Molecular Psychiatry (2007) 12, 854–869 & 2007 Nature Publishing Group All rights reserved 1359-4184/07 $30.00 www.nature.com/mp ORIGINAL ARTICLE

Allelic variation in GAD1 (GAD67) is associated with schizophrenia and influences cortical function and gene expression RE Straub, BK Lipska, MF Egan, TE Goldberg, JH Callicott, MB Mayhew, RK Vakkalanka, BS Kolachana, JE Kleinman and DR Weinberger Clinical Brain Disorders Branch, Genes, Cognition, and Psychosis Program, Intramural Research Program, National Institute of Mental Health, NIH, US Department of Health and Human Services, Bethesda, MD, USA

Cortical GABAergic dysfunction has been implicated as a key component of the pathophysiology of schizophrenia and decreased expression of the gamma-aminobutyric acid (GABA) synthetic

enzyme glutamic acid decarboxylase 67 (GAD67), encoded by GAD1, is found in schizophrenic post-mortem brain. We report evidence of distorted transmission of single-nucleotide polymorphism (SNP) alleles in two independent schizophrenia family-based samples. In both samples, allelic association was dependent on the gender of the affected offspring, and in the Clinical Brain Disorders Branch/National Institute of Mental Health (CBDB/NIMH) sample it was also dependent on catechol-O-methyltransferase (COMT) Val158Met genotype. Quantitative transmission disequilibrium test analyses revealed that variation in GAD1 influenced multiple domains of cognition, including declarative memory, attention and working memory. A 50 flanking SNP affecting cognition in the families was also associated in unrelated healthy individuals with inefficient BOLD functional magnetic resonance imaging activation of dorsal prefrontal cortex (PFC) during a working memory task, a physiologic phenotype associated with schizophrenia and altered cortical inhibition. In addition, a SNP in the 50 untranslated (and predicted promoter) region that also influenced cognition was associated with decreased expression of GAD1 mRNA in the PFC of schizophrenic brain. Finally, we observed evidence of statistical epistasis between two SNPs in COMT and SNPs in GAD1, suggesting a potential biological synergism leading to increased risk. These coincident results implicate GAD1 in the etiology of schizophrenia and suggest that the mechanism involves altered cortical GABA inhibitory activity, perhaps modulated by dopaminergic function. Molecular Psychiatry (2007) 12, 854–869; doi:10.1038/sj.mp.4001988; published online 1 May 2007

Keywords: GAD1; GAD67; schizophrenia; COMT; epistasis

Introduction Levinson3 and Owen et al.4 for review). In addition, meta-analyses have highlighted additional promising Evidence supporting specific genetic factors in the regions.5,6 More importantly, genetic associations to etiology of schizophrenia has strengthened dramati- genes in these linkage regions have been confirmed cally in the past few years. While archival twin and recently in multiple samples.7–9 These genes include, adoption studies had long established that family risk among others, DISC1 on 1q,10–12 DTNBP1 (dysbindin) patterns were largely due to inheritance, concerns on 6p,13,14 NRG1 on 8p,15 catechol-O-methyltransfer- about the complexity of the risk architecture and ase (COMT) on 22q,16,17 DAOA (G72) on 13q18,19 and variability in early linkage studies had in some RGS4 on 1q.20–22 The linkage findings represented an quarters raised premature doubts that risk genes important step, enabling targeted searches within could be identified.1,2 In the last few years, however, defined regions of the genome. Each of the suscept- the momentum of the positive studies has increased ibility genes identified thus far in these regions, when tremendously. Several genome-wide linkage studies analyzed alone, accounts for only a small increase in have identified many regions that are now supported risk, usually less than twofold. Linkage signals may by data from multiple independent studies (see be due to single susceptibility genes with relatively large effect sizes and/or operating in a relatively large Correspondence: Dr RE Straub, Genes, Cognition, and Psychosis percentage of families, but more likely they are due to Program, National Institute of Mental Health, National Institutes the combined signals from multiple genes in the of Health, 10 Center Drive, Clinical Center, Building 10, Room region, each of lesser impact and prevalence. 4D18, MSC 1385, Bethesda, MD 20892-1379, USA. E-mail: [email protected] Linkage methods alone are underpowered to re- Received 4 May 2006; revised 7 February 2007; accepted 11 liably provide the initial detection of signals from March 2007; published online 1 May 2007 single genes of lesser effect or genes influencing GAD1 polymorphisms and schizophrenia RE Straub et al 855 liability in only a small percentage of families.1 Thus, of intracellular GABA reserves for metabolic activity the more powerful strategy has evolved of direct and other functions, rather than for vesicular release. examination of allelic variation in candidate genes, GAD67, but not GAD65, protein levels can be influ- those selected because of their functional character- enced by intraneuronal GABA levels and this occurs 53,54 istics or protein binding partners. This strategy has without a change in the level of GAD67 mRNA. resulted in evidence of association of schizophrenia There also appears to be isoform-specific physiologi- with many other genes that do not map to prominent cal feedback, in that GAD67 (but not GAD65) mRNA linkage regions, for example, GRM3 on 7q,23–25 AKT1 and protein levels were increased in cortical neurons 26,27 28 55,56 on 14q and CHRNA7 on 15q. after sensory learning. GAD67 deleted homozygous Given the low a priori odds that any gene will be a mice die immediately after birth and have less than risk factor, the interpretation and validity of candi- 10% of normal brain GABA levels.57 During develop- date gene results is critically dependent on existing ment, it appears that GABA influences multiple information about the biology of the gene relative to processes, including proliferation, neuroblast migra- the biology of the disease. Abnormal glutamatergic tion and dendritic arborization, acting in a paracrine, signaling has been a prominent ‘hypothesis’ about the non-synaptic mode of action.58 In fact, it now appears pathophysiology of schizophrenia,29,30 and it has been that dynamic remodeling of dendrites in GABAergic suggested that a number of the genes currently interneurons continues to occur in the adult visual implicated in schizophrenia are related to NMDA cortex.59 Finally, there is evidence that in adult mouse (N-methyl-D-aspartate) signaling in the brain.31–33 An hippocampal slices, GABAergic synaptic inputs de- equally prominent hypothesis involves gamma-ami- polarize proliferating progenitor cells, contributing to nobutyric acid (GABA) neuronal function,34–37 but their later differentiation and functional incorpora- this avenue has been relatively unexplored from a tion into the dentate gyrus.60,61 Thus, improper genetic perspective. The recent demonstration that regulation of GAD67, either during development or loss of NRG1/ERBB4 signaling alters the tangential adulthood, has the potential to produce a variety of migration of cortical interneurons and reduces the phenotypic results, some of which may increase the number of GABAergic interneurons in post-natal risk of schizophrenia. cortex38 is potentially a quite important mechanistic There have been three prior reports of GAD1 lead, since statistical evidence suggests that these two variation and risk for psychosis. A case–control study genes may synergistically increase the risk for of association with schizophrenia using a single SNP schizophrenia.39 (rs769404, marker M13 in this study) was negative.62 The evidence of GABA dysfunction in schizophre- A second study63 reported case–control results for a nia is abundant. From circumstantial evidence of an Danish bipolar affective disorder (BPAD) sample and association of schizophrenia with a lowered thresh- Scottish BPAD and schizophrenia samples for eight old for seizures,40 recent studies have found direct SNPs, six of which we tested here (M03, M04, M09, evidence of impaired cortical inhibition (i.e. M13, M16 and M19 in Table 1). They found no enhanced excitability),41 and reduced transcortical P-values < 0.05 in the Scottish schizophrenia or inhibition of motor circuitry.42 Studies of molecular BPAD samples, and in the Danish BPAD sample markers of GABA signaling in post-mortem brain of found genotype-wise P-values of 0.062, 0.016 and patients with schizophrenia have provided consistent 0.096 for SNPs M03, M04 and M09, respectively. evidence of diminished GABA activity.43,44 The Based on the evidence of a role of GABA in findings include diminished expression of SLC6A1 schizophrenia and the consistent findings of reduced 34 (GAT-1) protein on specific axon terminals, in- GAD67 expression in schizophrenic brain, we per- creased GABA receptors containing the alpha2 sub- formed a family-based association study of variation unit, which correlate with reductions in GAT-1 in the GAD1 gene and risk for schizophrenia in two expression45 and reduced expression of glutamic independent samples containing parent–child trios. acid decarboxylase GAD1 mRNA and of GAD67 Because the post-mortem data have implicated 46–49 protein, a critical enzyme in GABA biosynthesis. reduced expression of GAD67, we concentrated on Reduced expression of GAD67 in the neocortex of the SNPs in and around the promoter region. We report schizophrenic brain is one of the more consistent evidence that variations in the GAD1 gene are findings reported in post-mortem studies of schizo- associated with schizophrenia in both of these phrenia.50 samples. After our initial observation of association,

GAD67 is one of two major isoforms of GAD, an we sought to confirm these results in another clinical enzyme that converts glutamate to GABA.51 The other data set, the National Institute of Mental Health isoform, GAD65, encoded by the gene GAD2, has (NIMH) childhood onset series. While we continued generally not been observed to be altered in schizo- to pursue the series of investigations reported herein phrenic brain. GAD67 is encoded by the 17 exon, 46 kb in an effort to elucidate the mechanism of the genetic gene GAD1 located in 2q31. GAD67 exists as both association, the results in this third clinical study homo- and heterodimers in both soluble and mem- were reported separately.64 Thus, we now also report brane-bound forms, primarily within the cell soma that variations in this gene are associated with but also to a limited degree in terminals.52 In the abnormalities in cortical function that are prominent adult, it is thought to be critical for the maintenance in schizophrenia and in healthy siblings at risk for

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 856 Table 1 Marker and map information

SNP NCBI Coding CBDB CBDB NIMH-GI Location Chromosomal Intermarker Distance dbSNP rs# strand case control case positionb distancec from M01 allelesa MAF MAF MAF

M01 rs10432420 G/A 0.30 0.31 0.30 50 Flanking 171485819 0 0 M02 rs7557793 T/C 0.23 0.27 0.24 50 Flanking 171490832 5013 5013 M03 rs1978340 G/A 0.32 0.29 0.32 50 Flanking 171495628 4796 9809 M04 rs872123 T/C 0.24 0.25 0.26 50 Flanking 171496673 1045 10854 M05 rs2356236 A/G 0.34 0.34 0.36 50 Flanking 171497191 518 11372 M06 rs3762556 C/G 0.34 0.33 0.37 50 Flanking 171497585 394 11766 M07 rs3762555 G/C 0.23 0.25 0.24 50 Flanking 171497902 317 12083 M08 rs3749035 A/C 0.43 0.42 0.39 50 Flanking 171498287 385 12468

