 5HTT TPH1 MnSOD NOS1 NOS3 NQO1 GPX1 GSTM1 GSTT1 GSTP1

8 2 0 1 0 0 4 2 0 0 0 1 0 1 1 0 1 0 0

12 2 4 3 1 1 3 1 2 3 1 4 2 0 2 1 1 1 1

(+) ND ND ND ND ND (+) ND ND ND ND ND ND ND ND ND ND ND ND

31

Clement Zai 1.3 DOPAMINE 1.3.1 Dopamine Signalling

I. Introduction

Dopamine is a catecholamine neurotransmitter that is synthesized from the amino acid tyrosine. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that converts tyrosine into Ldihydroxyphenylalanine (L-DOPA), which then gets decarboxylated to dopamine. Dopamine can be converted to norepinephrine (NE) by dopamine -hydroxylase, or it can be metabolized by COMT or MAO to homovanillic acid (HVA). It can also be taken up presynaptically by dopamine transporter (DAT1). The brain is thought to have four dopaminergic projections: mesolimbic, mesocortical, nigrostriatal, and tuberoinfundibular pathways (Lindvall and Bjrklund, 1978). The tuberoinfundibular pathway originates from the arcuate nucleus in the hypothalamus and terminates in the anterior pituitary where dopamine regulates the secretion of prolactin (Porter et al, 1990). The mesolimbic, and mesocortical pathways originate from the ventral tegmental area. The mesolimbic pathway projects to limbic areas including ventral striatum, amgdala, and hippocampus. The mesocortical pathway projects to cortical areas, including orbitofrontal, medial prefrontal, cingulated, dorsolateral prefrontal, temporal, and parietal cortices. The mesolimbic and mesocortical tracts are involved in emotions including reward, motivation, and attention (Majovski et al, 1981). The nigrostriatal tract projects from the substantia nigra to the dorsal striatum, where it integrates cognitive signals, coordinates sensorimotor information, and initiates movement (Majovski et al, 1981). Five dopamine receptors have been cloned. They can be grouped into two categories with regards to the type of G-proteins they interact with: D1-like receptors (D1 and D5) that stimulate adenylate cyclase via the activation of Gs and Golf, and D2-like DA receptors (D2, D3, and D4) that inhibit adenylate cyclase via the activation of Gi/o (Neve et al, 2004). The dopamine

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I. Introduction

receptors differ in anatomical localization in the brain (Meador-Woodruff et al, 1996). D1 is most widely expressed, with distribution in the cortex and striatum (Missale et al, 1998). The D2 DA receptor is expressed mainly in the striatum, though weak expression had been detected in the cortical regions (Missale et al, 1998). D3 is more restricted to the islands of Calleja and ventral striatum (Suzuki et al, 1998), particularly in the nucleus accumbens. D4 is expressed in the prefrontal cortex and hippocampus, but not in the striatum (Lahti et al, 1998). D5 is expressed mainly in the hippocampus and the entorhinal cortex (Missale et al, 1998). Thus, from an anatomical location perspective, the genes coding for the D1, D2, and D3 dopamine receptors are most attractive as candidates for TD studies.

1.3.2 The Dopamine D2 Receptor Gene The DRD2 gene was identified from DNA fragments that are homologous to the 2adrenergic receptor and later isolated from rat brain cDNA library (Bunzow et al, 1988). The human DRD2 gene was mapped to chromosome 11q23. It spans approximately 65kb with eight exons. Structurally, the D2 DA receptor protein is a seven-transmembrane receptor with a large third cytoplasmic loop and a short carboxyl terminus, structural features that are characteristic of Gi/o-coupled receptors. The downstream effectors of D2 DA receptor signalling include adenylate cyclase inhibition, phosphoinositol hydrolysis and calcium mobilization, and potassium ion influx. Mice homozygous for Drd2 gene deletion displayed Parkinsons disease-like phenotypes (Baik et al, 1995). Type 5 adenylate cyclase (AC5) and DARPP32 (dopamine and cAMPregulated phosphoprotein) appear to be critical for D2 DA receptor signalling. Cataleptic doses of antipsychotics haloperidol and sulpiride did not suppress motor activity in mice lacking Ac5 as they did in wildtype mice (Lee et al, 2002). Similarly, the D2 DA receptor antagonist raclopride

33

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I. Introduction

was cataleptic in wildtype mice, but not in mice lacking Darpp32 (Fienberg et al, 1998). Although Ac5-deficient and Darpp32-deficient mice did not display obvious phenotypes, their decreased motor response to antipsychotics and dopamine antagonists resembled that of Drd2lacking mice (Kelly et al, 1998). The D2 DA receptor has two known isoforms, with the short isoform lacking 29 amino acids in its third intracellular loop. The short isoform is predominantly expressed presynaptically, while the long isoform mediates most of the postsynaptic effects of D2 DA receptor (Usiello et al, 2000). As mentioned earlier, experiments on rodents have shown increased D2 DA receptor levels due to various neonatal stressors, including hippocampal lesions, Caesarian sections, amphetamine, and phencyclidine administrations (Seeman et al, 2005). In human SCZ patients, Wong et al (1986) found elevated caudate nucleus D2 DA receptor levels regardless of whether the patients were on antipsychotic medication or not. Farde et al (1990) found no such difference in drug-naive SCZ patients. The mixed results could be due to the age of the subjects and the use of different D2 DA receptor ligands. More recent studies using PET and SPECT have revealed elevated striatal D2 DA receptor density in SCZ patients not undergoing antipsychotic treatments, with a recent meta-analysis showing an overall 12% increase (Guillin and Laruelle, 2007). Genetically, DRD2 has been examined relatively extensively in SCZ and other neuropsychiatric conditions. The Del allele at position 141 was associated with lower promoter activity in vitro (Arinami et al, 1997). Two subsequent studies reported either an increase (Jonsson et al, 1999b) or no difference (Ritchie and Noble, 2003) in D2 DA receptor binding of the Del allele compared to the Ins allele. The mixed findings could be due to different ethnicities being studied. The TaqIA polymorphism approximately 9.5kb downstream of DRD2 was originally thought to be in the DRD2 gene; it has now been shown to be in the ANKK1 (ankyrin 34

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I. Introduction

repeat and kinase domain containing 1) gene (Neville et al, 2004). However, The TaqIA A1 allele has been associated with reduced D2 DA receptor levels in several studies (Jonsson et al, 1999b; Noble et al, 1991; Pohjalainen et al, 1998; Ritchie and Noble, 2003; Thompson et al, 1997). Laruelle and co-workers (1998) did not find a significant association between TaqIA and D2 DA receptor expression level, a finding that could be influenced by population stratification as subjects with four different ethnic backgrounds were included. The C957T polymorphism does not affect the amino acid sequence of the D2 DA receptor protein (Pro319Pro), but its T allele has been associated with decreased D2 DA receptor binding in vivo (Hirvonen et al, 2004), possibly through its effect on mRNA stability (Duan et al, 2003). TaqIA has been associated with alcoholism in a recent meta-analysis (Munafo et al, 2007), and SCZ (Golimbet et al, 1998; Parsons et al, 2007). Many studies have also focussed on the S311C, TaqIA, and 141C Ins/Del polymorphisms in SCZ. The non-synonymous S311C polymorphism was found to be associated with SCZ (Arinami et al, 1994; Shaikh et al, 1994), or of various subtypes of SCZ (Arinami et al, 1996; Serretti et al, 1998). Although several studies did not find a significant association (Crawford et al, 1996; Verga et al, 1997; Tanaka et al, 1996a; Hori et al, 2001b), a recent meta-analysis of 27 samples comprising of 3707 SCZ patients and 5363 controls found the Cys allele to confer a 40% increased risk of SCZ compared to the Ser allele (Glatt and Jonsson, 2006). The C957T was associated with SCZ in a number of recent studies, with the C allele to appearing over-represented in the SCZ groups compared to controls (Lawford et al, 2005; Kukreti et al, 2006; Hanninen et al, 2006; Hoenicka et al, 2006). Positive findings have also been reported for -141C Ins/Del (Arinami et al, 1997; Breen et al, 1999; Inada et al, 1999; Jonsson et al, 1999a). The Del allele at position 141 is underrepresented in SCZ patient group compared to the control group (Arinami et al, 1997). Breen et al (1999) found the opposite allele (Ins) to be underrepresented in their Caucasian sample. Several other studies, 35

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I. Introduction

however, could not replicate the DRD2 141C Ins/Del findings in SCZ (Stober et al, 1998; Tallerico et al, 1999; Hori et al, 2001b; Glatt et al, 2004). These results suggest a trend toward the 3 region of DRD2, encompassing the TaqIA, S311C, and C957T polymorphisms, to be associated with SCZ. A thorough examination of the 3 region of DRD2 is required to better understand this association. AKT1 was recently shown to be regulated specifically by D2 DA receptor in vivo (Beaulieu et al, 2007). AKT1, also known as protein kinase B, is an intracellular signalling Serine/Threonine kinase with multiple substrates. It has been historically linked to growth factor receptors, and it is involved in cell-cycle progression and survival (reviewed in Nicholson et al, 2002). For example, AKT1 phosphorylates GSK3, a target of the bipolar disorder medication lithium, at Ser9 and inactivates it. Its increased expression has been documented in multiple forms of cancers, but its role in neurotransmitter signalling has only recently been uncovered. Mice deficient in D2 DA receptor had significantly increased activating phosphorylation of AKT1 at Thr308 compared to their wildtype littermates (Beaulieu et al, 2007). Its interaction with the D2 DA receptor prompted investigations of the AKT1 gene, localized at 14q22-32, in SCZ. Emamian et al (2004) found AKT1 levels reduced in SCZ patients compared to controls in transformed lymphocytes and two independent post-mortem brain collections. They also found an AKT1 haplotype, which they associated with decreased AKT1 levels, to be preferentially transmitted to SCZ probands. The antipsychotic haloperidol increased the activating phosphorylation of AKT1 at Ser473 and Thr308. The association was replicated in a number of studies (Bajestan et al, 2006; Ikeda et al, 2004), but not others (Ide et al, 2006; Ohtsuki et al, 2006; Turunen et al, 2007).

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Clement Zai 1.3.3 The Dopamine D3 Receptor Gene

I. Introduction

The D3 DA receptor-coding DRD3 gene has seven exons and is mapped to the chromosomal region 3q13.3. The Ser9Gly polymorphism was studied previously because of an initial report of its association with TD (Badri et al, 1996), and an association of the glycine variant with increased D3 DA receptor affinity for dopamine as revealed in cell culture (Lundstrom and Turpin, 1996). Also, the glycine version showed increased activation of mitogen-activated protein kinase (MAPK) and inhibition of cAMP synthesis (Jeanneteau et al, 2006). The glycine variant has further been demonstrated in vitro to result in a shift in D3 signaling from inhibition of adenylate cyclase to inhibition of prostaglandin production (Hellstrand et al, 2004). Numerous association studies on DRD3 in SCZ have also been published with mixed results (Jonsson et al, 2004). The major limitation of previous DRD3 genetic studies, especially in TD, has been the use of only one polymorphism, Ser9Gly within the amino-terminal extracellular loop. Until recently, promoter polymorphisms have been largely ignored. Brain derived neurotrophic factor (BDNF) was found to be required for the expression of D3 in the ventral striatum in vivo (Guillin et al, 2001). 6-OHDA lesion of dopamine neurons projecting to the nucleus accumbens downregulated D3 DA receptor, and not D1 or D2, expression in that area (Guillin et al, 2001). Bdnf gene deletion mimicked the effect of the lesion on D3 DA receptor expression. Local BDNF infusion restored D3 DA receptor expression. BDNF is expressed in the hippocampus and cerebral cortex (Metsis et al, 1993). BDNF proteins are packaged in neuronal vesicles, and are released upon membrane depolarization of the neuron (Mowla et al, 1999). They bind receptor tyrosine kinase TrkB (Urfer et al, 1995), which transduces signals downstream through MAPK (Jovanovic et al, 2000; Suzuki et al, 2004), altering postsynaptic response to neurotransmitters (Levine et al, 1998). BDNF also has trophic properties. Dopamine neurons, in particular, require BDNF for establishment in the nigrostriatal 37

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I. Introduction

system (Baquet et al, 2005) and stimulation of dopamine release (Altar et al, 1998). Decreased BDNF expression has been reported in patients with Parkinsons Disease and Alzheimers Disease (Howells et al, 2000; Holsinger et al, 2000). The BDNF gene, located on 11p13, contains a 3 coding exon and a number of alternatively spliced upstream exons (Liu et al, 2005b). The use of alternative upstream exons determines the tissue-specific expression of BDNF (Metsis et al, 1993). A number of putative functional polymorphisms have been reported. Magnetic resonance imaging scans showed that the Met66 allele carriers had significantly lower average hippocampal volume compared to Val66/Val66 (Pezawas et al, 2004). The C270T polymorphism was shown to affect BDNF mRNA stability (Tongiorgi et al, 2006). The promoter C-281A polymorphism was shown to decrease in vitro DNA-binding activity and basal reporter gene activity in cultured neurons (Jiang et al, 2005). Meta-analyses supported a role of the Val66Met polymorphism in Eating disorders and substance use disorders (Gratacos et al, 2007), but not in Parkinsons Disease (Zintzaras and Hadjigeorgiou, 2005). Genetic studies of BDNF in depression have yielded mixed results (Strauss et al, 2004; 2005; Ribeiro et al, 2007). While BDNF Val66Met and C270T polymorphisms do not appear to confer a strong risk for SCZ (negative meta-analyses: Xu et al, 2007; Zintzaras et al, 2007; Kanazawa et al, 2007; positive meta-analysis: Gratacos et al, 2007), only the Val66Met polymorphism has been tested in one genetic study of TD (Liou et al, 2004) where they did not find a significant association.

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Clement Zai 1.4 GABA 1.4.1 GABA signalling

I. Introduction

GABA is the major inhibitory neurotransmitter in the central nervous system. It is synthesized from glutamate by the enzyme Glutamic Acid Decarboxylase (GAD67). GABA signals through its GABAA ionotropic and GABAB metabotropic receptors. Inhibiton by GABA is mediated through its GABAA receptors in postsynaptic neurons, resulting in the opening of Clchannels. The influx of the negative Cl- ions shunts excitatory currents, decreasing the probability of action potential initiation. GABAergic interneurons regulate firing rate from pyramidal neurons that mediate cortical signals for information processing in learning, memory, and perception (Mountcastle, 1997; McBain and Fisahn, 2001; Freund, 2003; Buzski, 2001; Mhler, 2006; Rao et al, 2000; Benes and Berretta, 2001; Sawaguchi and Iba, 2001). Long-term changes in GABAergic signalling have been associated with seizures, sedation, or coma (Benes and Berretta, 2001; Costa et al, 2001; Hensch and Stryker, 2004; Fagiolini et al, 2004).

1.4.2 The GABAA Receptor 2 Subunit Gene The GABRG2 gene, mapped on chromosome 5q31.1-33.1, has nine exons spanning 85kb. The long isoform has an additional eight amino acids in exon 9 that contains an additional regulatory phosphorylation site by Protein Kinase C (Krishek et al, 1994), and may mediate the effects of ethanol (Cheng et al, 1997). In a study with very small sample size, Huntsman and coworkers (1998) found in 5 SCZ patients reduced levels of the short isoform of GABRG2 that lacks a PKC regulatory phosphorylation site, in comparison to those in matched healthy controls. An NciI polymorphism of GABRG2 has been associated with prefrontal activity in a study of event-related potentials (Winterer et al., 2000). The NciI polymorphism is situated near the

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Clement Zai

I. Introduction

alternatively spliced exon 9 (Cheng et al, 1997). GABRG2 has been associated with febrile seizures (Wallace et al, 2001), a condition that is linked to a 44% increased risk of SCZ (Vestergaard et al, 2005).

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Clement Zai 1.5 METHODOLOGIES GENETIC ASSOCIATION STUDIES: 1.5.1 Case-control association tests

I. Introduction

For single marker allelic association tests with dichotomous variables on matched casecontrol samples, such as presence or absence of SCZ, suicide attempts, or TD, contingency tables for observed allelic frequency distribution are tabulated. From the row and column sums, tables of expected allelic frequency distributions are derived. Pearson 2 is then calculated as the sum of the differences between the observed and expected values in proportion to the expected values. Genotype association tests are done similarly. In cases where a contingency table has at least one cell count of less than five, Fishers Exact Tests are performed. For analysis of continuous variables such as AIMS and suicide behaviour, analysis of variance (ANOVA) is performed to compare these variables among genotypes of each marker. In cases where the ANOVA assumption of equal variances among comparison groups is violated using the Levenes Test, Kruskal-Wallis tests are performed with ranked variables.

1.5.2 Transmission Disequilibrium Tests and Family-based association tests (FBAT) For single marker allelic association tests with dichotomous variables on small nuclear family samples, Transmission Disequilibrium Tests (TDTs) can be performed (Spielman et al, 1993) using the Haploview program (Barrett et al, 2005). TDT is a modified 2 test that measures the degree of biased transmission of one allele over another from the parents to the affected offspring. It is calculated as [(transmitted-untransmitted)2/(transmitted+untransmitted)]. Familial data can also be analyzed with the FBAT program (Laird et al, 2000), which takes into consideration a wider range of family structures in addition to triads (parents and a proband; including diads, sibling pairs, and more distant relatives as well). FBAT outputs Z-scores, and effectively increases the sample size and power to detect associations. FBAT can also analyze 41

Clement Zai

I. Introduction

continuous variables. Family studies are more robust against population stratification because family members share a common genetic background (Evangelou et al, 2006). However, larger samples need to be recruited to derive the same amount of information as case-control studies. Results from case-control and family samples can be combined in cases where the same trend for over-representation and over-transmission of an allele is observed respectively. In one method, the case-control data in the contingency table is first converted to proportions to calculate the corresponding Z-score. Then the Z-score from the case-control sample (z1) is combined with that from FBAT (z2) using the formula: [(z1+z2)/2] (Hedges and Olkin, 1985). For haplotype analyses with case-control samples, the UNPHASED software is used for both dichotomous and continuous variables using the COCA-PHASE and QT-PHASE programs respectively in a sliding-window approach (Dudbridge et al, 2003). To prevent the influence of spurious fluctuations from rare observations, haplotypes with frequencies of less than 5% are omitted from analysis of individual haplotypes within a two-marker window as well as global test of the two-marker window. For family samples, HBAT command from the FBAT software allows testing haplotype windows across genes with both individual haplotype tests and global tests for each two-marker window. Power calculations for case-control samples are performed with Primer software, and power of the family sample is computed using Genetic Power Calculator (Purcell et al, 2003). For example, a sample size of 200 cases and 200 controls can detect an odds ratio as low as 1.86 with 80% power.

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I. Introduction

1.6 RATIONALE From epidemiological and genetic studies, it is clear that the etiopathophysiology of SCZ is complex. More specifically, the symptoms of SCZ can vary widely among patients. Findings from genetic association and linkage studies have been mixed. One possible reason for the mixed findings could be that many genetic association studies of SCZ and TD only examined a few polymorphisms within the candidate genes and lacked a comprehensive analysis that include polymorphisms that may affect the promoter activity or splicing efficiency. Another possible reason could be that genes do not act in isolation but in pathways. It is possible for variations in genes within signalling pathways to interact in influencing the risk of SCZ. We examined the dopamine hypothesis and the GABA hypothesis of SCZ using human genetic association design. Investigating SCZ patients with shared characteristics may reduce the heterogeneity of the sample, so we examined two SCZ-associated phenotypes, suicidal behaviour and the antipsychotic-induced motor side effect TD. (1) The GABAA 2 subunit gene GABRG2 has been investigated in SCZ in the past, mainly with negative results, however most of the previous studies did not use both family and casecontrol samples, and none has investigated suicidal behaviour in SCZ patients. We hypothesized that variations in the GABRG2 gene coding for the GABAA receptor 2 subunit are involved in SCZ per se and/or suicidal behaviour in SCZ patients. (2) The Dopamine D3 receptor gene DRD3 has been investigated in relation to SCZ and TD, both with mixed results, however most of the previous studies only tested the functional Ser9Gly polymorphism. We hypothesized that other genetic variations in DRD3 in addition to Ser9Gly are associated with TD and SCZ. We also hypothesized that variations in the BDNF

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I. Introduction

gene coding for Brain Derived Neurotrophic Factor are involved in TD and SCZ partly because BDNF controls the expression of D3 (Guillin et al, 2001). (3) The Dopamine D2 receptor gene DRD2 has been investigated in relation to TD, with mixed results. Many of the previous studies investigated only a few polymorphisms in DRD2, and in small samples. We hypothesized that genetic variations spanning DRD2, other than the markers previously studied, are associated with TD. We also reviewed previous studies between DRD2 and TD by performing a meta-analysis of the two most studied and putative functional DRD2 polymorphisms. We also hypothesized that polymorphisms spanning the AKT1 gene that codes for Protein Kinase B are associated with TD because AKT1 acts downstream of D2.

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Zai et al CHAPTER 2

II. GABRG2 in Schizophrenia

THE GAMMA-AMINOBUTYRIC ACID TYPE A RECEPTOR 2 SUBUNIT GENE IS ASSOCIATED WITH SCHIZOPHRENIA AND SUICIDALITY

Clement C. Zai(1,2), Nicole King(1), Vincenzo De Luca(1,3), Greg W. H. Wong(1), James L. Kennedy(1,2,3)

(1) Neurogenetics Section, Centre for Addiction and Mental Health, Toronto, Ontario M5T 1R8 Canada (2) Institute of Medical Science, University of Toronto, Toronto, Ontario M5S 1A8 Canada (3) Department of Psychiatry, University of Toronto, Toronto, Ontario M5T 1R8 Canada

Mr. Zai designed the experiment (with guidance from faculty), performed all of the genotyping for the GABRG2 gene polymorphisms, corresponded with the clinical collaborators to refine the details of the phenotype, performed all the statistical analyses, and wrote the manuscript.

Key words: Schizophrenia, GABRG2, genetics, suicidal behaviour, haplotype analysis

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Zai et al 2.1 ABSTRACT

II. GABRG2 in Schizophrenia

Schizophrenia (SCZ) is a severe neuropsychiatric disorder with a strong genetic basis. Because gamma-aminobutyric acid (GABA) is produced in areas of the brain implicated in SCZ, and it has been shown to interact with the dopamine system, GABA may be an important factor in the susceptibility to SCZ development. GABA acts mostly through its ionotropic GABAA receptor, and several genes coding for GABAA subunits, including GABRG2 encoding the 2 subunit, are clustered at 5q31-q35, a chromosomal region associated with SCZ in genome scan studies. We tested five polymorphisms spanning GABRG2 for association with SCZ and also suicidal behaviour. The sample consisted of 109 small nuclear families and 229 SCZ cases paired with 229 healthy controls. rs183294 in the 5 region of GABRG2 was found associated with SCZ in both samples with the C allele over-represented in SCZ cases and over-transmitted in SCZ families (combined p=3 10-3). Preliminary data also showed rs209356 to be associated with suicidal behaviour in SCZ patients (p=0.04). Taken together, the results of the present study suggest GABRG2 may be involved in SCZ susceptibility, but further studies are required.

