Department of Psychiatry, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA; 2

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1 ORIGINAL RESEARCH ARTICLE (2005) 10, & 2005 Nature Publishing Group All rights reserved /05 $ Catechol-O-methyl transferase Val 158 Met gene polymorphism in schizophrenia: working memory, frontal lobe MRI morphology and frontal cerebral blood flow B-C Ho 1, TH Wassink 1, DS O Leary 1, VC Sheffield 2 and NC Andreasen 1,3,4 1 Department of Psychiatry, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA, USA; 2 Department of Pediatrics, Division of Molecular Genetics, Howard Hughes Medical Institute, University of Iowa, Iowa City, IA, USA; 3 The MIND Institute, Albuquerque, NM, USA; 4 Department of Psychiatry, University of New Mexico, Albuquerque, NM, USA The catechol-o-methyl transferase (COMT) gene is considered a leading schizophrenia candidate gene. Although its role in increasing schizophrenia susceptibility has been conflicting, recent studies suggest the valine allele may contribute to poor cognitive function in schizophrenia. V 158 M COMT genotype was obtained on 159 schizophrenia patients and 84 healthy controls. The effects of COMT genotype on four measures of working memory/ executive functions (Wisconsin Card Sorting, digit span backward, Trail Making and N-back tests) and on MRI frontal brain volumes were examined. Genotype distributions were not significantly different between patients and controls. There were no significant genotype or genotype-by-group effects on any working memory/executive function measures. No genotype or genotype-by-diagnosis interaction effects were found with MRI frontal lobe volumes. Randomization analyses using [ 15 O]H 2 O positron emission tomography (PET) cerebral blood flow data found Val/Val patients had higher frontal lobe activation than Met/Met patients while performing the one-back task. Overall, these findings do not support a major role for COMT in increasing susceptibility for schizophrenia or in mediating frontal lobe function. Age-related changes and phenotypic heterogeneity of schizophrenia may influence the complex relationships between COMT genotype and cognition. (2005) 10, doi: /sj.mp Published online 25 January 2005 Keywords: endophenotype; dopamine; MRI; candidate gene; association study; PET Correspondence: Dr B-C Ho, Department of Psychiatry 2880 JPP, University of Iowa, 200 Hawkins Drive, Iowa City, IA 52252, USA. beng-ho@uiowa.edu Received 18 May 2004; revised 9 August 2004; accepted 4 October 2004 Schizophrenia is a severe and disabling neuropsychiatric disorder. Although a century of genetic epidemiological research has provided strong evidence for a genetic basis, success in the search for schizophrenia susceptibility genes has thus far been limited. Risks for developing schizophrenia appear to be related to complex interactions between multiple genes, and between genes and environmental factors. The phenotype of schizophrenia is equally complex, and encompasses deficits in multiple domains of brain function, thereby presenting an elusive and difficult target for research into the neurobiological and genetic underpinnings of schizophrenia. 1,2 One strategy to address the challenges from genetic complexity and phenotypic heterogeneity has been to study endophenotypes. 3,4 Endophenotypes can be neurocognitive, neurophysiological, biochemical or neuroanatomical traits that correlate with the disease and/or disease severity. 5 These traits are heritable and can be measured in unaffected relatives. Studying endophenotypes (vs using psychiatric diagnostic categories) may potentially provide more power for genetic studies. 6,7 Endophenotypes are presumably biologically closer to the gene, transmitted in a less complex manner and can be more accurately assessed. Using such an approach, investigations into putative endophenotypes, such as smooth-pursuit eye movements and prepulse inhibition, have already identified potential schizophrenia susceptibility genetic loci and candidate genes Working memory is widely considered to be an endophenotype for schizophrenia. Working memory refers to the neuropsychological construct of temporary storage and manipulation of the information necessary for such complex cognitive tasks as language comprehension, learning and reasoning. 12 Schizophrenia patients as well as their unaffected relatives perform poorly on tasks that assess working memory function Working memory is moderately heritable with 43 49% of its variability accounted by genetic factors 24 In a series of studies involving a population isolate, a significant additive genetic heritability estimate was found in verbal working memory, and for visual working memory at a trend level. 20,21,25 Spatial working memory performance was more highly correlated in monozygotic twins than in