Start of transcript NM_000817 171498707 12888 M09 rs3749034 G/A 0.23 0.24 0.18 Exon 1, 50 UTR 171498982 695 13163 M10 rs3762553 C/G 0.30 0.29 0.32 Intron 1 171499605 623 13786 M11 rs2270335 C/T 0.24 0.25 0.26 Intron 1 171500203 598 14384

Start codon (Met) of transcript NM_000817 171500609–11 14790–92 M12 rs2241165 T/C 0.24 0.25 0.24 Intron 2 171503886 3683 18067 M13 rs16823181 T/C 0.41 0.41 0.38 Exon 3, His37His 171504132 246 18313 M14 rs3828275 C/T 0.43 0.42 0.39 Intron 3 171508247 4115 22428 M15 rs10191129 C/G 0.24 0.23 0.25 Intron 5 171514100 5853 28281 M16 rs769390 A/C 0.27 0.27 0.27 Intron 6 171518962 4862 33143 M17 rs701492 C/T 0.30 0.29 0.30 Intron 9 171527987 9025 42168 M18 rs3791850 G/A 0.21 0.22 0.22 Intron 12 171533607 5620 47788 M19 rs769395 A/G 0.27 0.29 0.27 Exon 17 (30 UTR) 171542310 8703 56491

Abbreviations: CBDB, Clinical Brain Disorders Branch; MAF, minor allele frequency; NCBI, National Center for Biotechnology Information; NIMH-GI, National Institute of Mental Health Genetics Initiative; SNP, single-nucleotide polymorphism; UTR, untranslated region. aMajor/minor. bFrom UCSC May 2004. cIn base pairs.

schizophrenia, and that a 50 untranslated region for schizophrenia.68 Subjects in the CBDB/NIMH (UTR) SNP affects expression in human prefrontal sample included patients with schizophrenia, their cortex (PFC), with a similar trend in hippocampus. available siblings (most of whom were unaffected) Moreover, because alterations in GABA activity in and controls. Participants were from 18 to 60 years of brain are likely to impact on the excitability of age, were above 70 in IQ and gave written informed neurons in cortical microcircuits implicated in schi- consent of an Institutional Review Boards (IRB)- zophrenia,65,66 we explored the potential interaction approved protocol. Exclusionary criteria included of GAD1 risk alleles with a functional variation in significant medical problems, history of loss of COMT, another putative susceptibility gene that consciousness for greater than 5 min, alcohol or drug impacts on cortical neuronal excitability. We present abuse within the last 12 months and electroconvul- preliminary evidence of an epistatic interaction of sive therapy within the last 6 months. All subjects these genes. Since specific genetic variations that were interviewed and examined by a neurologist to increase risk for complex genetic disorders such as rule out neurological comorbidities that might con- schizophrenia often provide relatively weak statisti- found the study. All subjects spoke English since at cal evidence, we have taken the approach that the least age 4, and over 98% were born and educated in interpretation of such associations can be enhanced the United States. Self-described ethnicity was 6.3% by providing coincident biological evidence for African American, 89.8% European American. DNA functional effects on pathophysiological mechanisms genotyping was available for 309 European American that are implicated in the illness.67 patients with schizophrenia (243 male, 66 female), 372 of their siblings (156 male, 216 female), 317 control subjects (163 male, 154 female) and 454 Materials and methods parents of probands. Neuropsychological testing and Human subjects functional magnetic resonance imaging (fMRI) were Subjects were recruited for the Clinical Brain Dis- acquired for subgroups, as described below. For all orders Branch’s (CBDB) ‘sibling study’, a study of analyses shown here, only European American neurobiological abnormalities related to genetic risk (labeled ‘Caucasian’ below) families were used.

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 857 An independent sample for follow-up studies to comprehension (using the Wide Range Achievement confirm family-based association with schizophrenia Test, or WRAT, which may be considered a measure of was obtained from the NIMH Genetics Initiative premorbid IQ) were also included to assess levels of (NIMH-GI) data set consisting of only European general intelligence. American (‘Caucasian’) families (N = 67). This sam- ple, which is a subset of the entire data set, consisted Imaging data acquisition and analysis of DNA from selected members of nuclear families Whole brain BOLD fMRI data were collected on a 3 T (average size = 3.04) including one or two siblings Signa scanner using a GE-EPI RT pulse sequence (GE (average 1.87) with a diagnosis of schizophrenia or Medical Systems, Milwaukee, WI, USA).75–77 All fMRI schizoaffective disorder (depressed subtype only), data were processed and spatially normalized to a and available parents. The subset was chosen to common stereotactic space (Montreal Neurologic maximize the number of potential offspring–parent Institute or MNI template) and analyzed using trios for family-based association analyses (details SPM99 software (Wellcome Dept. Cogn Neurol., available on request). Details about collection and http://www.fil.ion.ucl.ac.uk/spm/). Briefly, we gener- diagnostic procedures have been described pre- ated single-subject contrast maps by contrasting the viously.69–71 active condition against its control condition, for example, 2B (two back) versus 0B (no back) for the Psychiatric diagnosis N back. These first level analyses for each normal All subjects in the CBDB/NIMH sample were inter- control were one sample t-tests. Reported loci for viewed by a research psychiatrist who conducted the genotype effects on task-related activation exceed Structured Clinical Interview for DSM-IV (SCID-1, significance levels of at P < 0.05 (corrected for multi- -II). For probands, data from psychiatric records and ple voxel comparisons within a region of interest family members were evaluated before enrollment. encompassing the region of PFC showing a main task All diagnoses were reviewed by 1–2 additional effect) and are reported with reference to the MNI clinicians. Inter-rater reliability for two raters based standard space within SPM99 as in prior reports.17,78 on 20 randomly selected probands was 100% (for diagnosis of schizophrenia or schizoaffective disor- Functional magnetic resonance imaging der). For all analyses, subjects with the following To test whether allelic variation in GAD1 influenced diagnoses were included as probands: schizophrenia, brain function independent of manifest behavior, we schizoaffective disorder, simple schizophrenia, psy- used fMRI to measure BOLD activation during the N chosis NOS, delusional disorder or schizoid, schizo- back task, a measure of short-term (working) memory, typal or paranoid personality disorder. This is similar in a sample of normal subjects. This task has been to a ‘broad’ diagnostic criteria used by other investi- shown to activate the PFC in normal subjects, and gators.72 To reduce potential confounders in the patients with schizophrenia show abnormal engage- cognitive analyses, we excluded a priori subjects ment of PFC.75,76 The PFC phenotype has been with a history of ADD (attention deficit disorder) and imputed to altered cortical inhibition.75,79,80 We used dyslexia. This had no effect on the statistical a simple N back block design alternating between a significance of the genetic association findings two back (2B) working memory condition and a no reported below. back (0B) control condition. Subjects were instructed to recall the stimuli (visually presented digits 1–4) Cognitive testing seen ‘n’ previously – two prior for 2B and the Subjects performed an extensive cognitive test battery currently presented digit for 0B. Visual stimuli were as described previously.73,74 To reduce the number of presented via a back-projection screen and perfor- statistical tests, effects of GAD1 genotype were mance (accuracy and reaction time) recorded via the examined on 15 measures, most of which we have use of a button box.76 All of the normal subjects used previously reported to be related to genetic risk for in this analysis were matched for N back performance schizophrenia.73,74 These included (1) four tests of and reaction time, handedness (all right), age, gender episodic memory (Logical memory I and II and Verbal and IQ. For M02, we compared 1/1s (N = 50) with Paired Associates from the Wechsler Memory Scale, 2-allele carriers (N = 52). Genotype groups sometimes revised version (WMSR) and 1–5 SS scores from differed slightly (P < 0.05) in age; therefore, an addi- California Verbal Learning Test (CVLT)); (2) five tional SPM (SPM99: www.fil.ion.ucl.ac.uk/spm/) measures of working memory (t-scores for categories analysis using age as a covariate was performed, and and perseverative errors from the Wisconsin Card we found that it did not alter the results. Availability Sorting Task (WCST) and accuracy on the N back task of the MRI scanner was the only factor determining (N back one, N back two, N back three); (3) one whether fMRI was performed. measure of attention (D0 distractible condition from the Gordon Continuous Performance Task, CPT); (4) Genotyping verbal fluency for letters; (5) Trails A & B (Ocular DNA was extracted from white blood cells or scanning, speed, attention; Trails B also involves set cerebellar brain tissue using standard methods. For switching); (6) IQ (from the Wechsler Adult Intelli- clinical samples, blood was collected from all gence Scale, revised edition, or WAIS-R) and reading subjects as well as all available parents of patients