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Zai et al 2.2 INTRODUCTION

II. GABRG2 in Schizophrenia

Schizophrenia (SCZ) is a severe debilitating neuropsychiatric disorder characterized by a variety of symptoms ranging from positive symptoms such as paranoia and auditory hallucinations to negative symptoms including thought poverty, anhedonia, and social withdrawal, as well as cognitive impairment. It affects approximately 1% of the general population. Findings from family, twin, and adoption studies support a genetic basis for this disorder (reviewed in McGuffin, 2004), but its etiology is still unclear. Hypothesis driven candidate gene studies have examined on the dopaminergic (COMT, DRD2, DRD3) and the serotonergic (HTR2A) systems among others. Chromosomal breakpoints or deletions point to the DISC1 gene and 22q11. Genome scans of SCZ families have found chromosomal regions that showed evidence of linkage and these regions are being examined for specific genes. These genes raise possibilities of dysfunction of other pathways, including intracellular mechanisms (DTNBP1, NRG1), myelination (OLIG2), and the glutamatergic system (GRM3, G72). However, in addition to multiple positive findings, there were also negative results for each of these genes, suggesting that the mechanism of SCZ etiology is complex and may have variable overlap among different populations (reviewed in Harrison and Weinberger, 2005). A growing body of evidence suggests that alterations in -aminobutyric acid (GABA) neurotransmission may underlie the mechanism of pathophysiology in a subset of SCZ cases (Benes et al., 1992; Delini-Stula and Berdah-Tordjman, 1996; Huntsman et al., 1998; Dean et al., 1999; Wassef et al., 1999; Guidotti et al., 2005). GABA is the major inhibitory neurotransmitter in the brain. It is produced mainly by interneurons in the hippocampus, limbic system, and cerebral cortex. The locations of its synthesis and receptors, as well as neurobiologic studies, suggest that it regulates other neurotransmitter pathways including dopamine, serotonin, and

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norepinephrine, all of which have been implicated in SCZ (Jones and Hendry, 1986; Fiber and Etgen, 1997; Carlsson et al, 1999; Matsumoto et al, 2003; Clements and Schreck, 2004). About 25% to 50% of neurons use GABA (Young and Chu, 1990), and GABA exerts its effects partly via the ionotropic GABAA receptors. The GABAA receptor is a heteropentameric chloride channel with 20 mammalian subunits identified thus far (Barnard et al., 1998). Pharmacological studies have shed light on the role of GABA in regulating the excitability of various neuronal circuits, including those affecting memory, learning, anxiety, and cognition (Olsen and Avoli, 1997; Fritschy et al., 1999; Pratt, 1992; Sarter et al., 1988; Izquierdo and Medina, 1991). Benzodiazepines are frequently prescribed in psychiatry, especially in more severe and chronic patients (Veronese et al, 2007). Its use in SCZ in combination with neuroleptics appears beneficial in temporarily increasing the responsiveness to therapy, especially in neuroleptic-resistant patients (Volz et al, 2007). Benzodiazepines work partly by binding to GABAA receptors allosterically (Smith and Olsen, 1995). In SCZ patients, the density of these receptors and the benzodiazepine-binding sites on GABAA receptors appear to be increased (Benes et al., 1996, 1997; Dean et al., 1999). GABAA receptor density was reportedly increased in the dorsolateral prefrontal cortex of SCZ patients (Benes et al., 1996), while a reduction or no change in the number of benzodiazepine-binding sites on GABAA receptors has also been reported (Squires et al., 1993; Pandey et al., 1997). The variable results could be explained by differences in methodologies and different brain regions examined. GABAA subunit genes are clustered in the genome, with 1, 6, 2, and 2 gathered around 5q32-q35 (Johnson et al., 1992; Wilcox et al., 1992; Hicks et al., 1994), a chromosomal region associated with schizophrenia in a number of genome scan studies (Sklar et al., 2004; Lewis et al., 2003). The , , and subunits make up most GABAA receptors (Wafford et al., 1993a, b; Pritchett et

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al., 1989; Pritchett and Seeburg, 1990; von Blankenfeld et al., 1990; Angelotti and MacDonald, 1993; Benke et al., 1996). The subunits determine the selectivity of benzodiazepines, and the 2 subunit is responsible for high-affinity benzodiazepine binding. The 2 subunit, in particular, regulates the binding of benzodiazepines to GABAA receptors (Knoflach et al., 1991; Wafford et al., 1991). The second intracellular loop of the 2 subunit has been shown to interact directly with the carboxyl terminus of the dopamine D5 receptor, thus providing a direct link between the two neurotransmission systems (Liu et al., 2000). A major contributor to mortality in SCZ is suicide, which accounts for about 10% of deaths in these patients (Meltzer, 2002). Cheetham et al. (1988) found an increased number of benzodiazepine binding sites in the frontal cortex of suicide victims. The results were consistent with the Pandey et al. (1997) study where an increased number of benzodiazepine binding sites were found in the prefrontal cortex of suicide victims. However, Pandey et al. (1997) did not find significantly different benzodiazepine sites between suicide and non-suicide post-mortem brain tissue in schizophrenia sample. The results with the SCZ patient samples may have been confounded by neuroleptic treatment because SCZ patients on neuroleptics had lower benzodiazepine binding than neuroleptic-free SCZ patients (Pandey et al, 1997). The 2 subunit is of particular interest in SCZ. Huntsman and coworkers found in 5 SCZ patients reduced levels of the short isoform of GABRG2 in comparison to those in matched healthy controls (Huntsman et al., 1998). An NciI polymorphism of GABRG2 has been associated with prefrontal activity in a study of event-related potentials (Winterer et al., 2000). GABRG2 has been associated with febrile seizures (Wallace et al, 2001), a condition that is linked to a 44% increased risk of SCZ (Vestergaard et al, 2005). Several recent studies reported mixed findings for GABRG2 and SCZ using case-control samples and small nuclear families

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(Turunen et al., 2003; Ambrosio et al., 2004; Petryshen et al., 2005; Ikeda et al., 2005; Nishiyama et al., 2005). Turunen and coworkers (2003) first reported an association of GABRG2 with SCZ. It was followed with a number of negative findings. The small number of polymorphisms examined (Ambrosio et al, 2005), as well as the use of only family or casecontrol samples alone and not a combination of both (Ambrosio et al, 2005; Nishiyama et al, 2005; Ikeda et al, 2005) could have contributed to the negative findings. In addition, none of the previous studies has studied the role of GABRG2 in suicidal behaviours. In the present study, we tested for the association of five polymorphisms as well as their haplotypes in GABRG2 with SCZ using independent paired case-control and small nuclear family samples, including examination of suicidal behaviour.

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Zai et al 2.3 PATIENTS AND METHODS 2.3.1 Subjects

II. GABRG2 in Schizophrenia

Two samples were analyzed in the present study, 229 SCZ unrelated patients paired with 229 healthy controls matched for sex, ethnicity, and where possible, age, as well as 109 SCZ probands with available first-degree relatives. The small nuclear families consist of 31 triads, 10 triads plus a sibling where 3 of the siblings are affected, 49 diads, 8 families of a proband with a single parent and a sibling where all siblings are unaffected, as well as 11 families with an affected proband plus a sibling where 2 of the siblings are affected. The Structured Clinical Interview for DSM-IV (SCID-I; First et al., 1997) was used as the primary diagnostic tool. A clinical summary of detailed information about sequence, context, and severity of symptoms was prepared for each patient (Maxwell, 1992). A best estimate diagnostic consensus was reached after the clinical summary and SCID-I response were reviewed and discussed among research psychiatrists. Patients who satisfied the DSM-IV diagnostic criteria for SCZ or schizoaffective disorder were included (APA, 1994), while patients with history of major neurological disorders, major substance abuse, and head injury with significant loss of consciousness were excluded from the study. Out of SCZ probands, 378 had data on suicide along a scale as follows: 0 being absent, 1 as having thoughts of ones own death, 2 as having suicidal ideation, 3 as having planned suicide(s), 4 as having attempted suicide(s), and 5 as having attempted violent suicide(s).

2.3.2 DNA isolation and polymorphism genotyping Blood specimens of probands and their family members were obtained by venipuncture into two 10mL EDTA tubes. Genomic DNA was purified from blood lymphocytes as described

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previously (Lahiri and Nurnberger, 1991). Five polymorphisms along the GABRG2 genes, the NciI site (rs211013), rs12520992, rs766349, rs209356, and rs183294, were genotyped using TaqMan allele-specific assays on the ABI Prism 7000 Sequence Detection System with the Allelic Discrimination program within the ABI software (Applied Biosystems, Foster City, CA).

2.3.3 Statistical Analyses Adherence to Hardy-Weinberg equilibrium was determined using the chi-square test in Haploview version 3.2 (Barrett et al., 2005). Genetic association with the paired case-control sample was done using chi-square test both in terms of allele frequencies and genotype frequencies. Two-tailed Fishers Exact Tests were used where expected cell counts were less than five (URL: http://home.clara.net/sisa/fiveby2.htm). Association within the family sample was done using the family based association test (FBAT) under the additive model (Hovarth et al., 2001). The quantitative suicide scores were analysed allele-wise using the student t-test and genotype-wise using One-way ANOVA (SPSS v10.0.7, 2000). Linkage disequilibrium across GABRG2 was determined using Haploview. Haplotype analyses were done using COCAPHASE (UNPHASED) for the case-control sample, FBAT for the family sample, and QTPHASE (UNPHASED) for the SCZ cases with scores of suicide behaviour (Dudbridge, 2003). Haplotypes with frequencies of less than 5% were excluded from the analyses. P-values from family and case-control samples were combined using the formula [(z1+z2)/2] (Hedges and Olkin, 1985).

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Zai et al 2.4 RESULTS 2.4.1 Sample Characteristics

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Both the paired case-control and family samples did not differ significantly from HardyWeinberg equilibrium (p>0.1).

2.4.2 The 5 region of GABRG2 may be associated with SCZ Upon testing for allele and genotype frequency distribution differences in our matched SCZ case-control sample, allele 2 (C) and genotype 22 (CC) of rs183294 are over-represented in SCZ patients compared to controls (p=0.01; ORC=1.42 [CI: 1.08-1.86]; p=0.04; ORCC=1.57 [CI: 1.06-2.31]; Table 3). From FBAT analysis on our sample of small nuclear families, we observed a trend for over-transmission of rs183294 allele 2 (C) (p=0.09) and genotype 22 (CC) (p=0.05) (Table 4). The significance level increased by combining the family and case-control samples (z=3.0; p=3 10-3) (Hedges and Olkin, 1985). rs183294 and rs209356 were in strong linkage disequilibrium (Figure 2); therefore, we conducted association analyses using two-marker haplotypes across GABRG2. Two-marker haplotypes containing rs183294 and rs209356 were significantly associated with SCZ in the case-control sample (p=8 10-3). More specifically, the 2-2 (C-A) haplotype was present more often in SCZ patients than expected (p=9 10-3; ORCA=1.62

[CI: 1.12-2.34]). The same haplotypes were not significant in the nuclear family sample

(p=0.33). However, combining the family and case-control samples yielded more significant results (z=9.18; p<1 10-3).

2.4.3 The 5 region of GABRG2 may be associated with Suicidal behaviour in SCZ patients To test for an association of suicidal behaviour in SCZ patients, we compared the allele

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and genotype frequency distributions of SCZ patients who had had at least one suicide attempt with those who had not (Table 5). For rs209356, allele G (p=0.05) and genotype G/G were overrepresented in the suicide group with genotype frequency distribution reaching statistical significance (among the three genotype groups: p=0.04; GG versus A-carriers: p=0.01). In line with the qualitative analyses, comparison of the quantitative suicide behaviour scale among the three genotypes for rs209356 yielded a trend in the suicide behaviour score in patients with the G/G genotype to be higher than patients with other genotypes (ANOVA p=0.095; t-test [GG versus A-carriers] p=0.06).

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Zai et al 2.5 DISCUSSION

II. GABRG2 in Schizophrenia

The current study reports a possible association between the GABRG2 gene and SCZ. The results from the case-control sample were corroborated by the independent family sample in that rs183294 in the 5 end of GABRG2 is associated with SCZ. The positive results are different from two recent studies of the GABAA receptor gene cluster at chromosomal region 5q31-q35 on Japanese and Portuguese samples (Ikeda et al., 2005; Nishiyama et al., 2005; Petryshen et al., 2005), and the associated 5 region of GABRG2 is different from the 3 region found associated in a Finnish family study sample (Turunen et al., 2003). The mixed results could be due to different sets of polymorphisms being tested (Ikeda et al., 2005). They could also be due to different selection and analysis procedures. Our cases were more stringently selected, each being paired with a control subject matched for age, sex, and ethnicity. As for our independent family sample, we used FBAT that takes advantage of the availability of family structures other than triads, thus increasing the sample size and power. It should be noted that even though majority of our subjects are Caucasians, ethnic differences in SCZ susceptibility and linkage disequilibrium block structure could have influenced the results. If only Caucasians were included in the analyses, the results remained significant for rs183294 in the SCZ case-control (p=8x10-3) and suicidal behaviour (p=0.02) data, but those from the family sample became less significant, possibly due to low power of the reduced sample size. It is especially true if the increased risk of SCZ conferred by rs183294 is small. It is important to note that the p-values reported in the current study were not corrected for multiple testing. Nonetheless, the results for rs183294 would remain significant after Bonferroni correction for the five tested markers. This is the first report of a possible association between GABRG2 and suicidal behaviour in SCZ. A quantitative mRNA analysis showed several GABAA subunits to be reduced in suicide post-

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mortem brains compared to non-suicide brains (Merali et al., 2004). Although the authors did not find a significant difference in the 2 subunit between suicide and non-suicide brains, the 2 subunit may influence the susceptibility of SCZ and suicidal behaviour through its regulatory activity on other GABAA subunits. Examining the other GABAA receptor subunit genes at 5q31q35 may help to resolve the mixed findings in SCZ. The present findings encourage further investigation of GABRG2 SCZ susceptibility and suicidal behaviour.

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Table 3. Genetic analysis of GABRG2 markers and schizophrenia using paired case-control samples. NC p-value Polymorphism Genotypes SCZ NC p-value* Allele SCZ (Allele) Assay (Genotype) rs183294 1/1 (T/T) 25 39 1 154 191 0.04 0.01 (C3167701) 1/2 (T/C) 104 113 2 290 253 1=T; 2=C 2/2 (C/C) 93 70 rs209356 1/1 (G/G) 52 51 0.88 1 213 207 0.24 (C3167710) 1/2 (G/A) 109 105 2 241 247 1=G; 2=A 2/2 (A/A) 66 71 rs766349 1/1 (T/T) 165 172 0.46# 1 378 388 0.30 (C985685) 1/2 (T/C) 48 44 2 58 48 1=T; 2=C 2/2 (C/C) 5 2 rs12520992 1/1 (C/C) 5 2 0.26# 1 51 56 0.61 (C3169571) 1/2 (C/A) 41 52 2 395 390 1=C; 2=A 2/2 (A/A) 177 169 NciI, rs211013 1/1 (A/A) 54 49 0.83 1 213 222 0.54 (C3169568) 1/2 (A/G) 105 104 2 215 206 1=A, 2=G 2/2 (G/G) 55 51 Haplotype SCZ NC HaplotypeGlobal p (10,000 permutations) specific p rs183294C-A 84 56 9 10-3 8 10-3 rs209356 Not C-A 358 386 rs2093560.70 rs766349 rs7663490.52 rs12520992 rs125209920.74 rs211013 *2-tailed Fishers Exact Tests # Haplotype analysis using COCA-PHASE.

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Table 4. Family-based association test using FBAT for GABRG2 polymorphisms and haplotypes. S E(S) (allele Var(S) Z (allele 1) P-value Polymorphism #informative families (allele 1/2) ) rs183294 42 35/55 41/49 14.2 -1.71 0.09 rs209356 38 40/40 35/45 13.5 1.44 0.15 rs766349 13 19/7 19/7 3.69 0.13 0.90 rs12520992 17 13/23 15/21 5.6 -0.94 0.35 rs211013 38 45/35 45/35 13.6 -0.09 0.93 Haplotype frequency #families HaplotypeP-value (<10,000 permutations) specific p rs183294C-G 0.39 38 0.11 0.33 rs209356 T-A 0.34 40 0.10 C-A 0.25 35 0.94 rs2093560.55 rs766349 rs7663490.89 rs12520992 rs125209920.73 rs211013

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Figure 2. Linkage disequilibrium plot among the five GABRG2 gene polymorphisms used. Shown are values for D and 95% Confidence Intervals, with the top right triangle derived from case-control data, and bottom left triangle derived from family data). rs209356 rs766349 rs12520992 rs211013 Polymorphism rs183294 0.94 0.96 0.32 0.09 rs183294 0.89-0.97 0.81-0.99 0.14-0.48 0.00-0.20 0.91 0.70 0.17 0.12 rs209356 0.77-0.97 0.53-0.82 0.02-0.38 0.03-0.21 0.74 0.20 1.00 0.94 rs766349 0.33-0.91 0.02-0.47 0.62-1.00 0.82-0.99 1.00 0.49 0.35 0.61 rs12520992 0.27-0.66 0.05-0.63 0.07-0.89 0.90-1.00 0.12 0.31 0.93 1.00 rs211013 0.01-0.31 0.15-0.44 0.69-0.99 0.79-1.00

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Table 5. Results considering suicidal behaviour in SCZ patients and GABRG2 polymorphisms. SNP Genotypes Suicide attempt(s) p-value Suicide p-value specifier Alleles Yes No rs183294 1/1 13 31 0.16 2.1+/-1.7 0.07 1/2 44 120 1.6+/-1.8 2/2 60 104 1.9+/-1.8 1 70 182 0.12 2 164 328 rs209356 1/1 28 35 2.2+/-1.9 0.04 0.10 1/2 54 137 1.6+/-1.8 2/2 32 81 1.8+/-1.7 1 110 207 0.06 2 118 299 rs766349 1/1 84 183 *0.90 1.8+/-1.8 0.94 1/2 28 67 1.7+/-1.8 2/2 1 2 2.0+/-1.7 1 196 433 0.77 2 30 71 # 0.67 rs12520992 1/1 1 9 *0.34 1.2+/-1.6 1/2 26 51 1.7+/-1.9 2/2 87 195 1.8+/-1.8 1 28 69 0.64 2 200 441 rs211013 1/1 33 77 0.80 1.7+/-1.8 0.82 1/2 52 118 1.8+/-1.8 2/2 29 56 1.8+/-1.8 1 118 272 0.54 2 110 230 *p-value calculated from two-tailed Fishers Exact Test. # p-value from Kruskal-Wallis Test.

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III. BDNF & DRD3 in Schizophrenia CHAPTER 3

ASSOCIATION STUDY OF BDNF AND DRD3 GENES IN SCHIZOPHRENIA

Clement C. Zai(1,2), Julien Renou(1), Vincenzo De Luca(1,3), Greg W. H. Wong(1), Bernard Le Foll(1), James L. Kennedy(1,2,3)

(1) Neurogenetics Section, Centre for Addiction and Mental Health, Toronto, Ontario M5T 1R8 Canada (2) Institute of Medical Science, University of Toronto, Toronto, Ontario M5S 1A8 Canada (3) Department of Psychiatry, University of Toronto, Toronto, Ontario M5T 1R8 Canada

Mr. Zai designed the experiment (with guidance from faculty), performed all of the genotyping for the BDNF and DRD3 gene polymorphisms, corresponded with the clinical collaborators to refine the details of the phenotype, performed all the statistical analyses, and wrote the manuscript.

Key words: Schizophrenia, DRD3, BNDF, genetics, suicidal behaviour, haplotype analysis

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Zai et al 3.1 ABSTRACT

III. BDNF & DRD3 in Schizophrenia

Schizophrenia (SCZ) is a severe neuropsychiatric disorder with prominent genetic etiologic factors. The dopamine receptor DRD3 gene is a strong candidate in genetic studies of SCZ because of the dopamine hypothesis of SCZ and the selective expression of D3 in areas of the limbic system implicated in the disease. We examined ten single-nucleotide polymorphisms (SNPs) in DRD3 in our sample of 109 small nuclear SCZ families and 229 paired case-controls. We also examined six BDNF SNPs in our samples because of evidence for BDNF regulation of DRD3 expression (Guillin et al, 2001). Further, we examined the SNPs of both genes for association with suicidal behaviors in the SCZ patients. We did not find a significant association between DRD3 or BDNF polymorphisms with SCZ. However, we found a possible interaction between BDNF Val66Met and DRD3 Ser9Gly in determining the history of suicide attempt(s). Specifically, a larger proportion of SCZ patients who were heterozygous for Val66Met and Ser9Gly have attempted suicides compared to patients with other genotypes (p=0.007). Taken together, the results from the present study suggest that BDNF and DRD3 may not be involved in SCZ susceptibility, but further studies are required, especially for the role of BDNF and DRD3 in suicidal behaviour.

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Zai et al 3.2 INTRODUCTION

III. BDNF & DRD3 in Schizophrenia

Schizophrenia (SCZ) is a severe disabling neuropsychiatric disorder characterized by a collection of symptoms that may include positive symptoms such as paranoia and auditory hallucinations, negative symptoms including thought poverty, anhedonia, and social withdrawal, as well as cognitive impairment. It affects approximately 1% of the general population. Findings from family, twin, and adoption studies support a genetic basis for SCZ (reviewed in McGuffin, 2004), but its etiology is still unclear. Dopamine hypothesis of SCZ arose from the observations that dopamine-mimetic agents including amphetamine induces SCZ associated symptoms, and that all antipsychotic medications have a certain degree of dopamine-blocking capacity. D3 is a G-protein coupled receptor (GPCR) that, upon binding of dopamine, transduces signal via inhibition of cAMP synthesis. It is encoded by the DRD3 gene, which is located on chromosomal region 3q13.3. It is primarily expressed in the striatum, with the highest expression in the ventral portion. These brain regions have been implicated in SCZ pathophysiology. Ser9Gly is the only polymorphism that has been studied for its effects on D3 function. The Gly allele has been associated with increased binding affinity of dopamine (Lundstrom and Turpin, 1996), as well as increased dopamine-induced stimulation of ERK and inhibiton of cAMP synthesis (Jeanneteau et al, 2006). Researchers found decreased DRD3 (Sokoloff et al, 1990) mRNA expression in postmortem brain samples (Schmauss et al, 1993) and peripheral blood lymphocytes (Vogel et al, 2004) of SCZ patients compared to those of controls. The decrease could be due to increased splicing of the 3 region of the DRD3 pre-mRNA (Schmauss et al, 1996), leading to an increased ratio of truncated (D3nf) to full-length (D3) mRNA. In contrast, Ilani et al (2001) found decreased

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DRD3 mRNA levels in SCZ patients blood lymphocytes compared to controls. The mixed results could be due to different housekeeping genes used (Ilani et al, 2001; Vogel et al, 2004). They could also be due to the influence of antipsychotics at time of death. Gurevich et al (1997) found increased D3 levels in the ventral striatum of antipsychotic-free SCZ patients at time of death, but similar D3 levels in SCZ patients taking antipsychotics up to time of death. These results suggest that while D3 is up in SCZ, antipsychotics down-regulate it. In the original genetic study of DRD3 in SCZ, Crocq et al (1992) found Ser9Gly to be associated with SCZ using two matched case-control samples from France and the UK. Mant and coworkers from the same research group updated the positive findings with additional subjects, with an excess of Ser allele and Ser/Ser genotype in SCZ cases compared to controls (Mant et al, 1994). Our laboratory replicated the original positive findings using two independent samples from North America and Italy (Kennedy et al, 1995). The positive findings were also replicated in other independent samples (Shaikh et al, 1996; Spurlock et al, 1998; Ishiguro et al, 2000). However, they were challenged by results on many other case-control

samples, where the authors did not find a significant association between Ser9Gly and SCZ (Yang et al, 1993; Nanko et al, 1993; Saha et al, 1994; Inada et al, 1995; Ohara et al, 1996; Tanaka et al, 1996b; Chen et al, 1997b; Hawi et al, 1998a; Cordeiro et al, 2001; Nimgaonkar et al, 1993; Nothen et al, 1993; Di Bella et al, 1994; Laurent et al, 1994; Gaitonde et al, 1996; Rietschel et al, 1996; Dollfus et al, 1996; Krebs et al, 1998; Joober et al, 2000; Virgos et al, 2001; Lorenzo et al, 2007). Transmission disequilibrium tests (Spielman and Ewens, 1993) were used in a number of studies with no significant findings (Rothschild et al, 1996; Prasad et al, 1999; Kremer et al, 2000; Ambrosio et al, 2004). To sort through the mixed results, several reviews and meta-analyses have been conducted. Shaikh et al (1996) found the Ser allele to

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confer increased risk of SCZ in ten previous studies, and Dubertret et al (1998) found the Ser/Ser genotype to be associated with SCZ in 13 studies on European Caucasians. More recently, Jonsson et al (2003) conducted a meta-analysis using results from 40 non-overlapping samples and found homozygosity to marginally increase the risk of SCZ. However, after adding four additional samples, the results were no longer statistically significant (Jonsson et al, 2004). Polymorphisms in addition to Ser9Gly have been examined in SCZ genetic studies. For example, the MspI and PvuII polymorphisms were not linked to SCZ in French families (Sabate et al, 1994). Ishiguro et al (2000) examined three polymorphisms (G-712C, A-205G, Ala38Thr) in addition to Ser9Gly in two independent Japanese case-control samples; they found Ser9 and haplotypes containing G-712C, A-205G, and Ser9Gly to be significantly associated with SCZ. Staddon and coworkers (2005) analyzed two other polymorphisms (G-205A, G-7685C) in addition to Ser9Gly, and found 7685C to be over-represented in SCZ in an isolate from Northern Spain. Baritaki et al (2004) reported marginally significant findings with the A-206/A206 genotype in SCZ Greeks. Despite having accumulated genotype data from over 10,000 SCZ patients across different studies, with most studies having tested only the Ser9Gly polymorphism within this 40kb gene, the role of DRD3 in SCZ remains elusive. Recently, Talkowski et al (2006) investigated 11 polymorphisms evenly distributed within and around DRD3 for association with SCZ in two family samples (US and Indian) and a case-control sample. They found consistent association between Ser9Gly and SCZ in both family samples, but only against a common haplotype background (Talkowski et al, 2006b). Dominguez and coworkers (2007) genotyped 17 polymorphisms spanning DRD3 and found haplotypes in the 3 portion of the gene to be significantly associated in a Galician isolate population. These latest findings suggest that

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Ser9Gly may be a marker for other polymorphism(s) that confer risk to SCZ and encourage further more comprehensive examination of DRD3 in SCZ. The brain-derived neurotrophic factor (BDNF) plays a critical role in dopaminergic neuronal establishment (Baquet et al, 2005). The number of tyrosine hydroxylase-expressing dopaminergic neurons was reduced in the midbrain-hindbrain regions where the BDNF gene was selectively deleted (Baquet et al, 2005). BDNF also specifically regulates the in vivo expression of DRD3 in the nucleus accumbens both during development and in adulthood (Guillin et al, 2001). Postmortem studies in the brains of SCZ patients showed decreases in BDNF protein (Iritani et al, 2003; Weickert et al, 2003) and mRNA levels (Weickert et al, 2003), while Buckley et al (2007) and Palomino et al (2006) recently found decreased plasma BDNF levels in firstepisode patients. However, Takahashi et al (2000) also found increased BDNF protein levels in the hippocampus and nucleus accumbens in SCZ subjects. SCZ patients who were chronically on antipsychotic medications showed increased (Gama et al, 2007) or decreased (Grillo et al, 2007; Toyooka et al, 2002) serum BDNF levels compared to healthy controls. Similar BDNF levels between the chronically treated SCZ and controls were also reported (Shimizu et al, 2003). The mixed results could be due to a number of factors, including insufficient sample sizes, different patient populations, different brain regions examined, or different antipsychotics at different doses. Genetically, our laboratory found modest association between the promoter GTdinucleotide repeat polymorphism and SCZ, especially with an excess of maternal transmission of the 170-bp allele (Muglia et al, 2003). However, other studies in various ethnic groups did not replicate the positive findings (Hawi et al, 1998; Sasaki et al, 1997; Wassink et al, 1999). A modest association was found with the T allele of C270T polymorphism in the 5 noncoding region (Nanko et al, 2003; Szekeres et al, 2003); the association was not replicated in other

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studies (Galderisi et al, 2005; Szczepankiewicz et al, 2005; Xu et al, 2007). Association studies have shown Val66 to be overrepresented in SCZ cases (Neves-Pereira et al, 2005) and overtransmitted to affected members in small nuclear families (Rosa et al, 2006). Other studies did not show association of Val66Met with SCZ in European Caucasians (Hawi et al, 1998; Skibinska et al, 2004; Galderisi et al, 2005; de Krom et al, 2005), and East Asians (Chen et al, 2006; Tochigi et al, 2006; Watanabe et al, 2006; Xu et al, 2007; Naoe et al, 2007). Individuals with at least one Met allele performed more poorly on memory and learning tasks, with abnormal hippocampal activation (Egan et al, 2003), and decreased volume of the hippocampal formation (Szeszko et al, 2005). Ho et al (2006) found the Met allele to be associated SCZ-specific visuospatial impairment and correlated decreases in gray matter volumes in the temporal and occipital lobes. Five recent meta-analyses on Val66Met did not find a significant association with SCZ (Naoe et al, 2007; Qian et al, 2007; Xu et al, 2007; Kanazawa et al, 2007; Zintzaras et al, 2007). The C270T polymorphism has also been analyzed in two meta-analyses, which found weak (Zintzaras et al, 2007) or no (Xu et al, 2007) association with SCZ. However, the role of the BDNF gene could not be excluded as Qian et al (2007) found a four-marker haplotype to be protective against SCZ in their Chinese sample. A major contributor to mortality in SCZ is suicide, which accounts for approximately 10% of deaths in these patients (Meltzer, 2003). Twin studies support a genetic basis of suicidal behaviour (Voracek and Loibl, 2007). A meta-analysis of excess mortality rates in SCZ by Brown (1997) found suicides to be the leading cause of excess deaths in SCZ, accounting for 28% of the excess SCZ deaths. BDNF mRNA (Dwivedi et al, 2003) and protein (Dwivedi et al, 2003; Karege et al, 2005) levels were reduced in the prefrontal cortex and hippocampus of suicidal victims compared to controls. Plasma BDNF levels were reduced in major depressive

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disorder patients who attempted suicide (Kim et al, 2007). BDNF was not significantly different between suicidal and non-suicidal SCZ patients in one study (Huang et al, 2006). One genetic study of Val66Met and suicide in mood disorder patients yielded negative results (Hong et al, 2003). Neither the role of the D3 protein nor the DRD3 gene has been examined in suicidal

behaviour. Although expression studies have pointed to a possible role of BDNF gene in suicidal behaviours, none has studied the combined role of BDNF and DRD3 genes in suicidal behaviours in SCZ patients. In the present study, we tested for the association of ten polymorphisms in DRD3 and six in BDNF. We analyzed individual polymorphisms and two-marker haplotypes in the sliding window approach on our paired case-control and small nuclear family SCZ samples. We also examined single-marker interactions between DRD3 and BDNF in SCZ. We further explored the effect of DRD3 and BDNF on suicidal behaviour of the SCZ patients.

