Association of DRD2 gene variant with schizophrenia

Neuroscience Letters 392 (2006) 68–71

Association of DRD2 gene variant with schizophrenia Ritushree Kukreti a,b,∗ , Sudipta Tripathi a , Pallav Bhatnagar a , Simone Gupta b , Chitra Chauhan b , Shobhana Kubendran c , Y.C. Janardhan Reddy c , Sanjeev Jain c , Samir K. Brahmachari b a

GenoMed Lab (Gene Quest Laboratory, Nicholas Piramal India Ltd) at Institute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi 110007, India b Institute of Genomics and Integrative Biology (Council of Scientific and Industrial Research), Mall Road, Delhi 110 007, India c Department of Psychiatry, National Institute of Mental Health and Neuro Sciences, Hosur Road, Bangalore 560029, India Received 24 May 2005; received in revised form 24 August 2005; accepted 27 August 2005

Abstract Schizophrenia is a complex multifactorial disorder for which the pathobiology still remains elusive. Dysfunction of the dopamine D2 receptor signaling has been associated with the illness, but numerous studies provide confounding results. This study investigates the association of synonymous polymorphisms (His313 and Pro319) in the dopamine D2 receptor gene with schizophrenia using a case–control approach, with 101 cases and 145 controls. Our results demonstrated that genotype distribution for the His313 polymorphism was significantly different between schizophrenia patients and control subjects (p = 0.0012), while the Pro319 polymorphism did not show any association with the disease. The results suggest that the synonymous SNP His313 in DRD2 may be associated with the illness. However, there is a need for further replication studies with larger sample sets. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Schizophrenia; Dopamine D2 receptor; Synonymous mutation; Association study

Schizophrenia is a multifactorial disease involving genetic and environmental factors, with a worldwide lifetime prevalence of 0.5–1% [21,29,27]. The disease is highly heritable, but the genetic mechanisms involved in the pathogenesis still remain elusive. High genetic risk for schizophrenia has led to considerable research efforts aimed at identifying susceptibility genes, including several linkage studies [26,37]. Significant linkages with specific chromosomal regions have been identified [30], but no mutations or disease-predisposing polymorphisms have been consistently identified. The mode of inheritance of schizophrenia is likely to be polygenic/multifactorial [21], and association studies are ideally suited to investigate the candidate genes implicated in this disorder. Although the biological basis of schizophrenia is unknown, altered dopamine function is thought to underlie several symptoms and also the action of antipsychotic medication. Genes involved in dopaminergic pathways are thus being studied as candidate genes for schizophrenia [16]. There is also evidence of excessive dopaminergic activity in the illness [19]. It has been suggested that overactivity of the

∗

Corresponding author. Tel.: +91 11 27666156/7x135; fax: +91 11 27667471. E-mail address: [email protected] (R. Kukreti).

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.08.059

dopaminergic system in the mesolimbic pathway may contribute to delusions, hallucinations and thought disorder in schizophrenia whereas dysfunction of the dopaminergic system in the mesocortical pathway may be responsible for apathy, withdrawal and impaired attention [38]. Five distinct subtypes of G-proteincoupled dopamine receptors mediate the actions of dopamine, three of which belong to the dopamine D2 family: the dopamine D2 receptor (DRD2), the dopamine D3 receptor (DRD3) and the dopamine D4 receptor (DRD4) [24]. The dopamine D2 gene (DRD2), located on chromosome 11q22-q23 [2,11] has been implicated as a possible candidate gene for schizophrenia. All antipsychotic medications are either antagonists or partial agonists of the dopamine D2 receptor, which is the primary site of action for these medications [25,31,35]. Recently, meta-analyses have supported the involvement of the DRD2 in the pathogenesis of schizophrenia [16]. Several studies thus suggest the central role of the dopamine receptor in the risk of schizophrenia [4,22]. A large number of groups have attempted to find association of DRD2 polymorphisms to validate the influence of DRD2 on the disease etiology. However, association studies, to date, have not been able to yield consistent results across different ethnic groups [3,4,10,12,17,34,36,39]. An allelic association has been identified between the Taq1-A polymorphism of the DRD2 and

