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Association study of polymorphisms in the excitatory amino acid transporter 2 gene (SLC1A2) with schizophrenia

Association study of polymorphisms in the excitatory amino acid transporter 2 gene (SLC1A2) with... Background: The glutamatergic dysfunction hypothesis of schizophrenia suggests that genes involved in glutametergic transmission are candidates for schizophrenic susceptibility genes. We have been performing systematic association studies of schizophrenia with the glutamate receptor and transporter genes. In this study we report an association study of the excitatory amino acid transporter 2 gene, SLC1A2 with schizophrenia. Methods: We genotyped 100 Japanese schizophrenics and 100 controls recruited from the Kyushu area for 11 single nucleotide polymorphism (SNP) markers distributed in the SLC1A2 region using the direct sequencing and pyrosequencing methods, and examined allele, genotype and haplotype association with schizophrenia.The positive finding observed in the Kyushu samples was re-examined using 100 Japanese schizophrenics and 100 controls recruited from the Aichi area. Results: We found significant differences in genotype and allele frequencies of SNP2 between cases and controls (P = 0.013 and 0.008, respectively). After Bonferroni corrections, the two significant differences disappeared. We tested haplotype associations for all possible combinations -5 of SNP pairs. SNP2 showed significant haplotype associations with the disease (P = 9.4 × 10 , P = 0.0052 with Bonferroni correction, at the lowest) in 8 combinations. Moreover, the significant haplotype association of SNP2-SNP7 was replicated in the cumulative analysis of our two sample sets. Conclusion: We concluded that at least one susceptibility locus for schizophrenia is probably located within or nearby SLC1A2 in the Japanese population. ious cognitive impairments. The life-time prevalence is Background Schizophrenia is a severe mental disorder characterized by about 1%, and genetic factors were known to play a criti- hallucinations, delusions, disorganized thoughts, and var- cal role in its pathogenesis [1]. Based on the fact that Page 1 of 6 (page number not for citation purposes) BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 phencyclidine (PCP) induces schizophreniform psycho- unrelated controls (mean age 39.9; 45% female) were col- sis, a glutamatergic dysfunction hypothesis has been pro- lected in the Aichi area about 600 km east of Fukuoka. All posed for the pathogenesis of schizophrenia [2-4]. This patients fulfilled the DSM-IV criteria for schizophrenia hypothesis has been supported by recent multiple reports [19]. All of the case and control samples are ethnically Jap- of association of schizophrenia with glutamate receptor anese. DNA samples were purified from whole peripheral genes and with the genes related to glutamatergic trans- blood by the method previously described [20]. This mission, such as G72 and NRG1 [5-10]. study was approved by the Ethics Committee of Kyushu University, Faculty of Medicine and the Fujita Health Uni- In addition, other synaptic elements related to glutamate, versity Ethics Committee. such as excitatory amino acid transporters (EAATs), also SNP selection in the SLC1A2 region potentially affect glutamatergic neurotransmission. EAATs maintain extracellular glutamate concentrations within We retrieved the primary SNP information from the physiological levels by reuptaking the synaptically dbSNP database http://www.ncbi.nlm.nih.gov/SNP/. released glutamate. A deficient uptake has been impli- Assuming the same size of the half length of linkage dise- cated in the pathogenesis of ischemic brain damage [11] quilibrium (LD) (60 kb) as reported in Caucasians [21], and may be involved in neurodegenerative diseases such we initially intended to select common SNPs every 50 kb as amyotrophic lateral sclerosis (ALS) [12]. Recently sig- in SLC1A2. We tested 22 candidate SNPs including all of nificant increases of mRNA expression of EAAT1 and the exonic SNPs, in the 16 healthy Japanese samples by EAAT2 have been reported in the thalamus of schizo- the direct sequencing method. Out of the 22 SNPs we phrenics, suggesting the possibility that an excessive gluta- selected the following 7 common SNPs with minor allele mate uptake is involved in schizophrenia [13]. On the frequencies over 10% for further analyses: SNP1, other hand, a significant decrease of EAAT2 mRNA expres- rs1923295; SNP3, rs4534557; SNP6, rs1885343; SNP8, sion was observed in the parahippocampal gyrus of schiz- rs752949; SNP9, rs1042113; SNP10, rs3838796; SNP11, ophrenics [14]. Therefore the EAAT genes are reasonable rs1570216. We also identified a novel SNP, SNP7, in candidates for schizophrenia, as well as glutamate recep- intron 1 (conting location: 34105026). After the LD anal- tor genes. yses described below, we noticed LD gaps (D' < 0.3) of the initial SNP set and examined additional 20 candidate The EAATs family consists