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Zai et al 3.3 PATIENTS AND METHODS 3.3.1 Subjects

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Two samples were analyzed in the present study, 229 SCZ unrelated patients paired with 229 healthy controls matched for sex, ethnicity and where possible, age, as well as 109 SCZ probands with available first-degree relatives. The small nuclear families consist of 31 triads, 10 triads plus a sibling where 3 of the siblings are affected, 49 diads, 8 families of a proband with a single parent and a sibling where all siblings are unaffected, as well as 11 families with an affected proband plus a sibling where 2 of the siblings are affected. The Structured Clinical Interview for DSM-IV (SCID-I; First et al., 1997) was used as the primary diagnostic tool. A clinical summary of detailed information about sequence, context, and severity of symptoms was prepared for each patient (Maxwell, 1992). A best estimate diagnostic consensus was reached after the clinical summary and SCID-I response were reviewed and discussed among research psychiatrists. Patients who satisfied the DSM-IV diagnostic criteria for SCZ or schizoaffective disorder were included (APA, 1994), while patients with history of major neurological disorders, major substance abuse, and head injury with significant loss of consciousness were excluded from the study. Out of SCZ probands, 378 had data on suicide along a scale as follows: 0 being absent, 1 as having thoughts of ones own death, 2 as having suicidal ideation, 3 as having planned suicide(s), 4 as having attempted suicide(s), and 5 as having attempted violent suicide(s).

3.3.2 DNA isolation and polymorphism genotyping Blood specimens of probands and their family members were obtained by venipuncture into two 10mL EDTA tubes. Genomic DNA was purified from blood lymphocytes as described

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previously (Lahiri and Nurnberger, 1991). 10 polymorphisms along the DRD3 genes, the rs905568, rs2399504, rs7611535, rs6762200, rs1394016, rs6280 (MscI, Ser9Gly), rs167770, rs2134655, rs2087017, and rs1025398, as well as six BDNF gene polymorphisms, Promoter C/A, C270T, BDNF_4, BDNF_3, BDNF_2, and Val66Met, were genotyped using TaqMan allele-specific assays on the ABI Prism 7500 Sequence Detection System with the Allelic Discrimination program within the ABI software (Applied Biosystems, Foster City, CA). The positions of the DRD3 and BDNF polymorphisms are indicated in Figures 3a and 3b (also see Table 9).

3.3.3 Statistical Analyses Adherence to Hardy-Weinberg equilibrium was determined using the chi-square test in Haploview version 3.3 (Barrett et al., 2005). Genetic association with the paired case-control sample was done using chi-square test both in terms of allele frequencies and genotype frequencies. Two-tailed Fishers Exact Tests were used where expected cell counts were less than five (URL: http://home.clara.net/sisa/fiveby2.htm). Association within the family sample was done using the family based association test (FBAT) under the additive model (Hovarth et al., 2001). The quantitative suicide scores were analysed allele-wise using the student t-test and genotype-wise using One-way ANOVA (SPSS version 14.0, 2005). Linkage disequilibrium across DRD3 and BDNF was determined using Haploview (Figures 4a, 4b). Haplotype analyses were done using COCA-PHASE (UNPHASED) for the case-control sample, FBAT for the family sample, and QT-PHASE (UNPHASED) for the SCZ cases with scores of suicide behaviour (Dudbridge, 2003). Haplotypes with frequencies of less than 5% were excluded from the analyses. Gene-gene interaction analysis was performed using HELIXTREE (GoldenHelix),

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and post-hoc analyses of the continuous variable were carried out using univariate general linear model in SPSS.

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Both the paired case-control and family samples did not differ significantly from HardyWeinberg equilibrium (p>0.1) except for rs1025398 for the case-control sample (p=0.02).

4.4.2 DRD3 and BDNF are not associated with SCZ Upon testing for allele and genotype frequency distribution differences in our matched SCZ case-control sample, none of the ten DRD3 and six BDNF polymorphisms was associated with SCZ (Tables 6a, 6b). From FBAT analysis on our sample of small nuclear families, we did not observe biased transmission of alleles in any of the 16 polymorphisms tested (Table 7).

4.4.3 DRD3 and BDNF are not associated with Suicidal behaviour in SCZ patients To test for an association of suicidal behaviour in SCZ patients, we compared the allele and genotype frequency distributions of SCZ patients who had had at least one suicide attempt with those who had not (Table 8). None of the ten DRD3 and six BDNF polymorphisms was associated with suicidality in SCZ. In line with the qualitative analyses, comparison of the quantitative suicide behaviour scale among genotypes did not yield significant results in any of the DRD3 and BDNF polymorphisms.

4.4.4 BDNF-DRD3 interaction in SCZ and Suicidal behaviour in SCZ patients Because of the functional relationship between BDNF and DRD3 in vivo, we performed interaction analysis with polymorphisms between BDNF and DRD3 using HELIXTREE. We did not find significant association between any BDNF-DRD3 two-marker combinations in SCZ

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diagnosis (Figure 5), but we observed a significant association between BDNF Val66Met and DRD3 Ser9Gly in suicide specifier (Figure 6; Bonferroni p<0.05). More specifically, SCZ patients who are heterozygous for both Val66Met and Ser9Gly appeared to be more likely to have attempted suicide(s) than those with other genotype combinations (28/50 or 56% versus 87/314 or 28%; OR=2.02 CI: 1.20-3.40).

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Zai et al 4.5 DISCUSSION

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The current study on a small nuclear family sample and an independent paired casecontrol sample reports no significant association of the DRD3 and BDNF genes with SCZ. These results are different from a recent study of the DRD3 gene (Talkowski et al, 2006b), where the authors found Ser9Gly to be consistently associated with SCZ. The mixed results could be due to more stringent criteria for our case-control sample in which each SCZ subject was matched with a control subject on age, sex, and ethnicity. As for our family sample, we used FBAT that takes advantage of the availability of family structures other than triads, thus increasing the sample size and power. It should be noted that even though the majority of our subjects are Caucasians, ethnic differences in SCZ susceptibility and linkage disequilibrium block structure could have influenced the results. When only Caucasians were included in the analyses, rs6762200 became marginally significant (p<0.05). It is also important to note that, except for the HELIXTREE gene-gene interaction analysis, the p-values reported in the current study were not corrected for multiple testing. The findings suggest that the selected polymorphisms may not influence schizophrenia risk or that the current sample size is too small to have enough power to detect a small effect size. This is the first reported study of DRD3 and BDNF with suicidal behaviour in SCZ. Although we did not find a significant association with individual polymorphisms, we found a significant interaction between the functional polymorphisms Val66Met and Ser9Gly in the history of suicide attempt(s). One interpretation is that since BDNF has been associated with depression (Strauss et al, 2004; 2005; Martinowich et al, 2007; Ribeiro et al, 2007), and DRD3 has been associated with impulsivity (Retz et al, 2003), suicide attempts may require the interaction between the depressive and impulsive trait (Mann, 2003). Therefore, even though

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BDNF and DRD3 did not confer risk of suicide individually, the combination of the two genes did. Further replication studies and functional analyses are required to better understand this interaction. The role of other confounding factors in suicidal behaviour, including alcohol use, should also be considered (Sher, 2006). The present findings do not support a role of DRD3 and BDNF in SCZ development, but they encourage more studies of BDNF and DRD3 in SCZassociated phenotypes including suicidal behaviour.

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rs2399504 5 ~13kb ~20kb rs905568

rs6762200 rs1394016

rs2134655 rs167770 rs2087017 3

~256kb rs1025398

kb

rs7611535

rs6280, MscI, BalI, Ser9Gly

Figure 3a. Schematic diagram of the DRD3 gene with its exons and introns. The positions of the 12 polymorphisms used for the present study are indicated within the gene. See Table 9 on page 95 for more details.

P1CA 5

C270T

BDNF4

BDNF3

BDNF2

Val66Met 3

Figure 3b. Schematic diagram of the BDNF gene with its exons and introns. The positions of the 6 polymorphisms used for the present study are indicated within the gene. See Table 9 on page 95 for more details.

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Table 6a. Genetic analysis of DRD3 markers and SCZ using paired case-control samples.
Polymorphism Assay rs905568 Genotypes 1/1 (C/C) 1/2 (C/G) 2/2 (G/G) P 1/1 (G/G) 1/2 (G/A) 2/2 (A/A) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (C/C) 1/2 (C/T) 2/2 (T/T) P 1/1(A/A) 1/2(A/G) 2/2(G/G) P 1/1 (C/C) 1/2 (C/T) 2/2 (T/T) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (C/C) 1/2 (C/T) 2/2 (T/T) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P SCZ(Y/N) 64/71 120/108 39/44 0.52 149/146 71/66 4/12 0.12 11/18 92/90 122/117 0.40 32/33 109/100 78/86 0.67 73/72 104/107 46/44 0.95 87/94 101/91 35/38 0.63 21/27 97/92 104/103 0.64 13/14 80/88 133/124 0.69 50/43 104/112 69/68 0.66 94/107 91/90 39/27 0.22 Allele Allele 1 (C) Allele2 (G) P Allele 1 (G) Allele2 (A) P Allele 1 (A) Allele2 (G) P Allele 1 (A) Allele2 (G) P Allele 1 (C) Allele2 (T) P Allele 1 (A) Allele 2 (G) P Allele 1 (C) Allele2 (T) P Allele 1 (A) Allele2 (G) P Allele 1 (C) Allele 2 (T) P Allele 1 (A) Allele2 (G) P SCZ(Y/N) 248/250 198/196 0.89 369/358 79/90 0.68 0.35 114/126 336/324 0.70 0.37 173/166 265/272 0.40 0.63 250/251 196/195 0.80 0.95 275/279 171/167 0.25 0.78 139/146 305/298 0.49 0.62 106/116 346/336 0.69 0.44 204/198 242/248 0.31 0.69 279/304 169/144 0.08 p-value (Haplotype) 0.41

rs2399504

rs7611535

rs6762200

rs1394016

Ser9Gly rs6280 MscI BanI rs167770

rs2134655

rs2087017

rs1025398

*2-tailed Fishers Exact Tests # Sliding-window two-marker haplotype analysis using COCA-PHASE.

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Table 6b. Genetic analysis of BDNF markers and SCZ using paired case-control samples.
Polymorphism Assay Promoter C/A rs28383487 C-281A C270T HinfI BDNF_4 rs7103411 BDNF_3 rs2049045 BDNF_2 rs11030104 Val66Met rs6265 Genotypes 1/1 (A/A) 1/2 (A/C) 2/2 (C/C) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (C/C) 1/2 (C/G) 2/2 (G/G) P 1/1 (C/C) 1/2 (C/T) 2/2 (T/T) P 1/1(A/A) 1/2(A/G) 2/2(G/G) P SCZ(Y/N) 0/1 6/5 221/221 1.00* 201/209 24/16 1/1 0.43* 131/126 79/85 15/14 0.84 158/146 59/72 9/8 0.40 16/15 75/82 135/129 0.79 15/12 68/77 140/134 0.60 Allele Allele 1 (A) Allele2 (C) P Allele 1 (A) Allele2 (G) P Allele 1 (A) Allele 2 (G) P Allele 1 (C) Allele2 (G) P Allele 1 (C) Allele2 (T) P Allele 1 (A) Allele 2 (G) P SCZ(Y/N) 6/7 448/447 0.78 426/434 26/18 0.43 0.22 341/337 109/113 0.53 0.76 375/364 77/88 0.57 0.34 107/112 345/340 0.73 0.70 98/101 348/345 0.81 p-value# 0.23

*2-tailed Fishers Exact Tests # Sliding-window two-marker haplotype analysis using COCA-PHASE.

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Table 7. Family-based association test using FBAT for DRD3 and BDNF single-nucleotide polymorphisms and HBAT for two-marker haplotypes.
Polymorphism rs905568 rs2399504 rs7611535 rs6762200 rs1394016 Ser9Gly rs167770 rs2134655 rs2087017 rs1025398 Promoter C/A C270T BDNF_4 BDNF_3 BDNF_2 Val66Met #informative families 38 34 41 41 42 39 39 31 40 35 3 11 31 28 31 28 S (allele 1/2) 46/36 47/23 35/51 42/46 45/45 45/37 34/46 17/49 41/39 45/31 ND 16/8 38/26 41/21 26/36 23/39 E(S) (allele 1/2) 43/39 51/20 29/58 35/53 50/40 48/34 30/50 22/44 36/44 44/32 ND 17/7 39/25 41/21 23/39 23/39 Var(S) 13.9 9.7 13.2 12.7 13.8 13.5 12.8 9.4 13.5 12.7 ND 3.5 9.7 8.4 9.2 8.7 Z (allele 1) 0.68 -1.12 1.79 1.90 -1.30 -0.88 1.07 -1.72 1.28 0.23 ND -0.45 -0.19 -0.03 0.85 -0.14 P-value 0.50 0.26 0.20 0.07 0.14 0.06 0.19 0.19 0.44 0.38 0.55 0.28 0.19 0.09 0.16 0.20 0.47 0.82 NA ND 0.88 0.65 0.91 0.85 0.98 0.98 0.85 0.39 0.98 0.89 P (Haplotypes) 0.63

ND Not determined.

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rs2399504

rs7611535

rs6762200

rs1394016

rs2134655

rs2087017

rs905568 rs2399504 rs7611535 rs6762200 rs1394016 Ser9Gly rs167770 rs2134655 rs2087017 rs1025398 0.06 0.00-0.23

0 . 1 6 0 . 1 0 0.007 0 . 0 2 0 . 0 9 0 . 2 4 0 . 7 0 0 . 1 6 0 . 2 1 0.01-0.41 0.00-0.32 -0.01-0.16 -0.01-0.19 0.00-0.26 0.06-0.41 0.50-0.83 0.04-0.28 0.05-0.37 1 . 0 0 1 . 0 0 0 . 9 6 0 . 9 7 0 . 9 7 0 . 9 1 0 . 5 3 0.004 0.93-1.00 0.90-1.00 0.83-1.00 0.85-1.00 0.87-1.00 0.60-0.98 0.25-0.73 -0.01-0.24 0.98 0.97 0.79 0.76 0.93 0.18 0.02 0.89-1.00 0.88-1.00 0.66-0.88 0.64-0.84 0.67-0.98 0.02-0.42 -0.01-0.20 0.99 0.87 0.87 0.91 0.24 0.05 0.93-1.00 0.80-0.92 0.78-0.93 0.73-0.97 0.07-0.40 -0.01-0.20 0.98 1.00 0.96 0.28 0.08 0.92-1.00 0.94-1.00 0.81-0.99 0.11-0.43 0.00-0.24 1.00 1.00 0.41 0.11 0.95-1.00 0.86-1.00 0.22-0.57 0.01-0.26 1.00 0.35 0.01 0.83-1.00 0.14-0.53 -0.01-0.17 0.96 0.19 0.82-1.00 0.02-0.46 0.003 -0.01-0.02

0.03 0.99 -0.01-0.17 0.95-1.00 0.12 0.94 0.93 0.01-0.24 0.87-0.98 0.87-0.97

0.20 0.83 0.89 0.93 0.09-0.30 0.72-0.90 0.82-0.94 0.88-0.96 0.20 0.83 0.65 0.83 0.98 0.08-0.31 0.74-0.90 0.56-0.72 0.78-0.87 0.94-1.00

0.26 0.84 0.67 0.82 1.00 1.00 0.12-0.38 0.76-0.90 0.60-0.74 0.76-0.87 0.97-1.00 0.98-1.00 0.73 0.69 0.75 0.82 0.94 0.94 0.98 0.60-0.82 0.42-0.85 0.54-0.87 0.69-0.90 0.85-0.98 0.85-0.98 0.87-1.00

0.30 0.72 0.31 0.36 0.32 0.48 0.46 0.94 0.21-0.37 0.57-0.82 0.16-0.45 0.25-0.46 0.22-0.41 0.37-0.57 0.33-0.57 0.86-0.98 0 . 0 7 0.003 0 . 0 3 0 . 0 8 0 . 0 7 0 . 0 9 0 . 0 7 0 . 2 4 0 . 0 4 0.00-0.17 -0.01-0.14 -0.01-0.13 0.00-0.17 0.00-0.17 0.01-0.18 0.00-0.16 0.05-0.41 -0.01-0.17

Figure 4a. Linkage disequilibrium plot among the 10 DRD3 gene polymorphisms used in the present study. The numbers represents D values and the 95% confidence intervals of D, while the color darkness within each box corresponds to strength of linkage. Values in the upper right triangle were derived from the family sample, while values in the lower left triangle were derived from the case-control sample. The markers boxed in thick lines have the highest linkage disequilibrium given by D>0.90 and lower boundary of the 95% confidence intervals of D>0.70.

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rs905568

rs167770

Ser9Gly

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Promoter C/A

Promoter C/A C270T BDNF_4 BDNF_3 BDNF_2 Val66Met

1.00 1.00 1.00 1.00 1.00 0.05-0.98 0.13-0.99 0.09-1.00 0.13-0.99 0.11-0.99 0.46 0.04-0.96 1.00 1.00 0.22-1.00 0.23-0.99 1.00 1.00 0.97 0.13-0.99 0.16-0.99 0.93-1.00 1.00 1.00 0.98 0.99 0.21-1.00 0.31-1.00 0.95-1.00 0.95-1.00 1.00 1.00 0.98 0.97 1.00 0.18-1.00 0.28-1.00 0.94-1.00 0.92-0.99 0.98-1.00 1.00 1.00 1.00 1.00 0.35-1.00 0.30-1.00 0.36-1.00 0.26-1.00 0.95 0.96 0.96 0.85-0.99 0.91-0.99 0.88-0.99 1.00 1.00 0.93-1.00 0.93-1.00 1.00 0.94-1.00

Figure 4b. Linkage disequilibrium plot among the six BDNF gene polymorphisms used in the present study. The numbers represents D values and the 95% confidence intervals of D, while the color darkness within each box corresponds to strength of linkage. Values in the upper right triangle were derived from the family sample, while values in the lower left triangle were derived from the case-control sample. The markers boxed in thick lines have the highest linkage disequilibrium given by D>0.90 and lower boundary of the 95% confidence intervals of D>0.70.

Val66Met

BDNF_4

BDNF_3

BDNF_2

C270T

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Table 8. Results considering suicidal behaviour in SCZ patients with BDNF and DRD3 polymorphisms.
Polymorphism Assay rs905568 Genotypes 1/1 (C/C) 1/2 (C/G) 2/2 (G/G) P 1/1 (G/G) 1/2 (G/A) 2/2 (A/A) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (C/C) 1/2 (C/T) 2/2 (T/T) P 1/1(A/A) 1/2(A/G) 2/2(G/G) P 1/1 (C/C) 1/2 (C/T) 2/2 (T/T) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (C/C) 1/2 (C/T) 2/2 (T/T) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (A/A) 1/2 (A/C) 2/2 (C/C) P SCZ(Y/N) 35/92 64/118 17/48 0.24 77/169 37/77 3/9 0.91* 12/20 45/95 59/141 0.63 21/46 59/117 36/93 0.58 31/79 65/114 21/60 0.16 37/92 66/117 13/43 0.13 10/33 57/108 49/113 0.34 4/18 44/85 68/155 0.32 21/53 65/123 30/80 0.36 44/112 58/106 15/36 0.36 0/0 4/5 113/255 0.47* Suicide Specifier 1.81+/-1.74 1.81+/-1.87 1.60+/-1.80 0.70 1.72+/-1.82 1.93+/-1.81 1.75+/-1.77 0.59 2.00+/-1.92 1.94+/-1.81 1.63+/-1.80 0.23 1.82+/-1.83 1.87+/-1.84 1.63+/-1.78 0.53 1.68+/-1.79 2.00+/-1.83 1.51+/-1.79 0.094 1.69+/-1.77 1.98+/-1.85 1.43+/-1.75 0.10 1.51+/-1.76 1.92+/-1.86 1.73+/-1.78 0.38 1.50+/-1.57 1.96+/-1.82 1.69+/-1.83 0.31 1.64+/-1.80 1.88+/-1.85 1.71+/-1.76 0.54 1.61+/-1.81 2.01+/-1.85 1.62+/-1.71 0.12 NA 2.33+/-1.87 1.76+/-1.81 0.35 Allele Allele 1 (C) Allele2 (G) P Allele 1 (G) Allele2 (A) P Allele 1 (A) Allele2 (G) P Allele 1 (A) Allele2 (G) P Allele 1 (C) Allele2 (T) P Allele 1 (A) Allele 2 (G) P Allele 1 (C) Allele2 (T) P Allele 1 (A) Allele2 (G) P Allele 1 (C) Allele 2 (T) P Allele 1 (A) Allele2 (G) P Allele 1 (A) Allele2 (C) P SCZ(Y/N) 134/302 98/214 0.84 191/415 43/95 0.93 69/135 163/377 0.34 101/209 131/303 0.49 127/272 107/234 0.90 140/301 92/203 0.87 77/174 155/334 0.78 52/121 180/395 0.76 107/229 125/283 0.72 146/330 88/178 0.50 4/5 230/515 0.47*

rs2399504

rs7611535

rs6762200

rs1394016

Ser9Gly, rs6280

rs167770

rs2134655

rs2087017

rs1025398

Promoter C/A rs28383487 C-281A

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C270T HinfI BDNF_4 rs7103411 BDNF_3 rs2049045 BDNF_2 rs11030104 Val66Met rs6265 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (A/A) 1/2 (A/G) 2/2 (G/G) P 1/1 (C/C) 1/2 (C/G) 2/2 (G/G) P 1/1 (C/C) 1/2 (C/T) 2/2 (T/T) P 1/1(A/A) 1/2(A/G) 2/2(G/G) P 103/227 13/31 1/1 0.76* 70/148 42/82 5/18 0.52 76/187 36/61 5/9 0.29* 7/16 41/78 68/159 0.69 6/15 39/72 71/169 0.56

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1.79+/-1.80 1.68+/-1.91 2.00+/-2.83 0.92 1.81+/-1.85 1.85+/-1.81 1.61+/-1.64 0.85 1.70+/-1.81 1.98+/-1.84 2.00+/-1.71 0.38 1.91+/-1.73 1.88+/-1.83 1.72+/-1.82 0.70 1.90+/-1.70 1.88+/-1.83 1.72+/-1.81 0.69 Allele 1 (A) Allele2 (G) P Allele 1 (A) Allele 2 (G) P Allele 1 (C) Allele2 (G) P Allele 1 (C) Allele2 (T) P Allele 1 (A) Allele 2 (G) P 219/485 15/33 0.98 182/378 52/118 0.64 188/435 46/79 0.15 55/110 177/396 0.55 51/102 181/410 0.52

*p-value calculated from two-tailed Fishers Exact Test. #p-value from Kruskal-Wallis Test.

