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Keywords:

  • transporters;
  • glutamate;
  • postmortem brain;
  • antipsychotics

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  8. Supporting Information

Glutamate is one of the key molecules involved in signal transduction in the brain, and dysfunction of glutamate signaling could be linked to schizophrenia. The SLC1A1 gene located at 9p24 encodes the glutamate transporter EAAT3/EAAC1. To investigate the association between the SLC1A1 gene and schizophrenia in the Japanese population, we genotyped 19 tagging single nucleotide polymorphisms (tagSNPs) in the SLC1A1 gene in 576 unrelated individuals with schizophrenia and 576 control subjects followed by replication in an independent case–control study of 1,344 individuals with schizophrenia and 1,344 control subjects. In addition, we determined the boundaries of the copy number variation (CNV) region in the first intron (Database of Genomic Variants, chr9:4516796-4520549) and directly genotyped the CNV because of significant deviation from the Hardy–Weinberg equilibrium. The CNV was not associated with schizophrenia. Four SNPs showed a possible association with schizophrenia in the screening subjects and the associations were replicated in the same direction (nominal allelic P < 0.05), and, among them, an association with rs7022369 was replicated even after Bonferroni correction (allelic nominal P = 5 × 10−5, allelic corrected P = 2.5 × 10−4, allelic odds ratio, 1.30; 95% CI: 1.14–1.47 in the combined subjects). Expression analysis quantified by the real-time quantitative polymerase chain reaction in the postmortem prefrontal cortex of 43 Japanese individuals with schizophrenia and 11 Japanese control subjects revealed increased SLC1A1 expression levels in individuals homozygous for the rs7022369 risk allele (P = 0.003). Our findings suggest the involvement of SLC1A1 in the pathogenesis of schizophrenia. © 2011 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  8. Supporting Information

Schizophrenia is one of the most mysterious and costliest mental disorders and it affects 0.30–0.66% of the population. Despite its high heritability estimates, the identification of specific molecular genetic variation has not been easy. Recent findings have suggested that a small proportion of schizophrenia incidence could be explained by rare structural variations [van Os and Kapur, 2009; Vacic et al., 2011].

Glutamate transporters (excitatory amino acid transporters, EAATs) play important roles in maintaining extracellular glutamate concentrations. To date, 5 subtypes of Na+-dependent glutamate transporters—EAAT1 (GLAST, SLC1A3), EAAT2 (GLT-1, SLC1A2), EAAT3 (SLC1A1), EAAT4 (SLC1A6), and EAAT5 (SLC1A7)—have been identified [Shigeri et al., 2004]. Removal of extracellular glutamate in the forebrain is controlled by three major EAATs, that is, EAAT1, EAAT2, and EAAT3 [Amara et al., 1998; Danbolt, 2001]. EAAT1 and EAAT2 are mainly glial and EAAT3 is mostly neuronal [Rothstein et al., 1994]. EAAT3 is encoded by the glutamate transporter, solute carrier family 1 gene (SLC1A1), which is located on chromosome 9p24. EAAT3 (termed EAAC1 in rodents) is predominantly expressed in the cerebral cortex, basal ganglia, and hippocampus.

On the basis of pharmacological evidence, dysfunctions of glutamate neurotransmission have been implicated in the pathophysiology of schizophrenia [Coyle, 2006; Tuominen et al., 2006]. EAAC1 may control activation of some subtypes of N-methyl-D-aspartate (NMDA) receptors and vice versa in the hippocampus [Waxman et al., 2007]. Environmental enrichment has been shown to decrease the mRNA expression of EAAC1 in the hippocampus [Andin et al., 2007] and EAAC1-deficient mice have shown reduced neuronal glutathione levels, and, with aging, they developed brain atrophy and behavioral changes including decreased spatial learning abilities and cognitive impairment [Aoyama et al., 2006]. It has also been suggested that EAAC1 deficiency leads to impaired neuronal glutathione metabolism and oxidative stress [Aoyama et al., 2006]. Thus, the glutamate hypothesis [Coyle, 2006], oxidative stress hypothesis [Sarandol et al., 2007], and parallel effects of environmental enrichment and antipsychotic treatment in schizophrenia [Andin et al., 2007] suggest the involvement of EAAT3 in schizophrenia.