R. Kukreti et al. / Neuroscience Letters 392 (2006) 68–71

schizophrenia [9], but this has not been confirmed by other investigators [39]. Another single nucleotide polymorphism in the DRD2 is the missense variant C/G in exon 7, which results in a Ser/Cys substitution at position 311 [14,33]. These polymorphisms may be directly or indirectly implicated in schizophrenia through dopaminergic or other systems [7]. The −141 C del/ins has shown a significant association with schizophrenia; however, these associations in British participants [5] are in contrast with Japanese and Scandinavian counterparts [3,28,15]. Two synonymous polymorphisms His313 and Pro319 are also reported in the exon 7 of DRD2. Synonymous mutations are supposed to have no effect on the amino acid sequence of the gene product, but the Pro319 polymorphism shows marked functional consequences as it affects the stability of mRNA, decreases translation and leads to reduction in dopamine-induced upregulation of DRD2 receptors [8]. A positive association of the Pro319 C/C variant with schizophrenia has been recently reported [20]. The goal of the present study is to examine the association between two synonymous polymorphisms (His313, Pro319) and schizophrenia. A total of 101 patients (M:F::56:45) who met the DSM-IV criteria for schizophrenia were recruited for this study from the clinical services of the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore. Age at onset was defined as the age at which the first symptom of schizophrenia was documented, and was 25.3 ± 7.3 years, while the average duration of illness was 3.4 ± 2.4 years. The 145 normal comparison subjects who appeared to be healthy, but were not screened to exclude personal or family history of psychiatric disease were also included in the study. All subjects were from southern India and informed consent was obtained from each individual. The study was approved by the institutional human ethics committee at NIMHANS. Genomic DNA was isolated from peripheral blood using a salt-precipitation method [23]. The desired polymorphic region of the DRD2 containing synonymous mutations (His313 and Pro319) was amplified by the standard polymerase chain reaction (PCR) with a primer pair of 5 -GAG GAT TGC CAT GGG AAA AAG GAC-3 (sense) and 5 -CTG CAG CCA TGG TTA GGA AGG AC-3 (antisense). The PCR product was 626 bp in length corresponding to nucleotides +11554 to +12179 of the human DRD2 sequence. PCR was performed in a PerkinElmer Gene Amp PCR System 9600 (Perkin-Elmer, Oak Brook, IL) in a reaction volume of 25 ␮l by using 200 ng of DNA, 10 pmol each of sense and antisense primer, 1.2 units Taq Polymerase (Bangalore Genei), MgCl2 and 200 ␮M deoxyribonucleoside triphosphate (dNTP) in a 10× PCR buffer (containing 100 mM Tris (pH 8.3), 500 mM KCl, and 0.1% Gelatin). Thermal cycling was performed by using the following thermal profile: denaturation at 95 ◦ C for 1 min 30 s, annealing at 65 ◦ C for 1 min and extension at 72 ◦ C for 1 min. This was continued for a total of 40 cycles, followed by a final extension step for 10 min at 72 ◦ C. Three microliters of PCR product was electrophoresed on 1% agarose gel and visualized with ethidium bromide staining and UV illumination to check PCR amplification. The PCR products were purified with the Qiaquick PCR purification Kit (Qiagen), and DNA sequencing was performed