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Raw p-values rs1025398 rs2087017 rs2134655 rs167770 Ser9Gly rs1394016 rs6762200 rs7611535 rs905568 Val66Met BDNF_2 BDNF_3 BDNF_4 C270T P1CA rs1025398 rs2087017 rs2134655 rs167770 Ser9Gly rs1394016 rs6762200 rs7611535 rs905568 Val66Met BDNF_2 BDNF_3 BDNF_4 C270T P1CA Figure 5. P-values from analyses of two-marker interactions between BDNF and DRD3 polymorphisms in relation to SCZ diagnosis given by HELIXTREE program. Top left triangle indicates significance with the raw p-values, while the bottom right triangle indicates significance with Bonferroni adjusted p-values. 84 10-8 10-7 Bonferroni p-values 10-6 10-5 10-4 10-3 10-2 10-1 1-

Clement Zai

II. GABRG2 and Schizophrenia

Raw p-values rs1025398 rs2087017 rs2134655 rs167770 *Ser9Gly rs1394016 rs6762200 rs7611535 rs905568 *Val66Met BDNF_2 BDNF_3 BDNF_4 C270T P1CA rs1025398 rs2087017 rs2134655 rs167770 *Ser9Gly rs1394016 rs6762200 rs7611535 rs905568 *Val66Met BDNF_2 BDNF_3 BDNF_4 C270T P1CA Figure 6. P-values from analyses of two-marker interactions between BDNF and DRD3 polymorphisms in association with the history of suicide attempt(s) given by HELIXTREE program. Top left triangle indicates significance with the raw p-values, while the bottom right triangle indicates significance with Bonferroni adjusted p-values. *The interaction between BDNF Val66Met and DRD3 Ser9Gly was significant in history of suicide attempt(s). Page 85 10-8 10-7 Bonferroni p-values 10-6 10-5 10-4 10-3 10-2 10-1 1-

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IV. BDNF & DRD3 in Tardive Dyskinesia CHAPTER 4

GENETIC STUDY OF BDNF, DRD3, AND THEIR INTERACTION IN TARDIVE DYSKINESIA

Manuscript to be submitted

Clement C. Zai(1,2), Vincenzo De Luca(1,2), Daniel J. Mller(1,3), Natalie Bulgin (1), Nicole King (1), Aristotle N. Voineskos(1), Herbert Y. Meltzer(4), Jeffrey A. Lieberman(5), Steven G. Potkin(6), Gary Remington(1), James L. Kennedy(1,2)

(1) Centre for Addiction and Mental Health, Toronto, Ontario, CANADA (2) Institute of Medical Science, University of Toronto, Toronto, Ontario, CANADA (3) Department of Psychiatry, Charit University Medicine Berlin, Campus Charit Mitte, Berlin, Germany (4) Psychiatric Hospital at Vanderbilt University, Nashville, Tennessee, USA (5) New York State Psychiatric Institute, Columbia University Medical Centre, New York City, New York, USA (6) Brain Imaging Center, Irvine Hall, University of California at Irvine, California, USA

Mr. Zai designed the experiment (with guidance from faculty), performed all the genotyping for the BDNF and ZNF80 gene polymorphisms and over 90% of the genotyping for the DRD3 gene polymorphisms (50% of the Ser9Gly genotypes were

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reported previously in Basile et al (1999), and the remaining 50% of the Ser9Gly genotypes were performed by Ms. Nancy Chung, a former summer student working with Dr. Vincenzo De Luca). Mr. Zai corresponded with the clinical collaborators to refine the details of the phenotype, performed all the statistical analyses, and wrote the manuscript.

Keywords: Schizophrenia, tardive dyskinesia, genetics, BDNF, DRD3, Abnormal Involuntary Movement Scale (AIMS)

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Zai et al 4.1 ABSTRACT

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Tardive dyskinesia (TD) is a movement adverse effect of long-term antipsychotic medication. The pathophysiology is unclear, but dopamine neurotransmission system changes have been suggested to be involved. Thus, a number of studies have focused on the association of dopamine system gene polymorphisms and TD and most consistent findings have been focused on the Ser9Gly polymorphism of the DRD3 gene. However, as there were negative results and only one polymorphism within DRD3 has been tested thus far, the role of DRD3 in TD is still unclear. Further, Brain derived neurotrophic factor (BDNF), a neuronal growth and survival factor, regulates DRD3 expression and may be involved in neuronal degeneration observed in TD. In the present study, we investigated 10 polymorphisms spanning the DRD3 gene and 6 polymorphisms spanning the BDNF gene for association with TD in our sample (N=223). The rs905568 was found to be associated with TD diagnosis (p=0.015) and AIMS scores (p=0.007) in our European Caucasian sample. Taken together, the present study suggests that DRD3 may be involved in TD development in Caucasian population, though further studies are warranted.

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Zai et al 4.2 INTRODUCTION

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Tardive dyskinesia (TD) is a potentially irreversible motor side effect that develops in about 25% of SCZ patients who are undergoing long-term antipsychotic treatment (reviewed in Tarsy and Baldessarini, 2006). Patients with this condition display orofacial movements that are athetoid, choreoform, or rhythmic in nature. In more severe cases, the movements may involve the trunk and limbs. Due to motor difficulties, patients with TD often struggle with treatment adherence, discrimination, and poorer quality of life (Marsalek, 2000; Gerlach, 2002), so predicting which patients are vulnerable to TD remains a high priority for psychiatrists in treatment selection. The etiology of TD is complex and remains unclear. Age, gender, and ethnicity are all suggested risk factors for TD. More specifically, Woerner and coworkers found patients above the age of 50 were three to five folds more likely to develop TD than younger patients in other studies (Woerner et al, 1998; Kane et al, 1988; Morgenstern and Glazer, 1993). A review of 13 studies found females to be almost 70% more likely to develop TD (Smith and Dunn, 1979), though other studies reported the opposite findings (Morgenstern et al, 1987; van Os et al, 1997). The difference was attributed to possible selection bias for more severe cases in the female gender group in earlier studies. Jeste and coworkers reported the yearly TD incidence of over 45% for African-Americans compared to 27% in Caucasians (Jeste et al, 2000), but socioeconomic factors could have contributed to the findings. Smoking, drinking, and using street drugs could further increase the risk for TD (Menza et al, 1991; Bailey et al, 1997; Olivera et al, 1990). TD appears to be more common among first-degree relatives, thus providing evidence for a genetic component of TD (Mller et al, 2001). A number of mechanisms leading to TD

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have been hypothesized, with the best known hypothesis postulating TD as being caused by hypersensitivity of dopamine receptors induced by dopamine receptor blocking medication including antipsychotics (Tarsy and Baldessarini, 1977; Klawans et al, 1980; Gerlach and Casey, 1988; Abilio et al, 2003). The dopamine D3 receptor is a member of the G-protein coupled receptor superfamily. By coupling to inhibitory G-proteins, it regulates the production of cyclic AMP in response to dopamine binding. Functionally, D3 is localized to the ventral striatum and putamen of the basal ganglia, an area of the brain that is involved in locomotor control (Joyce and Meador-Woodruff, 1997; Suzuki et al, 1998). D3 levels were increased in response to chronic haloperidol administration in rat brains (Buckland et al, 1992). The increase was also observed in human postmortem schizophrenia patient basal ganglia after neuroleptic treatment (DSouza et al, 1997). R-(+)-7-OH-DPAT, a D3-selective agonist, inhibited locomotor activity when injected into the nucleus accumbens of rats (Kling-Petersen et al, 1995), while D3 antagonists increased motor activity (Kling-Petersen et al, 1995; Fink-Jensen et al, 1998; Gendreau et al, 1997; Van Hartesveldt, 1997). The findings were corroborated in mice lacking functional D3; they were hyperactive (Accili et al, 1996). The D3-coding DRD3 gene has seven exons and is mapped to the chromosomal region 3q13.3. The Ser9Gly polymorphism in the second exon was studied previously because of our initial report of its association with TD (Badri et al, 1996), and an association of the glycine variant with increased D3 affinity for dopamine as revealed in CHO cells (Lundstrom and Turpin, 1996), accompanied by increased activation of ERK and inhibition of cAMP synthesis (Jeanneteau et al, 2006). The glycine variant has also

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been demonstrated in vitro to result in a shift in D3 signaling from inhibition of adenylate cyclase to inhibition of prostaglandin production (Hellstrand et al, 2004). The Ser9Gly polymorphism has been examined for possible association with schizophrenia in numerous studies, with a recent meta-analysis determining the odds ratio to be around 1.10 for homozygosity (Jnsson et al, 2003). DRD3 has received much attention in TD studies in light of several positive association results. Specifically, the Gly variant was found to be associated with increased risk of TD (Badri et al, 1996; Steen et al, 1997; Segman et al, 1999; Basile et al, 1999; Lovlie et al, 2000; Liao et al, 2001). Although these findings could not be replicated in other studies (Inada et al, 1997; Rietschel et al, 2000; Garcia-Barcelo et al, 2001), the Gly allele (or Gly/Gly genotype) has been found associated with TD in a combined analysis involving 780 patients, and two meta-analyses, with the latest reported odds ratio for the glycine allele being 1.17 (Lerer et al., 2002; Bakker et al, 2006). Small sample sizes could have contributed to some of the negative findings, especially if the glycine allele is exerting a small effect on TD risk and severity. The mixed results could also be due to different ethnic backgrounds of the study sample populations. Different variants may contribute to TD in different ethnic groups. However, a comprehensive analysis of the DRD3 gene in TD is still lacking. Brain-derived neurotrophic factor (BDNF) is known to exert wide-ranging effects on the nervous system, including growth, differentiation, and survival of neurons (Lewin and Barde, 1996; Altar and DiStefano, 1998), and support the activities of dopaminergic, glutaminergic, cholinergic, and serotonergic neurons (Altar, 1999). More specifically, the involvement of BDNF in the dopaminergic system (Hyman et al, 1991; Thoenen,

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1995) includes its stimulation of forebrain dopamine release (Altar et al, 1998) and prevention of damage of dopaminergic neurons by neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) (Ebadi et al, 1998). BDNF is required for neuronal establishment in the nigrostriatal dopaminergic system that is implicated in TD (Nishio et al, 1998; Murer et al, 2001; Baquet et al, 2005). BDNF from dopaminergic neurons was demonstrated in vivo to be required for D3 receptor expression in the nucleus accumbens during development and adulthood (Guillin et al, 2001). Several studies found BDNF mRNA expression to be decreased in the rat hippocampus after haloperidol treatment while clozapine appeared to increase BDNF (Chlan-Fourney et al, 2002; Bai et al, 2003), but others did not report the differences between typical and atypical antipsychotics on BDNF levels (Angelucci et al, 2000; Lipska et al, 2001). Recently, Tan and coworkers found decreased plasma BDNF levels in schizophrenia patients with TD compared to those without (Tan et al, 2005). The BDNF gene, located on 11p13, contains a number of putative functional polymorphisms. Magnetic resonance imaging scans showed that the Met66 allele carriers had significantly lower average hippocampal volume compared to Val66/Val66 (Pezawas et al, 2004). The C270T polymorphism was shown to affect BDNF mRNA stability (Tongiorgi et al, 2006). The promoter C-281A polymorphism was shown to decrease in vitro DNA-binding activity and basal reporter gene activity in cultured neurons (Jiang et al, 2005). While BDNF Val66Met and C270T polymorphisms do not appear to confer a strong risk for schizophrenia (Xu et al, 2007; Zintzaras et al, 2007; Kanazawa et al, 2007), only the Val66Met polymorphism has been tested in one genetic study of TD (Liou et al, 2004).

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Because BDNF is required for D3 expression, and neither BDNF nor DRD3 has been extensively examined in TD, we investigated the possible role of polymorphisms spanning the DRD3 and BDNF genes as well as their interactions in TD risk and severity.

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Zai et al 4.3 PATIENTS AND METHODS 4.3.1 Subjects

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Subjects were recruited from four clinical sites in North America: Center for Addiction and Mental Health in Toronto, Ontario (Dr. G Remington, N=92); Case Western Reserve University in Cleveland, Ohio (Dr. HY Meltzer, N=69); Hillside Hospital in Glen Oaks, New York (Dr. JA Lieberman, N=50); University of California at Irvine, California (Dr. SG Potkin, N=12). Subjects were selected based on their diagnoses for Schizophrenia or Schizoaffective Disorder according to DSM-III-R or IV (APA, 2000). All patients have undergone at least one year of treatment with typical or atypical antipsychotics. The presence of TD was assessed using the Abnormal Involuntary Movement Scale (AIMS) or the modified Hillside Simpson Dyskinesia Scale (HSDS) for 50 patients recruited from the Hillside Hospital (Guy, 1976; Schooler and Kane, 1982; Basile et al, 1999). AIMS scores were available for 161 patients. In all, 223 schizophrenia patients were studied. 193 of them were European Caucasians, of which 76 were positive for the diagnosis of TD. The remaining 30 were African-Americans, of which 11 were positive for TD. Because of small sample size, the African-Americans were only used in the test for allele frequency association with TD.

4.3.2 Gene polymorphism analysis Genomic DNA was purified from whole blood samples using non-enzymatic method previously described (Lahiri and Nurnburger, 1991). 10L Polymerase Chain Reactions on 20ng genomic DNA were performed using Assays-on-Demand (ABI) with the following conditions: 95oC 10min, followed by 50 cycles of 92oC 15sec, 60oC 1min.

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Genotyping was done after the subjects have completed the follow-up in which all laboratory staff was blind to the AIMS scores. The assays with their corresponding polymorphisms and locations are shown in Table 1 and Figure 1. They are as follows: for DRD3: rs905568, rs2399504, rs7611535, rs6762200, rs1394016, rs6280 (Ser9Gly), rs167770, rs2134655, rs2087017, rs1025398; for BDNF: Promoter C/A (C-281A), C270T (HinfI), rs7103411 (BDNF_4), rs2049045 (BDNF_3), rs11030104 (BDNF_2), rs6265 (Val66Met); for ZNF80: rs6438191 (R201H), rs3732781 (Y245stop), rs3732782 (D253A). Allelic discrimination was performed using ABI7000. More details about the polymorphisms used in the present study are presented in Table 9.

4.3.3 Statistics Statistical analyses were conducted using the SPSS program Student version 14.0, Haploview version 3.3 (Barrett et al, 2005), and UNPHASED version 2.0 (Dudbridge, 2003). Odds ratio calculations were conducted using Program 2BY2 version 2 written by Jurg Ott. Genotype frequency distribution was tested for fitness to Hardy-Weinberg equilibrium using Haploview. The association of genotype frequencies with age and AIMS was assessed using ANOVA, and where the variances of AIMS scores among genotypes differed significantly using the Levenes Test for Homogeneity of Variances, AIMS was tested with the Kruskal-Wallis test on SPSS. Gender differences in genotype frequencies were assessed using the 2 test on SPSS. The differences in allele and genotype frequencies between patients with and without TD were analyzed by 2 test on SPSS. For contingency tables with at least one expected cell count of less than five, twotailed Fishers Exact Tests were performed (URL: http://home.clara.net/sisa/fiveby2.

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htm). Haplotype analyses and linkage disequilibrium calculations were conducted using UNPHASED and Haploview respectively. Gene-gene interaction analysis was conducted using HELIXTREE program (GoldenHelix). .

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The genotype distributions of all 19 polymorphisms in the ZNF80, DRD3, and BDNF genes in the European Caucasian samples did not differ significantly from HardyWeinberg equilibrium (p>0.10). We did not observe significantly different average age among the genotypes for any of the 19 polymorphisms. A significant association was found between genotype frequencies of three DRD3 polymorphisms and sex (rs2399504, rs7611535, rs167770; p<0.05, Table 2a). Analyzing the presence or absence of TD and AIMS in males and females separately did not yield significant results.

4.4.2 Association study of DRD3 polymorphisms and haplotypes with TD and AIMS With our Caucasian sample, we found the rs905568 genotype and allele frequencies to be significantly associated with TD diagnoses (p=0.015, Table 10a, and p=0.002, Table 11). Specifically, the G allele is under-represented in the TD-positive group of patients (ORG = 0.49, CI: 0.32-0.77). Similarly, fewer TD-positive patients were observed with the GG genotype than expected (ORGG = 0.32, CI: 0.11-0.88). The other DRD3 polymorphisms analyzed did not show significant allele or genotype association with TD. We compared the average total AIMS scores among the genotype groups for each polymorphism. rs905568 showed significantly different AIMS scores among its genotype groups (p=0.007, Table 10a), with patients having the GG genotype also having the lowest average total AIMS scores compared to patients with other genotypes.

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Because the rs905568 polymorphism is located adjacent to the gene coding for a putative zinc-finger protein (ZNF80), we investigated whether the rs905568 association with TD may have been due to an association of ZNF80 with TD. We genotyped three non-synonymous polymorphisms in ZNF80, and found that the ZNF80 polymorphisms were not associated with TD diagnosis or AIMS scores (Table 10b). Using the Haploview program, strong evidence for linkage was found between markers (Figure 7a). As an exploratory test, we analyzed two-marker haplotypes across ZNF80 and DRD3 using the sliding window approach in UNPHASED. Only windows containing rs905568 showed significant association with TD and AIMS (Table 12).

4.4.3 Association study of BDNF polymorphisms and haplotypes with TD and AIMS For BDNF, the promoter C/A genotype and allele distributions were associated with TD, but the results did not reach statistical significance (Tables 10b and 11). The other BDNF polymorphisms did not show significant association with TD diagnosis or AIMS scores (Table 10b and 11). The two-marker haplotypes of BDNF were not significantly associated with TD in our European Caucasian sample (Table 12).

4.4.4 Interaction analysis of BDNF and DRD3 polymorphisms in TD Because of the functional relationship between BDNF and DRD3 in vivo, we performed interaction analysis with polymorphisms between BDNF and DRD3 using HELIXTREE. We did not find significant association between any BDNF-DRD3 twomarker combinations in TD occurrence or in AIMS (Figure 8).

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For the African-American sample, preliminary results indicated a significant association between TD and alleles of the rs6762200 polymorphism (p=0.017) and rs1394016 (p=0.048)(Table 11). Neither Ser9Gly nor rs905568 was associated with TD in our African American sample.

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Zai et al 4.5 DISCUSSION

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The present study reports that the region about 50kb upstream of the DRD3 gene is associated with TD. The region does not appear to extend into the ZNF80 gene because the ZNF80 polymorphisms are not associated with TD in our sample. The present study also reports that BDNF is not associated with TD. The rs905568 association with TD might be a false-positive result from multiple testing; however, this possibility is unlikely because the rs905568 results remained significant after correction for testing 10 DRD3 markers. Nevertheless, larger sample sizes are required to detect small effects of genotypes on TD risk and severity, especially for our AfricanAmerican sample, where two other polymorphisms in the 5 region of DRD3 are associated with TD. Even though Ser9Gly was not significantly associated with TD in our African American sample, there is a trend for the Gly allele to be the risk allele for TD, which is in agreement with previous studies in which African Americans have a higher frequency of the Gly allele (Basile et al, 1999). Age, sex, and ethnicity are suggested risk factors for TD (Basile et al, 1999; Jeste, 2000; van Os et al, 1997; Kaiser et al, 2002). Age was not likely responsible for the positive findings in this study, because mean age did not differ significantly among the genotypes of any of the polymorphisms in the present study. Also, rs905568 could be in linkage disequilibrium with a nearby polymorphism that directly affects TD, possibly through variations in transcription factor binding sites. The present study has several limitations. Firstly, not all clinical data was available for our study. These include medication history like antipsychotic dose and duration, schizophrenia disease history such as clinical subtypes, psychopathology, and co-morbidities. These factors or variables are also associated with TD according to

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previous studies (reviewed in Mller et al, 2004). It was noted that medications taken by patients for other adverse effects could have masked the TD phenotype (Shale and Tanner, 1996; Egan et al, 1997; Glazer, 2000). Moreover, environmental risk factors such as smoking, alcohol and substance use could increase the risk of TD and contribute to our findings, though we do not have the information available for our entire sample (Menza et al, 1991). Further, our sample was drawn from four different clinical sites. Even though the study Caucasian population was in Hardy-Weinberg equilibrium for all 10 polymorphisms and the sex ratios among the four geographical groups do not differ significantly (p>0.1), the mean ages differ significantly among them (p<0.001). Therefore, the possibility of ascertainment bias cannot be ignored. However, when we analyzed the rs905568 polymorphism in subjects recruited from the US and Canada separately, the same trend remained for both sub-samples. The DRD3 gene is unlikely to be the only genetically determined factor for TD, as other genes have been found associated with TD. Meta-analyses have identified DRD2 and HTR2A to be associated with TD (Zai et al, 2007b; Lerer et al, 2005). Studies in other genes such as HTR2C, CYP1A2, and manganese superoxide dismutase require replication studies to confirm their association (Basile et al, 2000; Segman et al, 2000; Schulze et al, 2001; Hori et al, 2000). As all antipsychotics target more than one receptor, it is likely that TD is a polygenic condition with each gene contributing a small proportion of the risk to the disorder. Additional gene-gene interaction studies may help in identifying and clarifying pathways that contribute to TD. TD risk is also likely to be influenced by many environmental factors. Acquiring these information will help immensely in limiting effects of potential confounders in genetic studies of TD. The

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present study encourages further studies into the rs905568 and adjacent polymorphisms surrounding the 5 region of the DRD3 gene in TD.

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Table 9. ABI Assays-on-Demand and Assays-by-Design with information on their corresponding BDNF and DRD3 polymorphisms used in the study. (See Figures 3a, 3b on page 72) Gene
DRD3 DRD3 DRD3 DRD3 DRD3 DRD3 DRD3 DRD3 DRD3 DRD3 BDNF BDNF BDNF BDNF BDNF BDNF ZNF80 ZNF80 ZNF80

Assay-onDemand / -by-Desgn
rs905568 rs2399504 rs7611535 rs6762200 rs1394016 rs6280 rs167770 rs2134655 rs2087017 rs1025398 rs6265 rs7103411 rs2049045 rs11030104 HinfI rs28383487 rs6438191 rs3732781 rs3732782

Allele FAM C G A A C A(S) C A C A A(M) A C C G A

Allele VIC G A G G T G(G) T G T G G(V) G G T A C

A(H) G(R) G(Stop) T(Y) A(D) C(A)

Polymorphism Location in DRD3 Name(s) gene CG Promoter CT Promoter CT Promoter CT Promoter AG Promoter Ser9Gly Exon 2 A11277G Intron 2 C32638T Intron 5 A48826G 3 CT 3 Val66Met Exon 2 BDNF_4 Intron 1 BDNF_3 Intron 1 BDNF_2 Intron 1 C270T Intron 1 C-281A, Promoter P1CA His201Arg Exon 1 Tyr245Stop Exon 1 Ala253Asp Exon 1

References Talkowski et al, 2006 Talkowski et al, 2006 Talkowski et al, 2006 Talkowski et al, 2006 Talkowski et al, 2006 Pezawas et al, 2004

Tongiorgi et al, 2006 Jiang et al, 2005

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Table 10a. Statistical analyses on demographics (sex, age) as well as total AIMS scores and TD diagnoses with DRD3 genotypes.
DRD3 markers rs905568 N (M/F) Age (years) Total AIMS score 1/1 (C/C) 62(41/21) 38.08+/-9.03 7.33+/-7.32 1/2 (C/G) 103(64/39) 37.73+/-10.43 6.28+/-8.06 2/2 (G/G) 26(22/4) 37.08+/-10.04 2.64+/-4.55 P 0.910 0.095 0.007! rs2399504 1/1 (G/G) 122(88/34) 38.31+/-9.95 6.09+/-8.14 1/2 (G/A) 65(38/27) 36.98+/-10.25 6.22+/-6.40 2/2 (A/A) 5(2/3) 37.80+/-6.65 4.50+/-4.20 P 0.689 0.909 0.060* rs7611535 1/1 (A/A) 14(5/9) 35.64+/-9.24 5.10+/-4.75 1/2 (A/G) 82(56/26) 37.82+/-9.89 5.81+/-7.16 2/2 (G/G) 96(67/29) 38.20+/-10.18 6.44+/-8.11 P 0.671 0.800 0.045* rs6762200 1/1 (A/A) 30(16/14) 34.87+/-8.82 6.13+/-6.38 1/2 (A/G) 95(65/30) 38.03+/-9.46 5.18+/-6.87 2/2 (G/G) 65(45/20) 39.05+/-10.59 7.50+/-8.67 P 0.259 0.152 0.261! rs1394016 1/1 (C/C) 58(40/18) 38.67+/-10.84 7.44+/-8.85 1/2 (C/T) 102(71/31) 37.75+/-9.97 5.07+/-6.44 2/2 (T/T) 33(18/15) 36.67+/-8.13 6.65+/-7.86 P 0.256 0.648 0.370! Ser9Gly 1/1(A/A) 71(51/20) 38.46+/-10.19 6.87+/-8.50 1/2(A/G) 93(62/31) 37.03+/-10.68 5.44+/-6.85 2/2(G/G) 24(12/12) 37.58+/-7.74 5.22+/-6.99 P 0.147 0.671 0.490 rs167770 1/1 (C/C) 16(5/11) 37.88+/-7.96 7.00+/-8.22 1/2 (C/T) 87(60/27) 37.92+/-10.33 5.77+/-6.34 2/2 (T/T) 89(63/26) 37.78+/-10.01 6.24+/-8.42 P 0.995 0.848 0.007 rs2134655 1/1 (A/A) 9(5/4) 38.33+/-14.98 6.50+/-5.68 1/2 (A/G) 69(48/21) 38.38+/-9.34 6.19+/-7.51 2/2 (G/G) 112(75/37) 37.41+/-9.86 5.86+/-7.69 P 0.619* 0.807 0.954 rs2087017 1/1 (C/C) 36(26/10) 38.69+/-9.47 6.55+/-8.65 1/2 (C/T) 95(64/31) 36.31+/-9.83 6.00+/-7.64 2/2 (T/T) 60(37/23) 40.15+/-9.81 6.08+/-6.71 P 0.551 0.942 0.053 rs1025398 1/1 (A/A) 67(44/23) 38.85+/-9.23 6.68+/-7.12 1/2 (A/G) 96(67/29) 36.92+/-10.20 6.04+/-7.79 2/2 (G/G) 28(16/12) 39.07+/-10.74 5.09+/-7.82 P 0.452 0.381 0.698 * With at least 1 expected cell count <5; Fisher Exact Test used. ! Variances among comparisons groups differ significantly; Kruskal-Wallis test used. Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05. TD (Yes/No) 32/30 39/64 5/21 0.015 48/74 27/38 1/4 0.769* 6/8 31/51 39/57 0.898 12/18 33/62 31/34 0.259 26/32 39/63 11/22 0.527 32/39 35/58 6/18 0.207 5/11 34/53 37/52 0.733 5/4 28/41 41/71 0.489* 13/23 42/53 21/39 0.460 30/37 38/58 8/20 0.338