of five members (EAAT1- SNPs. Out of the 20 SNPs, we selected the following 3 EAAT5). Their cellular localizations are different: EAAT1 SNPs to fill the LD gaps: SNP2, rs4755404; SNP4, and EAAT2 are astroglial, whereas EAAT3 EAAT4 and rs4756224; SNP5, rs1923298. The locations of the total EAAT5 are neuronal [25]. Since EAAT2 accounts for 11 SNPs are shown in Figure 1. approximately 90% of glutamate reuptake in the rodent forebrain [16,17], we focused on the EAAT2 gene Genotyping (SLC1A2) in association studies of schizophrenia. SLC1A2 Eleven SNPs were amplified as 11 individual fragments by has been mapped to 11p13-p12 [18] and consists of 11 PCR using the primers shown in Table 1 - additional file exons spanning over 165 kb. In this study we tested asso- 1. The reaction mixture for PCR was prepared in a total ciations of schizophrenia with 11 SNPs distributed in volume 10 µl with 5 ng of genomic DNA, 10 pmol of each SLC1A2 with an average interval of 15.9 kb. To enhance primer (4 pmol of SNP3), 2.5 mM of MgCl , 0.2 mM of each dNTP and 0.25 U of Taq DNA polymerase. An initial the detection power of the study, we also examined the haplotype associations of the SNPs with the disease. denaturing step of 1 min at 94°C was followed by 30, 35 or 40 cycles of 94°C for 30 sec, appropriate annealing Methods temperature for 30 sec and 72°C for 30 sec. A final exten- Human subjects sion step was carried out at 72°C for 7 min. The nucle- Blood samples were obtained from unrelated Japanese otide sequences of each primer, PCR conditions and individuals who had provided written informed consent. genotyping methods for each SNP are shown in Table 1 - We used two Japanese sample sets in this study. In the first additional file 1. We genotyped SNP3 by pyrosequencing one, Kyushu samples, 100 schizophrenia patients (mean analysis on a PSQ™96MA Pyrosequencer according to the age 49.5; 44.0% female) were recruited from hospital in manufacturer's specifications with a biotinylated reverse the Fukuoka and Oita areas and 100 healthy unrelated primer (5'-CGCCTACTCCTGGTGACTTC-3'), and the controls (mean age 51.2; 44.0% female) were recruited sequencing primer (5'-CGCCCCCATGTGT-3'). The other from the Fukuoka area. In the initial SNP selection proc- 10 SNPs were genotyped by direct sequencing, as previ- ess, we used another 16 Japanese samples which are ously described [7]. The raw data of direct sequencing recruited in the Fukuoka area and informed in the same were compiled on PolyPhred [22]. way. In the second one, Aichi samples, 100 schizophrenia patients (mean age 34.4; 44% female) and 100 healthy Page 2 of 6 (page number not for citation purposes) BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 SNP7 SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP8 SNP9 SNP10 SNP11 16.9 kb 11.7 kb 22.6 kb 7.0 kb 7.8 kb 35.6 kb 13.2 kb 19.3 kb 9.0 kb 17.0 kb 1 2 3 4 5 6 7 8 9 10 11 10 kb Genomic or Figure 1 ganization of SLC1A2 and locations of the SNPs Genomic organization of SLC1A2 and locations of the SNPs. Exons are shown as vertical bars with exon numbers. Eleven SNPs are indicated by circles. Distances between the SNPs are indicated above with kb. Statistical analyses size of 40–50 individuals to a precision equal to 10–20% To control genotyping errors, Hardy-Weinberg equilib- of the asymptotic limit [19]. We observed relatively strong rium (HWE) was checked in the control samples by the LD (D' > 0.8) in the seven combinations: SNP4-SNP5 (D' χ -test (d.f. = 1). We evaluated the statistical differences in = 0.800), SNP7-SNP8 (D' = 0.877), SNP8-SNP9 (D' = genotype and allele frequencies between cases and con- 0.925), SNP4-SNP11 (D' = 0.838), SNP5-SNP11(D' = trols by the χ -test (d.f. = 2) and the Fisher's exact proba- 0.999), SNP7-SNP11 (D' = 0.816), SNP9-SNP11 (D' = bility test (d.f. = 1), respectively. The magnitude of LD was 0.819). Modest LD (D' > 0.4) was observed in the combi- evaluated in D' and r using the haplotype frequencies nations of adjacent SNPs except for SNP5-SNP6 (D' = estimated by the EH program, version 1.14 [23]. Statistical 0.286) in the control samples. However, modest LD was analysis of the haplotype association was carried out as detected in cases in the SNP5-SNP6 combination (D' = previously described [24]. The significance level for all sta- 0.497). tistical tests was 0.05. We constructed pairwise haplotypes for all of the 55 pos- sible SNP pairs (Table 3 - additional file 3. , lower diago- Results Genotyping and SNP association analysis nal). We observed significant associations with We selected 11 SNPs at average interval of 15.9 kb to cover schizophrenia in eight combinations: SNP2-SNP3 (P = the entire SLC1A2 region with LD as described in Materi- 0.0021), SNP2-SNP4 (P = 0.0274), SNP2-SNP5 (P = als and Methods. Table 2 - additional file 2. shows the 0.0054), SNP2-SNP6 (P = 0.0178), SNP2-SNP7 (P = 9.4 × -5 results of genotype and allele frequencies of SNPs in case 10 ), SNP2-SNP9 (P = 0.0354), SNP2-SNP10 (P = and control samples. No significant