No association of GRIP1 gene polymorphisms with schizophrenia in Chinese population

Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 752 – 755 www.elsevier.com/locate/pnpbp

No association of GRIP1 gene polymorphisms with schizophrenia in Chinese population Shih-Jen Tsai a,b,⁎, Ying-Jay Liou c,d,e , Ding-Lieh Liao e,f , Chih-Ya Cheng c , Chen-Jee Hong a,b a

Department of Psychiatry, Taipei Veterans General Hospital, Taipei, Taiwan Division of Psychiatry, School of Medicine, National Yang-Ming University, Taipei, Taiwan c Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan d Department of Psychiatry, Yuli Veterans Hospital, Hualien, Taiwan Division of Mental Health and Substance Abuse Research, National Health Research Institutes, Miaoli, Taiwan f Department of Psychiatry, Executive Yuan Department of Health, Pali Psychiatric Hospital, Taipei, Taiwan b

e

Received 21 August 2006; received in revised form 9 January 2007; accepted 10 January 2007 Available online 20 January 2007

Abstract Disturbance in glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia. Since glutamate receptor interacting protein 1 (GRIP1), a modular protein that enables anchoring of AMPA receptors via its PDZ (postsynaptic density-95/discs large/zona occludens1) domain and modulates D-serine release, plays an important role in glutamatergic function, this study tests the hypothesis that GRIP1 genetic variants confer susceptibility to schizophrenia. This study investigated whether GRIP1 genetic polymorphisms (rs1038923 and rs4913301) cause a predisposal to schizophrenia. Two GRIP1 polymorphisms were studied in a sample population of 252 people with schizophrenia and 207 normal controls. Significant linkage disequilibrium was obtained between the two polymorphisms. Results demonstrated that neither single marker nor haplotype analysis revealed an association between variants at the GRIP1 locus and schizophrenia, suggesting that it is unlikely that the GRIP1 polymorphisms investigated play a substantial role in conferring susceptibility to schizophrenia. However, association between schizophrenia and other polymorphisms in the GRIP1 gene cannot be totally ruled out as the whole gene was not covered by the two polymorphisms studied. © 2007 Elsevier Inc. All rights reserved. Keywords: Association study; Glutamate receptor interacting protein 1; Haplotype; Polymorphism; Schizophrenia

1. Introduction Schizophrenia is a complex psychiatric disorder involving dysfunction in several brain regions and neurotransmitter systems, such as glutamatergic abnormalities. Evidence of glutamatergic abnormalities has been inferred from several observations in post-mortem studies as well as from pharmacological findings (Belsham, 2001; Weickert and Kleinman, 1998).

Abbreviations: ABP, AMPA receptor-binding protein; AOO, age of onset; GRIP1, glutamate receptor interacting protein 1; htSNP, haplotype-tagging and SNP; LD, linkage disequilibrium; PDZ, postsynaptic density-95/discs large/ zone occludens-1; PICK1, protein interacting with C kinase 1. ⁎ Corresponding author. Department of Psychiatry, Taipei Veterans General Hospital, No. 201, Shih-Pai Road, Sec. 2, 11217, Taipei, Taiwan. Tel.:+886 2 2875 7027x269; fax: +886 2 2872 5643. E-mail address: [email protected] (S.-J. Tsai). 0278-5846/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2007.01.015

For example, previous post-mortem studies have reported a decrease in AMPA receptor subunit protein levels in the medial temporal lobe and the hippocampal formation of schizophrenia patients (Eastwood et al., 1997a,b). The AMPA and NMDA classes of ionotropic glutamate receptors are concentrated at postsynaptic sites in excitatory synapses. Glutamate neurotransmission involves not only glutamate receptors but also intracellular molecules enriched in postsynaptic proteins. Recent studies have indicated that the targeting of AMPA- and NMDA-type glutamate receptors to synapses in the central nervous system is essential for efficient excitatory synaptic transmission, and that protein–protein interactions of these receptors with synaptic proteins that contain a common motif, called the postsynaptic density-95/discs large/zone occludens-1 (PDZ) domain, are crucial for receptor targeting (O'Brien et al., 1998). Among various postsynaptic PDZ-domain-containing proteins, the postsynaptic density

