Analysis of genetic variations in the RGS9 gene and antipsychotic-induced tardive dyskinesia in schizophrenia.код для вставкиСкачать
RESEARCH ARTICLE Neuropsychiatric Genetics Analysis of Genetic Variations in the RGS9 Gene and Antipsychotic-Induced Tardive Dyskinesia in Schizophrenia Ying-Jay Liou,1,2 Mao-Liang Chen,3 Ying-Chieh Wang,3,4 Jen-Yeu Chen,3 Ding-Lieh Liao,5 Ya-Mei Bai,1 Chao-Cheng Lin,6 Tzu-Ting Chen,3 Geng-Han Mo,3 and I-Ching Lai3,4* 1 Department of Psychiatry, Taipei Veterans General Hospital, Taipei, Taiwan Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan 2 3 Department of Psychiatry, Yuli Mental Health Research Center, Yuli Veterans Hospital, Yuli, Hualien, Taiwan 4 Institute of Medical Science, Tzu Chi University, Hualien, Taiwan Department of Psychiatry, Pali-Psychiatric Hospital, Taipei, Taiwan 5 6 Department of Psychiatry, National Taiwan University Hospital, Taipei, Taiwan Received 4 March 2008; Accepted 29 April 2008 Some patients treated chronically with antipsychotics develop tardive dyskinesia (TD), an abnormal involuntary movement disorder. Typical antipsychotics block D2 dopamine receptors (D2DR) and produce D2DR supersensitivity. On contrary, regulators of G-protein signaling (RGS) can enhance the signal termination of G-protein-coupled D2DR. Besides, after prolonged inhibition of dopaminergic transmission, dopaminergic agonists induced severe dyskinesia only in RGS9 knock-out mice but not in normal mice. Therefore, variety in the human RGS9 gene may be related to susceptibility to TD. In this study, schizophrenic inpatients receiving long-term antipsychotic treatment were assessed using the Abnormal Involuntary Movement Scale twice over a 3-month interval. Only patients in whom abnormal involuntary movements were absent (non-TD group) and those who showed persistent TD (TD group) were enrolled. There were 407 patients in the study sample (TD ¼ 252; nonTD ¼ 155) and seven single nucleus polymorphisms (SNPs) in the RGS9 gene were genotyped for each subject. Genotype and allelic distributions of SNPs did not differ between the TD and non-TD groups in this study, with the exception that a weak trend of allelic association was seen with rs4790953 (P ¼ 0.0399). In the haplotype analysis, a significant association of the AGG haplotype (rs8077696–rs8070231–rs2292593) of the RGS9 gene was found (permutation P ¼ 0.007), and this is worthy of replication and further study. Ó 2008 Wiley-Liss, Inc. Key words: RGS; tardive dyskinesia; schizophrenia; antipsychotic; haplotype INTRODUCTION Tardive dyskinesia (TD) is an abnormal involuntary movement disorder characterized by a variable combination of the following: persistent orofacial and lingual dyskinesia, tics, grimacing, truncal Ó 2008 Wiley-Liss, Inc. How to Cite this Article: Liou Y-J, Chen M-L, Wang Y-C, Chen J-Y, Liao D-L, Bai Y-M, Lin C-C, Chen T-T, Mo G-H, Lai I-C. 2009. Analysis of Genetic Variations in the RGS9 Gene and Antipsychotic-Induced Tardive Dyskinesia in Schizophrenia. Am J Med Genet Part B 150B:239–242. or axial muscle involvement, chorea and athetosis. Approximately 13–36% of patients treated chronically with antipsychotics, but less than 2% of psychiatric patients who have never been exposed to antipsychotics, develop TD [Woerner et al., 1991]. Typical antipsychotics act by blocking D2 dopamine receptors (D2DR); therefore, the pathophysiology of TD may be hidden in the signal pathway of dopamine transmission in the brain. Chronic inhibition of dopamine signaling along the nigrostriatal dopamine pathway can increase the proportion of high-affinity D2DR and produce D2DR supersensitivity [Rubinstein et al., 1990], leading to abnormal signaling and abnormal involuntary movements. Grant