deviation from HWE 0.0089) and SNP2-SNP11 (P = 0.0216). The combination in control samples was observed (data not shown). SNP2 of SNP2-SNP7 was the only one remained significant after showed significant differences in genotype (P = 0.013) Bonferroni correction (P = 0.0052). corr and allele (P = 0.008) frequencies between cases and con- trols. After Bonferroni corrections, these two P values Cumulative analysis using the second sample set became non-significance levels (P = 0.143, P = In this study, we detected significant associations of one corr corr 0.088, respectively). haplotype in the SLC1A2 region with schizophrenia in the Kyushu samples. To confirm the positive finding, we Pairwise linkage disequilibrium and haplotype association investigated the second Japanese sample set recruited analyses from the Aichi area. Although significant association of We compared the magnitude of LD for all possible pairs the disease was observed with neither genotype, allele fre- of the 11 SNPs in controls by calculating D' and r (Table quencies of SNP2 (P = 0.195, P = 0.178, respectively), nor 3 - additional file 3. , upper diagonal), because LD around haplotypes of SNP2-SNP7 (P = 0.084) in the second sam- common alleles can be measured with a modest sample ple set, the significant haplotype association of SNP2- Page 3 of 6 (page number not for citation purposes) BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 SNP7 was replicated in the cumulative analysis including -4 the two sample sets (P = 5.0 × 10 ) (Table 4 - additional file 4. ). Discussion SLC1A2 is located on the chromosomal region of 11p13- p12, to which no evidence has been reported for linkage of schizophrenia, [25,26]. However, there is still a possi- bility that SLC1A2 is a candidate for schizophrenia sus- ceptibility genes, because linkage studies could only detect genes with the large genotype relative risk [27]. We carried out the genotyping of 100 cases and 100 controls for 11 SNPs, which were selected to cover the entire Physical distance (kb) SLC1A2 region with LD. Since minor allele frequencies of each SNP we tested ranges from 0.220 to 0.485, the expected detection power of our case-control study is from 0.89 to 0.94 for the susceptibility gene assuming 2 A plot of pa tance between th Figure 2 irwise linkag e SNPs in e dise the quilibrium (LD) SLC1A2 regionvs. physical dis- for genotype relative risk [28]. A plot of pairwise linkage disequilibrium (LD) vs. physical distance between the SNPs in the SLC1A2 region. D' were plotted with filled diamonds, and r with Modest LD (D' = 0.925 ~ 0.409) was observed in the com- open diamonds. From the regression line, the half-length of binations of neighboring SNPs except for SNP5-SNP6 (D' LD was estimated to be 31.8 kb in the SLC1A2 region. = 0.286) in the control samples, suggesting that there may be a recombination hot spot present in the small region (7.8 kb) between the two SNPs (Table 3 - additional file 3. ). We plotted the magnitude of LD with the physical dis- tance for each pair of the SNPs, and estimated the average half-length of LD to be 31.8 kb by assuming a linear analyses of the two sample sets, however, provide the rep- regression (Fig. 2). This is approximately half of the lication of the significant haplotype association of SNP2- -4 previously estimated size 60 kb in a United States popula- SNP7 with schizophrenia (P = 5.0 × 10 ). The frequency tion of north-European descent [21]. of the G-C haplotype in schizophrenics (26.6%) was nota- bly higher than in controls (5.6%), suggesting that the G- Significant associations of schizophrenia with genotype C haplotype may be a risk haplotype for schizophrenia. (P = 0.013) and allele (P = 0.008) frequencies of SNP2 We observed that the G-C haplotype frequency of schizo- (rs4755404) were detected (Table 2 - additional file 2. ). phrenics (20.0%) was only slightly higher than controls However, none of these "single-marker" associations sur- (14.2%) in the Aichi sample, suggesting a less contribu- vived after Bonferroni corrections. An A-G transition in tion of this locus on schizophrenia pathogenesis in the codon 206, causing a substitution of serine for asparagine, Aichi sample, although no apparent difference in clinical was identified in the exon 5 of SLC1A2 in a heterozygous subtypes between both sample sets studied in this paper. sporadic ALS patient [29]. Since located in a putative gly- The positive association reported here needs to be vali- cosylation site, the nonsynonymous SNP is potentially dated in larger sample sets, and it would also be worth- involved in the pathophysiology of schizophrenia while to search for functional SNPs in the region spanning through affecting the glycosylation status and the trans- SNP2-SNP7. port activity of SLC1A2 [30]. No occurrence of the G allele of the SNP in 124 Italian schizophrenic and 50 control Conclusion subjects has been reported [30]. We found also only A We concluded that at least one susceptibility locus for allele of the SNP in the 