2 288 dizygotic twin pairs. Greater familial loading was associated with greater impairment in working memory performance. Working memory also appears to be a stable trait. Deficits have been demonstrated throughout the course of schizophrenia, 26,27 in neuroleptic-naïve, medicated and unmedicated patients, and are relatively unaffected by antipsychotic treatment. 16,28 30 Dopamine neurotransmission in the dorsolateral prefrontal cortex (DLPFC) plays a pivotal role in working memory. Single unit recording studies in non-human primates as well as functional neuroimaging studies in humans have consistently demonstrated this relationship. 31,32 The mesocortical dopamine system, which ascends from the ventral tegmental area, provides extensive connections to the neocortex, 33,34 where D1 receptors are the most highly expressed of the dopamine receptors. 35,36 In nonhuman primates studied using delayed response paradigms, stimulation of D1 receptors in the DLPFC enhances working memory. 37,38 These observations, together with the association between working memory deficits and aberrant DLPFC activation in schizophrenia patients, provide convergent evidence that disruptions in DLPFC dopamine neurotransmission may underlie working memory deficits in schizophrenia. 29,39 43 Elimination of extracellular dopamine relies on synaptic reuptake (via dopamine transporter and norepinephrine transporter) as well degradation by monoamine oxidase and catechol-o-methyl transferase (COMT) Owing to its role in the catabolism of dopamine, the COMT gene has been extensively studied as a schizophrenia candidate gene. In humans, there is a common V 158 M functional polymorphism 48 where a methionine (Met) substituted for a valine (Val) leads to a four-fold reduction in COMT enzyme activity. 49 Furthermore, the COMT gene is localized to chromosome 22q11, a region which has been implicated in schizophrenia linkage studies. 50 Deletion of chromosome 22q11 (as seen in velocardiofacial syndrome) is associated with high rates of schizophrenia-like psychosis. 51 A recent study has generated renewed interest in this gene, especially with regard to its role in mediating working memory and cognition. 52 The association between low-activity Met allele and better cognitive performance may be related to increased prefrontal dopamine bioavailability. 52,53 However, even though the COMT gene is an appealing candidate gene in schizophrenia, case control as well as family-based association studies to date have had mixed findings with regard to its contribution to schizophrenia susceptibility. 52,54 63 The aim of this study is to examine the role of V 158 M COMT polymorphism in working memory in schizophrenia. This study integrates data from multimodal investigational approaches, including neuropsychology, molecular genetics, morphometric and functional neuroimaging, to probe the genetic and phenotypic complexities of schizophrenia. This study extends the growing literature on COMT by examining several concurrent measures of working memory/ executive function in a sample of recent-onset schizophrenia patients (as compared to the more chronically ill patients examined in previous studies). In addition, no prior studies have examined the relationships between V 158 M COMT genotype and MRI frontal lobe morphology, nor have they compared prefrontal cerebral blood flow between schizophrenia patients with different COMT genotypes. Materials and methods Subjects In all, 159 schizophrenia patients and 84 healthy volunteers were obtained through the University of Iowa Mental Health Clinical Research Center (MHCRC). These subjects have participated in various MHCRC research studies approved by the University of Iowa institution review board. All subjects gave written informed consent to undergo research assessments. Patients were evaluated using a semistructured interview instrument, Comprehensive Assessment of Symptoms and History (CASH), 64 from which schizophrenia diagnoses meeting DSM-III-R or DSM-IV criteria were based. Healthy volunteers were recruited from the community through newspaper advertisements. They were initially screened by telephone, and further evaluated using an abbreviated version of the CASH to exclude subjects with current or past medical, neurological or psychiatric illnesses. To minimize the likelihood of spurious association arising from ethnic stratification, only subjects of Caucasian ancestry were included into this report. Sociodemographic characteristics of the sample are summarized in Table 1. Both groups were of comparable age. A significantly greater proportion of patients were male. Patients also had fewer years of education Table 1 Sociodemographics of the sample Controls Patients Statistic P-value N (% males) 84 (40.48) 159 (74.21) w 2 ¼ 26.7 o0.001 Age (years) 27.0 (7.03) 26.5 (7.35) t 241 ¼ Education (years) 14.8 (1.55) 12.9 (1.94) t 241 ¼ 8.51 o Full scale IQ 109 (12.05) 91.0 (12.45) t 241 ¼ o Parental education (years) 13.6 (2.18) 13.7 (2.63) t 241 ¼

