D 2 receptors binding potential is not affected by Taq1 polymorphism at the D 2 receptor gene

Transcription

1 Molecular Psychiatry (1998) 3, Stockton Press All rights reserved /98 $12.00 ORIGINAL RESEARCH ARTICLE D 2 receptors binding potential is not affected by Taq1 polymorphism at the D 2 receptor gene M Laruelle 1,2, J Gelernter 1,2 and RB Innis 1,2 1 Departments of Psychiatry, Yale University School of Medicine, New Haven, CT; 2 Veterans Administration Medical Center, West Haven, CT, USA Keywords: dopamine; D 2 receptors; gene; SPECT; schizophrenia Alcoholism and substance abuse have been associated with a polymorphism in a noncoding region of the D 2 receptor gene (the A1 allele of the Taq1 A system) in several, 1 9 but not all studies. In addition, the presence of the A1 allele has been associated with lower density of D 2 receptors in the caudate nucleus in one postmortem study. 14 If the Taq1 A1 allele is in linkage disequilibrium with a mutation that decreases the expression of the D 2 receptor gene, it is conceivable that subjects with the A1 allele might be predisposed to behaviors which stimulate dopamine transmission, such as alcoholism or substance abuse. In this study, we attempted to confirm the association between the A1 allele and lower D 2 receptor density, and explored a possible association between the B1 allele and D 2 receptor expression. Genotypes at the Taq1 A and B systems were determined in 70 subjects who underwent in vivo measurement of D 2 receptors-binding potential with single photon emission computerized tomography (SPECT) and the selective dopamine D 2 receptor radiotracer [ 123 I]IBZM. [ 123 I]IBZM-binding potential was identical in A1 carriers (ie, subjects heterozygous or homozygous for that allele) (230 ± 85 ml g 1, n = 27) and A1 noncarriers (231 ± 70 ml g 1, n = 43). Similarly, we found no effect of the B1 allele on [ 123 I]IBZM-binding potential. In conclusion, the results of this study failed to replicate the previously reported association between A1 allele and lower D 2 receptor expression. In 1990, Blum et al 1 reported an association between alcoholism and a polymorphism in a noncoding region of the D 2 receptor gene (the A1 allele of the Taq1 A system). This association has been supported in several, 2 5 but not all subsequent studies The A1 allele was also found to be associated with prolonged P300 latency, reduced visuospatial preference, and low cerebral glucose utilization, all putative markers of alcoholism and dopamine function deficiency In addition, the A1 allele has also been associated with substance abuse, 6 9 which led to its conceptualization as a reward gene. Together, these data suggest that this polymorphism might affect dopamine transmission in a way that predisposes subjects to reward seeking behavior, i.e. behaviors aiming at increasing stimulation of dopamine transmission. The mechanism underlying the association between the A1 allele and vulnerability to addictive behaviors is unclear, since the Taq1 A polymorphism is not located in the coding region of the gene and a direct effect of this polymorphism on the structure of the D 2 receptor gene has been excluded by denaturing gradient gel electrophoresis mutational analysis study. 18 Noble et al 14 described an association between the A1 allele and decreased D 2 receptor density expressed in postmortem striatal samples from alcoholic and nonalcoholic subjects. If replicated, this observation might suggest that this polymorphism is in linkage disequilibrium with an unknown mutation that might affect the expression of the D 2 receptor gene, and that the lower D 2 receptor density associated with the A1 allele might lead to a deficiency in D 2 receptor transmission which, in turn, might trigger behaviors aiming at enhancing dopamine. The Taq1 B system is in strong linkage disequilibrium with the A system. 19 While the A polymorphic site is located 3 to the coding sequence of the gene, the B system is within the gene, 5 to exon The B1 allele has also been shown to be associated with substance abuse. 