100 controls and 100 cases of the schizophrenia is probably located within or nearby Kyushu samples (data not shown). SLC1A2 in the Japanese population. In pairwise haplotype association analyses, SNP2 consist- Competing interests ently showed significant haplotype associations. The P None declared. value of the combination SNP2-SNP7 was still significant -5 even after Bonferroni correction (P = 9.4 × 10 , P = List of abbreviations used corr 0.0052). In our second sample set, the Aichi sample, no SNP; single nucleotide polymorphism significant association of SNP2 was observed in any of the analyses of genotypes, alleles and haplotypes. Cumulative Page 4 of 6 (page number not for citation purposes) LD BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 DSM-IV; dianostic and statistical manual of mental disor- References th 1. McGuffin P, Owen MJ, Farmer AE: Genetic basis of ders, 4 edn schizophrenia. Lancet 1995, 346:678-682. 2. Luby ED, Cohen BD, Rosenbaum G, Gottlieb JS, Kelley R: Study of PCR; polymerase chain reaction a new schizophrenomimetic drug, sernyl. Arch Neurol Psychiatr 1959, 81:363-369. 3. Javitt DC, Zukin SR: Recent advances in the phencyclidine HWE; Hardy-Weinberg equilibrium model of schizophrenia. Am J Psychiat 1991, 148:1301-1308. 4. Mohn AR, Gainetdinov RR, Caron MG, Koller BH: Mice with reduced NMDA receptor expression display behaviors LD; linkage disequilibrium related to schizophrenia. Cell 1999, 98:427-436. 5. Begni S, Popoli M, Moraschi S, Bignotti S, Tura GB, Gennarelli M: Association between the ionotropic glutamate receptor kai- EAAT; excitatory amino acid transporter nate 3 (GRIK3) ser310ala polymorphism and schizophrenia. Mol Psychiatr 2002, 7:416-418. Authors' contributions 6. Begni S, Moraschi S, Bignotti S, Fumagalli F, Rillosi L, Perez J, Gen- narelli M: Association between the G1001C polymorphism in XD carried out genotyping, statistical analyses and drafted the GRIN1 gene promoter region and schizophrenia. Biol the manuscript: HS participated in design of this study Psychiatr 2003, 53:617-619. 7. Makino C, Fujii Y, Kikuta R, Hirata N, Tani A, Shibata A, Ninomiya H, and statistical analyses: HN, NT, NI and NO participated Tashiro N, Shibata H, Fukumaki Y: Positive association of the in collecting specimens and clinical data: YF conceived of AMPA receptor subunit GluR4 gene (GRIA4) haplotype with the study and participated in its design and coordination. schizophrenia. Am J Med Genet 2003, 116B:17-22. 8. Fujii Y, Shibata H, Kikuta R, Makino C, Tani A, Hirata N, Shibata A, Ninomiya H, Tashiro N, Fukumaki Y: Positive associations of pol- Additional material ymorphisms in the metabotropic glutamate receptor type 3 gene (GRM3) with schizophrenia. Psychiatr Genet 2003, 13:71-76. 9. Takaki H, Kikuta R, Shibata H, Ninomiya H, Tashiro N, Fukumaki Y: Positive associations of polymorphisms in the metabotropic Additional file 1 glutamate receptor type 8 gene (GRM8) with schizophrenia. Table 1 Am J Med Genet 2004, 128:6-14. PCR primers for genotyping of SNPs in SLC1A2. 10. Owen MJ, Williams NM, O'Donovan MC: The molecular genetics Click here for file of schizophrenia: new findings promise new insights. Mol [http://www.biomedcentral.com/content/supplementary/1471- Psychiatr 2004, 9:14-27. 11. Kuwahara O, Mitsumoto Y, Chiba K, Mohri T: Characterization of 244X-4-21-S1.xls] D-aspartic acid uptake by rat hippocampal slices and effect of ischemic conditions. J Neurochem 1992, 59:616-621. Additional file 2 12. Rothstein JD, Kykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, Welty DF: Knock- Table 2 out of glutamate transporters reveals a major role for astro- Genotype and allele frequencies of SNPs in SLC1A2 in Kyushu samples. glial transport in excitoxicity and clearance of a glutamate. Click here for file Neuron 1996, 16:675-686. [http://www.biomedcentral.com/content/supplementary/1471- 13. Smith RE, Haroutunian V, Davis KL, Meador-Woodruff JH: Expres- 244X-4-21-S2.xls] sion of excitatory amino acid transporter transcripts in the thalamus of subjects with schizophrenia. Am J Psychiat 2001, 158:1393-1399. Additional file 3 14. Ohnuma T, Tessler S, Arai H, Faull RL, McKenna PJ, Emson PC: Gene Table 3 expression of metabotropic glutamate receptor 5 and exci- Pairwise linkage disequilibrium and haplotype association in SLC1A2. tatory amino acid transporter 2 in the schizophrenic Click here for file hippocampus. Mol Brain Res 2000, 85:24-31. 15. Gegelashvili G, Schousboe A: Cellular distribution and kinetic [http://www.biomedcentral.com/content/supplementary/1471- properties of high-affinity glutamate transporters. Brain Res 244X-4-21-S3.xls] Bull 1998, 45:233-238. 16. Rothstein JD, Van Kammen M, Levey AI, Martin LJ, Kuncl RW: Selec- Additional file 4 tive loss of glial glutamate transporter GLT-1 in amyo- trophic lateral sclerosis. Ann Neurol 1995, 38:73-84. Table 4 17. Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Tkahashi K, Association analysis of the SNP2-SNP7 haplotype using two sample sets. Iwama H, Nishikawa T, Ichihara N, Kikuchi T, Okuyama S, Kawashima Click here for file N, Hori S, Takimoto M, Wada K: Epilepsy and exacerbation of [http://www.biomedcentral.com/content/supplementary/1471- brain injury in mice lacking the glutamate transporter GLT- 244X-4-21-S4.xls] 1. Science 1997, 276:1699-1702. 18. Li X, Francke U: Assignment of the gene SLC1A2 coding for the human glutamate transporter EAAT2 to human chromo- some 11 bands p13-p12. Cytogenet Cell Genet 1995, 71:212-213. 19. American Psychiatric Association: DSM-IV: Diagnostic and Statistical Manual of Mental Disorders American Psychiatric Press, Washington; Acknowledgements We are grateful to all the medical staff involved in collecting specimens. This 20. Lahiri DK, Nurnberger JI Jr: A rapid non-enzymatic method for work was supported in part by a Grant-in Aid for Scientific Research on Pri- the preparation of HMW DNA from blood for RFLP studies. ority Areas "Medical Genome Science" and other grants from the Ministry Nucleic Acids Res 1991, 19:5444. 21. Reich DE, Cargill M, Bolk S, Ireland J, Sabeti PC, Richter DJ, Lavery T, of Education, Culture, Sports, Science and Technology and the Ministry of Kouyoumjian R, Farhadian SF, Ward R, Lander ES: Linkage disqui- Health, Labor and Welfare of Japan. librium in the human genome. Nature 2001, 411:199-204. 22. Nickerson DA, Tobe VO, Taylor SL: Polyphred: substitutions using fluorescence-based resequencing. Nucleic Acids Res 1997, 25:2745-2751. Page 5 of 6 (page number not for citation purposes) BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 23. Xie X, Ott J: Testing linkage disequilibrium between a disease gene and marker loci. Am J Hum Genet 1993, 53:1107. 24. Sham P: Statistics in Human Genetics Oxford University Press, New York; 1998. 25. Berry N, Jobanputra V, Pal H: Molecular genetics of schizophre- nia: a critical review. J Psychiatr Neurosci 2003, 28:415-429. 26. Japanese Schizophrenia Sib-Pair Linkage Group: Initial genome- wide scan for linkage with schizophrenia in the Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) families. Am J Med Genet 2003, 120B:22-28. 27. Risch NJ: Searching for genetic determinants in the new millennium. Nature 2000, 405:847-856. 28. Ohashi J, Yamamoto S, Tsuchiya N, Hatta Y, Komata T, Matsushita M, Tokunaga K: Comparison of statistical power between 2 × 2 allele frequency and positivity tables in case-control studies of complex disease genes. Ann Hum Genet 2001, 65:197-206. 29. Aoki M, Lin CL, Rothstein JD, Geller BA, Hosler BA, Munsat TL, Hor- vitz HR, Brown RH: Mutations in the glutamate transporter EAAT2 gene do not cause abnormal EAAT2 transcripts in amyotrophic lateral sclerosis. Ann Neurol 1998, 43:645-653. 30. Catalano M, Lorenzi C, Bocchio L, Racagni G: No occurrence of the glutamate transporter EAAT2 A206G polymorphism in schizophrenic subjects. Mol Psychiatr 2002, 7:671-672. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-244X/4/21/pre pub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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Association study of polymorphisms in the excitatory amino acid transporter 2 gene (SLC1A2) with schizophrenia

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Springer Journals
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Copyright © 2004 by Deng et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Psychiatry; Psychotherapy
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1471-244X
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15296513
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Abstract

Background: The glutamatergic dysfunction hypothesis of schizophrenia suggests that genes involved in glutametergic transmission are candidates for schizophrenic susceptibility genes. We have been performing systematic association studies of schizophrenia with the glutamate receptor and transporter genes. In this study we report an association study of the excitatory amino acid transporter 2 gene, SLC1A2 with schizophrenia. Methods: We genotyped 100 Japanese schizophrenics and 100 controls recruited from the Kyushu area for 11 single nucleotide polymorphism (SNP) markers distributed in the SLC1A2 region using the direct sequencing and pyrosequencing methods, and examined allele, genotype and haplotype association with schizophrenia.The positive finding observed in the Kyushu samples was re-examined using 100 Japanese schizophrenics and 100 controls recruited from the Aichi area. Results: We found significant differences in genotype and allele frequencies of SNP2 between cases and controls (P = 0.013 and 0.008, respectively). After Bonferroni corrections, the two significant differences disappeared. We tested haplotype associations for all possible combinations -5 of SNP pairs. SNP2 showed significant haplotype associations with the disease (P = 9.4 × 10 , P = 0.0052 with Bonferroni correction, at the lowest) in 8 combinations. Moreover, the significant haplotype association of SNP2-SNP7 was replicated in the cumulative analysis of our two sample