S.-J. Tsai et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 31 (2007) 752–755

protein PSD-95 family of proteins has been found to bind to NMDA receptors, whereas the glutamate receptor interacting protein 1 (GRIP1; previously termed GRIP) (Dong et al., 1997), AMPA receptor-binding protein (ABP) (Srivastava et al., 1998), and protein interacting with C kinase 1 (PICK1) (Xia et al., 1999) interact with AMPA receptors. Since glutamatergic dysfunction may play an important role in the pathogenesis of schizophrenia, these AMPA/NMDA related PSD molecules may play a role in the pathogenesis of schizophrenia, and genes of these proteins have thus become attractive candidates for genetic association studies. Recently, in a case–control association study, the authors have demonstrated an association of the PICK1 gene with schizophrenia in 225 schizophrenia and 260 controls (Hong et al., 2004), which is further supported by a recent study of the Japanese population (Fujii et al., 2006). Among the other AMPA/NMDA related PSD proteins, gene coding for GRIP1 is an attractive candidate gene for the study of schizophrenia susceptibility because, in addition to controlling AMPA receptor dynamics and synaptic plasticity, recent study has demonstrated that GRIP1 physiologically binds serine racemase (an enzyme that converts L-serine to D-serine), augmenting serine racemase activity and D-serine release (Kim et al., 2005). D-serine is one of the endogenous ligands implicated in the activation of NMDA receptors. Treatment of schizophrenia with D-serine together with antipsychotics alleviates schizophrenic symptoms in patients (Tsai et al., 1998, 1999). Furthermore, serum levels of D-serine in the patients with schizophrenia were significantly lower than those of healthy controls (Hashimoto et al., 2003). The human GRIP1, located on chromosome 12q14.2, is about 330 kb in length and includes 23 exons. To investigate the pathological role of the GRIP1 gene in schizophrenia, the association between the disorder and genetic variants in the GRIP1 gene was examined in a case–control study. Linkage disequilibrium (LD) measurement among the GRIP1 polymorphisms and haplotype analysis between patient and control groups was conducted to assess the association between markers within the GRIP1 gene and schizophrenia. 2. Methods

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orders, drug or alcohol abuse were not enrolled in the study. These patients had been reported in our previous association study of the PICK1 gene with schizophrenia (Hong et al., 2004). A total of 207 normal comparison subjects (mean age 41.1 ± 12.0 years; male/female: 109/98) who had been interviewed by a psychiatric staff member to rule out major psychiatric disorders, were enrolled as controls. Controls and patients were matched for age and sex. Control subjects were recruited from medical staff and volunteers living in the same area as the patients. The entire sample consisted of Taiwanese ethnic Han Chinese. The study was approved by the Ethics Committee of the Pali Psychiatric Hospital. Written, informed consent was obtained from all subjects. 2.2. Genotyping analysis Two polymorphisms in GRIP1 were genotyped. These markers were chosen from the public database (dbSNP home page; http://www.ncbi.nlm.nih.gov/projects/SNP/) based on position and allele frequencies. These two SNPs included rs1038923 (intron 9) and rs4913301 (intron 9), which cover 7.2 kb in the GRIP1 gene. The exact location of the two studied polymorphisms was obtained from the dbSNP (http://www. ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=4913301,XM_290559). Exon 9 of the GRIP1 gene encodes amino acids coded from 292 to 347, while amino acids from 262 to 333 contain a PDZ domain (from website database search http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt= full_report&list_uids=23426). In silico haplotype-tagging and SNP (htSNP) selection using the SNP tagger program indicated that in order to cover more than 90% of haplotype diversity, both rs1038923 and rs4913301 should be selected as htSNPs (Ke and Cardon, 2003). For determination of these two GRIP1 polymorphisms, peripheral venous blood was withdrawn from the study subjects after informed consent had been obtained. Genomic DNA was isolated by using the PUREGENE DNA purification system (Gentra Systems). GRIP1 polymorphism genotyping was performed using high-throughput MALDI-TOF mass spectrometry.