6 9 Because of the importance of this question for the genetics of addiction, we measured both Taq1 A and B polymorphism in 70 subjects who underwent in vivo quantitation of D 2 receptor-binding potential with single photon emission computerized tomography (SPECT) and the selective dopamine D 2 receptor radiotracer [ 123 I](S)-( )-3-iodo-2-hydroxy-6-methoxy-N-[(1- ethyl-2-pyrrolidinyl)methyl]benzamide ([ 123 I]IBZM). 20 The sampled population included 47 healthy controls and 23 subjects with schizophrenia. None of the subjects had a history of alcohol or substance abuse or dependence. Forty-three subjects were A2A2 homozygotes, twenty-three were A1A2 heterozygotes, and four subjects were A1A1 homozygotes. This distribution corresponded to an allele frequency of f(a1) = The frequency of A1 carriers was identical in schizophrenics (39%) and controls (38%, Chi-squared P = 0.98). We observed a significant effect of age on [ 123 I]IBZM-binding potential (P 0.05), but no effect of clinical status (controls vs schizophrenic subjects) and no effect of the Taq1 A allele status. [ 123 I]IBZM-binding potential was identical in A1 carriers (230 ± 85 ml g 1, n = 27) and A1 noncarriers (231 ± 70 ml g 1, n = 43) (Table 1). The four A1A1 homozygotes had [ 123 I]IBZM-binding potential in the high range (284 ± 61 mg g 1 ; ANOVA age-corrected values: 270 ± 75 mg g 1 ). We observed no diagnosis allele interaction on [ 123 I]IBZM-binding potential (P = 0.23). The results of these analyses were similar when we restricted the analysis to European American (EA) subjects ([ 123 I]IBZM-binding potential: EA A1 carriers: 234 ± 102 ml g 1, n = 16; EA A1 noncarriers: 233 ± 70 ml g 1, n = 34; ns). Fifty-one subjects were B2B2 homozygotes, seventeen were B1B2 heterozygotes, and two subjects were B1B1 homozygotes (f(b1) = 0.16). A1 allele and B1 allele were highly correlated (Chi-square P ).

2 262 Table 1 Effect of Taq1 A polymorphism, age and diagnosis on D 2 receptor-binding potential (ml g 1 ), measured with SPECT [ 123 I]IBZM Diagnosis A1(+) A1( ) Average by diagnosis Healthy controls 215 ± 65 (18) 236 ± 70 (29) 228 ± 68 (47) Schizophrenics 259 ± 114 (9) 219 ± 73 (14) 235 ± 90 (23) Average by A1 230 ± 85 (27) 231 ± 70 (43) 230 ± 76 (70) status Ancova Source df F P Age Diagnosis Allele Data are given as mean ± s.d. (n). All A2A2 subjects were B2B2 subjects. Among A1 carriers, 70% were B1 carriers and 30% were not B1 carriers. The frequency of A1 carriers was not significantly different in schizophrenics (17%) and controls (31%, Chi-squared, P = 0.19). We observed an effect of age on [ 123 I]IBZM-binding potential (P 0.05), no effect of the clinical status and no effect of the Taq1 B allele status. [ 123 I]IBZM-binding potential was identical in B1 carriers (230 ± 93 ml g 1, n = 19) and B1 noncarriers (230 ± 69 ml g 1, n = 51). The two B1B1 homozygotes had [ 123 I]IBZM-binding potential in the high range (267 ± 72 mg g 1 ; age-corrected values: 247 ± 76 mg g 1 ). We observed a diagnosis alelle interaction on [ 123 I]IBZM-binding potential (P = 0.021). Schizophrenic subjects with the B1 allele had high [ 123 I]IBZM-binding potential values (307 ± 156 ml g 1, n = 4; age-corrected values: 324 ± 72 ml g 1 ) compared to schizophrenics without the B1 allele (219 ± 67 ml g 1, n = 19; age-corrected ml g 1 ), and this was not true in controls (see Table 2). However, this finding should be Table 2 Effect of Taq1 B polymorphism, age and diagnosis on D 2 receptor-binding potential (ml g 1 ), measured with SPECT [ 123 I]IBZM Diagnosis B1(+) B1( ) Average by diagnosis Healthy controls 209 ± 61 (15) 237 ± 71 (32) 228 ± 68 (47) Schizophrenics 307 ± 156 (4) 219 ± 67 (19) 235 ± 90 (23) Average by B1 230 ± 93 (19) 230 ± 69 (51) 230 ± 76 (70) status Ancova Source df F P Age Diagnosis Allele Data are given as mean s.d. (n). regarded with caution because of the small number of schizophrenics with the B1 allele (n = 4). Results were similar when we restricted the analysis to EA subjects ([ 123 I]IBSM-binding potential: EA B1 carriers: 229 ± 70 ml g 1, n = 16; EA B1 noncarriers: 244 ± 108 ml g 1, n = 34; ns). Aging was associated with a loss of 18.2 ml g 1 of [ 123 I]IBZM-binding potential per decade. Expressed as a percentage of the intercept (296 ml g 1 ), this corresponded to a loss of 6% of receptors per decade. We did not observe a different rate of loss in D 2 receptors in schizophrenics and controls (age diagnosis interaction, P = 0.86). In this study we failed to replicate the earlier report of decreased expression of D 2 receptors associated with the Taq1 allele. 