sets. Conclusion: We concluded that at least one susceptibility locus for schizophrenia is probably located within or nearby SLC1A2 in the Japanese population. ious cognitive impairments. The life-time prevalence is Background Schizophrenia is a severe mental disorder characterized by about 1%, and genetic factors were known to play a criti- hallucinations, delusions, disorganized thoughts, and var- cal role in its pathogenesis [1]. Based on the fact that Page 1 of 6 (page number not for citation purposes) BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 phencyclidine (PCP) induces schizophreniform psycho- unrelated controls (mean age 39.9; 45% female) were col- sis, a glutamatergic dysfunction hypothesis has been pro- lected in the Aichi area about 600 km east of Fukuoka. All posed for the pathogenesis of schizophrenia [2-4]. This patients fulfilled the DSM-IV criteria for schizophrenia hypothesis has been supported by recent multiple reports [19]. All of the case and control samples are ethnically Jap- of association of schizophrenia with glutamate receptor anese. DNA samples were purified from whole peripheral genes and with the genes related to glutamatergic trans- blood by the method previously described [20]. This mission, such as G72 and NRG1 [5-10]. study was approved by the Ethics Committee of Kyushu University, Faculty of Medicine and the Fujita Health Uni- In addition, other synaptic elements related to glutamate, versity Ethics Committee. such as excitatory amino acid transporters (EAATs), also SNP selection in the SLC1A2 region potentially affect glutamatergic neurotransmission. EAATs maintain extracellular glutamate concentrations within We retrieved the primary SNP information from the physiological levels by reuptaking the synaptically dbSNP database http://www.ncbi.nlm.nih.gov/SNP/. released glutamate. A deficient uptake has been impli- Assuming the same size of the half length of linkage dise- cated in the pathogenesis of ischemic brain damage [11] quilibrium (LD) (60 kb) as reported in Caucasians [21], and may be involved in neurodegenerative diseases such we initially intended to select common SNPs every 50 kb as amyotrophic lateral sclerosis (ALS) [12]. Recently sig- in SLC1A2. We tested 22 candidate SNPs including all of nificant increases of mRNA expression of EAAT1 and the exonic SNPs, in the 16 healthy Japanese samples by EAAT2 have been reported in the thalamus of schizo- the direct sequencing method. Out of the 22 SNPs we phrenics, suggesting the possibility that an excessive gluta- selected the following 7 common SNPs with minor allele mate uptake is involved in schizophrenia [13]. On the frequencies over 10% for further analyses: SNP1, other hand, a significant decrease of EAAT2 mRNA expres- rs1923295; SNP3, rs4534557; SNP6, rs1885343; SNP8, sion was observed in the parahippocampal gyrus of schiz- rs752949; SNP9, rs1042113; SNP10, rs3838796; SNP11, ophrenics [14]. Therefore the EAAT genes are reasonable rs1570216. We also identified a novel SNP, SNP7, in candidates for schizophrenia, as well as glutamate recep- intron 1 (conting location: 34105026). After the LD anal- tor genes. yses described below, we noticed LD gaps (D' < 0.3) of the initial SNP set and examined additional 20 candidate The EAATs family consists of five members (EAAT1- SNPs. Out of the 20 SNPs, we selected the following 3 EAAT5). Their cellular localizations are different: EAAT1 SNPs to fill the LD gaps: SNP2, rs4755404; SNP4, and EAAT2 are astroglial, whereas EAAT3 EAAT4 and rs4756224; SNP5, rs1923298. The locations of the total EAAT5 are neuronal [25]. Since EAAT2 accounts for 11 SNPs are shown in Figure 1. approximately 90% of glutamate reuptake in the rodent forebrain [16,17], we focused on the EAAT2 gene Genotyping (SLC1A2) in association studies of schizophrenia. SLC1A2 Eleven SNPs were amplified as 11 individual fragments by has been mapped to 11p13-p12 [18] and consists of 11 PCR using the primers shown in Table 1 - additional file exons spanning over 165 kb. In this study we tested asso- 1. The reaction mixture for PCR was prepared in a total ciations of schizophrenia with 11 SNPs distributed in volume 10 µl with 5 ng of genomic DNA, 10 pmol of each SLC1A2 with an average interval of 15.9 kb. To enhance primer (4 pmol of SNP3), 2.5 mM of MgCl , 0.2 mM of each dNTP and 0.25 U of Taq DNA polymerase. An initial the detection power of the study, we also examined the haplotype associations of the SNPs with the disease. denaturing step of 1 min at 94°C was followed by 30, 35 or 40 cycles of 94°C for 30 sec, appropriate annealing Methods temperature for 30 sec and 72°C for 30 sec. A final exten- Human subjects sion step was carried out at 72°C for 7 min. The nucle- Blood samples were obtained from unrelated Japanese otide sequences of each primer, PCR conditions and individuals who had provided written informed consent. genotyping methods for each SNP are shown in Table 1 - We used two Japanese sample sets in this study. In the first additional