2.1. Subjects Two hundred and fifty-two schizophrenic patients (mean age 42.5 ± 10.3 years; male/female: 131/121), who met the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV; American Psychiatric Association, 1994; Code number: 295.XX), were recruited from the Pali Psychiatric Hospital, which is located in Northern Taiwan. The diagnoses were re-evaluated by a senior psychiatrist who interviewed each patient. Age at onset was determined from medical records and/ or interviews and was considered to be the age at which an individual had his/her first psychotic episode or a significant change in social-occupational function. All patients were assessed using the Brief Psychiatric Rating Scale (BPRS) (Overall and Gorham, 1962). Patients with associated diagnoses of mental retardation, organic brain disease, severe physical dis-

Table 1 Genotype distribution and allele frequencies for the GRIP1 genetic polymorphisms for schizophrenics (n = 252) and controls (n = 207) Genotype, n (%) rs1038923 Schizophrenics Controls rs4913301 Schizophrenics Controls a

A/A 77 (30.6) 59 (28.5) A/A 15 (6.0) 12 (5.8)

A/G 125 (49.6) 110 (53.1) A/G 89 (35.3) 75 (36.2)

Compared with control group.

Allele, n (%) G/G 50 (19.8) 38 (18.4) G/G 148 (58.7) 120 (58.0)

Pa 0.752

0.979

A 279 (55.3) 228 (55.1) A 119 (23.6) 99 (23.9)

G 225 (44.6) 186 (44.9) G 385 (76.4) 315 (76.1)

Pa 0.947

0.938

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Briefly, primers and probes were designed with SpectroDESIGNER software (Sequenom, San Diego). A multiplex polymerase chain reaction was performed, and unincorporated dNTPs were dephosphorylated with shrimp alkaline phosphatase (Hoffman-LaRoche, Basel) followed by primer extension. The purified primer extension reaction was spotted onto a 384element silicon chip (SpectroCHIP, Sequenom, San Diego) and analyzed in the Bruker Biflex III MALDI-TOF SpectroREADER mass spectrometer (Sequenom, San Diego). The resulting spectra were processed with SpectroTYPER (Sequenom, San Diego).

The two markers (rs1038923 and rs4913301) were found to be in strong LD to each other in both case (D′ = 1.0; r2 = 0.385; P b 0.001) and control groups (D′ = 0.96; r2 = 0.353; P b 0.001). The results of global case–control haplotype analysis and comparisons of individual haplotypes between groups are presented in Table 2. Global case–control haplotype analysis showed that there was no significant difference in haplotype distribution between the groups (P = 0.608). Individual haplotype analysis showed that the frequencies of all haplotypes are similar in the control and case groups (Table 2). 4. Discussion

2.3. Statistical analysis Statistical analysis was performed using the statistical package for social sciences (SPSS) 10.0. Differences in continuous variables were evaluated using Student's t-test or one-way analysis of variance, followed by the LSD multiple range test for between-group comparison. Categorical data were analyzed using the chi-square test, or Fisher's exact test if necessary. Data are presented as mean ± standard deviation (SD). The software SNP Alyze® V3.2 (Dynacom Co., Ltd. Kanagawa, Japan) was used to evaluate the status of pairwise LD for the studied polymorphisms, to infer the haplotype frequency and to determine whether haplotype frequency varied between groups. The significance level of these analyses obtained from the SNP Alyze® V3.2 was set as P value b 0.05 after 100,000 permutation tests. 3. Results These 252 schizophrenic patients were chronic inpatients and were receiving a mean of 608.7 ± 326.3 mg/day chlorpromazine equivalents. The mean education years were 9.1 ± 3.1 years and average BPRS score was 20.6 ± 8.6 for these patients. Mean age of onset of these patients was 21.4 ± 7.2 years. The genotype and allele distributions for the two GRIP1 polymorphisms for the schizophrenic patients and control subjects are presented in Table 1. For the GRIP1 polymorphisms investigated, genotype distributions did not deviate significantly from the expected values at the Hardy–Weinberg equilibrium for either cases or controls. For the two GRIP1 polymorphisms tested, no significant differences in the frequency of the genotype distribution between schizophrenia and controls were found (Table 1).