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 858 with schizophrenia. We selected 19 SNPs in GAD1 above and beyond the main effects. Odds ratios were from NCBI databases based on position and allele calculated relative to the 1/1 genotype at the GAD1 frequencies (Table 1). Genotyping was performed SNPs with either the 2/2 (G/G; Met/Met) genotype at using the Taqman 50-exonuclease allelic discrimina- rs4680 (Val158Met) or the 1/1 (A/A) genotype at tion assay81 (details available on request). Genotype rs2075507 (formerly rs2097603). reproducibility was routinely assessed by regenotyp- Two-tailed Student’s t-tests or one-way analyses of ing all samples for selected SNPs and was generally variance (ANOVAs) were used to test if GAD1 mRNA > 99%. Genotyping completion rate was > 95% and levels differed between the genotypic groups. Spear- genotyping errors were detected as Mendelization man’s coefficients of correlation were calculated to errors by the program TRANSMIT82,83 and as haplo- examine if mRNA levels were correlated with age, pH type inconsistencies by the program MERLIN,84 or post-mortem interval (PMI). In the case of sig- which identifies improbable recombination events nificant correlations, one-way analyses of covariance from dense SNP maps. (ANCOVAs) were performed co-varying for a corre- lated factor. To test the effects of sex or race on Statistical analyses dependent measures, two-way ANOVAs were used We measured linkage disequilibrium (LD) between with genotype and gender as independent factors. markers with the D0 and r2 statistics from unrelated case and control haplotypes in parallel by use of DNA sequence analysis the program LDMAX within the GOLD software We identified putative promoter regions and package.85 SNP effects on transcription factor binding sites For binary traits in families, single SNP association (TFBS) with the Genomatix software suite (http:// analysis was calculated using the TDTPHASE pro- www.genomatix.de), specifically the El Dorado gene gram version 2.37.86,87 We tested three-marker haplo- annotation database and the SNP Inspector tool of the types for association in a sliding window across the GEMS sequence analysis package. TFBS were pre- gene using the method implemented in TDTPHASE. determined in the El Dorado annotation by the The E-M algorithm in TDTPHASE was used for MatInspector program.93 unknown phase haplotype estimation. All P-values were computed empirically based on 100 000 permu- Human post-mortem tissue and RNA extraction tations. The FBAT,88 family-based association test, Detailed information about the CBDB/NIMH post- program also was used to test for individual marker mortem sample, including ascertainment and diag- association with the clinical phenotype, and P-values nostic procedures, neuropathology and toxicology are based on > 1000 permutations. screening, neuroleptic data, smoking history, manner The program COCAPHASE (version 2.404 from of death and agonal state, as well as tissue retrieval Frank Dudbridge’s web site http://www.hgmp.mrc. and processing and RNA extraction and quality ac.uk/~fdudbrid/software/unphased/) was used to assessment has been published.94 Post-mortem hip- test for disease association with case–control data. pocampal tissue (one hemisphere) from 76 brains of We carried out tests of association to quantitative normal controls (21 women, 55 men; 24 Caucasians, intermediate phenotypes using several approaches. 50 African Americans, two Hispanic) with mean age The program SNPHAP (version 1.0, available from 41.4715.4 years, PMI 31.3714.2 h and pH 6.5970.27 David Clayton’s web site http://www-gene.cimr. and dorsolateral prefrontal cortical (DLPFC) tissue cam.ac.uk/clayton/software/) was also used to infer (one hemisphere) from a subset of 67 brains of the haplotype frequencies in unrelated controls and in same normal controls (19 women, 48 men; 21 cases from the CBDB families. Very similar frequen- Caucasians, 44 African Americans, two Hispanic), cies were observed with the program PHASE.89 with mean age 40.9715.3 years, PMI 31.1713.5 h and Single SNP quantitative trait analysis (i.e. with pH 6.5970.24 were collected at the Clinical Brain cognitive phenotypes) was performed in parent– Disorders Branch, NIMH. An additional sample of affected child trios using the variance-components hippocampal and DLPFC tissue from the brains of 32 method from the version 2.4.3 of the program schizophrenic cases (10 women, 22 men; 14 Cauca- quantitative transmission disequilibrium test sians, 17 African Americans, one Hispanic), with (QTDT).90 For all QTDT analyses, the orthogonal mean age 45.6715.5 years, PMI 35.9715.6 h and pH model was used. In order to protect against possible 6.4970.28 was also used in this study. Thus, the total inaccuracies due to deviations from either normality sample comprised brain tissue from 108 subjects. The or selection on the trait, empirical P-values derived tissues were pulverized and stored at À801C. Total from 10 000 permutations are reported. RNA was extracted from approximately 300 mg of To test for epistasis, we used SPSS version 11.0 tissue using the TRIZOL Reagent (Life Technologies (SPSS Inc., Chicago, IL, USA) to script an uncondi- Inc., Grand Island, NY, USA) as described previously. tional logistic regression,91,92 which examined combi- The yield of total RNA was determined by absorbance nations of genotypes in COMT and GAD1, using at 260 nm. RNA quality was assessed with a high- affection status as the outcome variable. The ‘inter- resolution capillary electrophoresis (Agilent Techno- action-only’ P-value shown in Table 5 results from a logies, Palo Alto, CA, USA). RNA integrity number test of the significance of the interaction model terms (RIN) was used as an estimate of RNA quality (only

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 859 samples with RINX3.8 were included in the analy- and then incubated with an anti-GAD67 (0.5 g/ml) and sis). Total RNA (4 mg) was used in 50 ml of a reverse anti-b-actin (1:3000 dilution) antibodies. Blots were transcriptase reaction to synthesize cDNA, by using a rinsed in TBS-T, incubated in a peroxidase-conju-

SuperScript First-Strand Synthesis System for reverse gated rabbit (GAD67) or mouse (b-actin) secondary transcriptase-polymerase chain reaction (RT-PCR) antibody (1:10 000 dilution, Chemicon, Temecula, (Invitrogen, Carlsbad, CA, USA), according to the CA, USA) for 2 h in 5% normal goat serum in TBS-T manufacturer’s protocol. and rinsed in TBS-T. Blots were developed in ECL- plus (Amersham, Piscataway, NJ, USA) and exposed Real-time quantitative PCR to Kodak Bio-Max film. Films were digitized using a

GAD67 mRNA expression levels were measured by scanner, and the resulting images were analyzed real-time quantitative RT-PCR using an ABI Prism using gel plotting macros in the NIH Image software. 7900 sequence detection system with 384-well for- The values for all samples were expressed as a mat. Taqman probe and primers spanning exons percentage of the mean of controls on the same gel 11–12 were purchased from Applied Biosystems and also as ratios to b-actin. (Assay-on-Demand, Cat. no. Hs00241471_m1, Applied Biosystems, Foster City, CA, USA). Each 20 ml PCR contained 6 ml of cDNA, 1 mlof20Â primer/probe Results mixture and 10 ml of qPCR Mastermix Plus (Euro- gentec, Seraing, Belgium) containing Hot Goldstar GAD1 markers tested DNA polymerase, deoxyribonucleotide triphosphates Marker and map information for the 19 SNPs tested in with dUTP, uracil-N-glycosylase, passive reference and around GAD1 is shown in Table 1. We concen- and optimized buffer components. PCR cycling con- trated on the 50 flanking region, under the assumption ditions were as follows: 501C for 2 min, 951C for that since GAD1 expression is decreased in schizo- 10 min, 40 cycles of 951C for 15 s and 591C phrenic brain, at least some of the regulatory elements or 601C for 1 min. PCR data were obtained with involved in this dysregulation would be located there. the Sequence Detector Software (SDS version 2.0, Work on the mouse homolog had shown numerous Applied Biosystems, Foster City, CA, USA) and potential neuron-specific cis-regulatory elements well quantified by a standard curve method using serial upstream, and that 10.2 kb of upstream sequence was dilutions of pooled cDNA derived from RNA obtained sufficient to drive brain region-specific neuronal from hippocampi of 12 normal control subjects. SDS expression of a reporter gene in transgenic mice.95 software plotted real-time fluorescence intensity. The Therefore, we genotyped eight SNPs in the 12 kb threshold was set within the linear phase of the upstream of the transcription start site, a chromo- amplicon profiles. All measurements were performed somal region for which there are no known transcripts. in triplicates. None of the GAD1 SNPs or COMT SNPs (described below) deviated from Hardy–Weinberg equilibrium. Immunoblotting Supplementary Table 1 shows the pairwise dis- Human (100–130 mg) DLPFC tissue samples (1 g equilibrium coefficients from the program LDMAX tissue: 10 ml buffer) were homogenized in a protease when applied to genotypes from 309 Caucasian cases inhibitor-Tris-glycerol extraction buffer (AEBSF, from the CBDB/NIMH Sibling study sample. There 4-(2-Aminoethyl) benzenesulfonyl fluoride hydro- are quite variable levels of moderate to high LD chloride, 0.024%, aprotinin 0.005%, leupeptin between the markers typed. Results were similar 0.001%, pepstatin A 0.001%, glycerol 50%, Tris when either controls or the parents of the cases were 0.6%). Protein concentration was determined using used (data not shown). the Bradford assay. Homogenates containing 3 mgof total protein content per 10 ml were prepared for immunoblotting by diluting the homogenized sam- Clinical association, CBDB sample, effect of affected ples with water and adding NuPage LDS sample offspring gender buffer (Invitrogen, Carlsbad, CA, USA). Samples were The family-based TDT results from the CBDB sample then heated to 801C for 5 min to denature proteins. using the clinical phenotype are shown in Table 2. Ten microliters of each sample was loaded onto The program TDTPHASE was used first, and to precast 10% Bis-Tris polyacrylamide gels (Invitrogen, mitigate against potential errors, we followed up with Carlsbad, CA, USA), and proteins were separated by FBAT and we present the results of both analyses for electrophoresis at 200 V for 1 h using a single power comparison. When transmissions to both male source. Each gel contained a molecular weight marker and female affected offspring were considered, all ladder SeeBlue Plus 2 (Invitrogen Carlsbad, CA, USA) P-values were > 0.15. When only transmissions to and 14 samples from schizophrenics and controls. A affected female offspring were considered by setting total of four separate gels were used in one experi- male affected offspring phenotypes to unknown, three ment (n = 53 samples). Gels were transferred onto markers were positive. More strikingly, when male nitrocellulose membranes at 85 V for 1 h. Membranes offspring are considered alone, there is also no were blocked for an hour in 10% goat serum in Tris evidence of association, even though they outnumber buffered saline (TBS) with 0.1% Tween-20 (TBS-T), female cases nearly 4 to 1 (data not shown).

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 860 Table 2 TDT results from the CBDB family sample – effects of affected offspring gender and COMT Val158Met genotype

M# TDTPHASE resultsa FBAT resultsa

All 269 families Family subsetsb Family subsetsb

Val/Val Val/Met Met/Met All 269 Val/Val Val/Met Met/Met families All Offspring Female offspring only N =76 N = 135 N =58 N =76 N = 135 N =58

P-value T/nTc P-value T/nT Alleled P-value Allele P-value P-value P-value P-value Allele P-value P-value

M01 0.31 407/424 0.03 41/23 2 0.08 M02 0.76 447/451 0.14 M03 0.74 390/396 0.05 2 0.68 M04 0.95 451/452 0.44 M05 0.24 322/341 0.01 2 0.89 0.006 2 0.05 M06 0.45 313/325 0.01 2 0.77 0.008 2 0.02 M07 0.98 396/395 0.10 M08 0.40 244/230 0.05 77/59 1 0.02 1 0.61 0.003 1 M09 0.88 447/444 0.54 M10 0.97 291/290 0.75 M11 0.99 388/387 0.26 M12 0.97 313/312 0.33 M13 0.30 336/316 0.02 77/56 1 0.01 1 0.33 0.002 1 M14 0.67 325/317 0.62 M15 0.88 427/424 0.61 M16 0.81 423/419 0.03 2 0.68 0.02 2 0.02 M17 0.56 402/411 0.006 2 0.61 0.005 2 0.02 M18 0.16 381/360 0.30 M19 0.90 411/409 0.02 2 0.73 0.04 2 0.05

Abbreviations: CBDB, Clinical Brain Disorders Branch; COMT, catechol-O-methyltransferase; FBAT, family-based association test; TDT, transmission disequilibrium test. aP-values not shown are P > 0.05. bBoth genders included. cTransmissions and non-transmissions of the 1 allele. dAllele overtransmitted to affected individuals.