sponsor: National Science Council, Taiwan; Grant numbers: NSC952314-B480-002-MY3, NSC96-2314-B-480-002; Grant sponsor: Yu-Li Veterans Hospital, Hualien, Taiwan; Grant numbers: VHYL-97-02, VHYL-97-03. *Correspondence to: Dr. I-Ching Lai, Department of Psychiatry, Yuli Veterans Hospital, No. 91, Shin-Shin St., Yuli, Hualien 981, Taiwan. E-mail: email@example.com Published online 11 June 2008 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/ajmg.b.30796 239 240 The receptors for dopamine are members of the G-proteincoupled receptor (GPCR) superfamily [Missale et al., 1998], and GPCR signal termination can be enhanced by members of a family of proteins called regulators of G-protein signaling (RGSs) [Berman and Gilman, 1998]. Mice with a null mutation in the RGS9 gene showed augmented locomotor responses to psychostimulants, but viral-mediated overexpression of RGS9-2, the RGS9 splice variant, specifically reduced locomotor response to D2DR agonists [Rahman et al., 2003]. The expression of RGS9-2 is selectively confined to the striatum [Thomas et al., 1998; Rahman et al., 1999], the region involved in antipsychotic-induced TD. Furthermore, the DEP (Disheveled, EGL-10, Pleckstrin homology) domain of RGS9-2 mediates subcellular colocolization with D2DR, and such localization may imply a role of RGS9-2 in the suppression of antipsychotic-induced extrapyramidal symptoms [Kovoor et al., 2005]. In RGS9 knock-out mice, direct treatment with dopaminergic agonists only produces minor increases in locomotion; however, after prolonged inhibition of dopaminergic transmission, dopaminergic agonists induce severe dyskinesia exclusively in RGS9 knockout mice but not in normal mice [Kovoor et al., 2005]. Similarly, only a few drug-naive schizophrenic patients develop dyskinesia, but after prolonged antipsychotic treatment, a large proportion of schizophrenic patients develop TD. Hence, RGS9-2 is very likely to play an important role in the susceptibility of patients to antipsychotic-induced TD. It is impossible to knock out the RGS9 gene in humans to prove the evidence found in animal models. The percentage of schizophrenics with antipsychotic-induced TD is 13–36%, which is unlikely resulted from the whole gene deletion of RGS9 in humans. Besides, the severity of antipsychotic-induced TD varies between affected individuals; therefore, susceptibility to antipsychoticinduced TD is highly suspected as being associated with genetic variances in RGS9. To the best of our knowledge, there are no previous studies of this gene epidemiology, and so we performed the present study to explore the relationship between the RSG9 gene and antipsychotic-induced TD in Chinese schizophrenic patients. MATERIALS AND METHODS Clinical Assessment Two senior board-certificated psychiatrists verified the diagnosis of each inpatient according the criteria of DSM-IV before he/she entered the study, based on interview, an oral report from family and/or caregivers, clinical observation and chart records. Demographic and clinical variables were also measured, including gender, age, cumulative duration of antipsychotic exposure, and daily dosage of antipsychotics (converted to chlorpromazine (CPZ) equivalents). The inclusion criteria were: a diagnosis of schizophrenia, persistent treatment with typical antipsychotics over the past 2 years, maintenance on a stable dosage of antipsychotics for at least 6 months. The exclusion criteria were: aged over 65 or under 18 years old; combination with an organic mental disorder, mood disorder, neurological illness or diabetes mellitus; a history of substance use (alcohol, amphetamines, or opiates); and previous exposure to atypical or second-generation antipsychotic treatment. AMERICAN