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by the ABI Prism 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA) using dye terminator chemistry. The sequencing reaction consisted of 30 cycles, with denaturation at 96 ◦ C for 10 s and annealing at 60 ◦ C for 4 min. The PCR product was shown to be identical to the exonic region of the DRD2 sequence in the database (GenBank Accession Number: NM 000795). Sequences were aligned with the corresponding wild-type sequences using Factura and Sequence Navigator software programs. The Hardy–Weinberg equilibrium and the differences in the genotype distribution and allele frequencies between the schizophrenia patients and control group were tested using the χ2 -test. Odds ratios (OR) and confidence interval (95% CI) were calculated for the alleles and genotypes. Linkage disequilibrium (LD) between two synonymous SNPs (His313 and Pro319) was assessed in all 145 control individuals by calculating r2 and D using 2 LD program (http://linkage.rockefeller.edu). Haplotype based case control association analysis was also performed by PHASE software using genotype information of these two synonymous polymorphisms. Two variable synonymous SNP sites at position +11897 (His313) and at position +11915 (Pro319) corresponding to nucleotides of the human DRD2 sequence were identified by sequencing 626 bp of DRD2. A case–control study was designed to analyze the association of His313 and Pro319 polymorphisms of the DRD2 with schizophrenia. The study population consisted of 101 cases and 145 control samples, and the distributions of the genotypes in the control population were in Hardy–Weinberg equilibrium. The observed genotype frequency for His313 polymorphism shows a significant association with the illness (χ2 = 13.48, d.f. = 2, p = 0.0012) as shown in Table 1. In comparison with the cases, the control group shows a much higher frequency of heterozygotes (CT). It is evident from Table 1 that there is no difference in the frequency of the alleles between the cases and controls (χ2 = 0.37, d.f. = 1, p = 0.571). We have observed a significant association in a recessive model (TT versus TC and CC; p = 0.007) whereas we did not find any association in dominant model (CC versus TT and TC; p = 0.215) showing T as a recessive allele (Table 1). The observed genotype and allele frequency for Pro319 polymorphism shows no association between the cases and controls (Genotype: χ2 = 1.55, d.f. = 2, p = 0.459; Allele: χ2 = 0.87, d.f. = 1, p = 0.356) (Table 1). In assessing the strength of LD between His313 and Pro319 polymorphisms of the control population, we found that these SNPs are in moderate LD (r2 = 0.21, D = 0.67). Haplotype construction by PHASE showed four different haplotypes in our case–control groups. Haplotype frequencies among case and control groups are shown in Table 2 and none of the haplotypes have shown any association with the disease (global p-value of 0.12). The present case–control association study between synonymous SNPs (His313 and Pro319) of the DRD2 and schizophrenia showed that the genotypic distribution for His313 is significantly different between patients and controls (Table 1). These results suggest that in addition to D2 receptor being centrally involved in the action of antipsychotic medication [18], genetically determined differences in D2 upregulation may be partially

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Table 1 Genotypic and allelic distribution of the His313 and Pro319 polymorphisms of the DRD2 Genotype count (frequency) T/Ca

χ2

p

Allele count (frequency)

p

His313His Cases (n = 101) Controls (n = 145)

TT 26 (25.7) 18 (12.4)

TC 28 (27.7) 71 (49.0)

CC 47 (46.6) 56 (38.6)

13.48

0.0012

T 80 (39.6) 107 (36.9)

C 122 (60.4) 183 (63.1)

0.571

Pro319Pro C/Tb Cases (n = 101) Controls (n = 145)

CC 41 (40.6) 48 (33.1)

CT 38 (37.6) 64 (44.1)

TT 22 (21.8) 33 (22.8)

1.55

0.459

C 120 (59.4) 160 (55.2)

T 82 (40.6) 130 (44.8)

0.356

Genotype and allele frequencies (in parentheses) are expressed as percentages (%). a Allele: d.f. = 1, OR = 0.891, 95% CI = 0.616–1.29, Genotype (TT vs. TC and CC): χ2 = 7.20, d.f. = 1, OR = 0.408, 95% CI = 0.21–0.79, p = 0.007, Genotype (CC vs. TT and TC): χ2 = 1.53, d.f. = 1, OR = 1.383, 95% CI = 0.82–2.31, p = 0.215. b Allele: d.f. = 1, OR = 0.841, 95% CI = 0.584–1.21. Genotype (CC vs. CT and TT): χ2 = 1.45, d.f. = 1, OR = 0.724, 95% CI = 0.42–1.22, p = 0.229, Genotype (TT vs. CC and CT): χ2 = 0.03, d.f. = 1, OR = 0.945, 95% CI = 0.51–1.74, p = 0.856.