3 and lower full scale IQ. However, parental educational attainment was comparable between the groups. Patients were evaluated relatively early in the course of their illness. The mean duration of illness (since the onset of first psychiatric hospitalization) was 2.5 years (SD ¼ 4.73). A total of 79 patients (48%) were evaluated during their first psychiatric hospitalization, and 57 (35%) were neuroleptic naïve. The median duration of lifetime neuroleptic treatment was 2 months (25th and 75th interquartile range ¼ 12 months). Genetic analyses DNA was prepared by high-salt extraction from whole blood. 65 The COMT codon 108/158 polymorphism was determined by restriction fragment length polymorphism analysis. PCR amplification was performed on a 165 bp fragment of the COMT gene using the primers 5 0 -GGGCCTACTGTGGCTACTCA-3 0 (forward) and 5 0 -GGCCCTTTTTCCAGGTCTGACA-3 0 (backward). Each 20 ml PCR reaction contained 20 ng genomic DNA, 5.0 pm of each primer, 400 mm of each dntp, 0.1 U of Taq DNA polymerase, 2.0 ml PCR buffer (100 mm Tris-HCl (ph 8.8), 500 mm KCl, 15 mm MgCl 2, 0.01% gelatin (w/v)), 4.0 ml betaine and 10.5 ml water. Cycling conditions were as follows: initial denaturation was at 941C for 3 min, followed by 40 temperature cycles consisting of 30 s at 941C for denaturation, 30 s at 601C for annealing and 30 s at 721C for extension, followed by a final elongation at 721C for 5 min. The PCR products were then digested by restriction enzyme NlaIII at 371C for 3 h. Digestion products were electrophoresed on 4% NuSieve agarose gel, and visualized by ethidium bromide staining under ultraviolet light. Genotyping was performed blind to subject s clinical status. Working memory/executive function assessment Although working memory performance has been widely studied, there remains considerable debate with regard to its definition and how to best quantify the construct. 66 Some investigations emphasize the temporary storage component of working memory, while others stress the importance of measuring the executive function component of working memory (ie both holding information online as well as manipulation of stored information). Until a better consensus has been reached with regard to its definition and measurement, working memory performance may be best assessed by using a combination of different tests. 66,67 In this study, working memory assessment consists of a battery of four tests: Wisconsin Card Sorting Test (WCST), 68 WAIS-R digit span backward, Trail Making test and N-back test. The first three working memory tests are part of a standard neuropsychological test battery, while the N-back test was administered during a positron emission tomography (PET) study on a subset of the sample (see below). WCST raw test scores for the number of perseverative errors were normalized for age and educational attainment, and transformed to t-scores based on population norms 68 (population mean ¼ 50, SD ¼ 10, higher scores indicate better performance). The WAIS-R digit span subtest typically combines forward and backward performance into a single score. In this study, only the total raw score of the number of correct items from the backward portion of the test is used, since previous studies suggest that the backward span task is a more robust measure of working memory. 17,66 The Trail Making test is a measure of executive functioning and set alternation. 69 Trail A involves connecting numbers in succession, while Trail B requires subjects to connect numbers and letters alternately in successive order. Working memory load in the Trail Making test consists of keeping the next target in mind while searching for the target on the sheet. By subtracting Trail A from Trail B, it is possible to assess frontal lobe functions by eliminating motor speed. 70 The time difference (s) taken to connect all items correctly on Trails A and B is used in the analysis. MRI acquisition and image processing Images were obtained on a 1.5-T GE Signa MR scanner. Three different MR sequences were acquired for each subject (ie T1-weighted spoiled grass, proton density (PD) and T2-weighted images). The imaging parameters have been described previously. 71 The images were processed using the locally developed BRAINS (Brain Research: Analysis of Images, Networks, and Systems) software package. Detailed descriptions of image analysis methods have been provided elsewhere In brief, the T1-weighted images were spatially normalized and resampled so that the anterior posterior axis of the brain was realigned parallel to the anterior posterior commissure line, and the interhemispheric fissure was aligned on the other two axes. The T2- and PDweighted images were aligned to the spatially normalized T1-weighted image using an automated image registration program. 75 These images were then subjected to a linear transformation into standardized stereotaxic Talairach atlas space 76 to generate automated measurements of frontal, temporal, parietal, and occipital lobes, cerebellum, and subcortical regions. 