14 Using [ 3 H]spiperone, Noble et al 14 measured D 2 receptor B max and K D in caudate nuclei from 66 brains (33 from alcoholic subjects and 33 from nonalcoholic subjects). Taq1 A1 allele carriers had lower B max compared to non A1 carriers (P = 0.021), while no differences were observed in the K D (P = 0.68). A trend was observed towards lower binding potential in the A1 carriers (P = 0.085). No differences were observed in D 2 receptor parameters between alcoholic and nonalcoholic subjects. No alcohol allele interaction was observed, suggesting that A1 allele affected D 2 receptors B max to the same extent in alcoholic subjects and controls. The reasons for the discrepancies between our study and that of Noble et al 14 are unclear. A first obvious difference is the methodology postmortem vs in vivo studies. Postmortem studies are subject to numerous artifacts, associated with agonal states, autolysis time, medications at the time of death, freezing methods and storage time. On the other hand, in vivo measurements are confounded by limited resolution and by the presence of endogenous dopamine. We previously showed that endogenous dopamine occupies in vivo about 20% of D 2 receptors, and that this factor introduces a significant error on the binding measurement. 21 Thus, each approach has its limitations and strengths, but it is unclear how these methodological problems could be related to the discrepancy in the results. A second difference is the clinical composition of the samples (alcoholics and controls vs schizophrenics and controls). However, since no interactions were reported between alcoholism and allele effect of D 2 receptor parameters, this difference cannot explain our failure to replicate the findings. Another difference between the studies is the D 2 receptor parameter used as outcome measure. While the postmortem study reported K D,B max and binding potential, our study was limited to the binding potential. To measure [ 123 I]IBZM B max and K D, an injection of pharmacological doses of [ 123 I]IBZM would be needed. Because of the lack of information about possible toxic effects of [ 123 I]IBZM at pharmacological doses, this procedure is not currently approved. Noble et al 14 reported an allele effect on the B max but not on the K D. Because the binding potential is the B max /K D ratio, theoretically, a lower B max and an unchanged K D should translate in a

3 lower binding potential. However, because of the well known inverse correlation between B max and K D, 22 differences in binding potential tend to be less pronounced than differences in B max, and this was the case in the Noble et al 14 study. Nevertheless, we did not observe even a trend toward lower binding potential in carriers of the A1 or B1 alleles. Because A1 are less frequent than the A2 alleles, A1A1 homozygotes are relatively rare, precluding meaningful statistical treatment of their data. This reason prompted us to pool A1A1 homozygotes with A1A2 heterozygotes in the statistical analysis. However, these subjects carry important information for the hypothesis. If the presence of the A1 allele is associated with a mutation inducing a lower expression of D 2 receptors, the effect should be greater in A1A1 homozygotes than in A1A2 heterozygotes. Accordingly, A1A1 homozygote subjects would be expected to have lower D 2 receptor density than the other two groups (A1A2 and A2A2). This effect was indeed observed in the study of Noble et al. 14 Yet, in our study, the A1A1 homozygotes subjects had [ 123 I]IBZM-binding potential in the high range, a finding that is difficult to reconcile with a putative association