involved in the underlying pathophysiology of schizophrenia. A moderate level of LD was observed between His313 and Pro319 polymorphisms in our sample. In our study, haplotype based case–control association analysis performed by PHASE software showed no haplotype association with the disease (Table 2). The study of the synonymous polymorphism His313 (change from T to C), is important due to several reasons. All synonymous polymorphisms are not nonfunctional or neutral. This is evident from studies that report a strong correlation between bias in synonymous codon usage and the gene expression level [1,13]. It has been suggested by Duan et al. [8] that DRD2 is an ideal candidate to investigate the possible functional consequences of synonymous SNPs [8]. Although our study does not include any functional studies of the His313 change, the disease association for His313 polymorphism at the genotypic level provides novel information to the field of schizophrenia association studies. Similar results have also been observed with respect to another DRD2 synonymous polymorphism (Pro319), that also has functional consequences [20]. By analogy, the silent mutation at codon 313 could also result in some functional changes, which need to be evaluated. There are reports suggesting the contribution of common genetic variation, to the structural and functional diversity of mRNA, thus providing an insight into fundamental mechanisms of human phenotypic variation, and perhaps facilitating studies of disease susceptibility and drug response [32]. The potential structural change in mRNA caused by synonymous mutations might be of considerable significance in context to the expression level of this receptor [6]. The change of mRNA folding structures by the synonymous polymorphisms in DRD2 suggests a plausible structural influence on the mRNA stability [8]. The strength of these results is tempered by small sample size and needs to be substantiated by another set of larger samTable 2 Haplotypes and their frequencies among case and control groups Haplotypes

Controls (frequency)

Cases (frequency)

TC CC TT CT

0.13 0.47 0.3 0.1

0.08 0.49 0.3 0.13

Global p-value is 0.12.

ples. Pro319 polymorphism does not show any association with the disease in the studied samples. To demonstrate the power of Pro319 polymorphism under the given sample size, power calculation was carried out by PAWE software (Power for Association With Error, version 1.2) (http://linkage.rockefeller.edu/pawe/). The results showed that our sample size has the power of 15 and 37%, to detect a significant difference in the case–control study for allelic and genotypic tests, respectively. Our sample size has limited statistical power for any conclusion to be drawn regarding the association between the studied synonymous polymorphisms and schizophrenia. However, if the results are replicated, our results may provide new insights into the pathophysiology of disease. There is a need to further study the functional consequences of the T/C change in a more detailed manner. The strategy for future studies on schizophrenia should seek to examine the measurable biological consequences of His313 genotypes to observe their potential pathophysiological effects by studying in vitro and in vivo gene function such as mRNA translation, mRNA stability and dopamine-induced upregulation of DRD2. It will be interesting to study other synonymous SNPs of the dopamine D2 receptor and their interaction in combination with His313 polymorphism. Future research should also explore whether antipsychotic treatment response is associated with His313 polymorphisms and examine its effect in other ethnic groups. Acknowledgments This study was financially supported by Nicholas Piramal India Ltd., India and Institute of Genomics and Integrative Biology (IGIB) (CSIR), Delhi, India. The authors thank Dr. Somesh Sharma (Chief Scientific Officer, NPIL) and Dr. Swapan Das (Scientist, IGIB) for their invaluable scientific suggestions. Authors sincerely thank the reviewers for their invaluable comments and suggestions. References [1] H. Akashi, Gene expression and molecular evolution, Curr. Opin. Genet. Dev. 11 (2001) 660–666. [2] K. Araki, R. Kuwano, K. Morii, S. Hayashi, S. Minoshima, N. Shimizu, T. Katagiri, H. Usui, T. Kumanishi, Y. Takahashi, Structure and expres-

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