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Table 10b. Statistical analyses on demographics (sex, age) as well as total AIMS scores and TD diagnoses with BDNF and ZNF80 genotypes.
Markers Promoter C/A N (M/F) Age (years) Total AIMS score 1/1 (/) 0(0/0) N/A N/A 1/2 (/) 7(6/1) 37.29+/-6.58 3.50+/-4.73 2/2 (/) 185(122/63) 37.87+/-10.08 6.16+/-7.58 P 0.428* 0.879 0.487 C270T 1/1 (/) 171(115/56) 37.68+/-9.86 5.86+/-7.38 1/2 (/) 18(10/8) 38.89+/-11.48 8.72+/-8.70 2/2 (/) 2(2/0) 45.50+/-3.54 2.00+/-2.83 P 0.438* 0.494 0.234 BDNF_4 1/1 (A/A) 118(83/35) 38.96+/-10.11 5.47+/-6.93 1/2 (A/G) 63(37/26) 36.17+/-9.65 6.57+/-7.86 2/2 (G/G) 9(7/2) 37.44+/-6.89 13.50+/-12.08 P 0.237* 0.194 0.035 BDNF_3 1/1 (C/C) 133(95/38) 38.05+/-10.07 5.70+/-7.17 1/2 (C/G) 55(30/25) 37.82+/-9.90 6.76+/-7.98 2/2 (G/G) 4(3/1) 31.50+/-5.51 12.00+/-14.18 P 0.060* 0.434 0.288 BDNF_2 1/1 (C/C) 10(8/2) 37.40+/-10.63 12.38+/-11.75 1/2 (C/T) 67(40/27) 36.67+/-9.96 6.26+/-7.27 2/2 (T/T) 115(80/35) 38.57+/-9.92 5.49+/-7.11 P 0.291* 0.459 0.211! Val66Met 1/1 (/) 4(4/0) 36.75+/-7.04 9.67+/-15.89 1/2 (/) 64(37/27) 36.86+/-10.08 6.90+/-8.04 2/2 (/) 125(88/37) 38.38+/-9.96 5.57+/-7.02 P 0.085* 0.595 0.651! rs6438191 1/1 (A/A) 90(62/28) 37.64+/-9.36 6.19+/-7.25 1/2 (A/G) 88(54/34) 37.86+/-10.72 6.41+/-8.01 2/2 (G/G) 13(11/2) 40.08+/-8.86 3.91+/-6.25 P 0.211* 0.713 0.592 rs3732781 1/1 (G/G) 10(4/6) 33.90+/-7.08 5.14+/-8.69 1/2 (G/T) 96(65/31) 38.42+/-9.47 6.41+/-6.82 2/2 (T/T) 85(58/27) 37.93+/-10.53 5.90+/-8.29 P 0.209* 0.388 0.862 rs3732782 1/1 (A/A) 14(11/3) 41.14+/-9.40 5.00+/-7.06 1/2 (A/C) 88(55/33) 37.88+/-10.52 6.12+/-7.91 2/2 (C/C) 88(61/27) 37.34+/-9.49 6.27+/-7.33 P 0.460* 0.417 0.865 * With at least 1 expected cell count <5; Fisher Exact Test used. ! Variances among comparisons groups differ significantly; Kruskal-Wallis test used. Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05. TD (Yes/No) 0/0 1/6 75/110 0.248* 65/106 10/8 1/1 0.331* 45/73 26/37 5/4 0.561* 52/81 22/33 2/2 0.954* 5/5 28/39 43/72 0.633* 1/3 27/37 48/77 0.748* 37/53 36/52 3/10 0.443 4/6 43/53 29/56 0.332 4/10 34/54 37/51 0.617

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Table 11. Results from 2 test of allele frequencies of each of the 19 polymorphisms versus TD diagnoses for our Caucasian and African-American samples.
DRD3 markers BDNF markers TD (Yes/No) TD (Yes/No) Caucasian Black Caucasian Black rs905568 Allele 1 (C) 99/120 18/23 Promoter Allele 1 () 0/6 0/0 Allele2 (G) 43/106 8/15 C/A Allele 2 () 146/226 22/38 P 0.476 P N/A 0.002 0.086* rs2399504 Allele 1 (G) 117/185 18/30 C270T Allele 1 () 136/220 21/34 Allele2 (A) 29/45 6/6 Allele 2 () 10/10 1/4 P 0.944 0.517* P 0.292 0.643* rs7611535 Allele 1 (A) 42/67 6/8 BDNF_4 Allele 1 (A) 113/177 1/1 Allele2 (G) 100/165 20/30 Allele 2 (G) 35/45 21/37 P 0.885 0.847 P 0.439 0.999* rs6762200 Allele 1 (A) 57/98 19/19 BDNF_3 Allele 1 (C) 120/193 1/1 Allele2 (G) 89/130 3/15 Allele 2 (G) 24/37 21/37 P 0.450 P 0.883 0.999* 0.017 rs1394016 Allele 1 (C) 84/127 2/10 BDNF_2 Allele 1 (C) 36/49 21/37 Allele2 (T) 58/105 24/26 Allele 2 (T) 110/183 1/1 P 0.404 P 0.423 0.999* 0.048 Ser9Gly Allele 1 (A) 98/134 4/9 Val66Met Allele 1 () 28/41 18/35 Allele 2 (G) 48/92 22/25 Allele 2 () 122/187 4/3 P 0.128 0.302 P 0.866 0.405* rs167770 Allele 1 (C) 43/75 13/24 ZNF80 markers Allele2 (T) 101/157 7/14 P 0.616 0.890 rs2134655 Allele 1 (A) 38/49 3/7 rs6437191 Allele 1 (A) 110/158 17/25 Allele2 (G) 110/183 23/31 Allele 2 (G) 42/72 5/13 P 0.303 0.510* P 0.443 0.350 rs2087017 Allele 1 (C) 66/97 14/18 rs3732781 Allele 1 (G) 51/65 2/4 Allele 2 (T) 80/131 8/20 Allele 2 (T) 101/165 20/34 P 0.613 0.224 P 0.271 0.999* rs1025398 Allele 1 (A) 93/132 18/25 rs3732782 Allele 1 (A) 42/74 4/13 Allele2 (G) 51/96 8/11 Allele 2 (C) 108/156 16/25 P 0.199 0.986 P 0.388 0.258 * with at least one expected cell count <5. Fisher Exact Test was used. Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05.

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rs6438191

rs3731781

rs3732782

rs2399504

rs7611535

rs6762200

rs1394016

rs2134655

rs2087017

rs6438191 rs3732781 rs3732782 rs905568 rs2399504 rs7611535 rs6762200 rs1394016 Ser9Gly rs167770 rs2134655 rs2087017 rs1025398

1.00 1.00 0.91 0.04 0.07 0.26 0.22 0.20 0.15 0.69 0.26 0.02 0.82-1.00 0.97-1.00 0.82-0.96 -0.01-0.25 0.00-0.43 0.05-0.47 0.03-0.44 0.02-0.44 0.01-0.44 0.37-0.86 0.09-0.41 -0.01-0.19 0.94 0.93 0.23 0.33 0.37 0.47 0.36 0.72-0.99 0.78-0.98 0.02-0.69 0.05-0.60 0.11-0.57 0.22-0.64 0.08-0.58 0.90 0.01 0.12 0.27 0.24 0.23 0.81-0.95 0.00-0.49 0.01-0.46 0.05-0.48 0.04-0.45 0.03-0.46 0.27 0.31 0.05 0.26 0.03-0.56 0.14-0.45 0.00-0.25 0.04-0.48 0.21 0.69 0.26 0.01 0.02-0.48 0.36-0.86 0.09-0.41 -0.01-0.27

0.31 0.19 0.03 0.05 0.06 0.23 0.82 0.15 0.15 0.04-0.60 0.02-0.43 -0.01-0.18 -0.01-0.20 0.00-0.21 0.04-0.44 0.60-0.92 0.03-0.28 0.02-0.28 1.00 1.00 0.94 0.92 0.91 0.67 0.75 0.32 0.94-1.00 0.91-1.00 0.81-0.98 0.79-0.97 0.80-0.97 0.21-0.88 0.51-0.88 0.06-0.58 1.00 0.94 0.73 0.76 0.76 0.18 0.12 0.95-1.00 0.85-0.98 0.61-0.82 0.66-0.83 0.45-0.90 0.02-0.37 0.00-0.34 0.92 0.88 0.87 0.68 0.39 0.05 0.86-0.96 0.81-0.93 0.77-0.93 0.43-0.82 0.23-0.53 0.00-0.25 0.99 0.98 1.00 0.42 0.01 0.93-1.00 0.91-1.00 0.88-1.00 0.26-0.55 -0.01-0.17 1.00 0.94 0.65 0.00 0.95-1.00 0.73-0.99 0.50-0.77 -0.01-0.16 0.92 0.53 0.06 0.64-0.98 0.34-0.68 0.00-0.30 0.96 0.43 0.81-0.99 0.13-0.65 0.04 -0.01-0.19

Figure 7a. Linkage disequilibrium plot among the three ZNF80 and ten DRD3 gene polymorphisms used in the present study. The numbers represents D values and the 95% confidence intervals of D, while the color darkness within each box corresponds to strength of linkage. The blocks (1, 2, and 3) encompass areas with highest linkage disequilibrium given by D>0.90 and lower boundary of the 95% confidence intervals of D>0.70.

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rs1025398

rs905568

rs167770

Ser9Gly

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Promoter C/A

Promoter C/A C270T BDNF_4 BDNF_3 BDNF_2 Val66Met

1.00 0.96 1.00 1.00 1.00 0.05-0.97 0.05-0.97 0.05-0.97 0.05-0.97 0.06-0.98 0.45 0.48 0.38 0.82 0.04-0.86 0.04-0.96 0.03-0.85 0.06-0.97 0.91 0.94 0.89 0.81-0.97 0.87-0.98 0.79-0.94 1.00 0.94 0.94-1.00 0.86-0.98 0.96 0.89-0.99

Figure 7b. Linkage disequilibrium plot among the six BDNF gene polymorphisms used in the present study. The numbers represents D values and the 95% confidence intervals of D, while the color darkness within each box corresponds to strength of linkage. The blocks (1, 2, and 3) encompass areas with highest linkage disequilibrium given by D>0.90 and lower boundary of the 95% confidence intervals of D>0.70.

Val66Met

BDNF_4

BDNF_3

BDNF_2

C270T

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Table 12. P-values from analyses of two-marker haplotypes across ZNF80, DRD3, and BDNF in association to TD and AIMS using COCA-PHASE and QT-PHASE respectively. Haplotype across ZNF80 and DRD3 rs6438191-rs3731781 rs3731781-rs3731782 rs3731782-rs905568 rs905568-rs2399504 rs2399504-rs7611535 rs7611535-rs6762200 rs6762200-rs1394016 rs1394016-rs6280 rs6280-rs167770 rs167770-rs2134655 rs2134655-rs2087017 rs2087017-rs1025398 Haplotype across BDNF P1CA-C270T C270T-BDNF_4 BDNF_4-BDNF_3 BDNF_3-BDNF_2 BDNF_2-Val66Met P-value (TD+/-) 0.533 0.458 0.005 0.052 0.983 0.318 0.300 0.202 0.234 0.651 0.640 0.217 P-value (TD+/-) 0.164 0.456 0.089 0.517 0.249 P-value (AIMS) 1.000 0.828 0.016 0.086 0.652 0.448 0.351 0.544 0.400 0.947 1.000 0.552 P-value (AIMS) 0.410 0.102 1.000 0.157 0.134

Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05.

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Raw p-values rs1025398 rs2087017 rs2134655 rs167770 Ser9Gly rs1394016 rs6762200 rs7611535 rs905568 rs3732782 rs3732781 rs6438191 Val66Met BDNF_2 BDNF_3 BDNF_4 C270T P1CA rs1025398 rs2087017 rs2134655 rs167770 Ser9Gly rs1394016 rs6762200 rs7611535 rs905568 rs3732782 rs3732781 rs6438191 Val66Met BDNF_2 BDNF_3 BDNF_4 C270T P1CA Figure 8. P-values from analyses of two-marker interactions between BDNF and DRD3 polymorphisms in association to AIMS given by HELIXTREE program. Top left triangle indicates significance with the raw p-values, while the bottom right triangle indicates significance with Bonferroni adjusted p-values Page 110 10-8 10-7 Bonferroni p-values 10-6 10-5 10-4 10-3 10-2 10-1 1-

Clement Zai CHAPTER 5

V. DRD2 and Tardive Dyskinesia

ASSOCIATION STUDY OF TARDIVE DYSKINESIA AND TWELVE DRD2 POLYMORPHISMS IN SCHIZOPHRENIA PATIENTS

Published in the International Journal of Neuropsychopharmacology as an original research paper

Clement C. Zai(1,2), Rudi W. Hwang(1,2), Vincenzo De Luca(1,2), Daniel J. Mller(1,3), Nicole King (1), Gwyneth C. Zai(1,2), Gary Remington(1), Herbert Y. Meltzer(4), Jeffrey A. Lieberman(5), Steven G. Potkin(6), James L. Kennedy(1,2)

(1) Centre for Addiction and Mental Health, Toronto, Ontario, CANADA (2) Institute of Medical Science, University of Toronto, Toronto, Ontario, CANADA (3) Department of Psychiatry, Charit University Medicine Berlin, Campus Charit Mitte, Berlin, Germany (4) Psychiatric Hospital at Vanderbilt University, Nashville, Tennessee, USA (5) New York State Psychiatric Institute, Columbia University Medical Centre, New York City, New York, USA (6) Brain Imaging Center, Irvine Hall, University of California at Irvine, California, USA

Mr. Zai designed the experiment (with guidance from faculty), performed genotyping on the DRD2 polymorphisms in approximately 50% of the tardive dyskinesia sample. Rudi Hwang,

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another graduate student in the lab, genotyped the remaining 50% of the sample and used them to analyze another phenotype, not tardive dyskinesia. Mr. Zai corresponded with the clinical collaborators to refine the details of the phenotype, performed all the statistical analyses, wrote the manuscript, responded to reviewers comments from the journal and edited the text accordingly.

Keywords: Schizophrenia, tardive dyskinesia, genetics, DRD2, Abnormal Involuntary Movement Scale (AIMS)

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Clement Zai 5.1 ABSTRACT

V. DRD2 and Tardive Dyskinesia

Tardive dyskinesia (TD) is a side effect of chronic antipsychotic medication. Abnormalities in dopaminergic activity in the nigro-striatal system have been most often suggested to be involved because the agents which cause TD share in common potent antagonism of dopamine D2 receptors (DRD2), that notably is not balanced by effects such as more potent serotonin 5HT2A antagonism. Thus, a number of studies have focused on the association of dopamine system gene polymorphisms and TD. The most consistent findings have been found with the Ser9Gly polymorphism of the DRD3 gene. Although the DRD2 has long been hypothesized to be the main target for antipsychotics, only a few polymorphisms in DRD2 have been investigated for their potential involvement in the etiology of TD. In the present study, we investigated 12 polymorphisms spanning the DRD2 gene and their association with TD in our European Caucasian (N=202) and African-American (N=30) samples. Genotype frequencies for a functional polymorphism, C957T (Duan et al, 2003; Hirvonen et al, 2004), and the adjacent C939T polymorphism were found to be significantly associated with TD (p=0.013 and p=0.022, respectively). DRD2 genotypes were not significantly associated with TD severity as measured by AIMS (Abnormal Involuntary Movement Scale) with the exception of a trend for C939T (p=0.071). Both TD and total AIMS scores were found to be significantly associated with two-marker haplotypes containing C939T and C957T (p=0.021 and p=0.0087, respectively). Preliminary results indicated that C957T was also associated with TD in our African-American sample (p=0.047). Taken together, the present study suggests that DRD2 may be involved in TD in the Caucasian population, though further studies are warranted.

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Clement Zai 5.2 INTRODUCTION

V. DRD2 and Tardive Dyskinesia

Tardive dyskinesia (TD) is a potentially irreversible motor side effect that develops in SCZ patients treated chronically with typical antipsychotic drugs. It is characterized by involuntary choreoathetotic movements mostly in the orofacial regions, with more severe cases involving the trunk as well as the upper and lower limbs. Its reported prevalence varies from 16% to 43%, with an annual incidence rate of around 5% (reviewed in Tarsy and Baldessarini, 2006). Patients with this condition often struggle with the immediate difficulties of motor function, but also with adhering to treatment, discrimination, and poorer quality of life (Marsalek, 2000; Gerlach, 2002), so predicting which patients are vulnerable to TD remains a high priority for psychiatrists in treatment selection. The etiology of TD is complex and remains unclear. Age, gender, and ethnicity are all suggested risk factors for TD. More specifically, Woerner and coworkers found patients above age 50 were three to five times more likely to develop TD than younger patients in other studies (Woerner et al, 1998; Kane et al, 1988; Morgenstern and Glazer, 1993). A review of 13 studies found females to be more likely to develop TD with an odds ratio of 1.69 (Smith and Dunn, 1979), though other studies reported the opposite findings (Morgenstern et al, 1987; van Os et al, 1997). The difference was attributed to possible selection bias for more severe cases in the female group in earlier studies. Jeste and coworkers reported the yearly TD incidence of over 45% for African-Americans compared to 27% in Caucasians (Jeste et al, 2000), but socioeconomic factors could have contributed to the findings. Environmental risk factors such as smoking as well as alcohol and recreational drug use could further increase the risk for TD (Menza et al, 1991; Bailey et al, 1997; Olivera et al, 1990). Concordance for the presence or absence of TD among first-degree relatives provided evidence for a genetic component of TD

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(Mller et al, 2001). A number of mechanisms leading to TD have been hypothesized, including GABA insufficiency (Casey, 2000), free radical-mediated neuronal injury (Andreasson and Jorgensen, 2000), and structural abnormalities in brain regions involved in motor function such as the caudate nucleus (Chakos et al, 1994; Corson et al, 1999). The best known hypothesis postulates TD as being caused by hypersensitivity of dopamine receptors induced by dopamine receptor blocking medication including antipsychotics (Tarsy and Baldessarini, 1977; Klawans et al, 1980; Gerlach and Casey, 1988; Abilio et al, 2003). This hypothesis is based on several observations. Persistent dyskinesia was induced by neuroleptic treatment in a non-human primate model of TD with significantly decreased dopamine turnover in the caudate and substantia nigra (Gunne et al, 1984). The dopamine D2 receptor is most densely expressed in the basal ganglia, an area of the central nervous system that regulates movements (Hall et al, 1994). Moreover, clinical studies (Kane et al, 1993; Beasley et al, 1999; Jeste et al, 1999a, b) and rodent vacuous chewing TD models (Johansson et al, 1986; Gao et al, 1998) have revealed that compared to atypical antipsychotics, TD is more likely to develop after treatment with typical antipsychotics that generally have higher affinities for D2 given by in vitro competition assays (Schwartz et al, 2000). Some antipsychotic drugs that have high affinities for D2 have a lower risk of causing TD, e.g. olanzapine, risperidone and ziprasidone. This appears to be due to them also being more potent 5HT2A antagonists (Meltzer et al, 2003). Thus, variation in D2 function and expression may contribute to the risk of TD development. The dopamine D2 receptor belongs to the G-protein coupled receptor family. It activates intracellular signaling by inhibiting the synthesis of cAMP. DRD2, the D2 encoding gene, consists of eight exons and spans approximately 65kb (Figure 1). Several in vivo and in vitro studies have focused on the effect that DRD2 polymorphisms have on D2 expression. The 141C

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Del allele has been reported to decrease DRD2 promoter activity in vitro (Arinami et al, 1997), while one in vivo study showed that the Del allele is associated with an increase in striatal D2 binding (Jnsson et al, 1999b); however, another in vivo study reported no significant difference (Ritchie and Noble, 2003). The B1 allele in intron 1 has been associated with decreased D2 binding in vivo (Jnsson et al, 1999b; Ritchie and Noble, 2003), as has T957 (Hirvonen et al, 2004). The latter has also been reported to decrease DRD2 mRNA stability in vitro (Duan et al, 2003). The TaqIA polymorphism has received much attention recently as it was discovered to

reside in an overlapping gene, ANKK1, and cause a non-synonymous coding polymorphism (Neville et al, 2004). The A1 allele has been associated with reduced D2 levels in several studies (Noble et al, 1991; Thompson et al, 1997; Pohjalainen et al, 1998; Jnsson et al, 1999b; Ritchie and Noble, 2003). Laruelle and coworkers (1998), however, did not find a significant association between TaqIA and D2 expression levels, but this negative finding could be the result of population stratification as subjects with four different ethnic backgrounds were included. DRD2 has been previously associated with schizophrenia. Several polymorphisms, including the 141C Ins, A2, and B2 variants, as well as their haplotypes, have been reported to be positively associated or over-transmitted in various schizophrenia samples (Arinami et al, 1997; Breen et al, 1999; Inada et al, 1999; Jnsson et al, 1999a; Golimbet et al, 1998; Dubertret et al, 2001). However, other investigators did not replicate the findings, suggesting that further studies are required (Hori et al, 2001a; Stober et al, 1998; Suzuki et al, 2000; Tallerico et al, 1999). DRD2 is one of the first genes to be tested in TD genetic studies. In all, ten studies have been conducted on DRD2 and TD. Specifically, the A-241G, 141C Ins/Del, TaqIB, TaqID, C939T, and TaqIA polymorphisms have been analyzed for association with TD (Chen et al, 1997a; Inada et al, 1997; 1999; Hori et al, 2001a, b; Kaiser et al, 2002; Chong et al, 2003a;

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Segman et al, 2003; Lattuada et al, 2004; de Leon et al, 2005, Srivastava et al, 2006). Chen et al (1997a) initially detected an association between the TaqIA marker and TD particularly in female schizophrenia patients. However, the DRD2 association with TD could not be replicated in most other studies. In the present study, we tested for the presence of an association between the DRD2 gene and TD using both continuous (AIMS) and dichotomous (TD occurrence) measures in relatively large samples of Caucasian and African-American patients with SCZ using 12 polymorphisms spanning the DRD2 gene: A-241G, -141C Ins/Del, TaqID, C939T, C957T, TaqIA, rs4648317, rs1125394, rs1079598, rs2242591, rs2242592, and rs2242593 all of which have recently been used in a detailed analysis of DRD2 in clozapine response (Hwang et al, 2005).

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Clement Zai 5.3 PATIENTS AND METHODS 5.3.1 Subjects

V. DRD2 and Tardive Dyskinesia

Subjects were recruited from four clinical sites in North America: Center for Addiction and Mental Health in Toronto, Ontario (Dr. G Remington, N=94); Case Western Reserve University in Cleveland, Ohio (Dr. HY Meltzer, N=77); Hillside Hospital in Glen Oaks, New York (Dr. JA Lieberman, N=49); University of California at Irvine, California (Dr. SG Potkin, N=12). Subjects were selected based on their diagnoses of SCZ or Schizoaffective Disorder according to DSM-III-R or IV (APA, 1994). All patients had undergone cumulatively at least one year of treatment with typical antipsychotics. For the Meltzer, Lieberman, and Potkin samples, the presence or absence of tardive dyskinesia was evaluated before any atypical antipsychotic administration, while patients were on mixed typical and atypical antipsychotics when TD was evaluated in the Remington sample. The presence of TD was assessed using the Abnormal Involuntary Movement Scale (AIMS) or the modified Hillside Simpson Dyskinesia Scale (HSDS) for 49 patients recruited from the Hillside Hospital (Guy, 1976; Schooler and Kane, 1982; Basile et al, 1999). The seven body area items and the overall global item of HSDS match those of AIMS, thus the assessment for the presence of TD was the equivalent for all four sites. All four clinicians (GR, HYM, JAL, SGP) are highly experienced in TD severity measurements of which the consistency was further enhanced by exchange visits across sites. In all, 232 patients were studied. 202 of them were European Caucasians, of which 80 were positive for the occurrence of TD. The remaining 30 were African-Americans, of which 11 were TD-positive. AIMS scores were available for 197 patients (171 Caucasians and 26 African Americans). Because of small sample size, the African-Americans were only used in the test for allele frequency association with TD.

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5.3.2 Gene polymorphism analysis Genomic DNA was purified from whole blood samples using high-salt method previously described (Lahiri and Nurnburger, 1991). 10L Polymerase Chain Reactions on 20ng genomic DNA were performed using TaqMan allele-specific assays with the following conditions: 95oC 10min, followed by 50 cycles of 92oC 15sec, 60oC 1min. Genotyping was done after the subjects completed the follow-up in which all laboratory staff was blind to the AIMS scores. The A-241G, 141C Ins/Del, TaqID, C939T, and TaqIA polymorphisms have been used in previous TD studies. The C957T polymorphism was studied because of its functional significance and its high minor allele frequency (Hirvonen et al, 2004; Duan et al, 2003). The remaining polymorphisms, rs4648317, rs1125394, rs1079598, rs2242591, rs2242592, and rs2242593, were selected based on their position within the DRD2 gene and the presence of sufficiently high minor allele frequencies (Hwang et al, 2005). The assays with their corresponding polymorphisms and locations are shown in Figure 9. Genotypes were determined using the ABI Prism 7000 Sequence Detection System with the Allelic Discrimination program within the ABI software (Applied Biosystems, Foster City, CA). All ambiguous genotypes were retyped, and if they remained ambiguous, they were taken out of the analysis.

5.3.3 Statistics Statistical analyses were conducted using the Statistical Package for the Social Sciences version 10.0.7, Haploview version 3.2, and UNPHASED version 2.402 (SPSS, 2000; Barrett et al, 2005; Dudbridge, 2003; Hwang et al, 2005). Odds ratio calculations were conducted using Program 2BY2 version 2 written by Jurg Ott. Genotype frequency distribution was tested for

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fitness to Hardy-Weinberg equilibrium using Haploview. The association of genotype frequencies with age and AIMS scores was assessed using ANOVA, and where the variances of AIMS scores among genotypes differed significantly using the Levene Test for Homogeneity of Variance, AIMS scores were examined with the Kruskal-Wallis test on SPSS. Gender differences in genotype frequencies were assessed using the 2 test on SPSS. The differences in allele and genotype frequencies between patients with and without TD were analyzed by 2 test. For contingency tables with at least one expected cell count of less than five, two-tailed Fishers Exact Tests were performed (URL: http://home.clara.net/sisa/fiveby2.htm). Haplotype analyses and linkage disequilibrium calculations were conducted using UNPHASED and Haploview, respectively.

ETHICAL CONSIDERATIONS The scientific work described in the present paper complies with the current laws of Canada and the US, as well as the ethical standards established in the 1964 Declaration of Helsinki. Informed consent was obtained before subjects participation, and this study was approved by the Ethics Committee of the Centre for Addiction and Mental Health.

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Clement Zai 5.4 RESULTS 5.4.1 Sample Characteristics

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The genotype distributions of all the DRD2 gene polymorphisms in the European Caucasian samples did not differ significantly from the Hardy-Weinberg equilibrium (p>0.10). rs1125394 was found not to be in Hardy-Weinberg equilibrium in our African-American sample (p<0.01). A significant increase in frequency of TD was found in females (p=0.009), while no significant association was found between gender and genotype frequencies (Table 1). We also found a significant positive correlation between AIMS scores and age (r=0.206; p=0.007).