between this allele and a lower D 2 receptor expression. The same observation was made for the Taq1 B system. In conclusion, Taq1 A and B polymorphisms were determined in 70 subjects for which in vivo measurements of striatal D 2 receptor-binding potential were available. In this retrospective study, we failed to replicate the observation that Taq1 polymorphism affects D 2 receptor expression. Additional studies are needed to reconcile the results of this study with previously published reports. Methods Subjects The study was approved by the Yale Institutional Review Board for Human Investigations. Seventy subjects participated in our study. All subjects gave written informed consent. The sampled population included 47 healthy controls and 23 subjects with schizophrenia. None of the subjects had a history of alcohol or substance abuse or dependence. The sample included 60 males and 10 females. Ethnic distribution of the sample was: 50 European Americans (EA), 16 African Americans (AA), three Hispanics and one Asian. Inclusion criteria for healthy controls were: (1) absence of past or present neurological or psychiatric illnesses; (2) no concomitant or past severe medical conditions; (3) no pregnancy. The absence of medical, neurological and psychiatric history (including alcohol and drug abuse) was assessed by history, review of systems, physical examination, routine blood tests, urine toxicology, and EKG. Inclusion criteria for patients with schizophrenia were as follows: (1) diagnosis of schizophrenia according to Diagnostic and Statistical Manual (DSM-IV); (2) no other DSM-IV axis I diagnosis; (3) no history of alcohol or substance abuse or dependence; (4) absence of any psychotropic medication for at least 21 days prior to the study (with the exception of lorazepam, which was allowed at a maximal dose of 3 mg per day up to 24 h prior to the study); (5) no concomitant or past severe medical conditions; (6) no pregnancy; (7) no current suicidal or homicidal ideation; (8) ability to provide informed consent. SPECT experiments SPECT experiments were carried out as previously described. 23 [ 123 I]IBZM with specific activity 5000 Ci mmol 1 and radiochemical purity 95% was prepared by direct electrophilic radioiodination of the desiodoprecursor IBZM. A total of [ 123 I]IBZM dose of 9.28 ± 3.12 mci was given as a bolus followed by a continuous infusion at a constant rate for the duration of the experiment. The ratio of the bolus to hourly infused activity was 3.9. This protocol of administration was previously shown to induce a state of sustained tracer equilibrium in the brain: both the specific and nonspecific activities remained at a constant level from 150 min to the end of the experiment. 23 SPECT data were acquired on two PRISM 3000 (Picker, Cleveland, OH, USA) with high resolution fan beam collimators. Eight consecutive acquisitions of 8 min each were obtained from 180 to 244 min. Plasma metabolite-corrected [ 123 I]IBZM steady-state concentration (C SS ) was measured by extraction followed by high pressure liquid chromatography on four venous samples collected at 20-min intervals from 180 to 240 min. SPECT images were reoriented to the cantho-meatal line as visualized by four external fiducial markers glued to the subject s head. The four slices with highest striatal uptake were summed and attenuation corrected assuming uniform attenuation. Standard region of interest profiles (striatum 556 mm 2, occipital 2204 mm 2 ) were positioned on the summed images. The occipital region was selected as the nonspecific region because: (1) the density of dopamine D 2 receptors is negligible in this region compared to the striatum; 24 (2) this region can be identified with greater reliability than the cerebellum; (3) in humans, [ 123 I]IBZM activity in the occipital region is equal to the nonspecific activity in the striatum. 