77 To further classify tissue volumes into gray matter (GM), white matter (WM) and cerebrospinal fluid (CSF), we employed a discriminant analysis method of tissue segmentation based on automated training class selection that utilized data from the T1, T2 and PD sequences. 78 In this study, we examined frontal lobe GM, WM and CSF volumes. PET data acquisition and image processing During image acquisition, subjects lay reclined and are oriented in the PET scanner with laser light guides aligned at the orbital meatal line of the brain. Stimuli for the one-back task were presented on a video monitor. The baseline condition consisted of a checkerboard flashing at a rate of 0.5 Hz for 40 s. Subjects were told to fixate on a dot at the center of the checkerboard. During the one-back condition, the 289

4 290 same 0.5 Hz flashing checkerboard stimulus was presented but each checkerboard had an asterisk. The location of the asterisk changed with each flash. The subject was instructed to press the index finger button if he/she saw the asterisk appear in the same location on two consecutive flashes. Although subjects were told that this may happen more than once, only one set of two consecutive flashes were presented 35 s into the stimulus. Reaction time (ms) in this one-back task was used for analysis. Regional cerebral blood flow (rcbf) was measured using the bolus [ 15 O]H 2 O method 79 with a GE4096- PLUS Scanner. A total of 15 slices (6.5 mm center-tocenter) were acquired with a 10-cm axial field of view. Images were acquired over a 100-s interval following venous injection of mci of [ 15 O]H 2 O. Images were reconstructed for a 40-s interval following bolus transit, determined by time activity curves from a region-of-interest over a cerebral artery. 80 Arterial blood sampling allowed calculation of tissue perfusion in ml/min/100 g tissue using the autoradiographic method. 80 The outlines of the PET images were automatically identified with an edge detection algorithm and the PET images for each condition for each subject were coregistered with their MR images using a variance minimization program. 75 An 18 mm Hanning filter was applied to the PET images for each condition to eliminate residual anatomical variability. The PET study was carried in 17 healthy volunteers and 16 schizophrenia patients to examine rcbf differences during working memory performance. Among the 17 healthy controls, two subjects had Met/Met genotype, 12 Met/Val and three Val/Val. Of the 16 schizophrenia patients, seven had Met/Met genotype, three Met/Val and six Val/Val. Owing to this genotype distribution, only the seven Met/Met and six Val/Val patients were used in subsequent PET analyses. Statistical analyses COMT genotype frequencies were compared between subjects and controls using w 2 tests. Analyses of the relationships between COMT genotype and the three measures of working memory/executive function (ie WCST, WAIS-R digit span backward and Trail Making test) and with MRI frontal lobe volumes (GM, WM and CSF) were performed using ANOVAs. In each general linear model, the respective trait measure was entered as the dependent measure with genotype and affectation status as independent variables. Intracranial volume and age were entered as covariates in the MRI frontal lobe morphology analyses. For the [ 15 O]H 2 O PET data, a nonparametric randomization analysis 81 was performed to compare the rcbf response of seven Met/Met patients and six Val/Val patients on the one-back task. This randomization analysis relies on across-task and across-group comparisons for one-back condition minus baseline condition in Met/Met patients as opposed to oneback condition minus baseline condition in Val/Val patients. We used an uncorrected alpha of in the PET randomization analysis. Results Genotype frequency distribution of the sample, and working memory/executive functioning and MRI frontal lobe volumes broken down by COMT genotype, are summarized in Table 2. Met allele frequency was 0.52 for the whole sample. Genotype distributions did not deviate from Hardy Weinberg expectations (w 2 ¼ 0.50 and 0.95 in healthy volunteer and patient groups respectively, df ¼ 2, P sz0.62). Although a greater proportion of schizophrenia patients had the Val/Val genotype (19.5 vs 14.2% in controls), the genotype frequency distributions were not significantly different between the two samples (w 2 ¼ 2.56, P ¼ 0.28). There were no significant genotype or genotype-by-group effects on age, educational attainment or full scale IQ (F sr1.27, df ¼ 2,243, P sz0.27). Effects of COMT genotype on working memory/ executive functions The effects of COMT genotype on WCST, digit span backwards and Trail Making tests were examined using ANCOVA analyses. Patients had significantly lower WCST perseverative errors t-scores than healthy volunteers (mean ¼ 52.3 (SD ¼ 13.56) vs 56.6 (SD ¼ 11.10); main effect of group F ¼ 5.14, df ¼ 1,243, P ¼ 0.02). There were no significant genotype or