file 1. We genotyped SNP3 by pyrosequencing one, Kyushu samples, 100 schizophrenia patients (mean analysis on a PSQ™96MA Pyrosequencer according to the age 49.5; 44.0% female) were recruited from hospital in manufacturer's specifications with a biotinylated reverse the Fukuoka and Oita areas and 100 healthy unrelated primer (5'-CGCCTACTCCTGGTGACTTC-3'), and the controls (mean age 51.2; 44.0% female) were recruited sequencing primer (5'-CGCCCCCATGTGT-3'). The other from the Fukuoka area. In the initial SNP selection proc- 10 SNPs were genotyped by direct sequencing, as previ- ess, we used another 16 Japanese samples which are ously described [7]. The raw data of direct sequencing recruited in the Fukuoka area and informed in the same were compiled on PolyPhred [22]. way. In the second one, Aichi samples, 100 schizophrenia patients (mean age 34.4; 44% female) and 100 healthy Page 2 of 6 (page number not for citation purposes) BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 SNP7 SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP8 SNP9 SNP10 SNP11 16.9 kb 11.7 kb 22.6 kb 7.0 kb 7.8 kb 35.6 kb 13.2 kb 19.3 kb 9.0 kb 17.0 kb 1 2 3 4 5 6 7 8 9 10 11 10 kb Genomic or Figure 1 ganization of SLC1A2 and locations of the SNPs Genomic organization of SLC1A2 and locations of the SNPs. Exons are shown as vertical bars with exon numbers. Eleven SNPs are indicated by circles. Distances between the SNPs are indicated above with kb. Statistical analyses size of 40–50 individuals to a precision equal to 10–20% To control genotyping errors, Hardy-Weinberg equilib- of the asymptotic limit [19]. We observed relatively strong rium (HWE) was checked in the control samples by the LD (D' > 0.8) in the seven combinations: SNP4-SNP5 (D' χ -test (d.f. = 1). We evaluated the statistical differences in = 0.800), SNP7-SNP8 (D' = 0.877), SNP8-SNP9 (D' = genotype and allele frequencies between cases and con- 0.925), SNP4-SNP11 (D' = 0.838), SNP5-SNP11(D' = trols by the χ -test (d.f. = 2) and the Fisher's exact proba- 0.999), SNP7-SNP11 (D' = 0.816), SNP9-SNP11 (D' = bility test (d.f. = 1), respectively. The magnitude of LD was 0.819). Modest LD (D' > 0.4) was observed in the combi- evaluated in D' and r using the haplotype frequencies nations of adjacent SNPs except for SNP5-SNP6 (D' = estimated by the EH program, version 1.14 [23]. Statistical 0.286) in the control samples. However, modest LD was analysis of the haplotype association was carried out as detected in cases in the SNP5-SNP6 combination (D' = previously described [24]. The significance level for all sta- 0.497). tistical tests was 0.05. We constructed pairwise haplotypes for all of the 55 pos- sible SNP pairs (Table 3 - additional file 3. , lower diago- Results Genotyping and SNP association analysis nal). We observed significant associations with We selected 11 SNPs at average interval of 15.9 kb to cover schizophrenia in eight combinations: SNP2-SNP3 (P = the entire SLC1A2 region with LD as described in Materi- 0.0021), SNP2-SNP4 (P = 0.0274), SNP2-SNP5 (P = als and Methods. Table 2 - additional file 2. shows the 0.0054), SNP2-SNP6 (P = 0.0178), SNP2-SNP7 (P = 9.4 × -5 results of genotype and allele frequencies of SNPs in case 10 ), SNP2-SNP9 (P = 0.0354), SNP2-SNP10 (P = and control samples. No significant deviation from HWE 0.0089) and SNP2-SNP11 (P = 0.0216). The combination in control samples was observed (data not shown). SNP2 of SNP2-SNP7 was the only one remained significant after showed significant differences in genotype (P = 0.013) Bonferroni correction (P = 0.0052). corr and allele (P = 0.008) frequencies between cases and con- trols. After Bonferroni corrections, these two P values Cumulative analysis using the second sample set became non-significance levels (P = 0.143, P = In this study, we detected significant associations of one corr corr 0.088, respectively). haplotype in the SLC1A2 region with schizophrenia in the Kyushu samples. To confirm the positive finding, we Pairwise linkage disequilibrium and haplotype association investigated the second Japanese sample set recruited analyses from the Aichi area. Although significant association of We compared the magnitude of LD for all possible pairs the disease was observed with neither genotype, allele fre- of the 11 SNPs in controls by calculating D' and r (Table quencies of SNP2 (P = 0.195, P = 0.178, respectively), nor 3 - additional file 3. , upper diagonal), because LD around haplotypes of SNP2-SNP7 (P = 0.084) in the second sam- common alleles can be measured with a modest sample ple set, the significant haplotype association of SNP2- Page 3 of 6 (page number not for citation purposes) BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 SNP7 was replicated in the cumulative analysis including -4 the two sample sets (P = 5.0 × 10 ) (Table 4 - additional file 4. ). Discussion SLC1A2 is located on the chromosomal region of 11p13- p12, to which no evidence has been reported for linkage of schizophrenia, [25,26]. However, there is still a