25 Right and left striatal regions were averaged. Striatal specific binding was calculated as striatal minus occipital activity. The [ 123 I]IBZM-binding potential (ml g 1 ), corresponding to the product of the free receptor density (B max,nmor pmol per g of brain tissue) and affinity (1/K D,nM 1,or ml of plasma per pmol), was calculated as the ratio of striatal specific binding ( Ci per g of brain tissue) to the steady-state free unmetabolized plasma tracer concentration (f 1 C SS, Ci per ml of plasma) measured during scanning session. 23 The free fraction of [ 123 I]IBZM in the plasma is very small (2 5% of total [ 123 I]IBZM concentration). Because of the low reliability of this measurement 26 and because groups did not show differences in [ 123 I]IBZM plasma free fraction (f1), the average f1 value measured in this study (3.3%) was used in the computation of the free plasma unmetabolized [ 123 I]IBZM for each subject. Under these experimental conditions, the average test/retest variability of 263

4 264 the [ 123 I]IBZM-binding potential measurement is 14 ± 10% (measured in six subjects, scanned at 1-week intervals). This reproducibility corresponds to a high intraclass correlation coefficient of Polymorphism determination DNA was extracted from whole blood by standard methods. We used PCR-formatted assays for the Taq1 A 27 and B 28 system. Statistical analysis Allele frequency is reported as f(a1) = [2(A1A1) + (A1A2)]/2N. Given the small number of A1A1 subjects, we followed the method of Noble et al, 14 and classified subjects into A1 carriers, pooling A1A2 and A1A1 subjects, and A1 noncarriers (ie A2A2 subjects). We followed the same method for the B system. However, given the importance of homozygotes for the tested hypotheses, we also provided the results in A1A1 and B1B1 homozygotes. The association between Taq1 A and B polymorphism and [ 123 I]IBZM-binding potential was evaluated by ANOVA with Taq1 A or B status, diagnosis and age as covariates. Since the association between Taq1 A and B polymorphism and substance abuse has been documented in subjects of European origin, but not in subjects of African origin, we also performed an analysis restricted to EA subjects. When relevant, we provided the ANCOVA agecorrected values. All tests were two-tailed, and P = 0.05 was chosen as significance value. Brain imaging data from subsets of this sample have been previously published. 21,29 Acknowledgements We thank Anissa Abi-Dargham, MD, Ronald M Baldwin, PhD, and John P Seibyl, MD for their help in carrying the imaging studies; Christine Fingado and the staff of the NeuroSpect laboratory at Yale-New Haven Hospital for technical assistance. Supported by a young investigator award (ML) from the National Alliance for Research on Schizophrenia and Depression, the Scottish Rite Schizophrenia Research Program, the Department of Veterans Affairs (Schizophrenia Research Center) and the Public Health Service (M01RR00125). References 1 Blum K, Noble EP, Sheridan PJ, Montgomery A, Ritchie T, Jagadeeswaran P et al. Allelic association of human dopamine D 2 receptor gene in alcoholism. JAMA 1990; 263: Parsian A, Todd RD, Devor EJ, O Malley KL, Suarez BK, Reich T et al. Alcoholism and alleles of the human D2 dopamine receptor locus. Studies of association and linkage. Arch Gen Psychiatry 1991; 48; Comings DE, Comings BG, Muhleman D, Dietz G, Shahbahrami B, Tast D et al. The dopamine D2 receptor locus as a modifying gene in neuropsychiatric disorders. JAMA 1991; 266: Blum K, Noble EP, Sheridan PJ, Montgomery A, Ritchie T, Ozkaragoz T et al. Genetic predisposition in alcoholism: association of the D2 dopamine receptor Taq1 RFLP with severe alcoholics. Alcohol 1993; 10: Blum K, Noble EP, Sheridan PJ, Finley O, Montgomery A, Ritchie T et al. Association of the A1 allele of the D2 dopamine receptor gene with severe alcoholism. Alcohol 1991; 8: O Hara BF, Smith SS, Bird G, Persico AM, Suarez BK, Cutting GR et al. Dopamine D2 receptor RFLPs, haplotypes and their association with substance use in black and Caucasian research volunteers. Hum Hered 1993; 43: Smith SS, O Hara BF, Persico AM, Gorelick DA, Newlin DB, Vlahov D et al. Genetic vulnerability to drug abuse. The D2 dopamine receptor TaqI B1 restriction fragment length polymorphism appears more frequently in polysubstance abusers. Arch