genotype-by-group effects on WCST performance (Table 2 and Figure 1; F s ¼ 1.74 and 0.32, respectively, df ¼ 2,243, P sz0.18). Similarly, patients performed more poorly on digit span (mean s ¼ 5.98 (SD ¼ 1.77) vs 8.12 (SD ¼ 2.20) in healthy volunteers) and on Trail Making test (mean s ¼ 49.2 (SD ¼ 36.06) and 28.7 (SD ¼ 15.03), respectively; main effect of group F sz20.8, df ¼ 1,200 or 1,211, P so0.0001). There were also no significant genotype or genotypeby-group effects on digit span or Trail Making performance (Table 2 and Figure 1; F sr0.53, df ¼ 2,200 or 2,211, P sz0.59). Since the patient group had a significantly greater preponderance of male subjects, fewer years of education and lower full scale IQ than healthy volunteers, these three variables were entered as additional covariates in the general linear models. The results remained unchanged, with no significant genotype or genotype-by-group effects on any of the three measures of working memory/executive functioning. Effects of COMT genotype on MRI frontal lobe morphology In all, 49 of the 84 healthy volunteers and 100 of the 168 patients had MRI frontal lobe volumetric data. The remaining 35 healthy volunteers and 68 patients did not have available MRI scans for a variety of reasons, including inability to undergo scanning procedure because of claustrophobia, poor quality

5 Table 2 Mean (SD) working memory/executive functioning and frontal lobe MRI volumes (cm 3 ) in controls and schizophrenia patients, and ANOVA analyses (F statistic (P)) on the main effects of diagnostic grouping, genotype and genotype-by-grouping (total brain compartment volume and age as covariates) Genotype by Diagnosis F (P) Genotype F (P) Controls Patients Diagnosis F (P) Met/Met Met/Val Val/Val Met/Met Met/Val Val/Val N Working memory/executive functions a WCST 53.3 (10.75) 56.7 (11.01) 60.8 (11.42) 50.7 (14.36) 52.5 (13.94) 53.7 (11.47) 5.14 (0.02) 1.74 (0.18) 0.32 (0.73) Digit span (backwards) 8.3 (2.03) 8.2 (2.79) 7.6 (2.15) 5.9 (1.88) 5.9 (2.03) 6.2 (2.08) 30.2 (o0.0001) 0.12 (0.89) 0.53 (0.59) Trail Making test 28.4 (11.80) 28.9 (14.68) 28.2 (21.18) 54.7 (42.70) 45.0 (29.32) 52.0 (44.32) 20.8 (o0.0001) 0.34 (0.71) 0.46 (0.63) Frontal lobe volumes (cm 3 ) GM (14.24) (13.67) (15.54) (16.51) (17.33) (11.49) 1.98 (0.16) 1.06 (0.35) 2.08 (0.13) WM (14.16) (16.24) (15.85) (15.60) (16.60) (13.95) 0.20 (0.65) 0.58 (0.56) 0.08 (0.92) CSF 32.2 (14.32) 34.5 (15.53) 45.0 (28.07) 36.5 (16.31) 45.4 (18.06) 38.9 (23.43) 0.37 (0.55) 1.43 (0.24) 2.58 (0.08) a WCST: Wisconsin Card Sorting Test (t-scores). WAIS-R digit span, backwards (number of correct, total raw score). Trail Making test (Trail B minus Trail A (s)). Figure 1 Working memory/executive functions performance in healthy volunteers and in schizophrenia patients broken down by COMT genotype. scans or scans obtained using noncomparable imaging parameters (either on an older or newer scanning protocol). There were no statistically significant differences between healthy volunteers with and without MR data with regard to gender composition, COMT genotype distribution or age (details of statistical analyses available upon request). Patients with MR data were also not significantly different from those without MR data on a variety of sociodemographic and clinical measures: gender composition, COMT genotype distribution, age and duration of illness or symptom severity (details of statistical analyses available upon request). The mean frontal lobe GM, WM and CSF volumes (adjusted for intracranial volume and age) for 49 healthy volunteers and 100 schizophrenia patients broken down by COMT genotype are summarized in Table 2 and Figure 2. There were no statistically significant effects of genotype on frontal lobe mor- 291

6 292 location on two consecutive flashes of the checkerboard. Met/Met allele patients had slightly longer reaction times than Val/Val patients, but this difference was not statistically significant (mean s ¼ and ms, respectively; t ¼ 0.18, df ¼ 10.2, P ¼ 0.86). Randomization analyses indicated that there were no regions within the DLPFC or within the anterior cingulate where Met/Met patients showed significantly greater rcbf than Val/Val patients. Table 3 and Figure 3 summarize brain regions where schizophrenia patients with Met/Met alleles had significantly lower rcbf than patients with Val/Val alleles. While performing the one-back task, Val/Val patients showed greater activation in the left dorsal lateral prefrontal cortex (Brodmann s area 47) and in the right mesial frontal lobe (Brodmann s areas 8 and 9). Figure 2 Mean frontal lobe GM, WM and CSF volumes for healthy volunteers and for schizophrenia patients broken down by COMT genotype. phology (F so1.43, df ¼ 2,148, P s40.24). No significant genotype by group interaction effects on frontal GM or WM (F so2.08, df ¼ 2,148, P s40.13). Genotype-by-group interaction effects approached but did not achieve statistical significance for frontal CSF volume (F s ¼ 2.58, df ¼ 2,148, P ¼ 0.08). Effects of COMT genotype on [ 15 O]H 2 O rcbf during N-back task performance All seven schizophrenia patients with Met/Met alleles and six patients with Val/Val alleles were able to perform the one-back version of the N-back task. Each subject responded correctly by pressing the button when the asterisk appeared at the same Discussion In this study, we found that V 158 M COMT genotype frequencies did not differ significantly between schizophrenia patients and healthy volunteers. We also investigated the effects of COMT genotype on a wide range of frontal lobe-related measures. Working memory/executive function performance (in WCST, digit span backwards and Trail Making tests) and MRI frontal lobe volumes (GM, WM and CSF) were not significantly different between genotype groupings. Patients with Val/Val genotype showed greater activation in the frontal lobes than Met/Met patients while performing the N-back task. Although this difference in rcbf, based on a small subset of the sample, may be taken to mean that subjects with valine allele have prefrontal cortical inefficiency, the overall findings from this study do not support a major role for COMT in increasing susceptibility for schizophrenia, or in mediating frontal lobe structure and functions. V 158 M COMT functional polymorphism is probably the most widely investigated candidate gene in schizophrenia research. The bulk of the evidence to date suggests that this polymorphism confers, at best, a small increase in susceptibility to the disorder. Most case control studies, as well as our study, have failed to find a significant association between COMT polymorphism and schizophrenia. 52,54,55,59 61,82 88 A handful of case control studies found that the high enzyme activity valine allele had a modest effect on conferring increased risk for schizophrenia. 58,63,89 Conversely, the methionine allele has also been reported to occur more frequently among schizophrenia patients than normal controls. 90,91 Family-based studies have had equally mixed results, with some studies finding preferential transmission of the valine allele in schizophrenia patients 52,56,57 while others have detected no significant associations These conflicting results with regard to the role of V 158 M COMT polymorphism in schizophrenia susceptibility may relate to inadequate statistical power of individual association studies or to variability in association between different ethnic populations.

7 Table 3 Regions where schizophrenia patients with Met/Met alleles had significantly lower rcbf than patients with Val/Val alleles while performing the one-back task 293 Brain region a Talairach coordinates Significance of t max (P) b Number of voxels c x y z Right cerebellum (0.0005) 1.2 Left DLPFC (Brodmann 47) (0.0014) 0.4 Right cerebellum (0.0018) 0.3 Left precuneus (Brodmann 30) (0.0011) 0.3 Right mesial frontal lobe (Brodmann 8) (0.0022) 0.2 Right mesial frontal lobe (Brodmann 8) (0.0018) 0.2 Left posterior inferior lobe cerebellum (0.0014) 0.2 Right middle temporal gyrus (0.0015) 0.1 a Regions were named from the inspection of coregistered magnetic resonance and PET images, as well as Talairach coordinates. These coordinates represent spatial orientation with respect to a point located in a horizontal plane through the anterior and posterior commissures (z ¼ 0), at the midline of the brain slice (x ¼ 0) and at the anterior commissure line (y ¼ 0). The x-axis was the distance in millimeters to the left (negative) and to the right (positive) of the midline. The y-axis was the distance in millimeters anterior (positive) or posterior (negative) to the anterior commissure line. The z-axis was the distance in millimeters above (positive) or below (negative) a horizontal plane through the anterior and posterior commissures. b t max is the highest t-value identified in the peak. c Number of voxels in the peaks that exceeded alpha ¼ Figure 3 Regions where cerebral blood flow among Met/ Met schizophrenia patients were significantly lower than those in Val/Val patients while performing the one-back task. When data from case control association studies were pooled, the two published meta-analyses indicate that V 158 M COMT allele variation does not contribute to increased vulnerability for schizophrenia. 62,95 Familybased studies, on the other hand, suggest that the valine allele may be a risk factor in schizophrenia patients of European ancestry. 62 To clarify the true role of the V 158 M COMT polymorphism in schizophrenia susceptibility, future studies will require larger sample sizes and will need to minimize the effects of population stratification. Beyond its potential as a susceptibility gene, there has also been a long-standing interest within schizophrenia research with regard to the phenotypic correlates of the COMT gene. The methionine allele has been associated with aggression, suicide, worse prognosis and greater positive symptom severity in schizophrenia. 