Clinical association, CBDB sample, effect of COMT controls, failed to yield positive results from GAD1 Val158Met genotype SNPs (data not shown). Table 2 also illustrates the effect on TDT results of stratifying families into three groups based on the Clinical association in CBDB case–control data set COMT Val158Met (SNP rs4680) genotype of the Genotype-wise results for individual SNPs from the affected offspring. Transmissions to both male and program COCAPHASE, comparing unrelated cases female affected offspring were included in these from the family data set to unrelated controls, are analyses. shown in Supplementary Table 2. All markers were When either the 135 families with Val/Met affected negative, as were haplotypes in sliding windows of offspring or the 58 families with Met/Met offspring two, three and four markers (data not shown). We were analyzed, none of the GAD1 SNPs were positive. generated haplotype estimations using the program In contrast, when the 76 Val/Val offspring families SNPHAP based on all 19 markers, run in parallel on were analyzed, eight of the 19 SNPs were positive. 309 cases and 317 controls. Three common haplo- The program FBAT gave a very similar pattern of types (frequencies 0.321, 0.172 and 0.166 in cases) results to those from TDTPHASE, but they tended to were found, and none of the 14 haplotypes with be more significant, and, in addition, five SNPs were frequency > 0.01 in both groups differed by more than positive in the Val/Met subset. A similar stratification 1% between groups. There were also no case–control scheme based on either the functional ‘l’ (long) and ‘s’ frequency differences in haplotypes composed of the (short) alleles at the SLC6A4 (serotonin transporter) 50 six markers (M01, M02, M04, M07, M09 and M11) variable number of tandem repeats (VNTR)96 or on the that showed the strongest LD with each other and that functional BDNF Val66Met SNP rs6265,78 both of individually had the strongest association with the which were tested specifically afterwards as negative cognitive phenotypes (data not shown).

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 861 Clinical association, NIMH-GI sample but especially so on three measures: a measure of Table 3 shows the FBAT results for the whole sample, attention (D0 distractible condition from the Gordon and parallel analyses where only transmissions to CPT), a measure of verbal list learning (CVLT 1–5 Std affected female or male offspring were considered. Scores) and a working memory task (N back). The Only SNP M03 is positive when all transmissions are clinical risk allele (allele 2 which is overtransmitted considered. It is more significant in the males-only to females) at M01 is associated with poorer attention analysis and it is the same (minor) allele that is and N back performance, but better verbal learning overtransmitted in the CBDB sample. In the NIMH-GI, scores. Overall, six of the eight SNPs that were six SNPs are positive in each of the gender-specific associated with the clinical phenotype in the analyses, but none are positive in both. Most Val/Val subset produced negative results with most importantly, seven of the eight positive SNPs in the or all of the 15 cognitive phenotypes. Val/Val family subset of the CBDB sample are positive in either the male or female analyses in the NIMH-GI Intermediate phenotype association – neuroimaging sample and for all seven SNPs the overtransmitted We tested for effects of three SNPs (M02, M09 and allele is the same. We only rarely observe such a M13) on the BOLD response with fMRI during the strong concordance in the pattern of positive results N back task in normal subjects. We tested SNP M02 between these two family samples, but given that the because it is strongly associated with a variety of stratification strategy was different (by Val/Met cognitive phenotypes but not with the clinical genotype versus by gender), the meaning of this is phenotype, SNP M09 because its 1 allele is associated unclear. with reduced GAD1 mRNA expression (see below) and also with the clinical phenotype in the NIMH-GI Intermediate phenotype association – cognitive and NIMH Childhood Onset samples64 and SNP M13 phenotypes because it is a coding polymorphism (His37His) We used the program QTDT to examine the relation- associated with the clinical phenotype in both the ship between individual alleles and 15 neurocogni- CBDB and NIMH-GI samples. SNPs M09 and M13 tive phenotypes in the families in the CBDB Sibling were not associated with differential activation. As Study and results from the 11 positive phenotypes are shown in Figure 1, for M02, 1/1 subjects (N = 50) shown in Table 4. A number of SNPs that were in showed greater activation in right dorsal PFC moderate to high LD with one another were asso- (Brodmann’s area 46/10; Talairach and Tornoux (xy ciated with a broad spectrum of cognitive domains, z) coordinates (30 41 20), Z = 2.27, P = 0.01, k = 12) compared with 2-allele carriers (N = 52). Because the genotype groups are matched on performance and Table 3 FBAT results from the NIMH-GI family sample – reaction time, this pattern of increased prefrontal effect of affected offspring gender engagement is qualitatively similar to the ‘inefficient’ prefrontal function phenotype associated with genet- M# All offspring Females Males ic risk for schizophrenia76 as well as with COMT Val alleles. It is intriguing that the 1 allele associated with a b b P-value Allele P-value Allele P-value Allele greater activation also predicts poorer performance on the CVLT, WAIS (IQ) and WRAT Reading tests but M01 0.85 better performance on D0 dist., N back, Trails and M02 1.00 WMSR-Logical Memory 1 tests. M03 0.03 2 0.005 2 M04 1.00 Statistical interaction between GAD1 and M05 0.51 0.02 2 M06 0.51 0.02 2 COMT – case–control results M07 0.68 To expand on the observations shown in Table 2 that M08 0.19 0.03 1 in the CBDB family sample the genotype of the M09 1.00 0.05 1 affected offspring at COMT Val158Met strongly M10 0.41 affected our ability to detect association with SNPs M11 0.85 in GAD1, we tested more directly for epistasis using M12 1.00 the same cases and our CBDB controls. We used M13 0.27 0.04 1 unconditional logistic regression91,92 to examine com- M14 0.24 0.03 1 binations of genotypes in COMT and GAD1, using M15 0.85 0.02 1 affection status as the outcome variable. Table 5 M16 0.82 0.04 2 M17 0.64 0.01 2 contains only the positive (P < 0.05) results found M18 0.83 0.01 1 after testing for interaction between 19 GAD1 SNPs M19 0.49 0.04 2 and either COMT Val158Met or the COMT SNP rs2075507 (formerly rs2097603), which is located in Abbreviations: FBAT, family-based association test; NIMH- a putative promoter region and has been shown to GI, National Institute of Mental Health Genetics Initiative. influence COMT enzyme activity.97 These two COMT aAllele overtransmitted to affected individuals. SNPs exhibit only minimal LD (r2 = 0.148) with each bP-values not shown are P > 0.05. other. Odds ratios were calculated relative to the 1/1

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 862 Table 4 QTDT results with the cognitive phenotypes from the CBDB family sample

D0 dist. CVLT1–5 N back –N back – TrailsA TrailsB WAIS Est. WMSR Log. WMSR Log. WMSR WRAT Std Score two three Full Scale Mem. 1 Mem. 2 VPA1 total Reading IQ

M01 0.0004 0.005 0.003 0.009 M02 0.0001 0.0004 0.001 0.003 0.022 0.009 0.007 0.016 0.008 M03 M04 0.0003 0.006 0.004 0.048 0.031 0.033 M05 M06 M07 0.001 0.005 0.028 0.030 0.031 M08 0.008 M09 0.001 0.008 0.008 M10 M11 0.006 0.004 0.043 0.014 0.038 0.036 M12 0.018 0.005 0.020 0.019 M13 0.027 0.029 M14 0.026 M15 0.046 M16 M17 M18 0.001 0.037 0.022 M19

Abbreviations: CBDB, Clinical Brain Disorders Branch; CVLT, California Verbal Learning Test; QTDT, quantitative transmission disequilibrium test; WMSR, Wechsler Memory Scale, revised version. P-values < 0.05 are shown.

Figure 1 Effect of SNP M02 (rs7557793) genotype on the BOLD fMRI response during the N back working memory task. Results are displayed on the ‘single subject T1’ canonical image within SPM99. Healthy individuals who were genotype 1/1 (N = 50) showed an apparent abnormal increase in activation relative to 2-allele carriers (N = 52).

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 863 Table 5 Results of GAD1–COMT interaction analysis

GAD1 GAD1 genotype COMT Genotype interaction Odds ratio 95% CI 95% CI Case N Control M# val158meta lower upper N (rs4680) genotype P-value Type

M05 1/1 (A/A) 1/1 (G/G; Val/Val) 0.042 Protective 0.43 0.186 0.969 21 35 M05 1/2 (A/G) 1/1 (G/G; Val/Val) 0.001 Risk 6.57 2.076 20.79 36 19 M06 1/2 (C/G) 1/1 (G/G; Val/Val) 0.008 Risk 5.00 1.521 16.434 36 18 M10 1/2 (C/G) 1/1 (G/G; Val/Val) 0.003 Risk 4.30 1.623 11.388 45 30 M10 1/2 (C/G) 2/2 (A/A; Met/ 0.029 Protective 0.45 0.22 0.923 24 42 Met)

GAD1 GAD1 genotype COMT rs2075507b Genotype interaction Odds ratio 95% CI 95% CI Case N Control M# genotype lower upper N P-value Type

M03 2/2 (A/A) 1/1 (A/A) 0.035 Risk 4.17 1.106 15.72 12 3 M03 2/2 (A/A) 1/2 (A/G) 0.026 Protective 0.17 0.034 0.806 10 17

M06 2/2 (G/G) 1/2 (A/G) 0.040 Protective 0.20 0.042 0.926 9 17

M08 1/1 (A/A) 1/2 (A/G) 0.020 Protective 0.45 0.227 0.883 28 50 M08 2/2 (C/C) 1/2 (A/G) 0.006 Risk 5.53 1.65 18.56 28 24

M13 1/1 (T/T; His37His) 1/2 (A/G) 0.002 Protective 0.38 0.207 0.711 32 63 M13 2/2 (C/C; His37His) 1/2 (A/G) 0.002 Risk 6.11 1.973 18.936 30 27 M13 2/2 (C/C; His37His) 1/1 (A/A) 0.027 Protective 0.36 0.144 0.889 9 19

Abbreviations: CI, confidence interval; COMT, catechol-O-methyltransferase; GAD, glutamic acid decarboxylase. P-values < 0.05 are shown. aThe Met (2, A) allele frequency was 0.48 in cases and 0.49 in controls. bThe 2 (G) allele frequency was 0.40 in cases and 0.42 in controls. This SNP is the same as rs2097603.