JOURNAL OF MEDICAL GENETICS PART B Yuli Veterans Hospital Institutional Review Board had approved this study in advance, and all patients were fully informed about the study before giving informed consent. The senior psychiatrists (Dr. Lai IC, Dr. Bai YM, Dr. Lin CC, Dr. Liao DL, and Dr. Chen JY) involved in the study were experienced in using the Abnormal Involuntary Movement Scale (AIMS). TD was defined as of a mild degree of involuntary movement in two or more body regions or of a moderate to severe degree in one or more areas of the body. For confirmation of the diagnosis of TD, all patients were rated twice over a 3-month interval based on the Research and Diagnostic Criteria for persistent TD [Schooler and Kane, 1982]. Non-TD was defined as the absence of any abnormal involuntary movements as assessed in two successive interviews. In order to maintain assessment consistency, each patient was evaluated by the same rater. All raters completed their assessments before DNA genotyping and were therefore blind to the genotypes of patients. Marker Selection and Genotyping We selected genotyped genetic markers in the combined HCB population from the International HapMap Project (http:// www.hapmap.org/). Seven single nucleotide polymorphisms (SNPs) have minor allele frequencies of greater than 10% (listed in Table I), including rs8077696, rs8070231, rs2292593, rs2292592, rs9916525, rs1122079, and rs4790953. Genomic DNA was extracted from the white blood cells of peripheral venous blood, and the genotype of each SNP was determined by TaqManÒ assay. TaqManÒ probes and Universal PCR Master Mix were obtained from Applied Biosystems (http:// www.appliedbiosystems.com). After amplification, the allelic specific fluorescence was measured using ABI PRISMÒ 7500 Sequence Detector Systems. Statistical, Single Marker, and Haplotype Analysis We used an independent t-test to evaluate the differences in the means of the continuous variables between groups, and the chisquare test to evaluate the independence between categorical variables. Deviation from Hardy–Weinberg Equilibrium for each SNP was checked by a chi-square goodness of fit test with the degree of freedom of 1. The threshold of significance was set as P < 0.05. All analyses were performed using SPSS 10.0 for Microsoft Windows. Information for pairwise linkage disequilibrium (LD) was evaluated using the SNP AlyzeÒ V3.2 program (Dynacom Co., Ltd., Kanagawa, Japan). We also used this program to infer the haplotype frequency and to examine the difference in haplotype frequencies between the TD and non-TD groups. The significance level of the analyses obtained from SNP AlyzeÒ V3.2 was set as P < 0.05 after 100,000 permutation tests. RESULTS After the 3-month interval evaluations, 407 patients with schizophrenia (252 with persistent TD and 155 without TD) were recruited to the study. There was no significant difference between the groups in the distribution of gender (male/female, LIOU ET AL. 241 TABLE I. Distribution of the RGS9 Genotypes and Alleles in the TD and Non-TD Groups Genotype SNP ID rs8077696 Allele 1/2 C/A Position Intron 3 rs8070231 A/G Intron 4 rs2292593 A/G Intron 4 rs2292592 C/T Intron 7 rs9916525 C/T Intron 8 rs1122079 T/A Intron 8 rs4790953 A/C 30 -flanking region Group TD (%) Non-TD (%) TD (%) Non-TD (%) TD (%) Non-TD (%) TD (%) Non-TD (%) TD (%) Non-TD (%) TD (%) Non-TD (%) TD (%) Non-TD (%) 1/1 107 (43.7) 62 (40.5) 98 (40.0) 54 (34.8) 107 (43.3) 53 (34.6) 119 (47.8) 68 (44.2) 109 (45.0) 61 (39.6) 120 (48.2) 67 (43.5) 130 (53.1) 66 (43.1) Allele 1/2 111 (45.3) 72 (47.1) 110 (44.9) 67 (43.2) 104 (42.1) 76 (49.7) 109 (43.8) 68 (44.2) 113 (46.7) 73 (47.4) 112 (45.0) 70 (45.5) 100 (40.8) 72 (47.1) TD ¼ 97/58, non-TD ¼ 153/99, P ¼ 0.753), age (years [mean SD], TD ¼ 47.9 9.2, non-TD ¼ 47.2 9.2, P ¼ 0.464), cumulative length of antipsychotic exposure (years [mean SD], TD ¼ 21.0 8.3, non-TD ¼ 20.2 7.9, P ¼ 0.325) or current daily antipsychotic dosage (mg/day [mean SD], TD ¼ 699.1 477.1, nonTD ¼ 778.1 456.3, P ¼ 0.128). The distributions of the RGS9 genotypes in the TD and non-TD groups were all in Hardy– Weinberg equilibrium. Genotype and allelic distributions of SNPs in the study did not differ between the TD and non-TD groups, with the exception of a weak trend of allelic association seen with rs4790953 (P ¼ 0.0399) (Table I). Pairwise LD analysis and haplotype block determination revealed that there were two blocks across the studied genomic region in which the RGS9 gene is located (Fig. 1). Measurement of pairwise LD showed that the first 3 SNPs were in strong LD with each other and were located in the sample block (haplotype block 1). Another block (haplotype block 2) was constructed by rs2292592, rs9916525, and rs1122079. In the haplotype-based analyses, the haplotypes in the two blocks were analyzed separately according to their locations in the specific block. The results of the comparisons of haplotype distribution in haplotype block 1 between TD and non-TD groups are listed in Table IIa. Individual haplotype analysis showed that the haplotype AGG was associated with non-TD (permutation P-value ¼ 0.007). For the genetic variation in block 2, no significant difference in haplotype distribution was found between the groups in global analysis (permutation P ¼ 0.162). 2/2 27 (11.0) 19 (12.4) 37 (15.1) 34 (21.9) 36 (14.6) 24 (15.7) 21 (8.4) 18 (11.7) 20 (8.3) 20 (13.0) 17 (6.8) 17 (11.0) 15 (6.1) 15 (9.8) P-value 0.802 0.200 0.217 0.518 0.255 0.239 0.109 1 325 (66.3) 196 (64.0) 306 (62.4) 175 (56.5) 318 (64.3) 182 (59.5) 347 (69.7) 204 (66.2) 331 (68.4) 195 (63.3) 352 (70.7) 204 (66.2) 360 (73.5) 204 (66.7) 2 165 (33.4) 110 (36.0) 184 (37.6) 135 (43.5) 176 (35.7) 124 (49.5) 151 (30.3) 104 (33.8) 153 (31.6) 113 (36.7) 146 (29.3) 104 (33.8) 130 (26.5) 102 (33.3) P-value 0.5116 0.0915 0.1645 0.3068 0.1403 0.1846 0.0399 striatum [Rahman et al., 2003; Kovoor et al., 2005]. RGS9 knockout mice show dyskinesia after antipsychotic treatment but seem to be normal while they are drug-nai?ve; therefore, RGS9 may be a modulator of the dopamine pathway. The reason why some patients develop TD after antipsychotic treatment and others do not is very likely due to genetic differences in RGS9. Our results showed that one SNP, rs4790953, was found to have a weak trend of allelic association with TD, but this was insigni- DISCUSSION Although the cause of TD remains obscure, the brain-specific protein, RGS9-2, is a good candidate for the pathogenesis of TD. Chronic administration of antipsychotics results in increased D2DR and dopamine sensitivity [Samaha et al., 2007] and increases the possibility of developing TD [Woerner et al., 1991]. RGS9-2 has been suggested as colocalizing with D2DR and terminating the signal transduction of G-protein in the dopamine pathway in the FIG. 1. Illustration of the organization of the haplotype blocks constructed by the seven studied single nucleotide polymorphisms (SNPs) in the RSG9 gene. 242 AMERICAN JOURNAL OF MEDICAL GENETICS PART B TABLE II. Haplotype Analysis of (a) Block 1 (rs8077696–rs8070231–rs2292593) and (b) Block 2 (rs2292592–rs9916525–rs1122079) of the RGS9 Gene of the TD and Non-TD Groups Haplotype Block 1 CAA AGA CAG CGG AGG Global Block 2 CCT TTA Global TD (%) Non-TD (%) Permutation P-value 54.4 9.0 7.5 7.0 22.1 62.9 0.0 0.0 0.0 37.1 0.276 0.124 0.243 0.403 0.007* 0.079 70.0 30.0 64.9 35.1 0.160 0.160 0.162 *P < 0.05. ficant after Bonferroni correction. In