91, Egan et al s finding of an association between the valine allele and poor WCST performance has generated renewed interest in this area of investigation, especially with regard to the effects of COMT in mediating cognition in schizophrenia. 52 This finding has been replicated in part by Joober et al, who found that genotype effects on WCST performance among schizophrenia patients was at a trend level of significance, and was nonsignificant in healthy volunteers. 59 In another study, healthy volunteers with the valine allele committed significantly more WCST perseverative errors than those with methionine allele. 101 The effects of V 158 M COMT polymorphism appear to extend beyond WCST performance, and have been implicated in other measures of prefrontal neurocognitive functions (such as N-back task 102 and prefrontal P300 amplitude and latency 103,104 ), and in mediating other cognitive domains (such as processing speed and attention 105 and cognitive stability and flexibility 106 ). All these studies have found an association between the valine allele and poorer cognitive performance, and postulate that the underlying mechanism may be related to lower prefrontal dopamine levels arising from higher dopamine catabolism mediated by the valine allele.

8 294 In contrast to these previous studies, we did not find a significant association between the high enzyme activity valine allele and poor WCST performance. In fact, among schizophrenia patients as well as healthy volunteers, none of the working memory/ executive function measures we examined differed significantly across genotype groupings. Our findings of no significant genotype effects are consistent with a recent study involving 200 healthy volunteers. 107 Fossella et al found that V 158 M COMT genotype status did not predict performance in any of the four measures of attention they examined. Interestingly, healthy volunteers with alleles expected to result in higher synaptic dopamine actually performed more poorly in the attention tasks. The four-repeat allele of the DRD4 exon III polymorphism and 10-repeat allele of the DAT 3 0 -VNTR polymorphism were both significantly associated with poorer executive attention scores. Although the COMT Met allele showed no significant associations, subjects with Met/Met genotype had numerically worse attention scores. The MAOA-LPR three-repeat allele, which would also be expected to result in higher synaptic dopamine levels, showed a trend towards poorer attention performance. The younger age, milder cognitive impairment and/ or shorter illness duration of our sample may potentially explain our nonreplication of the association between the valine allele and poor cognitive performance. Except for the Tsai et al study 104 (which studied Han Chinese female nursing students aged between 19 and 21 years), the mean age of subjects in previous studies 52,59, ,105,106,108 are substantially older than ours. Age-related loss in frontal lobe dopamine function has been repeatedly demonstrated in post-mortem as well as in vivo neuroimaging studies (eg Rinne, 109 de Keyser et al, 110 Suhara et al, 111 Kaasinen et al 112 and Volkow et al 113 ). This decline in dopamine function appears to begin early, 114 and has been estimated to occur at a rate of 11 13% per decade in age. 112 Thus, even though subjects from previous studies 52,59, ,105,106,108 were still in their mid-adulthood, and were only years older than our sample, these older subjects may already have considerably lower dopamine functional reserve than our younger subjects. Any further reduction in dopamine function among these older individuals, such as an increased dopamine catabolism associated with the presence of valine allele, may have more pronounced detrimental effects on frontal lobe functions than in younger individuals. Compared with the Egan et al and Bilder et al studies, 52,105 which examined similar neuropsychological measures, our sample was cognitively less impaired and our patients less chronically ill. The mean WCST t-scores for patients and controls in Egan et al study were 37.6 (712.6) and 49.4 (79.0), respectively 52 (vs 52.3 (712.2) and 56.6 (79.6) in our patients and controls subjects, respectively). In the Bilder et al study, 105 the mean number of perseverative errors among schizophrenia patients was 49.2 (vs 11.2 in our patients). The mean illness duration among patients in the Bilder et al study and in our study is 19.3 and 2.5 years, respectively. Although the Egan et al study did not report illness duration, the average length of illness would likely be years based on the mean age of the subjects. These differences in severity of cognitive impairment and in duration of illness may be indicative of greater heterogeneity in our patient sample. Even though all three studies utilized DSM diagnostic criteria to ascertain patients, it is well recognized that there can be substantial variability in patients who