Gen Psychiatry 1992; 49: Noble E, Blum K, Khalsa M, Rithchie T, Montgomery, Woods RC et al. Allelic association of the D2 dopamine receptor gene with cocaine dependence. Drug Alcohol Depend 1993; 33: Persico AM, Bird G, Gabbay FH, Uhl GR. D2 dopamine receptor gene Taq1 A1 and B1 restriction fragment length polymorphisms: enhanced frequencies in psychostimulant-preferring polysubstance abusers. Biol Psych 1996; 40: Gelernter J, O Malley S, Risch N, Kranzler HR, Krystal J, Merikangas K et al. No association between an allele at the D2 dopamine receptor gene (DRD2) and alcoholism. JAMA 1991; 266: Bolos AM, Dean M, Lucas-Derse S, Ramsburg M, Brown GL, Goldman D. Population and pedigree studies reveal a lack of association between the dopamine D 2 receptor gene and alcoholism. JAMA 1990; 264: Turner E, Ewing J, Shilling P et al. Lack of association between an RFLP near the D2 receptor gene and severe alcoholism. Biol Psychiatry 1992; 31: Cook B, Wang Z, Crowe R, Hausser R, Freimer M. Alcoholism and the D 2 receptor gene. Alcohol Clin Exp Res 1992; 4: Noble EP, Blum K, Ritchie T, Montgomery A, Sheridan PJ. Allelic association of the D2 dopamine receptor gene with receptor-binding characteristics in alcoholism. Arch Gen Psychiatry 1991; 48: Noble EP, Berman SM, Ozkaragoz TZ, Ritchie T. Prolonged P300 latency in children with the D2 dopamine receptor A1 allele. Am J Hum Genet 1994; 54: Berman SM, Noble EP. Reduced visuospatial performance in children with the D2 receptor A1 allele. Behav Genet 1995; 25: Noble EP, Gottschalk LA, Fallon JH, Ritchie TL, Wu JC. D-2 dopamine receptor polymorphism and brain regional glucose metabolism. Am J Med Genet 1997; 74: Gejman PV, Ram A, Gelernter J, Friedman E, Cao Q, Pickar D et al. No structural mutation in the dopamine D2 receptor gene in alcoholism or schizophrenia. Analysis using denaturing gradient gel electrophoresis. JAMA 1994; 271: Hauge X, Grandy DK, Eubanks JH, vans GA, Civelli O, Litt M. Detection and characterization of additional DNA polymorphisms in the dopamine D2 receptor gene. Genomics 1991; 10: Kung HF, Guo Y-Z, Billings J, Xu X, Mach RH, Blau M et al. Preparation and biodistribution of [ 125 I]IBZM: a potential CNS D-2 dopamine receptor imaging agent. Nucl Med Biol 1988; 15: Laruelle M, D Souza CD, Baldwin RM, Abi-Dargham A, Kanes SJ, Fingado CL et al. Imaging D2 receptor occupancy by endogenous dopamine in humans. Neuropsychopharmacology 1997; 17: Farde L, Hall H, Pauli S, Halldin C. Variability in D2-dopamine receptor density and affinity: a PET study with [11C]raclopride in man. Synapse 1995; 20: Laruelle M, Abi-Dargham A, van Dyck CH, Rosenblatt W, Zea- Ponce Y, Zoghbi SS et al. SPECT imaging of striatal dopamine release after amphetamine challenge. J Nucl Med 1995; 36; Lidow MS, Goldman-Rakic PS, Rakic P, Innis RB. Dopamine D 2 receptors in the cerebral cortex: distribution and pharmacological characterization with [ 3 H]raclopride. Proc Natl Acad Sci 1989; 86: Seibyl J, Woods S, Zoghbi S, Baldwin R, Dey H, Goddard A et al. Dynamic SPECT imaging of D 2 receptors in human subjects with iodine-123-ibzm. J Nucl Med 1992; 33: Abi-Dargham A, Gandelman M, Zoghbi SS, Laruelle M, Baldwin RM, Randall P et al. Reproducibility of SPECT measurement of benzodiazepine receptors in human brain with iodine-123-iomazenil. J Nucl Med 1995; 36;

5 27 Grandy DK, Zhang Y, Civelli O. PCR detection of the TaqA RFLP at the DRD2 locus. Hum Mol Genet 1995; 2: Castiglione CM, Deinard AS, Speed WC, Sirugo G, Rosenbaum HC, Zhang Y et al. Evolution of haplotypes at the DRD2 locus. Am J Hum Genet 1995; 57; Laruelle M, Abi-Dargham A, van Dyck CH, Gil R, De Souza CD, Erdos J et al. Single photon emission computerized tomography imaging of amphetamine-induced dopamine release in drug free schizophrenic subjects. Proc Natl Acad Sci 1996; 93: Correspondence: M Laruelle, MD, Columbia University College of Physicians and Surgeons, New York State Psychiatric Institute, Unit 28, 722 West 168th Street, New York, New York 10032, USA. laruelle neuron.cpmc.columbia.edu Received 11 August 1997; revised and accepted 4 September