genotype at the GAD1 SNPs with either the Met/Met were strikingly negative with the cognitive pheno- genotype at Val158Met or the 1/1 genotype at types. rs2075507. No main effects of the individual SNPs As a negative control for interaction, we tested were observed, but some combinations of genotypes GAD1 SNPs with either the functional ‘l’ (long) and ‘s’ greatly increased or decreased the risk of schizophre- (short) alleles at the SLC6A4 (serotonin transporter) 50 nia. For example, odds ratios of 6.57 (95% confidence VNTR96 or with the functional BDNF Val66Met SNP interval (CI): 2.08–20.8; P = 0.001), 5.00 (95% CI: rs6265.78 As expected, they were both negative. 1.5–16.4; P = 0.008) and 4.30 (95% CI: 1.62–11.4; P = 0.003) were observed for the Val/Val genotype GAD1 expression in post-mortem brain with heterozygotes at GAD1 SNPs M05, M06 and We used the CBDB/NIMH post-mortem brain collec- M10, respectively. This pattern is consistent with the tion, which is independent from the CBDB/NIMH strong LD we observed between these three SNPs Sibling Study sample, to measure GAD67 transcripts. (r2 = 0.752–0.984). The putative COMT promoter SNP Cases and controls did not significantly differ in age rs2075507 produced results of similar significance (P = 0.2), PMI (P = 0.12), pH (P = 0.8), but did trend levels, but with largely different GAD1 SNPs (M03, toward significance in RIN (P = 0.08). Expression

M06, M08, M13) than those identified when Val158- levels of GAD67 mRNA normalized by reference Met was used. Notably, these included a coding SNP gene b-2-microglobulin (B2M) were inversely corre- (M13, His37His) with an odds ratio of 6.11 (95% Cl: lated with age (R = À0.277, P = 0.004) and positively 1.97–18.9; P = 0.002). SNPs M03 and M06 are in fairly correlated with pH (R = 0.382, P < 0.0001) and RIN strong LD (r2 = 0.662), but they are only in weak LD (R = 0.3, P = 0.003) but not with PMI (R = À0.02, with the high LD (r2 = 0.874) pair of M08 and M13, an P > 0.8). There was a significant positive correlation indication that rs2075507 is perhaps interacting with between DLPFC GAD67 mRNA expression levels and more than one GAD1 haplotype to elevate risk. It is hippocampal GAD67 mRNA expression (R = 0.373, worth emphasizing that five of the six GAD1 SNPs P = 0.0002). identified as interacting with COMT SNPs were In the hippocampus, there were no significant clinical positives in the Val/Val subset, but all six differences between the diagnostic groups in the

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 864 expression of B2M (F(1,107) = 0.21, P > 0.5) or GAD67 contrast, family-based analysis based on the same mRNA normalized by B2M (F(1,107) = 0.08, P > 0.5). cases revealed a significant association within the This result is consistent with the report of Heckers et CBDB sample that became evident when analysis was al.98 In the DLPFC, however, schizophrenic patients restricted to females receiving the susceptibility

showed significantly reduced expression of GAD67 alleles. This observation may be due to the biology mRNA normalized by B2M (by 16%, F(1,98) = 4.5, of females with increased risk for schizophrenia. P = 0.036), whereas B2M expression was not different There is ample precedence that GABAergic mechan- between the groups (P > 0.3). In order to examine isms are at times sexually dimorphic, both during whether risk genotypes were associated with changes development and adulthood. For example, it appears

in GAD67 mRNA expression among controls and that GABA is responsible in part for the inhibition of schizophrenics, we performed a two-way ANOVA luteinizing hormone-releasing hormone release before with diagnosis and genotype as independent factors. the onset of puberty in female rhesus monkeys,99 and 0 The 5 UTR SNP M09 (rs3749034) trended toward sex differences in GABA turnover and GAD65 and significance in the hippocampus (F(1,104) = 3.1, GAD67 transcript levels have been observed in the rat P = 0.08) and reached significance in the DLPFC hypothalamus.100 Finally, in the olfactory bulb of (F(1,98) = 5.1, P = 0.026). There were significant diag- adult rats, a region of high synaptic plasticity, noses by genotype interactions in both the hippo- estrogen modulates in a cyclic manner, a truncated campus and DLPFC (F(1,104) = 8.6, P = 0.004 and version of the ‘embryonic’ isoform GAD25.101 It is also F(1,95) = 4.7, P = 0.0006, respectively). Post hoc ana- likely that the effects of alterations in GABA signaling lysis revealed that GAD1 mRNA expression normal- on cortical excitability differentially impact on cor- ized by B2M was decreased in both the hippocampi tical function in the males and females because of and DLPFC of schizophrenics with the 1/1 genotype other genetic and environmental factors. The poten- as compared with 2-allele carriers (both P < 0.01). We tial epistatic interaction we observed with COMT may then used ANCOVA with pH as a covariate to account further support this possibility, as COMT also has for differences in pH among genotypic groups, and been reported to show greater risk effects in females found that the schizophrenics carrying the 1/1 than in males.102 genotype still showed significantly lower expression Alternatively, since the affected males and females

of GAD67 mRNA compared with 2-allele carriers in the CBDB sample are from unrelated families, it is (P < 0.01 for both hippocampus and DLPFC). Similar possible that the gender differences observed may analysis of the expression of B2M was negative (all actually be secondary to some combination of biases P > 0.6). There were no significant effects of genotype in family ascertainment or environmental factors, or

on GAD67 mRNA expression (all P > 0.05) from M03, to genetic heterogeneity or differential epistasis. M05, M06, M08, M13, M16, M17 or M19. However, we also observed that the clinical associa- We then used a subset of DLPFC samples for the tion was dependent on gender in the NIMH-GI

assessment of GAD67 protein levels (27 controls, 26 sample, where the families often have both male schizophrenics) on Western blots. In this subset, and female affected offspring in the same sibship. controls did not differ from schizophrenics in pH or When we first observed positive association in both age (P > 0.5). A two-way ANOVA showed no signifi- the CBDB and NIMH-GI samples,103 and noted the cant effect of diagnosis or of the risk genotype at 50 concordance of positive SNPs and risk alleles, we UTR SNP M09, and no significant diagnosis by genotyped samples in the NIMH Childhood Onset

genotype interaction on GAD67 immunoreactivity Schizophrenia sample to seek additional confirma- (all F < 0.5, all P > 0.5). There was no significant tion. We found further evidence for association to correlation between protein and mRNA levels GAD1, but in that sample our negative markers M09, (r < 0.1, P > 0.6). M11 and M12 were positive.64 The reasons for such allelic differences between the all ‘Caucasian’ family samples we studied here and the Childhood Onset Discussion sample are at present unclear, although this may be We tested 19 SNPs in the GAD1 gene in two due in part to the latter being only about 50% independent samples for association with schizo- Caucasian. Likewise, the post-mortem sample is only phrenia, and for association with biological pheno- 29% Caucasian, which may explain in part the types related to schizophrenia. Since there have been observation that the SNPs that affect expression in

several reports of GAD67 transcript and protein post-mortem brain are not the SNPs that influence the downregulation in schizophrenia brain, we extended clinical and cognitive phenotypes in the CBDB our SNP coverage well into the 50 flanking region. sample. Of interest though, is that the 1 (G) allele at Moderate to high marker to marker LD was evident in the 50 UTR SNP M09 (rs3749034), which we found

a highly variable pattern, but three common 19 associated with reduced GAD67 transcript levels in marker haplotypes accounted for about 66% of the hippocampal and prefrontal regions of adult post- total. An additional nine haplotypes accounted for a mortem brains, is also contained in the four marker further 20%. Case–control analyses of single SNPs, as 1111 risk haplotype (our M04–M09–M11–M12) that is well as 19-marker and six-marker haplotypes using overtransmitted to affected individuals in the Child- the clinical phenotype were uniformly negative. In hood Onset sample.64

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 865 In addition to differences in ethnicity, the differing samples we have assembled, and that this will risk variants across studies are likely to reflect the use probably be characteristic of most studies unless it of underpowered samples combined with incomplete is actively controlled for. The statistical evidence for characterization of genetic variation, since most or of the GAD1 clinical association is not particularly all the SNPs and haplotypes tested here are probably strong, but perhaps this should be expected by now merely tags for a highly heterogeneous collection of when analyzing genes in isolation that are neither risk and protective haplotypes and diplotypes of necessary nor sufficient to generate the phenotype. varying frequencies and penetrances. In addition, The positive SNPs, and the particular risk alleles cross sample differences in associated variants may found in the CBDB and NIMH-GI samples are very also be due to the combined effects of genetic similar to each other, and given that the childhood heterogeneity, differential epistatic interactions with onset sample was also positive, it would appear that other susceptibility genes and variable environmental the signal in GAD1 from the CBDB sample is unlikely factors. to be due to chance. For example, when we stratified the CBDB families There is substantial variability in the specific risk on the COMT Val158Met genotype of the single SNPs, SNP alleles, haplotypes and diplotypes found affected offspring, the results changed dramatically. in studies of complex genetic disorders such as While none of the 19 GAD1 SNPs were positive in the mental illnesses. This has lead some observers to entire sample of 269 families, eight SNPs showed claim that such differences should be interpreted as association in the subset of 76 families containing discrepancies. However, such a categorization would Val/Val affected offspring. In addition, we found some require a specific model, based on a far greater level of evidence for an interaction between the Val/Val understanding of the variation present on risk genotype and three GAD1 SNPs in a logistic regres- chromosomes and their evolutionary history than is sion analysis of the case–control data set. An appeal- currently available. Such debates underscore the ing interpretation of this finding is that affected point that statistical association alone is an incom- individuals with the Val/Val genotype, who are plete strategy for identifying and confirming causative presumably relatively dopamine deficient in frontal genes.67 We have taken the approach that coincident cortex,65 are more susceptible to the biologic effects data about the effect of genetic variation on biologic of allelic variation in GAD1. The Val allele of COMT characteristics of the illness that are in turn plausibly has been consistently associated with prefrontal related to the biology of the gene can strengthen cognitive and physiologic deficits related to schizo- interpretation of such statistical results.65,73,109 Thus, phrenia.17,74,104,105 The mechanism of this effect is we have also tested the effects of variation in GAD1 likely related to altered dopamine signaling in PFC106 on cognitive and physiologic measures related to and altered excitability of glutamate and GABA schizophrenia and to GABA function in the cortex. neurons.66 Thus, broadly speaking, altered GABA We and others have consistently found that genes function might be expected to exaggerate the effects of associated with psychiatric illnesses show greater the COMT Val allele, and vice versa. Our statistical effects at these putative intermediate phenotypes than data suggesting an interaction of COMT and GAD1 are at the clinical diagnosis.25,28,110,111 The cognitive consistent with this prediction. While we have ruled domains of attention (CPT) and episodic and working out some of the more obvious artifacts (e.g. differ- memory (CVLT, N back) were both influenced by ences in sibship size, number of parents genotyped, genetic variation in GAD1. These are well recognized proportion female, order of ascertainment, etc.), the as core cognitive deficits associated with schizo- mundane possibility remains that stratifying the phrenia. The WRAT Reading Score, often cited as an families on COMT Val158Met genotype served also indicator of premorbid IQ, was also influenced. to stratify on some non-biological variable that Because cognition is a behavioral manifestation increases the detectability of the signal from GAD1 of information processing at a more basic level of in the Val/Val families. Such an artifact would apply cortical circuitry, we have also developed assays of to the case–control evidence for epistasis we found cortical function based on BOLD fMRI that relate to since the cases are probands from the families. physiologic abnormalities associated with schizo- Regardless of the underlying reasons, the data phrenia. With this approach, we examined the effect indicate that the power to detect an association of genetic variation in GAD1 in normal controls between GAD1 and schizophrenia may quite strongly matched for performance on the test administered depend, at least in part, on other characteristics of the during the fMRI experiment. Controlling for behavior sample tested, such as the frequency of alleles at other allows testing for gene effects at the level of cortical genes. We note also that, consistent with a number of information processing. We have shown that this other susceptibility genes we are studying, extensive strategy has greater power to reveal gene effects than testing of cases versus controls yielded no evidence of studies of the behavior mediated by these cir- association (even when stratifying this analysis on cuits.73,77,78,111 Val158Met genotype), whereas the TDT test using the In the BOLD fMRI assay during the prefrontally very same cases was positive. We assume that these dependent working memory task, the 1 allele of SNP type 2 errors reflect subtle but meaningful genetic M02 was clearly linked to inefficient activation of substructure107,108 in the ‘Caucasian’ case and control right dorsal PFC in normal subjects. This is the same