possi- bility that SLC1A2 is a candidate for schizophrenia sus- ceptibility genes, because linkage studies could only detect genes with the large genotype relative risk [27]. We carried out the genotyping of 100 cases and 100 controls for 11 SNPs, which were selected to cover the entire Physical distance (kb) SLC1A2 region with LD. Since minor allele frequencies of each SNP we tested ranges from 0.220 to 0.485, the expected detection power of our case-control study is from 0.89 to 0.94 for the susceptibility gene assuming 2 A plot of pa tance between th Figure 2 irwise linkag e SNPs in e dise the quilibrium (LD) SLC1A2 regionvs. physical dis- for genotype relative risk [28]. A plot of pairwise linkage disequilibrium (LD) vs. physical distance between the SNPs in the SLC1A2 region. D' were plotted with filled diamonds, and r with Modest LD (D' = 0.925 ~ 0.409) was observed in the com- open diamonds. From the regression line, the half-length of binations of neighboring SNPs except for SNP5-SNP6 (D' LD was estimated to be 31.8 kb in the SLC1A2 region. = 0.286) in the control samples, suggesting that there may be a recombination hot spot present in the small region (7.8 kb) between the two SNPs (Table 3 - additional file 3. ). We plotted the magnitude of LD with the physical dis- tance for each pair of the SNPs, and estimated the average half-length of LD to be 31.8 kb by assuming a linear analyses of the two sample sets, however, provide the rep- regression (Fig. 2). This is approximately half of the lication of the significant haplotype association of SNP2- -4 previously estimated size 60 kb in a United States popula- SNP7 with schizophrenia (P = 5.0 × 10 ). The frequency tion of north-European descent [21]. of the G-C haplotype in schizophrenics (26.6%) was nota- bly higher than in controls (5.6%), suggesting that the G- Significant associations of schizophrenia with genotype C haplotype may be a risk haplotype for schizophrenia. (P = 0.013) and allele (P = 0.008) frequencies of SNP2 We observed that the G-C haplotype frequency of schizo- (rs4755404) were detected (Table 2 - additional file 2. ). phrenics (20.0%) was only slightly higher than controls However, none of these "single-marker" associations sur- (14.2%) in the Aichi sample, suggesting a less contribu- vived after Bonferroni corrections. An A-G transition in tion of this locus on schizophrenia pathogenesis in the codon 206, causing a substitution of serine for asparagine, Aichi sample, although no apparent difference in clinical was identified in the exon 5 of SLC1A2 in a heterozygous subtypes between both sample sets studied in this paper. sporadic ALS patient [29]. Since located in a putative gly- The positive association reported here needs to be vali- cosylation site, the nonsynonymous SNP is potentially dated in larger sample sets, and it would also be worth- involved in the pathophysiology of schizophrenia while to search for functional SNPs in the region spanning through affecting the glycosylation status and the trans- SNP2-SNP7. port activity of SLC1A2 [30]. No occurrence of the G allele of the SNP in 124 Italian schizophrenic and 50 control Conclusion subjects has been reported [30]. We found also only A We concluded that at least one susceptibility locus for allele of the SNP in the 100 controls and 100 cases of the schizophrenia is probably located within or nearby Kyushu samples (data not shown). SLC1A2 in the Japanese population. In pairwise haplotype association analyses, SNP2 consist- Competing interests ently showed significant haplotype associations. The P None declared. value of the combination SNP2-SNP7 was still significant -5 even after Bonferroni correction (P = 9.4 × 10 , P = List of abbreviations used corr 0.0052). In our second sample set, the Aichi sample, no SNP; single nucleotide polymorphism significant association of SNP2 was observed in any of the analyses of genotypes, alleles and haplotypes. Cumulative Page 4 of 6 (page number not for citation purposes) LD BMC Psychiatry 2004, 4:21 http://www.biomedcentral.com/1471-244X/4/21 DSM-IV; dianostic and statistical manual of mental disor- References th 1. McGuffin P, Owen MJ, Farmer AE: Genetic basis of ders, 4 edn schizophrenia. Lancet 1995, 346:678-682. 2. Luby ED, Cohen BD, Rosenbaum G, Gottlieb JS, Kelley R: Study of PCR; polymerase chain reaction a new schizophrenomimetic drug, sernyl. Arch Neurol Psychiatr 1959, 81:363-369. 3. Javitt DC, Zukin SR: Recent advances in the phencyclidine HWE; Hardy-Weinberg equilibrium model of schizophrenia. 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Ann Neurol 1998, 43:645-653. 30. Catalano M, Lorenzi C, Bocchio L, Racagni G: No occurrence of the glutamate transporter EAAT2 A206G polymorphism in schizophrenic subjects. Mol Psychiatr 2002, 7:671-672. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-244X/4/21/pre pub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 6 of 6 (page number not for citation purposes)

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