5.4.2 Association study of individual polymorphisms with TD occurrence and AIMS With our Caucasian sample, we found the C957T allele frequencies to be significantly associated with TD occurrence (p=0.0135). Specifically, the T957 allele appears to be underrepresented in TD-positive patients compared to TD-negative patients (ORT957=0.59 [CI: 0.390.90]; Table 14). We also found a significant association for the C allele that appears to be present in a lower proportion of TD-positive patients compared to TD-negative patients (p=0.0085, ORC939=0.56 [CI: 0.36-0.86]; Table 14). The genotype frequencies of the C957T and C939T polymorphisms were also found to be significantly associated with TD (p=0.013, ORTT957=0.30 [CI: 0.13-0.69]; p=0.022, ORCC939=0.42 [CI: 0.23-0.79]; Table 13). We further tested for differences in average AIMS scores among genotypes with ANOVA. C957T and C939T showed the same trend as for TD analyses, with the differences being suggestive for C939T (p=0.150 and p=0.071, respectively; Table 13). Upon close inspection with student ttests, we found patients homozygous for C939 to have significantly lower total AIMS scores than patients carrying at least one copy of T939 (p=0.001). Patients homozygous for T957 had

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significantly lower total AIMS scores than patients carrying at least one copy of C957 (p=0.044). rs2242592 also showed significant or suggestive association with TD at the allelic and genotypic levels. The rs2242592 findings could be due to generally high linkage disequilibrium observed in the 3 region of DRD2 (Figure 10). The other polymorphisms analyzed did not show significant associations with TD either for allele, genotype, or AIMS scores analyses.

5.4.3 Association study of haplotypes with TD diagnosis and AIMS Using the Haploview program with linkage disequilibrium block defined by Gabriel et al (2002), strong evidence for linkage was found between C939T and C957T, prompting us to test for association between TD and two-marker haplotypes across DRD2 (Figure 9). rs2242593 and Taq1A were also found to be in linkage disequilibrium in our sample. COCA-PHASE analysis within UNPHASED revealed an association for the C939T-C957T haplotype and TD diagnosis (global p=0.021; Table 15). Specifically, the C939-T957 haplotype appeared to occur less often while the T939-C957 haplotype appeared more frequently in TD-positive patients (p=0.007, ORC939-T957=0.56 [CI: 0.37-0.86]; p=0.013, ORT939-C957=1.72 [CI: 1.11-2.65], respectively). QTPHASE analysis within the UNPHASED program using two-marker haplotypes also showed significant association of haplotypes containing C957T and C939T polymorphisms with AIMS scores (p=0.0087; Table 15). Patients carrying the C939-T957 haplotype had significantly lower average AIMS scores than other haplotypes, while those carrying the T939-C957 had significantly higher average AIMS scores (p=0.017 and p=0.0013, respectively). For the African-American sample, preliminary results indicated a marginally significant association between TD and alleles of the C957T (p=0.047), with the T957 allele appearing to be protective (ORT957=0.22 [CI: 0.04-1.08]). The linkage disequilibrium chart showing D values

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calculated using TD status on the 12 polymorphisms is provided as a reference in designing future genetic studies (Figure 14).

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Previous studies yielded conflicting results with regard to the DRD2 gene and TD. Besides the positive findings by Chen et al (1997a), none of the others have found a significant association using DRD2 polymorphisms. There are several reasons for these mixed results. First, different polymorphisms were used in many of the studies, and in most cases only a few polymorphisms were tested without haplotype analyses. Only Kaiser et al (2002) analyzed nine polymorphisms spanning the entire 65kb long DRD2 gene. Second, populations with different ethnic backgrounds were used in the studies, making findings difficult to compare due to potentially undetected stratification effects. Further, in some studies, the sample sizes were small, limiting the power to detect an association. Finally, many studies did not take into account statistical advantages of the continuous AIMS scores and only used the dichotomous TD occurrence for the analyses. The aim of our study has been to investigate twelve DRD2 gene polymorphisms for genetic association with TD in a relatively large sample involving haplotype analyses. Results from the present study are consistent with most previous studies in that the A241G, -141C Ins/Del, TaqID, and TaqIA polymorphisms were found to be not significantly associated with TD. The previous positive finding with TaqIA could be due to higher linkage disequilibrium in the 3 portion of DRD2 as shown in our sample and others (Figure 2; Kaiser et al, 2002; Ritchie and Noble, 2003), and that the causative variant may reside within DRD2. Indeed, we found a significant association between TD and the C957T polymorphism as well as its neighboring C939T polymorphism in our Caucasian sample. To our knowledge, the C957T polymorphism has not been investigated previously for its effect on TD. Although the polymorphism does not affect the amino acid sequence, the T variant has been associated with

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decreased striatal D2 levels in vivo and decreased DRD2 mRNA stability in vitro (Duan et al, 2003; Hirvonen et al, 2004). Using MFOLD, Duan and coworkers showed that the predicted mRNA folding structures were different between the two alleles (Duan et al, 2003). They hypothesized that T957 may decrease DRD2 expression through its effect on D2 mRNA secondary structure. The change in secondary structure may affect binding of mRNA stabilizing proteins at the 5-cap and 3-poly(A) as well as translation initiation factors, thus decreasing both translation efficiency and mRNA half-life (Duan et al, 2003; Perkins et al, 2005). An underrepresentation of the T allele in patients with TD and a decrease in TD severity in T-allele carriers suggest that decreased D2 levels may decrease TD susceptibility and severity. Our present study also reported a positive association with the nearby C939T, a polymorphism not found to be associated with TD in a previous study on a Japanese sample (Inada et al, 1997). It is possible, though, that TD susceptibility and risk factors may be different among different ethnic groups. Ethnic differences were reflected by findings in our African-American sample, in which a significant association was only detected between C957T alleles and TD. TD occurrence and severity were found to increase with age in the current sample, supporting previous studies from our laboratory and others (Basile et al, 1999; Jeste, 2000; van Os et al, 1997; Kaiser et al, 2002). Age and gender were not likely responsible for the positive findings in this study, because mean age and gender proportions did not differ significantly among the genotypes of C939T and C957T. Since both C939T and C957T are located in exon 7, it is possible that they are linked to a region that affects splicing around exon 6, resulting in a different ratio of the long (D2L) and short (D2S) isoforms. The two isoforms have distinct functions in vivo, and only the postsynaptic D2L isoform appears to be a major target of haloperidol (Centonze et al, 2004;

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Usiello et al, 2000). Thus, splicing changes could have contributed to changes in DRD2 function, expression levels and patterns, affecting antipsychotic response and adverse effects. Understanding the mechanisms that regulate splicing of the DRD2 gene will help answer the question of the locations of polymorphisms in the DRD2 gene that affect the splicing efficiency. From the epigenetics standpoint, the C957T polymorphism may have arisen from deamination of methylated C957 to T957. Though C957T has been reported to regulate D2 expression through its effect on mRNA stability, it is possible that C957 could be methylated in a subset of individuals. Methylated C957 may be functionally different from unmethylated C957 at the DNA level, thus opening another dimension of regulation of DRD2 expression and function. This may also be the case for other polymorphisms in DRD2, especially in the promoter region where methylation has been reported (Popendikyte et al, 1999). Because association studies of DRD2 have not considered CpG methylation and genetic polymorphisms do not capture the variability of regional CpG methylation (Flanagan et al, 2006), it may have given rise to the mixed results in previous findings. Understanding the role of epigenetics in the regulation of gene expression will help resolve at least some of the variability of results from genetics studies. The present study encourages further examinations into C957T, C939T, as well as adjacent polymorphisms and TD, but it has several limitations. First, not all clinical data were available for our study. These include medication history such as antipsychotic dose and duration, schizophrenia disease history including age of onset, clinical subtypes, psychopathology, and co-morbidities. These factors or variables have been previously associated with TD (reviewed in Mller et al, 2004). Medications taken by patients for other adverse effects such as parkinsonism could have masked the TD phenotype (Shale and Tanner, 1996; Egan et al, 1997; Glazer, 2000). Moreover, we did not have information on tobacco,

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alcohol, or substance use for our entire sample. Further, our sample was drawn from four different clinical sites. Even though the study Caucasian population was in Hardy-Weinberg equilibrium for all 12 polymorphisms and the gender ratios among the four geographical groups do not differ significantly (p=0.165), the mean ages differ significantly among them (p<0.001). Therefore, the possibility of ascertainment bias cannot be ignored. About half of the present sample has been analyzed previously for other genes with TD, with DRD3 findings being replicated (Basile et al, 1999; Lerer et al, 2002; Bakker et al, 2006). The other half of the sample has not been published previously for TD. Nonetheless, when the two halves were analyzed separately, the trend remained for C939T and C957T (data not shown). Also, heterogeneity of the TD phenotype could have confounding effects; only total AIMS scores, but not the subscores, were available for most of our sample for genetic analyses, preventing further dissection of the phenotype. Finally, the marginally significant association could be due to the possibility that the polymorphisms have only a small contributing effect to the risk for TD as expected in complex phenotypes. The sample size in the current study was not large enough to provide sufficient power to detect a significant difference in AIMS scores between the genotypes. Falsepositive results from multiple testing are possible; indeed, if we corrected for multiple testing taking linkage disequilibrium into account using the online SNPSpD program, the significance threshold () in order to keep the Type I error rate at 5% would have become 0.005 (Nyholt, 2004). As a result, only our findings from haplotype analyses would have remained statistically significant. Larger sample sizes are required to detect small effects of genotypes on TD risk and severity, especially for our AfricanAmerican sample where we did not detect significant associations between TD and any DRD2 polymorphisms after correction for multiple testing. With set at 0.005, the Caucasian portion of the sample used for the present study has only 18%

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power to detect the differences in AIMS scores observed among the C939T genotypes (Glantz, 1992). To detect such differences, a sample size of at least 120 per genotype group will be needed for future studies. The DRD2 gene is unlikely to be the only genetically determined factor for TD, as other genes have been associated with TD as well. Genetic studies have identified DRD3 to be reproducibly associated with TD (Steen et al, 1997; Basile et al, 1999; Segman et al, 1999; Lovlie et al, 2000; Liao et al, 2001; Garcia-Barcelo et al, 2001; Lerer et al, 2002). Studies in other genes such as HTR2A, HTR2C, CYP1A2, and manganese superoxide dismutase require further investigation (Hori et al, 2000; Basile et al, 2001; Segman et al, 2000; Segman et al, 2001; Schulze et al, 2001; Tan et al, 2001). As nearly all antipsychotics target more than one receptor, it is likely that TD is not related to one receptor gene, but rather it is a polygenic condition with each gene contributing a small proportion of the risk to the disorder. Gene-gene interaction studies may help in identifying and clarifying pathways that contribute to TD. TD risk is also likely to be influenced by several environmental factors (Mller et al, 2004), and acquiring this information will help immensely in increasing the statistical power and limiting the effects of potential confounders in genetic studies of TD.

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-141C Ins/Del rs4648317 5 1 A-241G

rs1079598 TaqID

rs2242591 C939T rs2242593 3

~15kb ~34kb rs1125394

7 C957T

~5kb TaqIA

1 kb

rs2242592

Figure 9. Schematic diagram of the DRD2 gene with its exons and introns. The positions of the 12 polymorphisms used for the present study are indicated within the gene.

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Table 13. Statistical analyses on demographics (gender, age) as well as total AIMS scores and TD diagnoses with genotypes of the 12 polymorphisms in DRD2.
DRD2 markers A-241G N (F/M) Age (years) Total AIMS score +/- SD (N) TD Yes 59 12 0.531 1 14 54 0.554* 56 12 2 0.581* 52 18 4 0.372* 4 16 55 0.277* 20 33 17 0.467 21 38 11 0.022 20 43 8 0.013 46 18 5 0.357* 8 40 24 0.070 47 20 4 0.291* 3 25 40 0.781* 3 47 71 84 30 2 9 49 59 80 33 3 24 59 35 59 47 11 30 50 39 2 32 84 83 31 2 87 26 2 1 18 96 No 102 16

1/1 (A/A) 161(54/107) 37.71+/-9.46 5.53+/-7.18 (131) 1/2 (A/G) 28(8/20) 37.25+/-11.72 7.56+/-8.75 (27) P 0.605 0.820 0.200 -141C Ins/Del 1/1 (Del/Del) 2(1/1) 38.00+/-1.41 6.50+/-6.36 (2) 1/2 (Ins/Del) 32(10/22) 39.06+/-10.76 5.71+/-7.84 (28) 2/2 (Ins/Ins) 150(49/101) 37.14+/-9.76 5.66+/-7.20 (123) P 0.927* 0.608 0.987 rs4648317 1/1 (C/C) 143(51/92) 37.83+/-10.16 5.78+/-7.21 (124) 1/2 (C/T) 38(8/30) 37.92+/-8.56 5.32+/-7.61 (28) 2/2 (T/T) 4(2/2) 26.25+/-4.35 5.00+/-7.07 (2) P 0.144* 0.947 0.066 rs1125394 1/1 (A/A) 135(42/93) 37.76+/-10.36 6.34+/-8.04 (116) 1/2 (A/G) 49(17/32) 38.31+/-8.55 4.97+/-5.89 (38) 2/2 (G/G) 6(2/4) 39.50+/-10.56 5.60+/-6.69 (5) P 0.907* 0.878 0.988! rs1079598 1/1 (C/C) 6(3/3) 36.33+/-11.73 6.20+/-6.65 (5) 1/2 (C/T) 48(16/32) 38.83+/-8.52 4.74+/-5.88 (39) 2/2 (T/T) 138(45/94) 37.34+/-10.33 6.34+/-7.96 (118) P 0.684* 0.632 0.514! TaqID 1/1 (C/C) 50(15/35) 37.42+/-9.35 6.31+/-8.00 (45) 1/2 (C/T) 83(30/53) 37.43+/-9.89 5.22+/-6.44 (64) 2/2 (T/T) 56(15/41) 37.21+/-10.30 6.16+/-8.34 (49) P 0.485 0.991 0.946! C939T 1/1 (C/C) 80(27/53) 37.51+/-8.69 3.80+/-4.91 (69) 1/2 (C/T) 85(29/56) 38.08+/-9.78 6.69+/-7.46 (68) 2/2 (T/T) 22(5/17) 34.36+/-11.21 9.85+/-11.77 (20) P 0.573 0.263 0.071! C957T 1/1 (C/C) 44(10/34) 36.59+/-10.94 7.29+/-9.42 (38) 1/2 (C/T) 102(38/64) 38.21+/-9.32 5.84+/-6.62 (82) 2/2 (T/T) 43(14/29) 37.95+/-9.09 3.89+/-5.86 (38) P 0.229 0.646 0.150! rs2242591 1/1 (A/A) 126(40/86) 37.49+/-10.32 6.14+/-8.06 (107) 1/2 (A/G) 51(16/35) 37.45+/-8.53 5.03+/-5.99 (40) 2/2 (G/G) 8(4/4) 38.50+/-11.16 5.14+/-5.90 (7) P 0.602* 0.960 0.705 rs2242592 1/1 (C/C) 17(3/14) 35.18+/-12.60 7.44+/-10.36 (16) 1/2 (C/T) 89(31/58) 37.69+/-9.80 6.99+/-7.94 (72) 2/2 (T/T) 79(29/54) 38.02+/-9.08 4.37+/-5.85 (70) P 0.355 0.547 0.135! rs2242593 1/1 (A/A) 131(42/89) 37.63+/-10.23 6.05+/-7.97 (111) 1/2 (A/G) 50(15/35) 37.98+/-8.71 5.27+/-5.92 (40) 2/2 (G/G) 6(3/3) 36.33+/-11.73 6.20+/-6.65 (5) P 0.588* 0.923 0.842 TaqIA 1/1 (T/T) 6(2/4) 38.33+/-10.35 6.12+/-8.16 (5) 1/2 (C/T) 72(27/45) 38.47+/-9.07 4.93+/-5.88 (60) 2/2 (C/C) 111(32/79) 36.63+/-10.38 5.20+/-6.87 (93) P 0.446* 0.458 0.960! * With at least 1 expected cell count <5; Fisher Exact Test used. ! Variances among comparisons groups differ significantly; Kruskal-Wallis test used. Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05.

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Table 14. Results from Chi-squared test of allele frequencies of each of the 12 DRD2 polymorphisms versus TD diagnoses for both Caucasian and African-American populations.
DRD2 markers Caucasian African American TD TD Yes No Yes No A-241G Allele 1 (A) 130 220 15 33 Allele2 (G) 12 16 7 5 P 0.548 0.102* -141C Ins/Del Allele 1 (Del/C) 16 20 9 16 Allele2 (Ins/CC) 122 210 13 22 P 0.365 0.928 rs4648317 Allele 1 (C) 124 200 19 34 Allele2 (T) 16 30 3 4 P 0.648 0.700* rs1125394 Allele 1 (A) 122 197 19 37 Allele2 (G) 26 35 3 1 P 0.521 0.135* rs1079598 Allele 1 (C) 24 36 2 0 Allele2 (T) 126 200 20 38 P 0.844 0.131* TaqID Allele 1 (C) 73 110 13 18 Allele2 (T) 67 128 9 20 P 0.266 0.381 C939T Allele 1 (C) 80 165 6 14 Allele2 (T) 60 69 16 24 P 0.449 0.0085 C957T Allele 1 (C) 83 107 20 26 Allele 2 (T) 59 129 2 12 P 0.0135 0.0472 rs2242591 Allele 1 (A) 110 193 20 34 Allele2 (G) 28 39 2 4 P 0.401 1.000* rs2242592 Allele 1 (C) 56 67 14 20 Allele2 (T) 88 167 8 18 P 0.687 0.039 rs2242593 Allele 1 (A) 114 198 21 37 Allele2 (G) 28 34 1 1 P 0.201 1.000* TaqIA Allele 1 (C) 31 53 4 8 Allele2 (T) 105 189 18 30 P 0.841 1.000* * with at least one expected cell count <5. Fisher Exact Test was used. Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05.

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rs4648317

rs1125394

rs1079598

rs2242591

rs2242592

rs2242593 0.04
0.00-0.28

-141C I/D

A-241G

C939T

C957T

TaqID

A-241G -141C Ins/Del rs4648317 rs1125394 rs1079598 TaqID C939T C957T rs2242591 rs2242592 rs2242593 TaqIA

1.00
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0.41
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Block 2

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1.00
0.85-1.00

1.00
0.77-1.00

1.00
0.95-1.00

1.00
0.73-1.00

Block 3

Figure 10. Linkage disequilibrium plot among the 12 DRD2 gene polymorphisms used in the present study. The numbers represents D values and the 95% confidence intervals of D, while the color darkness within each box corresponds to strength of linkage. The blocks (1, 2, and 3) encompass areas with highest linkage disequilibrium given by D>0.90 and lower boundary of the 95% confidence intervals of D>0.70 (Gabriel et al, 2002).

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TaqIA 0.14
0.01-0.41

0.45
0.05-0.80

0.07
0.00-0.27

0.91
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0.98
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0.37
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0.85
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0.95
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Clement Zai

V. DRD2 and Tardive Dyskinesia

Table 15. Global p-values from analyses of DRD2 two-marker haplotypes in association to TD and AIMS using COCA-PHASE and QT-PHASE respectively. Haplotypes with frequencies of less than 0.05 were excluded from the analyses. Haplotype P-value (TD+/-) P-value (AIMS) A-241G -141C I/D 0.358 0.326 -141C I/D rs4648317 0.616 0.924 rs4648317 rs1125394 0.771 0.726 rs1125394 rs1079598 0.869 0.362 rs1079598 TaqID 0.423 0.609 TaqID C939T 0.0569 0.00655 C939T C957T 0.0206 0.00868 C957T RS2242591 0.0339 0.0628 rs2242591 rs2242592 0.131 0.0341 rs2242592 rs2242593 0.122 0.0148 rs2242593 TaqIA 0.536 0.629
Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05.

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VI. DRD2-Tardive Dyskinesia meta-analysis CHAPTER 6

META-ANALYSIS OF TWO DOPAMINE D2 RECEPTOR GENE POLYMORPHISMS WITH TARDIVE DYSKINESIA IN SCHIZOPHRENIA PATIENTS

Published in Molecular Psychiatry

Clement C. Zai(1,2), Vincenzo De Luca(1,2), Rudi Hwang(1), Aristotle Voineskos(1), Daniel J. Mller(1,3), Gary Remington(1), James L. Kennedy(1,2)

(1) Centre for Addiction and Mental Health, Toronto, Ontario, CANADA (2) Institute of Medical Science, University of Toronto, Toronto, Ontario, CANADA (3) Department of Psychiatry, Charit University Medicine Berlin, Charit Campus Mitte, Berlin, Germany

Mr. Zai performed all the bibliographical searches for DRD2 gene association studies of tardive dyskinesia. He performed the meta-analysis under the guidance of Dr. Vincenzo De Luca, and wrote the manuscript.

RUNNING TITLE: Two Dopamine D2 Gene Polymorphisms in Tardive Dyskinesia, a Metaanalysis

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Clement Zai 6.1 INTRODUCTION

VI. DRD2-Tardive Dyskinesia meta-analysis

Tardive dyskinesia (TD) is a potentially irreversible movement disorder related to chronic antipsychotic medication exposure. The etiology is unknown, though genetic factors are likely contributors (Mller et al, 2001). Although the dopamine D2 receptor has been recognized as the main target for antipsychotics, studies investigating potential involvement of D2 gene (DRD2) polymorphisms in TD have yielded mixed results (Zai et al, 2007a). Of these polymorphisms, TaqIA and 141C Ins/Del have been studied most extensively. The 141C Del allele has been reported to decrease DRD2 promoter activity in vitro, while two in vivo studies showed the Del allele to be associated with either an increase in striatal D2 binding or no significant difference (Zai et al, 2007a). The TaqIA polymorphism was recently discovered to reside in the coding region of an overlapping gene coding for a Serine/Threonine kinase, and causes an amino acid change (Zai et al, 2007a). However, the A1 (T) allele has been associated with reduced D2 levels in most reported studies (Zai et al, 2007a).

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Clement Zai 6.2 METHODS 6.2.1 Publication Search

VI. DRD2-Tardive Dyskinesia meta-analysis

We carried out a computer search on the National Library of Medicines PubMed online search engine database for all papers published up to December 2006 on tardive dyskinesia and DRD2. In all, 12 genetic association studies were found reporting on TD and DRD2. Six included the number of patients with and without TD and genotypes for TaqIA, while five included the number of TD-positive and TD-negative patients with genotypes for the 141C Ins/Del polymorphism (Zai et al, 2007a; Chen et al, 1997a; Inada et al, 1999; Hori et al, 2001a; Chong et al, 2003a; Segman et al, 2003; Liou et al, 2006). All subjects were selected based on their diagnoses of Schizophrenia or Schizoaffective Disorder, according to DSM-III-R or IV, using case records with or without patient interviews. The presence of TD was assessed using the Abnormal Involuntary Movement Scale (AIMS), or the modified Hillside Simpson Dyskinesia Scale (HSDS) in the case of 49 patients from our study as described previously (Zai et al, 2007a). TD ratings were performed once for the majority of the studies. Quantitative AIMS scores were not available for most subjects and were not included in the analyses.

6.2.2 Sample Description In all, 1256 schizophrenia patients were genotyped for TaqIA. 507 of them were positive for the diagnosis of TD. 897 schizophrenia patients were genotyped for 141C Ins/Del, of which 328 were TD-positive.

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VI. DRD2-Tardive Dyskinesia meta-analysis

We analysed the data using the Stata Release 8 statistical software package (StataCorp. 2003. Stata Statistical Software: Release 8. College Station, TX: StataCorp LP). The odds ratios and standard errors of TaqIA and 141C I/D for TD from the individual studies were calculated using the metan command, with the pooled odds ratio and standard error calculated under the random effects model. The possible effects of ethnicity, age, and gender ratio on heterogeneity amongst the studies were assessed by meta-regression analysis using the metareg command. We tested for publication bias using the metabias command.

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Clement Zai 6.3 RESULTS AND DISCUSSION

VI. DRD2-Tardive Dyskinesia meta-analysis

Compared to TD-negative patients, TD-positive patients had a higher A2 allele frequency (p=0.003), with an effect size of 1.30 (95% CI: 1.09-1.55), and higher A2/A2 genotype frequency (p=0.001), with an effect size of 1.50 (95% CI: 1.17-1.92). The 141C Ins/Del alleles and genotypes were not associated with TD. There was no evidence of heterogeneity amongst the studies or publication bias for TaqIA or 141C Ins/Del (p>0.1). Ethnicity, gender ratio, or age did not contribute to the results observed for TaqIA (p>0.1). Results are summarized in Table 16. Despite heterogeneity amongst studies in terms of TD assessment, results from the present meta-analysis suggest that TaqIA is associated with TD and are in agreement with two of the previous studies (Chen et al, 1997a; Liou et al, 2006). The mixed results in other studies could be attributed to the lower A1 allele frequencies observed in European Caucasians compared to East Asians, as well as small sample sizes. The results reported here could be affected by potential confounding factors, including tobacco and substance use, antipsychotic dose, and years of antipsychotic exposure, information that was not available for most of the studies. False-positive results from multiple testing are possible, but association between TaqIA and TD from the present meta-analysis would have survived correction for testing two markers. The relatively low OR is consistent with the idea of contributions of multiple genetic variants in complex phenotypes, with DRD3 Ser9Gly as an example of another genetic risk factor for TD (Bakker et al, 2006). Nonetheless, the present study, the first meta-analysis of DRD2 polymorphisms in TD, encourages further examination of the role of TaqIA in TD.