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 866 allele that was significantly associated with poorer either the CBDB or NIMH-GI samples, and yet it still performance on the CVLT, WAIS-IQ and WRAT strongly influenced multiple CBDB intermediate Reading tests, but also apparently associated with phenotypes. It is likely that the SNPs tested are better performance on the D0 dist., N back, Trails and monitoring several haplotypes that impact cognitive WMSR Logical Memory 1 tests. Surprisingly, this SNP function, only some of which are relevant to the is not associated with the clinical phenotype in any clinical phenotype and that a considerable variety of sample tested. We are uncertain of the basis for this, haplotypes and diplotypes exist across samples. To but suspect that it reflects differences in statistical dissect this will require considerable additional work power, distributions of cognitive measures and of on much larger samples, and to facilitate higher other higher order sample characteristics, all in resolution genotyping we will resequence a large addition to what must be a variety of GAD1 number of cases. In addition, it is likely that the

haplotypes and diplotypes, each with potentially decreased GAD67 in schizophrenia is due also to differing cognitive and clinical effects, and some in factors both genetic and environmental that are differential epistasis with other genes. independent of variation in GAD1. While acknowl- We have performed many tests in this study, testing edging these various complexities and uncertainties, many SNPs and haplotypes on a variety of clinical the fact that all three samples that we have tested to and biological phenotypes, followed by a series of date produce positive clinical associations, and that tests of potential genetic interactions. Virtually none risk SNPs and alleles are so similar in the CBDB and of the P-values we report would survive correction for NIMH-GI samples and that genetic variation also all of these tests if they were treated as independent, impacts strongly on measures of cortical function and without prior probability of a finding, as would suggests that GAD1 may be a fairly common risk be the case upon application of any biologically factor for schizophrenia. Finally, our observation of a agnostic Bonferroni-type ‘correction’. For many rea- potential interaction with COMT adds biologic plau- sons, we do not think that such adjustment of sibility to assumptions about the mechanisms by P-values is an appropriate or productive approach to which genes increase risk for schizophrenia7 and deal with the problem of multiple testing. First, many demonstrates the considerable importance of epistatic of the SNPs are in substantial LD and thus are not effects in mediating the strength of genetic association independent, nor are many of the phenotypes. results in psychiatric disorders. Second, the prior probability of SNPs in this gene impacting on expression of this gene and on inter- Acknowledgments mediate phenotypes related to risk for schizophrenia is not null. Third, we have taken the approach of We thank our patients and their families, as well as including supposed negative controls to test for Mary Weirich (MSW) for her invaluable work in randomness, and found no evidence of interaction leading the recruitment team and Drs Llewellyn between GAD1 with two other functional polymorph- Bigelow and Thomas M Hyde for their tireless work isms implicated in psychiatric phenotypes, that is, a in the clinical evaluation of subjects in CBDB Sibling VNTR in the serotonin transporter and BDNF val66- Study and Drs Mary M Herman and Thomas M Hyde met. Nevertheless, our principal findings require for their invaluable assistance in post-mortem tissue confirmation in other, preferably larger, schizophre- processing. This work was supported by the Intra- nia samples, and other psychiatric phenotypes should mural Research Program (IRP) of the NIMH, NIH. be likewise explored. We undertook this genetic study of GAD1 because References of the evidence of reduced expression of GAD67 in schizophrenic brain tissue. We predicted that the 1 Risch N, Merikangas K. The future of genetic studies of complex clinically positive SNPs would affect expression, and human diseases. Science 1996; 273: 1516–1517. 2 Moldin SO. The maddening hunt for madness genes. Nat Genet our data in post-mortem schizophrenic brain tissue 1997; 17: 127–129. 0 lend support to this only in that the 1 allele at the 5 3 Levinson DF. Molecular genetics of schizophrenia: a review of the UTR SNP M09 is associated with decreased expres- recent literature. Curr Opin Psychiatry 2003; 16: 157–170. sion. Other SNPs showing association with the 4 Owen MJ, Williams NM, O’Donovan MC. The molecular genetics of schizophrenia: new findings promise new insights. Mol clinical phenotype did not affect expression during Psychiatry 2004; 9: 14–27. adulthood, at least in this post-mortem sample. By 5 Badner JA, Gershon ES. Meta-analysis of whole-genome linkage software prediction, we found that M09 was within a scans of bipolar disorder and schizophrenia. Mol Psychiatry

putative promoter region for the GAD67 transcript, 2002; 7: 405–411. and that the DNA with the 1 allele of M09 lacks the 6 Lewis CM, Levinson DF, Wise LH, DeLisi LE, Straub RE, Hovatta I et al. Genome scan meta-analysis of schizophrenia and bipolar TFBS for proteins AREB6 and MYOD1 that are disorder, part II: Schizophrenia. Am J Hum Genet 2003; 73:34–48. present on the DNA with the 2 allele. The validity 7 Harrison PJ, Weinberger DR. Schizophrenia genes, gene expres- of this purely in silico prediction of course remains to sion, and neuropathology: on the matter of their convergence. Mol be determined experimentally. The 1 allele in M09 Psychiatry 2005; 10: 40–68. 8 Norton N, Williams HJ, Owen MJ. An update on the genetics of associated with reduced expression of GAD67 mRNA schizophrenia. Curr Opin Psychiatry 2006; 19: 158–164. was also a clinical risk factor in the NIMH Childhood 9 Straub RE, Weinberger DR. Schizophrenia genes – famine to feast. onset cases. However, M09 was not a risk factor in Biol Psychiatry 2006; 60: 81–83.