share the same set of clinical symptoms of schizophrenia. Since our patients consisted of a mixture of first episode, recent-onset and chronically ill schizophrenia patients, our sample may be more representative of the average schizophrenia patient. However, this heterogeneity will likely add noise to the analyses, and may be less ideal for genetic association studies. On the other hand, the Egan et al and Bilder et al samples, being more chronically ill and having greater cognitive impairment, may be genetically more homogeneous, and therefore, more likely to detect effects of COMT genotype on cognitive performance. A third reason for our lack of significant associations between COMT genotype and working memory performance may be related to the secondary role of COMT in determining synaptic dopamine levels. There is limited human data with regard to the relative contributions of catecholamine (dopamine and norepinephrine) transporters, monoamine oxidase and COMT in influencing synaptic dopamine levels in the prefrontal cortex. However, animal studies indicate that COMT plays only a minimal role in the clearance of synaptic dopamine, even under conditions of low dopamine transporter activity Furthermore, COMT genotype appears to account for only a small amount (2 4%) of shared variance with WCST performance. 52,105 Thus, this small effect of COMT genotype on cognitive performance may be especially difficult to detect in younger and more heterogeneous sample of schizophrenia patients. To the best of our knowledge, this is the first study that has examined MRI brain morphometric correlates of COMT genotype. We did not find any significant associations between genotype and frontal GM, WM or CSF volumes. Although our group has previously reported associations between BDNF and NOTCH4 with these same measures of frontal lobe, 115,116 future studies on the relationships between COMT and brain morphology will need to examine specific regions within the frontal lobes, especially regions known to mediate working memory. Our finding of Val/Val patients having greater frontal lobe activation than Met/Met patients while performing the one-back working memory task is consistent with Egan et al study. 52 The authors found greater fmri BOLD response in the DLPFC and anterior cingulate among unaffected siblings of

9 schizophrenia patients with Val/Val genotype. Since our Val/Val patients had comparable reaction times as our Met/Met patients and the one-back task is a low load working memory test, higher frontal lobe rcbf among our Val/Val patients suggests that they may be working harder in order to achieve equal task performance. While relative prefrontal cortical inefficiency in the valine allele may be a plausible interpretation of these PET study findings, a diametrically opposite view could be that lower frontal lobe rcbf among Met/Met patients may represent hypofrontality relative to Val/Val patients. Both hypofrontality as well as hyperfrontality have been described in schizophrenia. Unfortunately, we did not have sufficient numbers of healthy volunteers with Met/ Met and Val/Val genotypes to assess the patients relative brain activations. As Manoach 117 has elegantly presented, the interpretation of functional neuroimaging findings in schizophrenia is complex, and a variety of reasons (ranging from methodological issues to heterogeneity of schizophrenia) may account for the seemingly discrepant findings of both hypofrontality as well as increased prefrontal activity in schizophrenia. Irrespective of how these PET findings may be interpreted, that Val/Val patients and Met/Met patients differed significantly on frontal lobe rcbf seem inconsistent with the rest of our findings of no significant COMT genotype effects on a variety of frontal lobe-related measures. Cerebral blood flow, being a more direct measure of neuronal function, may be more sensitive for assessing the effects of COMT genotype than are neuropsychological measures or MRI brain morphometry. Conversely, our PET study involved only 13 patients, and the larger sample size used in the rest of this study may provide results that can be better generalized to the average patient with schizophrenia. Future efforts to integrate functional neuroimaging toward better understanding the effects of COMT genotype on cognitive endophenotypes of schizophrenia will require large samples of both patients as well as healthy volunteers. Working memory load will also need to be varied in future functional neuroimaging studies so as to be better able to interpret differences in brain activation between COMT genotypes. Conclusion The complex genetics and phenotypic heterogeneity remain as major obstacles in schizophrenia research. Integrating multimodal investigational approaches will help address these challenges, and facilitate greater understanding of the neurobiological basis for this group of disorders. 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