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Table 16. Summary for meta-analysis of DRD2 TaqIA and 141C Ins/Del polymorphisms. SNP OR2 95% CI Wt. OR22 95% CI Wt. (allele) (allele) % (genotype) (genotype) % 0.60 0.29-1.21 19 0.58 0.26-1.33 24 -141C Inada 1999 1.52 0.79-2.91 22 1.88 0.88-3.99 27 Ins/Del Hori 2001a Segman 2003 1.06 0.37-3.01 9 1.06 0.36-3.13 16 (CC, Liou 2006 0.92 0.55-1.56 32 1.52 0.25-9.28 6 Ins) Zai 2006a 0.73 0.36-1.45 19 0.71 0.34-1.52 27 Overall 0.92 0.67-1.25 100 0.99 0.61-1.59 100 1.37 0.89-2.09 17 2.13 1.13-4.02 15 TaqIA Chen 1997a Hori 2001a 1.17 0.72-1.89 13 1.46 0.62-3.45 8 (C, Chong 2003a* 1.28 0.92-1.78 28 1.32 0.82-2.11 28 A2) Segman 2003 1.40 0.71-2.76 7 1.57 0.72-3.44 10 Liou 2006 1.57 1.10-2.24 24 1.87 1.10-3.20 22 Zai 2006a 0.95 0.57-1.57 12 1.01 0.55-1.84 17 Overall 1.30 1.09-1.55 100 1.50 1.17-1.92 100 *Allele and Genotype information were reported for Ser311Cys, but allele names, frequencies, and reference of Grandy et al, 1993 (Ritchie and Noble, 2003) matched TaqIA. Study

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GENETIC STUDY OF EIGHT AKT1 GENE POLYMORPHISMS AND THEIR INTERACTION WITH DRD2 GENE POLYMORPHISMS IN TARDIVE DYSKINESIA Submitted to Neuropsychopharmacology Clement C. Zai(1,2), Marco A. Romano-Silva(3), Rudi Hwang(1,2), Gwyneth C. Zai(1,2), Vincenzo DeLuca(1,2), Daniel Mller(4), Nicole King (1), Aristotle N. Voineskos(1,2), Herbert Y. Meltzer(5), Jeffrey A. Lieberman(6), Steven G. Potkin(7), Gary Remington(1), James L. Kennedy(1,2) (1) Centre for Addiction and Mental Health, Toronto, Ontario, CANADA (2) Institute of Medical Science, University of Toronto, Toronto, Ontario, CANADA (3) Laboratorio de Neurociencia, Dept. Saude Mental, Faculdade de Medicina, Universidade Federal de Minas Gerais, Brazil (4) Department of Psychiatry, Charit University Medicine Berlin, Campus Charit Mitte, Berlin, Germany (5) Psychiatric Hospital at Vanderbilt University, Nashville, Tennessee, USA (6) New York State Psychiatric Institute, Columbia University Medical Centre, New York City, New York, USA (7) Brain Imaging Center, Irvine Hall, University of California at Irvine, California, USA Mr. Zai designed the experiment (with guidance from faculty and Dr. Marco RomanoSilva, a collaborator), performed genotyping on the AKT1 gene polymorphisms in approximately 50% of the tardive dyskinesia sample. Dr. Marco Romano-Silva genotyped the remaining 50% of the sample and used them to analyze other phenotypes, not tardive dyskinesia. Mr. Zai corresponded with the clinical collaborators to refine the details of the phenotype, performed all the statistical analyses, and wrote the manuscript. Page 140

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Keywords: Schizophrenia, tardive dyskinesia, gene association, polymorphism, Protein Kinase B PKB/AKT1, Dopamine Receptor DRD2, Abnormal Involuntary Movement Scale (AIMS)

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Clement Zai 7.1 ABSTRACT

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Tardive dyskinesia (TD) represents a potentially irreversible motor side effect associated with chronic antipsychotic exposure. Dopamine neurotransmission system changes have been implicated, and a number of studies have focused on the association of dopamine system gene polymorphisms and TD; for example, we recently found an association between polymorphisms in the dopamine D2 receptor gene DRD2 and TD. The small odds ratio, though, suggests additional factors are involved in the etiopathology of TD. All antipsychotic drugs block the D2 receptors AKT1 acts downstream of the D2 receptor, and all antipsychotic drugs block the D2 receptor to some degree. Haloperidol has been shown to alter AKT1 activity. Although AKT1 has been identified as a candidate gene for schizophrenia, it has not been investigated in TD. Thus, in the present study, we examined eight polymorphisms spanning the AKT1 gene and their association with TD in our Caucasian (N=193) and African-American (N=30) samples. AKT1 polymorphisms and haplotypes were not significantly associated with TD occurrence or severity as measured by AIMS (Abnormal Involuntary Movement Scale). However, interaction analysis showed a significant interaction between rs6275 of DRD2 and rs3730358 of AKT1 (p<1X10-5). Taken together, the present study suggests that the interaction of DRD2 and AKT1 is involved in TD development, though further studies are required.

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Clement Zai 7.2 INTRODUCTION

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Tardive dyskinesia (TD) is a motor side effect linked to chronic antipsychotic treatment that affects between 16 and 43% of SCZ patients treated with conventional or typical antipsychotics (Tarsy and Baldessarini, 2006). Older age, female gender, and African American ethnicity have all been suggested to increase the risk and severity of TD (Kane et al, 1988; Woerner et al, 1998; Smith and Dunn, 1979; van Os et al, 1997; Jeste et al, 2000). The use of alcohol, tobacco, and recreational drugs can further the risk of TD (Menza et al, 1991; Bailey et al, 1997; Olivera et al, 1990). As a class the newer, atypical antipsychotics have been linked to a diminished risk of motor side effects, but this advantage has been tempered by substantially higher costs, questionable clinical superiority, and their own substantial side effects in the form of weight gain and metabolic disturbances (Lieberman et al, 2005). As a result, there has been a renewed interest in typical antipsychotic use once again. This, in combination with the fact that atypicals are not devoid of TD liability, requires that we continue in our pursuit to better understand TD and those at risk for this potentially irreversible side effect. Though the etiopathology of TD remains elusive, a number of mechanisms have been proposed. TD has been postulated to arise from a hypersensitivity of dopamine receptors secondary to chronic antipsychotic exposure, resulting in excessive function of dopamine in the central nervous system (CNS). This theory is compatible with the fact that all marketed antipsychotics block dopamine receptors, albeit to varying degrees (Tarsy and Baldessarini, 1977; Klawans et al, 1980; Gerlach and Casey, 1988; Abilio et al, 2003).

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Family studies have suggested a genetic component underlying TD development (Mller et al, 2001), and several studies have investigated the role of dopaminergic system genes in TD. The association between DRD3 Ser9Gly and TD (Badri et al, 1996; Steen et al, 1997) has been confirmed by two meta-analyses (Lerer et al, 2002; Bakker et al, 2006), while our laboratory recently analyzed twelve DRD2 polymorphisms in TD and found that haplotypes containing rs6275 and rs6277 were associated with TD (Zai et al, 2007a). A recent meta-analysis conducted by our laboratory (Zai et al, 2007b) also confirmed the association between DRD2 Taq1A and TD, detected initially by Chen et al (1997a). Nonetheless, the odds ratios obtained from the meta-analyses ranged from 1.1 to 1.5, supporting the notion that multiple genetic factors influence TD risk. Most TD genetic studies thus far have analyzed genes that directly affect dopamine metabolism or dopamine receptor function, but none have investigated the role of gene products that transduce signals downstream of dopamine receptors. AKT1, also known as protein kinase B (PKB), is a serine/threonine kinase that is involved in numerous signaling pathways (Nicholson and Anderson, 2002). It is important in the regulation of neuronal plasticity (Kim and Chung, 2002; Wang et al, 2003) and synaptic transmission (Beaulieu et al, 2005). Moreover, it has been linked to both SCZ and dopamine function. Specifically, the AKT1 gene, located at 14q32.32, has been associated with SCZ in some studies (Emamian et al, 2004; Ikeda et al, 2004; Schwab et al, 2005; Bajestan et al, 2006), but not others (Ohtsuki et al, 2004; Ide et al, 2006; Liu et al, 2006; Turunen et al, 2007). Beaulieu et al (2004) found that pharmacological or genetic upregulation of dopamine neurotransmission decreased the activating phosphorylation of AKT1; the decrease was blocked or attenuated by

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raclopride, a D2/D3 receptor antagonist, and not by SCH23390, a D1 receptor antagonist. The results were corroborated by the observation that activating phosphorylation of AKT1 was increased in mice deficient in D2 or D3 (Beaulieu et al, 2007), or by haloperidol treatment in mice (Emamian et al, 2004). The results suggest that AKT1 acts downstream of D2 receptors and may be relevant in dopamine related disorders including schizophrenia and TD. In the present study, we tested for an association between the AKT1 gene and TD in our samples of Caucasian and African American schizophrenia patients using eight polymorphisms spanning the AKT1 gene (Emamian et al, 2004; Schwab et al, 2005; Table 17, Figure 11). The polymorphisms were selected because they were used previously in schizophrenia genetic studies and they have high minor allele frequencies. The linkage disequilibrium chart showing D values on the eight polymorphisms is also provided as a reference in designing future genetic studies (Table 4). In addition, because AKT1 acts downstream of D2, we also tested for an interaction between DRD2 and AKT1 polymorphisms in TD.

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Clement Zai 7.3 PATIENTS AND METHODS 7.3.1 Subjects

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The sample for this study is largely the same as the one used in Zai et al (2007a). Subjects were recruited from four clinical sites in North America: Center for Addiction and Mental Health in Toronto, Ontario (Remington, N=92); Case Western Reserve University in Cleveland, Ohio (Meltzer, N=69); Hillside Hospital in Glen Oaks, New York (Lieberman, N=50); University of California at Irvine, California (Potkin, N=12). Subjects were selected based on their diagnoses for SCZ or schizoaffective Disorder according to DSM-III-R or DSM-IV (APA, 2000). All patients had undergone at least one year of treatment with typical or atypical antipsychotic treatment, and the presence of TD was evaluated using the Abnormal Involuntary Movement Scale (AIMS) (Guy, 1976; Schooler and Kane, 1982) or the modified Hillside Simpson Dyskinesia Scale (HSDS) for the 50 patients from the Hillside Hospital (Basile et al, 1999). In all, 223 SCZ patients were studied. Of this sample, 193 are Caucasians, with 76 of these subjects positive for the diagnosis of TD. The remaining 30 are AfricanAmericans, of which 11 are TD-positive. Because of the small sample size, the AfricanAmericans were used only in the allele frequency association tests.

7.3.2 Gene polymorphism analysis Genomic DNA was purified from whole blood samples using a non-enzymatic method previously described (Lahiri and Nurnburger, 1991). The 10L Polymerase Chain Reactions were performed on 20ng genomic DNA using ABI TaqMan genotyping assays under the following conditions: 95oC for 10min, followed by 50 cycles of 92oC for

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15sec and 60oC for 1min. The rs numbers for the markers and their relative positions in the AKT1 gene are given in Table 17 and Figure 11. Genotypes were determined using Allelic Discrimination software in the ABI Prism 7500 Sequence Detection System (Applied Biosystem, Foster City, CA, USA).

7.3.3 Statistics Statistical analyses of individual polymorphisms were conducted using the SPSS program v14.0. The 2 test was used for fit of genotypes to Hardy-Weinberg equilibrium and to test for gender differences. The association of genotype frequencies with age and AIMS was assessed using one-way ANOVA. Where the Levene Test for Homogeneity of Variance was violated, the Kruskal-Wallis test was performed. The differences in allele and genotype frequencies between patients with and without TD were analyzed by the 2 test. For contingency tables with at least one expected cell count of less than five, Fisher Exact Tests were performed (http://home.clara.net/sisa/fiveby2.htm). Haplotype analyses and linkage disequilibrium calculations were conducted using the UNPHASED v2.402 (Dudbridge et al, 2003) and Haploview v3.2 (Barrett et al, 2005) Programs respectively. Gene-gene interaction analysis was performed using HELIXTREE (GoldenHelix), and post-hoc analyses of the continuous variable (AIMS) were carried out using univariate general linear model in SPSS.

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The genotype distributions of all the AKT1 gene polymorphisms in the Caucasian and African American samples did not differ significantly from the Hardy-Weinberg equilibrium (p>0.10). There was a significant difference in genotype frequencies of rs2498784 in males versus females (Table 18), while age was not found to be associated with any of the eight polymorphisms. We assume that the association with sex is spurious; nonetheless, we included sex as a covariate in the ANCOVA for this marker.

7.4.2 Association analysis of individual polymorphisms with TD occurrence and AIMS In our Caucasian sample, we did not find a significant association between any of the eight AKT1 polymorphisms tested and TD, although we found a trend for allele 1 (G) of rs10149779 to be under-represented in the TD-positive group (p=0.08; Tables 18, 19). Next, we tested for an association between genotype frequencies and AIMS scores, but did not find a significant association with any of the eight polymorphisms. Using the Haploview program, strong evidence for linkage disequilibrium was found between rs3803304 and rs2494731, prompting us to test for association between TD and twomarker haplotypes across AKT1 (Figure 12). None of the two-marker haplotypes were associated with TD or AIMS scores (data not shown). For the African-American sample, preliminary results indicated a significant association between TD and rs2494738, as well as a trend for the adjacent rs3730358 (Table 19).

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7.4.3 Significant interaction between DRD2 and AKT1 polymorphisms in TD Using the Caucasian sample, we analyzed the interaction between the 12 DRD2 polymorphisms reported previously (Zai et al, 2007a) and the eight AKT1 polymorphisms in this study. Due to the large number of pairwise comparisons, we used the Bonferroni correction to account for multiple testing. Results are summarized in Figure 13. We found DRD2 rs6275 to interact with AKT1 rs3730358 (Bonferroni p=0.007), and to confirm the findings, we conducted a two-way ANOVA under the univariate general linear model analysis option using SPSS with AIMS as the dependent variable and with rs3730358 and rs6275 as fixed factors. A graphical representation of the interaction is given in Figure 14. The interaction between rs3730358 and rs6275 was significant (p<0.001). Because the assumption of equal variances for ANOVA was violated by the significant Levenes Test, we conducted the same analysis using ranked AIMS as dependent variable in place of AIMS. The results were equally significant (p=0.008). More detailed analysis showed that the average total AIMS scores of subjects with genotype 2/2 (T/T) at rs6275 were higher than subjects with genotype 1/2 (C/T), and those with genotype 1/2 (C/T) were higher than those with genotype 1/1 (C/C), as reported previously {linear regression: r=0.244, F=9.978, p(1,158)=0.002} (Zai et al, 2007a). While the same association was observed at rs6275 in patients with genotype 1/1 (C/C) at rs3730358 {linear regression: r=0.409, F=21.529, p(1,107)<0.001}, those of carriers with allele 2 (T) at rs3730358 did not follow the trend at rs6275. Instead, carriers of allele 2 (T) at rs3730358 had similar total average AIMS scores among the three rs6275 genotype groups {linear regression: r=0.061, F=0.156, p(1,42)=0.695}. When we examined rs6275 and rs3730358 in TD, we found a significant under-representation of

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the 1/1 (C/C) genotype at rs6275 in TD-positive patients with 1/1 (C/C) genotype at rs3730358 (p=0.006), but not in TD-positive patients carrying at least one copy of allele 2 (T) at rs3730358 (p=0.99).

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Clement Zai 7.5 DISCUSSION

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This is the first reported genetic association study between AKT1 and TD. While the results are not significant for AKT1 by itself in our Caucasian sample, we found that DRD2 rs6275 interacts with AKT1 rs3730358 in TD. Specifically, allele 2 (T) at rs3730358 restricts the genotypic effects of rs6275 on TD occurrence and severity. This finding is in agreement with the strong evidence that AKT1 is a downstream signal transducer of the D2 receptor (Beaulieu et al, 2007). While this positive gene-gene interaction result is intriguing, the methods for gene-gene interaction analyses are not well established, thus these results should therefore be considered preliminary. Nevertheless, the genetic interaction reported herein survived Bonferroni correction and encourages further investigations into the molecular mechanisms underlying this association. We recently reported an association of rs6275 and an adjacent functional polymorphism rs6277 with TD (Zai et al, 2007a). While rs6277 genotypes are associated with D2 expression (Duan et al, 2003; Hirvonen et al, 2004), they do not appear to interact strongly with AKT1 polymorphisms to affect AIMS score (Figure 3). rs6275 does not appear to be associated with D2 expression (Duan et al, 2003); nevertheless, it may influence RNA splicing at exon 6, thus changing the ratio of long and short D2 isoforms. AKT1 haplotypes containing the C allele at rs3730358 have been associated with altered AKT1 expression (Emamian et al, 2004). Interaction analysis using haplotypes may provide valuable information about the regions of biological interest within DRD2 and AKT1, but larger samples will be required for such extensive examinations.

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The lack of association between AKT1 and TD in our Caucasian sample could be a true negative result; alternatively, the polymorphisms may only have a small contributing effect to the risk for TD, and the sample size in the current study provides insufficient power to detect the small difference in AIMS observed among the genotypes. Under reasonable assumptions (=0.05, risk allele frequency=0.3), the current sample has 82.1% power to detect a risk ratio for TD of as low as 2.0. The association between rs2494738 and TD in our African-American sample, as well as the rs6275-rs3730358 interaction requires replication in larger samples. The present study has several limitations. First, not all relevant clinical data were available for our study. These include medication history (antipsychotics, dose, duration), schizophrenia disease history (onset, clinical subtypes, severity), and comorbidities. Medications taken by patients for other adverse effects (e.g., parkinsonism, anxiety) could mask TD (Shale and Tanner, 1996; Egan et al, 1997; Glazer, 2000). Conversely, environmental risk factors such as smoking have been identified as a risk factor for TD and could contribute to our findings, though we do not have such information available for our entire sample. Finally, different manifestations (e.g., facial versus truncal) of the TD phenotype could have different genetic contributions. We evaluated total AIMS scores because our sample sizes would have been too small to have enough power to detect an association with specific subtypes of TD. On the other hand, TD is likely less sensitive to design-related variables than presumably more complex phenotypes such as antipsychotic response. Meta-analyses support the association of DRD2 (Zai et al, 2007b), DRD3 (Lerer et al, 2002; Bakker et al, 2006), HTR2A (Lerer et al, 2005), and CYP2D6 (Patsopoulos et

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al, 2005) with TD. Studies of other genes such as HTR2C, CYP1A2, and manganese superoxide dismutase are preliminary and require replication to confirm their potential association with TD (Basile et al, 2000; Segman et al, 2000; Schulze et al, 2001; Hori et al, 2000). As all antipsychotics target more than one receptor and behavioural phenotypes are complex, it is likely that TD is a polygenic condition with each gene contributing a small proportion of the risk. TD risk is also likely to be influenced by many environmental factors (Menza et al, 1991; Bailey et al, 1997; Olivera et al, 1990). Acquiring this additional information for future samples will help in limiting effects of potential confounds in genetic studies of TD and, in doing so, increase the predictive value of future genetic tests for TD. The present study encourages further investigations of the interactions among genes along signaling pathways in deciphering the pathophysiology of TD and other complex genetic diseases.

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rs1130214 rs2494746 5 1 rs2498784 2 rs10149779

rs2494738 rs3730358

rs3803304

3 3 4 5 6 7 8 9 10
11 12 13 14

1 kb

rs2494731

Figure 11. Schematic diagram of the AKT1 gene with its exons and introns. The darker color indicates the coding region. The positions of the eight polymorphisms used for the present study are indicated within the gene.

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Table 17. ABI Assays-on-Demand with information on their corresponding AKT1 polymorphisms used in the present study. VIC and FAM are fluorescent dyes that are conjugated onto probes specific for the corresponding alleles of each polymorphism. AssayAllele Allele Polymorphism Location with References on1 2 Name(s) respect to Demand FAM VIC AKT1 gene rs2498784 C T SNP1a, G-5983A 5 Schwab et al, 2005 rs1130214 G T SNP2, C-754A Intron 1 Emamian et al, 2004 rs2494746 G C G1261C Intron 2 rs10149779 C T SNP2a, G7894A Intron 2 Schwab et al, 2005 rs2494738 C T G12294A Intron 2 rs3730358 C T SNP3, G12573A, Intron 3 Emamian et al, G3+18A 2004 rs3803304 G C G19834C, Intron 11 G11+69C rs2494731 G C G21300C Intron 12

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Table 18. Statistical analyses on demographics (sex, age) as well as total AIMS scores and TD occurrence with each of the eight AKT1 polymorphisms.
DRD2 markers rs2498784 N (M/F) Age (years) Total AIMS score TD (Yes/No) 162(116/46) 38.04+/-10.14 6.28+/-7.90 63/99 27(11/16) 37.63+/-8.84 5.50+/-4.85 13/14 3(1/2) 29.67+/-8.74 0.00+/-0.00 0/3 0.002* 0.353 0.177# 0.266* rs1130214 1/1 (G/G) 111(68/43) 37.81+/-10.13 5.58+/-7.36 38/73 1/2 (G/T) 59(44/15) 37.27+/-9.46 7.04+/-7.91 28/31 2/2 (T/T) 17(12/5) 38.71+/-10.90 6.38+/-7.58 7/10 P 0.201 0.863 0.544 0.239 rs2494746 1/1 (G/G) 149(106/43) 38.05+/-10.08 6.20+/-7.82 58/91 1/2 (G/C) 31(16/15) 37.97+/-9.05 4.58+/-4.37 12/19 2/2 (C/C) 4(2/2) 27.25+/-8.62 3.67+/-6.35 1/3 P 0.060* 0.101 0.749# 1.000* rs10149779 1/1 (C/C) 113(70/43) 37.53+/-10.09 5.52+/-7.35 39/74 1/2 (C/T) 57(41/16) 38.12+/-9.68 7.79+/-7.98 29/28 2/2 (T/T) 16(11/5) 37.75+/-10.98 6.00+/-7.46 7/9 P 0.417 0.936 0.239 0.116 rs2494738 1/1 (C/C) 172(115/57) 37.73+/-10.02 6.04+/-7.58 66/106 1/2 (C/T) 13(8/5) 39.31+/-9.40 2.91+/-2.66 4/9 2/2 (T/T) 1(1/0) 21.00 8.00 1/0 P 0.842* 0.211 0.498# 0.526* rs3730358 1/1 (C/C) 131(84/47) 38.31+/-9.91 6.04+/-7.87 51/80 1/2 (C/T) 55(39/16) 37.25+/-9.94 6.11+/-6.51 22/33 2/2 (T/T) 1(0/1) 25.00 7.00 1/0 P 0.221* 0.345 0.991 0.540* rs3803304 1/1 (G/G) 10(6/4) 31.60+/-9.12 3.90+/-3.70 4/6 1/2 (G/C) 76(51/25) 38.13+/-9.84 7.10+/-8.00 31/45 2/2 (C/C) 102(67/35) 38.02+/-10.06 5.81+/-7.59 40/62 P 0.873* 0.137 0.369 0.969* rs2494731 1/1 (G/G) 80(53/27) 38.01+/-10.21 6.00+/-7.98 32/48 1/2 (G/C) 88(60/28) 38.57+/-9.89 6.41+/-7.56 32/56 2/2 (C/C) 22(13/9) 33.95+/-9.12 5.84+/-6.07 12/10 P 0.722 0.149 0.931 0.298 * With at least 1 expected cell count <5; Fisher Exact Test was used. # Variances among comparisons groups differ significantly; Kruskal-Wallis test was used. Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05. 1/1 (C/C) 1/2 (C/T) P

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Table 19. Results from 2 tests of allele frequencies of each of the eight AKT1 polymorphisms versus TD occurrence for both Caucasian and African-American populations.
DRD2 markers TD (Yes/No) Caucasian African American rs2498784 Allele 1 (C) 139/212 16/31 Allele 2 (T) 13/20 6/7 P 0.981 0.520* rs1130214 Allele 1 (G) 104/177 15/22 Allele 2 (T) 42/51 7/14 P 0.165 0.587 rs2494746 Allele 1 (G) 128/201 12/22 Allele 2 (C) 14/25 10/16 P 0.714 0.801 rs10149779 Allele 1 (C) 107/176 15/21 Allele 2 (T) 43/46 7/15 P 0.453 0.080 rs2494738 Allele 1 (C) 136/221 18/34 Allele 2 (T) 6/9 4/0 P 0.882 0.020* rs3730358 Allele 1 (C) 124/193 17/24 Allele 2 (T) 24/33 3/14 P 0.672 0.082 rs3803304 Allele 1 (G) 39/57 4/9 Allele 2 (C) 111/169 18/29 P 0.865 0.751* rs2494731 Allele 1 (G) 96/152 11/24 Allele 2 (C) 56/76 11/14 P 0.482 0.319 * with at least one expected cell count <5. Fisher Exact Test was used. Bolded numbers indicate 0.05<p<0.10; bolded and italicized numbers indicate p<0.05.

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rs10149779

rs2498784

rs1130214

rs2494746

rs2494738

rs3730358

rs3803304

rs2498784 rs1130214 rs2494746 rs10149779 rs2494738 rs3730358 rs3803304 rs2494731

1.00 0.46-1.00

1.00 0.90-1.00 1.00 0.50-1.00

1.00 0.43-1.00 0.99 0.93-1.00 1.00 0.47-1.00

0.70 0.45-0.86 0.09 0.02-0.73 0.66 0.40-0.84 0.04 0.01-0.72

0.59 0.07-0.89 0.57 0.41-0.70 0.26 0.02-0.75 0.60 0.45-0.73 1.00 0.12-0.99

0.01 -0.01-0.27 0.28 0.15-0.40 0.05 0.01-0.55 0.28 0.14-0.41 1.00 0.12-0.99 0.77 0.61-0.87

0.55 0.26-0.75 0.26 0.09-0.40 0.47 0.21-0.67 0.20 0.04-0.36 0.72 0.30-0.90 0.73 0.53-0.85 1.00 0.95-1.00

Block 1

Figure 12. Linkage disequilibrium plot among the eight AKT1 gene polymorphisms used in the present study. The numbers represent D values and the 95% confidence intervals of D using the Haploview program. The color intensity within each box corresponds to strength of linkage, with the darkest having strong evidence for linkage, intermediate having uninformative results, and white having evidence for recombination. Block 1 encompasses an area with highest linkage disequilibrium given by D>0.90 and lower boundary of the 95% confidence intervals of D>0.70.

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Figure 13. p-values from analyses of two-marker interactions between DRD2 and AKT1 polymorphisms in association to AIMS given by HELIXTREE program. Top left triangle indicates significance with the raw p-values, while the bottom right triangle indicates significance with Bonferroni adjusted p-values. Note that AKT1 rs3730358 interacts with DRD2 rs6275 (Bonferroni p=7x10-3).

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Figure 14. Interaction between DRD2_rs6275 (C939T) and AKT1_rs3730358 in AIMS. We conducted a two-way ANOVA that showed a significant interaction between the two polymorphisms (p=0.008).