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 867 10 Fletcher JM, Evans K, Baillie D, Byrd P, Hanratty D, Leach S et al. schizophrenia to a chromosome 15 locus. Proc Natl Acad Sci Schizophrenia associated chromosome 11q21 translocation: USA 1997; 94: 587–592. identification of flanking markers and development of chromo- 29 Goff DC, Coyle JT. The emerging role of glutamate in the some 11q fragment hybrids as cloning and mapping resources. pathophysiology and treatment of schizophrenia. Am J Psychia- Am J Hum Genet 1993; 52: 478–490. try 2001; 158: 1367–1377. 11 Millar JK, Wilson-Annan JC, Anderson S, Christie S, Taylor MS, 30 Konradi C, Heckers S. Molecular aspects of glutamate dysregula- Semple CA et al. Disruption of two novel genes by a translocation tion: implications for schizophrenia and its treatment. Pharmacol co-segregating with schizophrenia. Hum Mol Genet 2000; 9: Ther 2003; 97: 153–179. 1415–1423. 31 Cloninger CR. The discovery of susceptibility genes for mental 12 Callicott JH, Straub RE, Pezawas L, Egan MF, Mattay VS, Hariri disorders. Proc Natl Acad Sci USA 2002; 99: 13365–13367. AR et al. Variation in DISC1 affects hippocampal structure and 32 Harrison PJ, Owen MJ. Genes for schizophrenia? Recent findings function and increases risk for schizophrenia. Proc Natl Acad Sci and their pathophysiological implications. Lancet 2003; 361: USA 2005; 102: 8627–8632. 417–419. 13 Straub RE, Jiang Y, MacLean CJ, Ma Y, Webb BT, Myakishev MV 33 Moghaddam B. Bringing order to the glutamate chaos in et al. Genetic variation in the 6p22.3 gene DTNBP1, the human schizophrenia. Neuron 2003; 40: 881–884. ortholog of the mouse dysbindin gene, is associated with 34 Lewis DA. GABAergic local circuit neurons and prefrontal schizophrenia. Am J Hum Genet 2002; 71: 337–348. cortical dysfunction in schizophrenia. Brain Res Brain Res Rev 14 Schwab SG, Knapp M, Mondabon S, Hallmayer J, Borrmann- 2000; 31: 270–276. Hassenbach M, Albus M et al. Support for association of 35 Benes FM, Berretta S. Gabaergic interneurons. Implications for schizophrenia with genetic variation in the 6p22.3 gene, understanding schizophrenia and bipolar disorder. Neuropsy- dysbindin, in sib-pair families with linkage and in an additional chopharmacology 2001; 25: 1–27. sample of triad families. Am J Hum Genet 2003; 72: 185–190. 36 Kalkman HO, Loetscher E. GAD(67): the link between the GABA- 15 Stefansson H, Sigurdsson E, Steinthorsdottir V, Bjornsdottir S, deficit hypothesis and the dopaminergic- and glutamatergic Sigmundsson T, Ghosh S et al. Neuregulin 1 and susceptibility to theories of psychosis. J Neural Transm 2003; 110: 803–812. schizophrenia. Am J Hum Genet 2002; 71: 877–892. 37 Wassef A, Baker J, Kochan LD. GABA and schizophrenia: a 16 Li T, Ball D, Zhao J, Murray RM, Liu X, Sham PC et al. Family- review of basic science and clinical studies. J Clin Psychophar- based linkage disequilibrium mapping using SNP marker macol 2003; 23: 601–640. haplotypes: application to a potential locus for schizophrenia at 38 Flames N, Long JE, Garratt AN, Fischer TM, Gassmann M, chromosome 22q11. Mol Psychiatry 2000; 5: 77–84. Birchmeier C et al. Short- and long-range attraction of cortical 17 Egan MF, Goldberg TE, Kolachana BS, Callicott JH, Mazzanti CM, GABAergic interneurons by neuregulin-1. Neuron 2004; 44: Straub RE et al. Effect of COMT Val108/158 Met genotype on 251–261. frontal lobe function and risk for schizophrenia. Proc Natl Acad 39 Norton N, Moskvina V, Morris DW, Bray NJ, Zammit S, Williams Sci USA 2001; 98: 6917–6922. NM et al. Evidence that interaction between neuregulin 1 and its 18 Chumakov I, Blumenfeld M, Guerassimenko O, Cavarec L, receptor erbB4 increases susceptibility to schizophrenia. Am J Palicio M, Abderrahim H et al. Genetic and physiological data Med Genet B Neuropsychiatr Genet 2006; 141: 96–101. implicating the new human gene G72 and the gene for D-amino 40 Hyde TM, Weinberger DR. Seizures and schizophrenia. Schi- acid oxidase in schizophrenia. Proc Natl Acad Sci USA 2002; 99: zophr Bull 1997; 23: 611–622. 13675–13680. 41 Daskalakis ZJ, Christensen BK, Chen R, Fitzgerald PB, Zipursky 19 Addington AM, Gornick M, Sporn AL, Gogtay N, Greenstein D, RB, Kapur S. Evidence for impaired cortical inhibition in Lenane M et al. Polymorphisms in the 13q33.2 gene G72/G30 are schizophrenia using transcranial magnetic stimulation. Arch associated with childhood-onset schizophrenia and psychosis Gen Psychiatry 2002; 59: 347–354. not otherwise specified. Biol Psychiatry 2004; 55: 976–980. 42 Mattay VS, Callicott JH, Bertolino A, Santha AK, Tallent KA, 20 Chowdari KV, Mirnics K, Semwal P, Wood J, Lawrence E, Bhatia Goldberg TE et al. Abnormal functional lateralization of the T et al. Association and linkage analyses of RGS4 polymorphisms sensorimotor cortex in patients with schizophrenia. Neuroreport in schizophrenia. Hum Mol Genet 2002; 11: 1373–1380. 1997; 8: 2977–2984. 21 Williams NM, Preece A, Spurlock G, Norton N, Williams HJ, 43 Costa E, Davis JM, Dong E, Grayson DR, Guidotti A, Tremolizzo L McCreadie RG et al. Support for RGS4 as a susceptibility gene for et al. A GABAergic cortical deficit dominates schizophrenia schizophrenia. Biol Psychiatry 2004; 55: 192–195. pathophysiology. Crit Rev Neurobiol 2004; 16: 1–23. 22 Talkowski ME, Seltman H, Bassett AS, Brzustowicz LM, Chen X, 44 Lewis DA, Hashimoto T, Volk DW. Cortical inhibitory neurons Chowdari KV et al. Evaluation of a susceptibility gene for and schizophrenia. Nat Rev Neurosci 2005; 6: 312–324. schizophrenia: genotype based meta-analysis of RGS4 poly- 45 Benes FM, Vincent SL, Marie A, Khan Y. Up-regulation morphisms from thirteen independent samples. Biol Psychiatry of GABAA receptor binding on neurons of the prefrontal 2006; 60: 152–162. cortex in schizophrenic subjects. Neuroscience 1996; 75: 23 Marti SB, Cichon S, Propping P, Nothen M. Metabotropic 1021–1031. glutamate receptor 3 (GRM3) gene variation is not associated 46 Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, Bunney Jr with schizophrenia or bipolar affective disorder in the German WE et al. Gene expression for glutamic acid decarboxylase is population. Am J Med Genet 2002; 114: 46–50. reduced without loss of neurons in prefrontal cortex of schizo- 24 Fujii Y, Shibata H, Kikuta R, Makino C, Tani A, Hirata N et al. phrenics. Arch Gen Psychiatry 1995; 52: 258–266. Positive associations of polymorphisms in the metabotropic 47 Hashimoto T, Volk DW, Eggan SM, Mirnics K, Pierri JN, Sun Z glutamate receptor type 3 gene (GRM3) with schizophrenia. et al. Gene expression deficits in a subclass of GABA neurons in Psychiatr Genet 2003; 13: 71–76. the prefrontal cortex of subjects with schizophrenia. J Neurosci 25 Egan MF, Straub RE, Goldberg TE, Yakub I, Callicott JH, Hariri AR 2003; 23: 6315–6326. et al. Variation in GRM3 affects cognition, prefrontal glutamate, 48 Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA. and risk for schizophrenia. Proc Natl Acad Sci USA 2004; 101: Decreased glutamic acid decarboxylase67 messenger RNA ex- 12604–12609. pression in a subset of prefrontal cortical gamma-aminobutyric 26 Emamian ES, Hall D, Birnbaum MJ, Karayiorgou M, Gogos JA. acid neurons in subjects with schizophrenia. Arch Gen Psychia- Convergent evidence for impaired AKT1-GSK3beta signaling in try 2000; 57: 237–245. schizophrenia. Nat Genet 2004; 36: 131–137. 49 Woo TU, Walsh JP, Benes FM. Density of glutamic acid 27 Schwab SG, Hoefgen B, Hanses C, Hassenbach MB, Albus M, decarboxylase 67 messenger RNA-containing neurons that ex- Lerer B et al. Further evidence for association of variants in the press the N-methyl-D-aspartate receptor subunit NR2A in the AKT1 gene with schizophrenia in a sample of European sib-pair anterior cingulate cortex in schizophrenia and bipolar disorder. families. Biol Psychiatry 2005; 58: 446–450. Arch Gen Psychiatry 2004; 61: 649–657. 28 Freedman R, Coon H, Myles-Worsley M, Orr-Urtreger A, Olincy 50 Torrey EF, Barci BM, Webster MJ, Bartko JJ, Meador-Woodruff JH, A, Davis A et al. Linkage of a neurophysiological deficit in Knable MB. Neurochemical markers for schizophrenia, bipolar