the haplotype analysis, a significant association of the AGG haplotype (rs8077696– rs8070231–rs2292593) of the RGS9 gene was found (permutation P ¼ 0.007), and this is worthy of replication and further study. The function of the AGG haplotype (rs8077696–rs8070231–rs2292593) of the RGS9 gene is unknown; it may alter the binding efficiency of RGS9-2 to DRD2 or other genetic variations near any one of these SNPs (rs8077696, rs8070231, and rs2292593), and may play an important role in the development of TD. However, it is difficult to replicate the present study in Taiwan. First, the non-TD control group patients must take typical antipsychotics chronically but have no symptoms of TD development; however, more and more schizophrenic patients take atypical antipsychotics as a first-line treatment, due to their condition being unmanageable with typical antipsychotics, or to the extrapyramidal symptoms caused by typical antipsychotics. Therefore, it is becoming increasingly difficult to recruit non-TD controls. Second, although persistent TD is not rare, it is not frequently seen. Finally, we have to recruit our samples from three large psychiatric institutes in order to obtain enough subjects for meaningful study. Because the risk of false positive is high in this kind of study design; therefore, we hope that our results study can be replicated in other countries or other populations. In conclusion, our findings support the involvement of genetic variations of the RGS9 gene in the susceptibility to antipsychoticinduced TD. ACKNOWLEDGMENTS This work was supported by grants NSC95-2314-B480-002-MY3 and NSC96-2314-B-480-002 from the National Science Council, Taiwan, and grants VHYL-97-02 and VHYL-97-03 from Yu-Li Veterans Hospital, Hualien, Taiwan. REFERENCES Berman DM, Gilman AG. 1998. Mammalian RGS proteins: Barbarians at the gate. J Biol Chem 273:1269–1272. Kovoor A, Seyffarth P, Ebert J, Barghshoon S, Chen CK, Schwarz S, Axelrod JD, Cheyette BN, Simon MI, Lester HA, Schwarz J. 2005. D2 dopamine receptors colocalize regulator of G-protein signaling 9-2(RGS9-2) via the RGS9 DEP domain, and RGS9 knock-out mice develop dyskinesias associated with dopamine pathways. J Neurosci 25:2157–2165. Missale C, Nash SR, Robinson SW, Jaber M, Caron MG. 1998. Dopamine receptors: From structure to function. Physiol Rev 78:189–225. Rahman Z, Gold SJ, Potenza MN, Cowan CW, Ni YG, He W, Wensel TG, Nestler EJ. 1999. Cloning and characterization of RGS9-2: A striatalenriched alternatively spliced product of the RGS9 gene. J Neurosci 19:2016–2026. Rahman Z, Schwarz J, Gold SJ, Zachariou V, Wein MN, Choi KH, Kovoor A, Chen CK, DiLeone RJ, Schwarz SC, Selley DE, Sim-Selley LJ, Barrot M, Luedtke RR, Self D, Neve RL, Lester HA, Simon MI, Nestler EJ. 2003. RGS9 modulates dopamine signaling in the basal ganglia. Neuron 38:941–952. Rubinstein M, Muschietti JP, Gershanik O, Flawia MM, Stefano FJ. 1990. Adaptive mechanisms of striatal D1 and D2 dopamine receptors in response to a prolonged reserpine treatment in mice. J Pharmacol Exp Ther 252:810–816. Samaha AN, Seeman P, Stewart J, Rajabi H, Kapur S. 2007. ‘‘Breakthrough’’ dopamine supersensitivity during ongoing antipsychotic treatment leads to treatment failure over time. J Neurosci 27:2979–2986. Schooler NR, Kane JM. 1982. Research diagnoses for tardive dyskinesia. Arch Gen Psychiatry 39:486–487. Thomas EA, Danielson PE, Sutcliffe JG. 1998. RG S9: A regulator of G-protein signalling with specific expression in rat and mouse striatum. J Neurosci Res 52:118–124. Woerner MG, Kane JM, Lieberman JA, Alvir J, Bergmann KJ, Borenstein M, Schooler NR, Mukherjee S, Rotrosen J, Rubinstein M. 1991. The prevalence of tardive dyskinesia. J Clin Psychopharmacol 11:34–42.