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Zai et al CHAPTER 8

VIII. Discussion

GENERAL DISCUSSION

8.1 SUMMARY OF FINDINGS AND IMPLICATIONS In the first manuscript, we investigated the GABRG2 gene in SCZ. It resides in the SCZ susceptibility region at 5q31-35 (Sklar et al, 2004; Lewis et al, 2003). It gene product also interacts physically with the D5 receptor in a mutually inhibitory manner (Liu et al, 2000). We tested five polymorphisms within GABRG2 for association with SCZ diagnosis and suicidal behaviour in the sixth manuscript. We found a nominally significantly association with rs183294 in the 5 region of GABRG2 in our case-control sample, and a trend for the same polymorphism in our independent family sample. The significance level increased when the case-control and family samples were combined. The results are not in agreement with previous studies showing GABRG2 not being associated with SCZ in Caucasian and East Asian samples. They are also not consistent with the previous positive finding where the 3 region of GABRG2 was found to be most significantly associated. The mixed results could be due to a number of factors. Sampling could have contributed to the positive findings in our sample. Only one other GABRG2 study utilized both case-control and family samples (Petryshen et al, 2005). Also, although our family and case-control samples were mostly Caucasians, the African American and East Indian subjects were also included. These additional ethnic groups have not previously been studied in GABRG2 and SCZ. However, when we analyzed only Caucasian subjects, our findings remained significant for the case-control sample. Another possible reason for the mixed findings could be the different sets of polymorphisms used in previous studies. A separate polymorphism in

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linkage disequilibrium with rs183294, and not rs183294 itself, may be conferring SCZ susceptibility because the results became more significant with haplotype analysis. Yet another explanation could be that our reported positive results were spurious. Nonetheless, our results would have survived multiple-testing correction for five markers. Replication in independent

samples with larger sizes is required, especially if the effect conferred by GABRG2 is small. Functional analysis of polymorphisms in GABRG2 is needed to find functional variants for more targeted genetic studies. The GABRG2-coded GABAA receptor 2 subunit interacts directly with the dopamine D5 receptor in a mutually inhibitory manner (Liu et al, 2000). Exploring the genetic interaction between GABRG2 and DRD5 polymorphisms may reveal a larger combined genetic effect, and shed some light on the discrepant findings on GABRG2. In the second manuscript, we investigated a member of the D2 dopamine receptor family, D3. Ser9Gly has been investigated in numerous SCZ genetic studies, and the results have been mixed (Jonsson et al, 2004). A number of meta-analyses have yielded both positive and negative findings with odds ratios of close to one (Jonsson et al, 2004). We also examined BDNF, which is required for the expression of D3 DA receptor in the striatum (Guillin et al, 2001). The previous findings for BDNF in SCZ were mixed, with three meta-analyses failing to find a significant effect of Val66Met or C270T in SCZ (Naoe et al, 2007; Qian et al, 2007; Xu et al, 2007; Kanazawa et al, 2007; Zintzaras et al, 2007). Most previous studies focussed on Ser9Gly in DRD3, and Val66Met and C270T in BDNF, and did not explore other regions of the DRD3 and BDNF genes. Therefore, we examined ten polymorphisms across and surrounding DRD3 and six polymorphisms spanning BDNF for association with SCZ. Our analyses of BDNF and DRD3 did not yield significant findings in either family or case-control samples, suggesting that neither BDNF nor DRD3 plays a major role in SCZ. Although the results were negative for the

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diagnosis of SCZ, BDNF and DRD3 may affect phenotypes that are related to SCZ. Age of onset was recently found to be affected by Ser9Gly in our sample in that the high-activity Gly allele is associated with earlier onset (Renou et al, 2007). We found a significant interaction between Ser9Gly and Val66met in the analysis of suicide data in our SCZ sample using the HELIXTREE program. Specifically, schizophrenia patients who are heterozygous at both Ser9Gly and Val66Met are at higher risk of attempting suicide. One possible explanation for this observation is heterosis, a term that refers to increased vigour as a result of outbreeding. It was first observed in maize where the hybrid maize offspring by cross-fertilization of two different inbred maize parents are 25% taller than the parental maize (Hochholdinger and Hoecker, 2007). On the other hand, heterozygous mRNA products may in some cases interfere with each other and cause overall reduction in gene expression (Wang et al, 1995). The mechanism behind this phenomenon is poorly understood. Smoking data is currently being analyzed (Le Blanc et al, in preparation). Additional phenotypes important in SCZ, including alcohol and substance use, should be analyzed in future studies. While it appears that the interaction between BDNF and DRD3 does not play a significant role in SCZ development, the observed interaction between Val66Met and Ser9Gly in suicide attempts warrants independent replication. In the third manuscript, we investigated the DRD3 gene in TD, a motor side effect of long-term antipsychotic treatment that occurs in a subset of SCZ patients. The association of Ser9Gly in TD has been replicated in two meta-analyses (Lerer et al, 2002; Bakker et al, 2006). However, its effect may be small given the small odds ratio estimated from the meta-analyses. Thus, we investigated ten polymorphisms spanning the DRD3 gene in search of additional variants that may contribute to TD development. We did not find Ser9Gly to be significantly associated with TD in our Caucasian or African-American sample. We found rs905568 in the 5 Page 163

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region to be associated with TD. This polymorphism is adjacent to a gene coding for a putative transcription factor further upstream, ZNF80. We genotyped three non-synonymous polymorphisms within the ZNF80 gene, and found them not to be significantly associated with TD. The results suggest that either rs905568 itself or a DNA variant in linkage disequilibrium with rs905568 influences TD risk. The function of rs905568 has not been investigated. It may be involved in enhancer elements that regulate the expression of DRD3. Preliminary consensus sequence analysis showed that the polymorphism alters the recognition for the transcription factor Pax3, a developmentally regulated transcription factor. The Pax3 consensus sequence is relatively common within the human genome (random chance of one Pax3 site for every 20000 bases); therefore, the functional consequence of rs905568 on Pax3 recognition should be viewed with caution. Pax3 is expressed in the neural tube and neural crest during embryogenesis (Pruitt et al, 2004). Another possible function for rs905568 may be the use of a rare and yet unidentified alternative promoter for DRD3. For example, the first intron in DRD2 is approximately 50kb in size, so it is not unreasonable to hypothesize the presence of additional DRD3 exon(s) in the intergenic region between DRD3 and ZNF80. To date, only 9kb of genomic sequence upstream of DRD3 gene has been investigated (Anney et al, 2002). More comprehensive analysis of the 5 region of DRD3 is required to answer these questions. We also investigated six polymorphisms spanning the BDNF gene in TD. We did not find significant association in single-marker or haplotype tests of BDNF, nor did we find significant gene-gene interactions between BDNF and DRD3 polymorphisms using HELIXTREE. Thus, the interaction of variations in the genes for BDNF and D3 does not appear to play a major role in the pathophysiology of TD. Investigation in independent samples is required before the role of BDNF in TD can be dismissed.

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In the fourth manuscript, we found an association between TD and two adjacent DRD2 gene polymorphisms, C939T and C957T. The association did not appear to be specific for either polymorphism, because the significance level increased with haplotype analysis. TD could be associated with a polymorphism in linkage disequilibrium with the two polymorphisms. In fact, our data showed generally high linkage disequilibrium observed in the 3 region of DRD2 that encompasses the TaqIA polymorphism. In the fifth manuscript, TaqIA, a polymorphism 3 to DRD2, was associated with TD, while 141C Ins/Del, a polymorphism 5 of DRD2 was not. Both TaqIA and C957T have been associated with altered D2 DA receptor expression (Jonsson et al, 1999b; Noble et al, 1991; Pohjalainen et al, 1998; Ritchie and Noble, 2003; Thompson et al, 1997; Hirvonen et al, 2004; Duan et al, 2003). T957 has been associated with decreased in vivo D2 DA receptor binding in healthy human subjects, possibly through its effect on D2 mRNA stability (Duan et al, 2003; Hirvonen et al, 2004). TaqIA is now identified as a non-synonymous polymorphism (K713E) in ANKK1, a novel Serine/Threonine protein kinase gene adjacent to DRD2, with its mRNA detected in the placenta and spinal cord (Neville et al, 2004). Functional studies of ANKK1 are required. Nonetheless, most studies have associated the A1 allele with decreased D2 levels (Ritchie and Noble, 2003). Both A1 and T957 alleles were associated with decreased TD risk, suggesting that decreased D2 levels may protect against TD occurrence. Conversely, increased D2 levels predict the occurrence of TD. This is in agreement with brain imaging studies showing increased occupancy at the D2 DA receptor is associated with TD (Kapur et al, 2000). In the sixth manuscript, we investigated the possible association between TD and eight AKT1 polymorphisms. We did not find a significant association. Since we investigated AKT1 due to its role in signalling downstream of the D2 DA receptor (Beaulieu et al, 2007), we conducted a gene-gene interaction analysis using pairs of DRD2 and AKT1 polymorphisms in the Page 165

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HELIXTREE program. Preliminary findings show the association of DRD2 C939T became more significant on the G allele background of AKT1 rs3730358 in determining TD severity. Haplotypes with the G allele at rs3730358 have been associated with changes in AKT1 levels (Emamian et al, 2004), suggesting that AKT1 signalling from the D2 DA receptor may be important for TD development. Another possible explanation for the association and interaction could be the proximity of the C957T and C939T polymorphisms to the alternatively spliced exon six in DRD2. C957T and C939T could be in linkage disequilibrium with polymorphisms that affect the splicing efficiency of exon six. Aberrant splicing has been demonstrated to be the mechanism behind the role of the microtubule-associated protein tau (MAPT) in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Mutations surrounding exon 10 of MAPT leads to decreased splicing efficiency, leading to impaired microtubule assembly and resultant neurofibrillary tangles and neurodegeneration (Lee VM et al, 2001). The mechanism of alternative splicing of exon six in DRD2 is unclear, and may be epigenetically regulated (Young et al, 2005). While the full-length long D2 isoform is predominantly located postsynaptically, splicing at exon six produces the short D2 DA receptor isoform that is predominantly presynaptic (Usiello et al, 2000). It is interesting to note that the two D2 DA receptor isoforms may bind or respond to antipsychotics differently (Xu et al, 2002; Centonze et al, 2004). Examining the association of C957T and C939T and their haplotypes on the expression and ratio of the D2 DA receptor isoforms will help clarify the role of D2 DA receptor in TD, as well as other conditions. Molecular studies are required to explore the role of AKT1 in signalling from the two D2 DA receptor isoforms. The DRD2-AKT1 interaction in TD demonstrates that single genes by themselves may not affect risk of TD and other complex diseases but may play a role within the context of particular signalling pathways. AKT1 is not associated with TD by itself possibly

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because it has multiple functions in multiple organ systems. However, its role in dopamine signalling may play a modifying role in TD susceptibility.

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Zai et al 8.2 LIMITATIONS AND CONSIDERATIONS 8.2.1 Sample Characteristics and Power

VIII. Discussion

Our TD results should be regarded with caution because even though the main sample that was analyzed consisted of European Caucasians only, with age and gender adequately matched, the medication histories of the subjects were mixed. As discussed in the introduction, medication type and period could influence TD occurrence and severity. Moreover, smoking is a risk factor for TD, as is the use of alcohol and illicit drugs. These factors could have confounded our TD findings in that these factors could have created spurious significant associations or masked associations that would have otherwise been significant. This point is clearly demonstrated by the inclusion of multiple factors in determining the risk of TD by Basile et al (2002). In the review, the authors combined the effects of pharmacodynamic (DRD3_Ser9Gly), pharmacokinetic (CYP1A2*F), as well as age, ethnicity, and sex, and showed that this group of variables accounted for over 50% of the risk for TD in their sample (Basile et al, 2002). The results from the meta-analysis of the DRD2 gene in TD should be viewed as the current status in the investigation of DRD2 in TD, until further markers or samples are investigated. Lack of significant heterogeneity in the meta-analysis could be due to the lack of power with the small number of studies included. Heterogeneity does exist among studies. For example, the assessment of TD was not the same among the studies. Some included persistent cases that were examined at least twice, while others included probable cases where patients were examined for TD only once. The background patient population could be different among studies in that some included only schizophrenia patients and others included schizophrenia and schizoaffective disorder patients. Medication histories of the study populations are also different. Some samples only underwent medication with typical antipsychotics, while others underwent

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both typical and atypical antipsychotic treatment. Additional studies with more homogeneous samples are required before firm conclusions can be drawn on the association of DRD2 with TD. The known ethnic heterogeneity within the case-control and family samples could have diluted any effects of the genetic variants or given rise to spurious positive findings of association between GABRG2 and SCZ. The significant findings in GABRG2 were maintained even after the exclusion of ethnicities other than Caucasian in the matched case-control sample. However, the findings in the family sample became less significant, possibly due to the loss of statistical power with the removal of over 20% of the sample. As for our entire TD sample, we analyzed the European Caucasians and African Americans separately because several DRD2 genotypes deviated from Hardy-Weinberg Equilibrium in the combined sample. Sample size remains a major limiting factor in genetic studies. Our TD sample, with approximately 80 SCZ patients with TD and 120 without, has 80% power to detect a genotypic relative risk for TD of as low as 1.9 if we genotype polymorphisms with minor allele frequencies of 20% and set the critical p-value at 0.05. With the marker minor allele frequency of 20% and at 0.05, our 229 SCZ case control sample pair has 80% power to detect genotypic relative risk for SCZ of as low as 1.8. While our TD and SCZ samples are moderate in size, they would be unlikely to detect the small relative risks of 1.2 to 1.5 that are commonly observed in complex diseases. In addition, as we are moving toward gene-gene interaction analyses, the samples will be divided into increasing number of comparison groups, making individual group sizes smaller. One way to overcome this limitation in addition to larger samples is to replicate the experiments in independent samples, and to follow up with meta-analyses.

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Zai et al 8.2.2 Multiple Testing

VIII. Discussion

For each single-marker test, we set the critical p-value at 0.05. In other words, we set the false-positive (type-I error) rate at 5% for each test. Because we are analyzing multiple markers in each study, the random chances of yielding a positive result is increased in the overall study. A common way of controlling for inflated false-positive rate of testing multiple markers is the Bonferroni correction. Traditionally, Bonferroni correction is used for post-hoc pair-wise comparisons among independent comparison groups after a global comparison among all comparison groups yields a significant result. It involves setting the critical p-value at a level calculated by dividing 0.05 by the number of pair-wise comparisons. It has been adopted for many genetic studies. For example, we tested 12 DRD2 markers in TD. The critical p-value threshold, below which we will consider the results significant, would have been 0.05/12, or 0.0042. Some argue that Bonferroni correction is over-conservative. In fact, if the 12 DRD2 markers were completely independent, statistically there would have been a 45% chance of getting a false-positive finding in the study. The critical p-value would have to be set at 0.0043 for each marker test in order to maintain the overall false-positive rate of the study at 0.05. Another issue with multiple testing corrections is that the markers in the study are often associated with one another. For example, the polymorphisms in the 3 region of DRD2 were in high linkage disequilibrium with one another given by the LD plot. Nyholt et al (2004) offered a solution by accounting for correlation between markers in the calculation of the critical p-value adjustment, thus decreasing the effective number of markers used in the study. However, the correction factor would change every time a different number of polymorphisms are tested (Perneger, 1998). Some researchers may simply minimize the number of markers tested or

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reported in order to avoid a large correction factor. This situation would prevent them from exploring many markers required to assess an entire gene of interest in an association study. Along the same line, how often are researchers willing to adjust their p-values every time they use the same clinical sample to study additional candidate genes and polymorphisms? Further, neither the Bonferroni nor Nyholt correction could explain the relationship among the polymorphisms in that the comparison groups are all derived from the same clinical data set. The single marker analyses can be considered as the same data set grouped differently according to genotypes at each polymorphism. A more recent approach to resolve this problem involves permutation. It is more computationally challenging because it involves iterative randomization of transmission status for family samples or randomization of affected status for case-control samples. The number of simulations is specified and the distribution of simulated data is compared to the observed data to derive the global permutation p-value (UNPHASED manual). This would control for the multiple testing issue because entire genes or sets of polymorphisms are considered, not just individual polymorphisms. Unfortunately, most computers available are not capable of running 10,000 permutations with multiple markers. Lastly, correction for multiple testing to minimize the chances of false-positive results will increase the chances of false-negative results, or type-II errors (Perneger, 1998). It is especially true for complex disorders where multiple factors of small effect sizes contribute to the conditions. As a result of the above limitations, a consensus on multiple testing correction has yet to be reached. Perhaps one option is to report the p-values both in their unadjusted raw form and also their corrected form (by whatever means the authors use), and leave the results up to the reader to decide whether the reported association is real or not. Replications in independent samples are usually the gold standard required to strengthen the genetic findings. It is also

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important to report raw data in publications to allow for future systematic reviews and metaanalyses using pooled samples to detect genetic factors of small effect sizes. Perhaps the key limitation for genetic association studies is that the results are correlative, and do not reflect necessarily a cause-and-effect relationship between the polymorphisms within the genes and TD or SCZ. This is especially true if the study is of retrospective design. Prospective cohort studies, where the size of each genotype group is matched before the emergence of the phenotype, be it SCZ or TD, are better at answering causality because it is less susceptible to sampling bias; however, prospective study design is very costly due to long followup periods and possibly high dropout rates. To establish causality, it would be necessary to determine the function of each significant polymorphism by cell culture and in-vitro molecular studies, followed by the use of genetically modified mice to determine the effects of the DNA variation on phenotypes of interest.

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Zai et al 8.3 FUTURE DIRECTIONS 8.3.1 Gene-gene interactions

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The manuscripts in the current thesis demonstrate the utility of using gene-gene interaction analysis to elucidate the genetic and molecular mechanism underlying complex disorders such as SCZ and TD. Future studies may include the interaction between other candidate genes of interest. Other genes in the DRD2-AKT1 signalling pathway could be added to the analysis to further explore the effects of this pathway on TD development. However, much larger sample sizes are required because the number of possible genotype combinations will be large, given that the number of possible genotype combinations is 3n with n being the number of biallelic markers examined. Another interaction of interest could be that between DRD5 and GABRG2. Association studies of DRD5 with SCZ have yielded mixed results (Muir et al, 2001; Hoogendoorn et al, 2005), and GABRG2 has not been associated with SCZ in most studies (Petryshen et al, 2005). Perhaps the interaction between the two genes, and not the individual genes, is important for SCZ development. There is evidence for genetic interaction in the case of BDNF Val66Met and DRD3 Ser9Gly in suicidal behaviour in SCZ patients. 8.3.2 Gene-environment interaction After decades of intense search for susceptibility loci of SCZ, the underlying genetic mechanism of SCZ is still far from resolved. SCZ development is also likely influenced by environmental factors, including marijuana use (Di Forti et al, 2007) and family stress (Phillips et al, 2007). For the purpose of discussion, we will focus on infectious agents as environmental risk factors. Several lines of evidence point to a role of prenatal immune challenge in SCZ development: epidemiological, molecular biological, genetic, and animal models. Obstetric complications such as premature birth, low birth weight, and hypoxia have been associated with SCZ (Cannon et al, 2002; Gilmore et al, 2000; Seeman et al, 2005). Winter and Page 173

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spring births appear to increase the risk of schizophrenia (Davies et al, 2003). Influenza epidemics have been associated with subsequent spikes in SCZ incidence (Mednick et al, 1988). Serological evidence of prenatal exposure to influenza, especially in the first trimester leads to a seven-fold increase in risk of the offspring developing SCZ (Brown et al, 2004). In these cases, serum autoantibodies were identified against brain structures and neurotransmitter receptors (Henneberg et al, 1994; Tanaka et al, 2003). IL-1, IL-2, and IL-4 levels were reported to be abnormal in CSF of SCZ patients (Licinio et al, 1993; Katila et al, 1994; Mittleman et al, 1997). A number of association studies and linkage studies have pointed to a role of the HLA region on chromosome 6p in SCZ (Wright et al, 2001). Specifically, the HLA-DRB1 locus has been consistently associated with SCZ in East Asian samples (Li et al, 2001; Akaho et al, 2000; Sasaki et al, 1999; Wright et al, 1996). Studies on the HLA-DQB1 gene have yielded mixed results (Chowdari et al, 2001; Gibson et al, 1999; Wright et al, 1996; Nimgaonkar et al, 1995a; Campion et al, 1992). Thus far, over twenty immune system genes have been examined in SCZ. IL10 (Chiavetto et al, 2002; Yu et al, 2004) and TNFA (Meira-Lima et al, 2003; Schwab et al, 2003; Tan et al, 2003; Boin et al, 2001; Duan et al, 2004; Riedel et al, 2002; Zai et al, 2006) genes were found to be associated with SCZ, while IL1B was found to be associated in a meta-analysis (Shirts et al, 2006). Other genes, including IL4 (Schwarz et al, 2006; Jun et al, 2003), IL2RB (Nimgaonkar et al, 1995b; Tatsumi et al, 1997), PLA2G1B (Strauss et al, 1999; Chowdari et al, 2001), and PNOC (Imai et al, 2001; Blaveri et al, 2001) should not be discounted due to repeated negative findings or mixed results, as none of these genes was examined thoroughly. The single positive findings in CCR5 (Rasmussen et al, 2006), and CTLA4 (Jun et al, 2002) genes require replication. Recently, a genome-wide association study identified IL3RA as a potential candidate gene for SCZ (Lencz et al, 2007a).

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Administration of human influenza virus (Patterson, 2002; Fatemi et al, 1999; Shi et al, 2003), and viral mimics (Borrell et al, 2002; Shi et al, 2003; Zuckerman et al, 2003; Meyer et al, 2005; Ozawa et al, 2006) in pregnant mice produced offspring that showed acoustic prepulse inhibition deficit and abnormal social interaction. These SCZ-associated phenotypes could be reversed by antipsychotics (Patterson, 2002; Fatemi et al, 1999; Shi et al, 2003). In addition to behavioral changes, decrease in reelin expression and increase in D2 DA receptor levels have been reported (Fatemi et al, 1999; Beraki et al, 2005; Ozawa et al, 2006) in offspring of immunechallenged pregnant mice. The observations are in agreement with the reported decrease in reelin expression in human postmortem SCZ brain samples (Guidotti et al, 2000). The current body of work is primarily focused on the dopamine hypothesis of SCZ. It has been suggested the dopamine system and the immune system may interact. All dopamine receptors have been detected by reverse-transcription PCR in peripheral blood lymphocytes from healthy individuals (Ostadali et al, 2004). D2 and D3 DA receptors, in particular, have been implicated in the dopamine induced increased T cell activation (Levite et al, 2001). The immune system, on the other hand, has been implicated in the regulation of the expression of D2 and D3 DA receptors. Specifically, IL-2, a TH1 cytokine that is found increased in SCZ patients, has been shown to induce the expression of BDNF and its receptor trkB (Besser and Wank, 1999). IL-10, a TH2 cytokine that has also been found increased in SCZ, has been linked to the induction of nerve growth factor (NGF) expression (Brodie C, 1996). NGF increases the expression of D2 DA receptor (Fiorentini et al, 2002), and BDNF increases the expression of D3 DA receptor (Guillin et al, 2001). Perhaps dysregulation, due to genetic predisposition or environmental triggers or both, in the interaction between the dopamine and immune systems leads to SCZ. Future genetic studies should introduce additional research data from medical records on the history of maternal infections and early life events in SCZ patients, so that the interaction Page 175

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between SCZ candidate genes and these environmental variables can be explored. In any case, having this environmental information will help minimize the heterogeneity within the study sample. 8.3.3 Whole Genome Association Recently, there is increasing interest in using genetic markers that are evenly and densely spaced across the entire genome to identify novel candidate genes. Lencz et al (2007a) published the initial findings from a genome-wide association study on SCZ and found a novel marker near CSF2RA and IL3RA to be significantly associated. However, this approach is very costly, so as a way to minimize expenses of individual genotyping, pooled sample whole genome association studies have been attempted. Steer et al (2007) were able to replicate association findings for known candidate genes PTPN22 and MAGI3 in rheumatoid arthritis. A novel candidate gene diacylglycerol kinase eta (DGKH) was implicated in bipolar disorder from a pooled sample whole genome association scan (Baum AE et al, 2007). There are likely additional candidate genes to be discovered for SCZ and TD, and using pooled sample whole genome association may provide new insights into these debilitating conditions. Also, genome-wide association studies are revealing deletions and copy-number variations that may be playing a role in SCZ (Lencz et al, 2007b). Future genome-wide studies could be extended to analyses of global epigenetic and variable-number repeats profiles.

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Zai et al 9.4 CONCLUDING REMARKS Through experiments on human genomic DNA, we conclude that:

VIII. Discussion

(1) Sequence variation in the GABAA receptor 2 subunit GABRG2 gene is associated with risk of SCZ. Other GABA genes in the vicinity of GABRG2 should also be investigated, as well as the interaction between the GABRG2 subunit and the D5 DA receptor. Neither BDNF, DRD3, nor their interaction is a major factor in SCZ, but the interaction between BDNF Val66Met and DRD3 Ser9Gly is associated with history of suicide attempt. Future studies should include investigations of subpopulations of SCZ patients that share specific SCZassociated phenotypes such as suicidal behaviour, smoking, age of onset, as well as antipsychotic response and side effects, in order to decrease the heterogeneity of the sample and increase the power to detect genetic associations. One SCZ-related phenotype that we are particularly interested in is Tardive Dyskinesia (TD). (2) Variations in the dopamine receptor DRD2 gene are associated with risk of TD, possibly by signalling via AKT1. The dopamine receptor DRD3 gene is associated with TD in some sample populations, but the interaction of BDNF and DRD3 genes does not appear to play a major role. Our association of the 5 region of DRD3 with TD encourages more comprehensive investigations of polymorphisms spanning and surrounding candidate genes in association studies. (3) Overall, the body of work presented in this thesis strengthens support for the dopamine hypothesis, in particular, the role of D2 and D3 dopamine receptors, in TD. Future genetic studies should involve analyzing more than one gene along pathways in association with TD (and other complex diseases) rather than testing single genes in isolation. Future studies should also include deriving genetically modified animals where multiple genes are mutated

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or inactivated to explore the biological effects caused by the genetic variations and their interactions.

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IX. References

REFERENCES

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