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 868 disorder, and major depression in postmortem brains. Biol phrenia Consortium: linkage analysis of African-American Psychiatry 2005; 57: 252–260. pedigrees. Am J Med Genet 1998; 81: 282–289. 51 Pinal CS, Tobin AJ. Uniqueness and redundancy in GABA 72 Straub RE, MacLean CJ, O’Neill FA, Burke J, Murphy B, Duke F production. Perspect Dev Neurobiol 1998; 5: 109–118. et al. A potential vulnerability locus for schizophrenia on 52 Kanaani J, Lissin D, Kash SF, Baekkeskov S. The hydrophilic chromosome 6p24–22: evidence for genetic heterogeneity. Nat isoform of glutamate decarboxylase, GAD67, is targeted to Genet 1995; 11: 287–293. membranes and nerve terminals independent of dimerization 73 Egan MF, Goldberg TE, Gscheidle T, Weirich M, Rawlings R, with the hydrophobic membrane-anchored isoform, GAD65. Hyde TM et al. Relative risk for cognitive impairments in siblings J Biol Chem 1999; 274: 37200–37209. of patients with schizophrenia. Biol Psychiatry 2001; 50: 98–107. 53 Rimvall K, Martin DL. The level of GAD67 protein is highly 74 Goldberg TE, Egan MF, Gscheidle T, Coppola R, Weickert T, sensitive to small increases in intraneuronal gamma-aminobu- Kolachana BS et al. Executive subprocesses in working tyric acid levels. J Neurochem 1994; 62: 1375–1381. memory: relationship to catechol-O-methyltransferase Val158Met 54 Rimvall K, Sheikh SN, Martin DL. Effects of increased gamma- genotype and schizophrenia. Arch Gen Psychiatry 2003; 60: aminobutyric acid levels on GAD67 protein and mRNA levels in 889–896. rat cerebral cortex. J Neurochem 1993; 60: 714–720. 75 Callicott JH, Mattay VS, Verchinski BA, Marenco S, Egan MF, 55 Gierdalski M, Jablonska B, Siucinska E, Lech M, Skibinska A, Weinberger DR. Complexity of prefrontal cortical dysfunction in Kossut M. Rapid regulation of GAD67 mRNA and protein level in schizophrenia: more than up or down. Am J Psychiatry 2003; 160: cortical neurons after sensory learning. Cereb Cortex 2001; 11: 2209–2215. 806–815. 76 Callicott JH, Egan MF, Mattay VS, Bertolino A, Bone AD, 56 Lech M, Skibinska A, Siucinska E, Kossut M. Learning-induced Verchinksi B et al. Abnormal FMRI response of the dorsolateral plasticity of cortical representations does not affect GAD65 prefrontal cortex in cognitively intact siblings of patients with mRNA expression and immunolabeling of cortical neuropil. schizophrenia. Am J Psychiatry 2003; 160: 709–719. Brain Res 2005; 1044: 266–271. 77 Hariri AR, Mattay VS, Tessitore A, Kolachana B, Fera F, Goldman 57 Asada H, Kawamura Y, Maruyama K, Kume H, Ding RG, Kanbara D et al. Serotonin transporter genetic variation and the response N et al. Cleft palate and decreased brain gamma-aminobutyric of the human amygdala. Science 2002; 297: 400–403. acid in mice lacking the 67-kDa isoform of glutamic acid 78 Egan MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, decarboxylase. Proc Natl Acad Sci USA 1997; 94: 6496–6499. Bertolino A et al. The BDNF val66met polymorphism affects 58 Represa A, Ben Ari Y. Trophic actions of GABA on neuronal activity-dependent secretion of BDNF and human memory and development. Trends Neurosci 2005; 28: 278–283. hippocampal function. Cell 2003; 112: 257–269. 59 Lee WC, Huang H, Feng G, Sanes JR, Brown EN, So PT et al. 79 Callicott JH, Ramsey NF, Tallent K, Bertolino A, Knable MB, Dynamic remodeling of dendritic arbors in GABAergic inter- Coppola R et al. Functional magnetic resonance imaging brain neurons of adult visual cortex. PLoS Biol 2006; 4: e29. mapping in psychiatry: methodological issues illustrated in a 60 Tozuka Y, Fukuda S, Namba T, Seki T, Hisatsune T. GABAergic study of working memory in schizophrenia. Neuropsycho- excitation promotes neuronal differentiation in adult hippocam- pharmacology 1998; 18: 186–196. pal progenitor cells. Neuron 2005; 47: 803–815. 80 Callicott JH. An expanded role for functional neuroimaging in 61 Ge S, Goh EL, Sailor KA, Kitabatake Y, Ming GL, Song H. GABA schizophrenia. Curr Opin Neurobiol 2003; 13: 256–260. regulates synaptic integration of newly generated neurons in the 81 McGuigan FE, Ralston SH. Single nucleotide polymorphism adult brain. Nature 2006; 439: 589–593. detection: allelic discrimination using TaqMan. Psychiatr Genet 62 De LV, Muglia P, Masellis M, Jane DE, Wong GW, Kennedy JL. 2002; 12: 133–136. Polymorphisms in glutamate decarboxylase genes: analysis in 82 Clayton D, Jones H. Transmission/disequilibrium tests for extended schizophrenia. Psychiatr Genet 2004; 14: 39–42. marker haplotypes. Am J Hum Genet 1999; 65: 1161–1169. 63 Lundorf MD, Buttenschon HN, Foldager L, Blackwood DH, Muir 83 Clayton D. A generalization of the transmission/disequilibrium WJ, Murray V et al. Mutational screening and association study of test for uncertain-haplotype transmission. Am J Hum Genet 1999; glutamate decarboxylase 1 as a candidate susceptibility gene for 65: 1170–1177. bipolar affective disorder and schizophrenia. Am J Med Genet B 84 Abecasis GR, Cherny SS, Cookson WO, Cardon LR. Merlin – Neuropsychiatr Genet 2005; 135: 94–101. rapid analysis of dense genetic maps using sparse gene flow trees. 64 Addington AM, Gornick M, Duckworth J, Sporn A, Gogtay N, Nat Genet 2002; 30: 97–101. Bobb A et al. GAD1 (2q31.1), which encodes glutamic acid 85 Abecasis GR, Cookson WO. GOLD – graphical overview of decarboxylase (GAD67), is associated with childhood-onset linkage disequilibrium. Bioinformatics 2000; 16: 182–183. schizophrenia and cortical gray matter volume loss. Mol 86 Dudbridge F, Koeleman BP, Todd JA, Clayton DG. Unbiased Psychiatry 2005; 10: 581–588. application of the transmission/disequilibrium test to multilocus 65 Weinberger DR, Egan MF, Bertolino A, Callicott JH, Mattay VS, haplotypes. Am J Hum Genet 2000; 66: 2009–2012. Lipska BK et al. Prefrontal neurons and the genetics of 87 Spielman RS, Ewens WJ. A sibship test for linkage in the schizophrenia. Biol Psychiatry 2001; 50: 825–844. presence of association: the sib transmission/disequilibrium test. 66 Winterer G, Weinberger DR. Genes, dopamine and cortical Am J Hum Genet 1998; 62: 450–458. signal-to-noise ratio in schizophrenia. Trends Neurosci 2004; 88 Horvath S, Xu X, Lake SL, Silverman EK, Weiss ST, Laird NM. 27: 683–690. Family-based tests for associating haplotypes with general 67 Weiss KM, Terwilliger JD. How many diseases does it take to map phenotype data: application to asthma genetics. Genet Epidemiol a gene with SNPs? Nat Genet 2000; 26: 151–157. 2004; 26: 61–69. 68 Egan MF, Goldberg TE, Gscheidle T, Weirich M, Bigelow LB, 89 Stephens M, Smith NJ, Donnelly P. A new statistical method for Weinberger DR. Relative risk of attention deficits in siblings haplotype reconstruction from population data. Am J Hum Genet of patients with schizophrenia. Am J Psychiatry 2000; 157: 2001; 68: 978–989. 1309–1316. 90 Abecasis GR, Cardon LR, Cookson WO. A general test of 69 Cloninger CR, Kaufmann CA, Faraone SV, Malaspina D, association for quantitative traits in nuclear families. Am J Hum Svrakic DM, Harkavy-Friedman J et al. Genome-wide search Genet 2000; 66: 279–292. for schizophrenia susceptibility loci: The NIMH Genetics 91 Cordell HJ, Todd JA, Hill NJ, Lord CJ, Lyons PA, Peterson LB et al. Initiative and Millennium Consortium. Am J Med Genet 1998; Statistical modeling of interlocus interactions in a complex 81: 275–281. disease: rejection of the multiplicative model of epistasis in type 70 Faraone SV, Matise T, Svrakic D, Pepple J, Malaspina D, Suarez B 1 diabetes. Genetics 2001; 158: 357–367. et al. Genome scan of European-American schizophrenia pedi- 92 Cordell HJ. Epistasis: what it means, what it doesn’t mean, and grees: results of the NIMH genetics initiative and millennium statistical methods to detect it in humans. Hum Mol Genet 2002; consortium. Am J Med Genet 1998; 81: 290–295. 11: 2463–2468. 71 Kaufmann CA, Suarez B, Malaspina D, Pepple J, Svrakic D, 93 Quandt K, Frech K, Karas H, Wingender E, Werner T. MatInd and Markel PD et al. NIMH Genetics Initiative Millennium Schizo- MatInspector: new fast and versatile tools for detection of

Molecular Psychiatry GAD1 polymorphisms and schizophrenia RE Straub et al 869 consensus matches in nucleotide sequence data. Nucleic Acids 103 Straub RE, Egan MF, Goldberg TE, Callicott JH, Hariri AR, Res 1995; 23: 4878–4884. Balkissoon R et al. GAD1, which encodes glutamate decarbox- 94 Lipska BK, Deep-Soboslay A, Shannon-Weickert C, Hyde TM, ylase 1 (GAD 67), is associated with adult onset schizophrenia in Martin CE, Herman MM et al. Critical factors in gene expression two independent samples. Am J Med Genet (Neuropsychiatr in postmortem human brain: focus on studies in schizophrenia. Genet) 2003; 122B:177. Biol Psychiatry 2006; 60: 650–658. 104 Mattay VS, Goldberg TE, Fera F, Hariri AR, Tessitore A, Egan MF 95 Kobayashi T, Ebihara S, Ishii K, Kobayashi T, Nishijima M, Endo et al. Catechol O-methyltransferase val158-met genotype and S et al. Structural and functional characterization of mouse individual variation in the brain response to amphetamine. Proc glutamate decarboxylase 67 gene promoter. Biochim Biophys Natl Acad Sci USA 2003; 100: 6186–6191. Acta 2003; 1628: 156–168. 105 Rosa A, Peralta V, Cuesta MJ, Zarzuela A, Serrano F, Martinez- 96 Collier DA, Stober G, Li T, Heils A, Catalano M, Di Bella D et al. Larrea A et al. New evidence of association between COMT gene A novel functional polymorphism within the promoter of the and prefrontal neurocognitive function in healthy individuals serotonin transporter gene: possible role in susceptibility to from sibling pairs discordant for psychosis. Am J Psychiatry affective disorders. Mol Psychiatry 1996; 1: 453–460. 2004; 161: 1110–1112. 97 Chen J, Lipska BK, Halim N, Ma QD, Matsumoto M, Melhem S 106 Tunbridge EM, Bannerman DM, Sharp T, Harrison PJ. Catechol-o- et al. Functional analysis of genetic variation in catechol-O- methyltransferase inhibition improves set-shifting performance methyltransferase (COMT): effects on mRNA, protein, and and elevates stimulated dopamine release in the rat prefrontal enzyme activity in postmortem human brain. Am J Hum Genet cortex. J Neurosci 2004; 24: 5331–5335. 2004; 75: 807–821. 107 Helgason A, Yngvadottir B, Hrafnkelsson B, Gulcher J, Stefansson 98 Heckers S, Stone D, Walsh J, Shick J, Koul P, Benes FM. K. An Icelandic example of the impact of population structure on Differential hippocampal expression of glutamic acid decarbox- association studies. Nat Genet 2005; 37: 90–95. ylase 65 and 67 messenger RNA in bipolar disorder and 108 Gardner M, Gonzalez-Neira A, Lao O, Calafell F, Bertranpetit J, schizophrenia. Arch Gen Psychiatry 2002; 59: 521–529. Comas D. Extreme population differences across Neuregulin 1 99 Terasawa E, Fernandez DL. Neurobiological mechanisms of the gene, with implications for association studies. Mol Psychiatry onset of puberty in primates. Endocr Rev 2001; 22: 111–151. 2006; 11: 66–75. 100 Searles RV, Yoo MJ, He JR, Shen WB, Selmanoff M. Sex 109 Egan MF, Goldberg TE. Intermediate cognitive phenotypes differences in GABA turnover and glutamic acid decarboxylase associated with schizophrenia. Methods Mol Med 2003; 77: (GAD(65) and GAD(67)) mRNA in the rat hypothalamus. Brain 163–197. Res 2000; 878: 11–19. 110 Freedman R, Olincy A, Ross RG, Waldo MC, Stevens KE, Adler 101 Krizbai IA, Katarova Z, Szabo G, Parducz A, Wolff JR. Modulation LE et al. The genetics of sensory gating deficits in schizophrenia. of the truncated GAD25 by estrogen in the olfactory bulb of adult Curr Psychiatry Rep 2003; 5: 155–161. rats. Neuroreport 2000; 11: 791–794. 111 Hariri AR, Goldberg TE, Mattay VS, Kolachana BS, Callicott JH, 102 Shifman S, Bronstein M, Sternfeld M, Pisante-Shalom A, Egan MF et al. Brain-derived neurotrophic factor val66met Lev-Lehman E, Weizman A et al. A highly significant association polymorphism affects human memory-related hippocampal between a COMT haplotype and schizophrenia. Am J Hum Genet activity and predicts memory performance. J Neurosci 2003; 23: 2002; 71: 1296–1302. 6690–6694.

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