Table 2 Inferred haplotype frequency in GRIP1 gene and haplotype comparison between schizophrenics and controls

AG GA GG AA Global

Schizophrenics

Controls

Permutation P value

0.55 0.23 0.22 0.005

0.55 0.24 0.21 0

0.927 0.737 0.808 0.153 0.608

The order of the markers (left to right) is rs1038923 and rs4913301.

This is the first study dealing with GRIP1 polymorphism in a population with schizophrenia. Our data indicate that no significant difference was observed between the controls and patients in allelic frequencies or genotypic distributions of the GRIP1 polymorphisms. Permutation tests showed no significant difference in estimated haplotype frequencies of the GRIP1 gene between the controls and patients. There are several implications of these findings. Firstly, the present study indicates that GRIP1 is not a common major locus for schizophrenic disorders in the population studied in this paper. This is in line with post-mortem study, which revealed that there were no changes in the levels of the GRIP1 protein in the prefrontal cortex or in the hippocampus of the schizophrenic patients (Toyooka et al., 2002). However, if GRIP1 is an uncommon disease locus or one of small effect, the power to detect a gene would be reduced. With the sample size in this study, 76.8% of power was needed to detect a risk allele with allele frequency of 23% in the control group with an odds ratio of 1.5 for schizophrenia susceptibility at the 5% significance level, but only 40.2% of power was needed to detect a risk allele with an odds ratio of 1.3. Secondly, the two studied SNPs, rs1038923 and rs4913301 are located in the intron 9 of GRIP1 gene. Therefore, the studied polymorphisms cover part (7.2 kb) of the whole gene. Although the current results do not provide evidence supporting the proposed relationship between schizophrenia and the GRIP1 polymorphisms investigated, an association with other GRIP1 polymorphisms, particularly functional ones, in the GRIP1 gene cannot be completely excluded. Thirdly, another possible explanation for our negative findings could stem from the clinical heterogeneity of schizophrenia, because schizophrenia probably comprises a group of disorders with heterogeneous aetiologies. Thus, there is still a possibility that associations occur in diagnostic subtypes of schizophrenic patients (e.g. paranoid vs. non-paranoid). Finally, although the current study cannot find an association between GRIP1 genetic variants and schizophrenia, other PDZ-domain-containing proteins that target glutamate receptors may be implicated in the pathogenesis of schizophrenia. For example, this study and others have demonstrated that PICK1 genetic variants, which play an important role in the targeting and clustering of AMPA receptors, are associated with schizophrenia (Fujii et al., 2006; Hong et al., 2004). Thus, the genetic variants of other PDZ-domain-containing scaffold proteins that target glutamate receptors in schizophrenic disorders may need further investigation. In addition, GRIP1 has recently been found to affect D-serine release in the brain (Kim et al., 2005). Clinical study has

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previously demonstrated that oral administration of D-serine can help to improve schizophrenic symptoms (Tsai et al., 1998, 1999). Whether the GRIP1 genetic variants may affect schizophrenic prognosis or antipsychotic treatment response may warrant further exploration. 5. Conclusion Our findings suggest that it is unlikely that the GRIP1 polymorphisms investigated play a substantial role in conferring susceptibility to schizophrenia in the Chinese population. However, association between schizophrenia and other genetic variants in the GRIP1 gene cannot be completely ruled out as the whole gene was not covered by the two polymorphisms investigated. Further studies with other GRIP1 variants, relating either to schizophrenia, psychotic symptoms or to therapeutic response in schizophrenia, are suggested. Acknowledgements This work was supported by grant NSC 92–2314-B-075– 087 from the National Science Council, Taiwan and grant VGH92–161 from the Taipei Veterans General Hospital. The authors thank Dr. Jer-Yuan Wu, of the National Genotyping Center and the National Clinical Core at Academia Sinica, Taiwan, for genotyping support. References Belsham B. Glutamate and its role in psychiatric illness. Hum Psychopharmacol 2001;16:139–46. Dong H, O'Brien RJ, Fung ET, Lanahan AA, Worley PF, Huganir RL. GRIP: a synaptic PDZ domain-containing protein that interacts with AMPA receptors. Nature 1997;386:279–84. Eastwood SL, Burnet PW, Harrison PJ. GluR2 glutamate receptor subunit flip and flop isoforms are decreased in the hippocampal formation in

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