Deng et al. [2007] genotyped eight even-spaced single nucleotide polymorphisms (SNPs) that were separated from each other by an average distance of 14 kb in the SLC1A1 gene in 100 Japanese patients with schizophrenia and 100 Japanese controls. Although a potential association between rs2228622 and schizophrenia was found, the association was not confirmed in an additional sample comprising 300 schizophrenics and 320 controls. Since the average summary odds ratio (OR) of nominally significant effects of 24 genetic variants in 16 different genes was shown to be ∼1.23 by systematic meta-analyses [Allen et al., 2008], large sample sizes are required to detect SNPs associated with schizophrenia. The present study aims to investigate associations between SNPs in the SLC1A1 gene and schizophrenia by a large case–control study of 1,920 Japanese schizophrenic patients and 1,920 Japanese control subjects.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  8. Supporting Information

Subjects

The screening groups were comprised 576 unrelated Japanese patients with schizophrenia and 576 mentally healthy unrelated Japanese control subjects. The replication groups were comprised 1,344 unrelated Japanese patients with schizophrenia and 1,344 mentally healthy unrelated Japanese control subjects. Patients with schizophrenia (1,055 men and 865 women; mean age ± standard deviation (SD), 48.2 ± 14.7 years) were diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV; American Psychiatric Association (APA), 2001) with consensus from at least 2 experienced psychiatrists, and the control subjects (1,051 men and 869 women; mean age ± SD, 47.6 ± 13.4 years) were those whose second-degree relatives were free of psychosis on the basis of self-reporting by the subjects. All the participants provided their written informed consent. The association analysis was approved by the Ethics Committees of the University of Tsukuba, Niigata University, Fujita Health University, Nagoya University, Okayama University, and Seiwa Hospital.

Human Postmortem Brains

Brain specimens were obtained from Japanese individuals of 43 schizophrenic patients and 11 age- and gender-matched controls. Tissue blocks were cut from gray matter in an area of the prefrontal cortex referred to as Brodmann's area 9 (BA9). The Japanese subjects met the DSM-III-R criteria for schizophrenia. The control subjects had no known history of psychiatric illness. The study was approved by the Ethics Committees of Niigata University, University of Tsukuba, Tokyo Metropolitan Matsuzawa Hospital, and the Tokyo Institute of Psychiatry.

SNP Selection and Genotyping

The selection of tagSNPs for genotyping in the SLC1A1 gene was conducted with the use of the International HapMap Project. A total of 19 tagSNPs were selected in this study (Fig. 1, Table I). The SNPs tagged by the selected 19 tagSNPs are shown in the Supplementary Table I.

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Figure 1. The results of SNP association with schizophrenia and the position of the CNV analyzed in the SLC1A1 gene. a: Results of the association study. Squares indicate the allelic P-value in the screening population. SNPs in bold letters were also analyzed in the confirmation population and squares of them are the allelic P-values in the combined populations. b: Schematic representation of SLC1A1. The 12 exons and 11 introns of the SLC1A1 gene and the approximate location of each polymorphism genotyped in the present study are shown here. The polymorphisms represented in bold showed a positive association in this study. The bold line indicates the copy number variation (CNV) region. c: Linkage disequilibrium and haplotype blocks in the SLC1A1 gene region. Each box represents the D′ value corresponding to each pair-wise single nucleotide polymorphism combination. D′ is color-coded; the red box indicates D′ = 1.0 between two loci. d: The sequence and position of breakpoints of the CNV. e: An example of genotypes of the CNV amplified by PCR with the primers A, B, and C shown in (d). The ladder marker on the left side lane is 2-Log DNA Ladder (New England BiolLabs, MA).

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Table I. Genotypic and Allelic Distributions of the SLC1A1 Gene Polymorphisms in the Screening Population
SNP No.dbSNP IDSubjectsnGenotype count (frequency)PgenotypicAllele count (frequency)PallelicHWE P
  • Pgenotypic, the Cochran–Armitage trend test; Palleric, Fisher's exact test.

  • a

    Permutation P-value = 0.02.

1rs2150192  AAAGGG AG  
 Sz569259 (0.46)238 (0.42)72 (0.13) 756 (0.66)382 (0.34) 0.138
 C576267 (0.46)253 (0.44)56 (0.10)0.28787 (0.68)365 (0.32)0.340.726
2rs1360329  TTTGGG GG  
 Sz568477 (0.84)85 (0.15)6 (0.01) 1039 (0.91)97 (0.09) 0.318
 C566472 (0.83)89 (0.16)5 (0.01)0.901033 (0.91)99 (0.09)0.860.724
3rs972519  GGGCCC GC  
 Sz574503 (0.88)66 (0.11)5 (0.01) 1072 (0.93)76 (0.07) 0.093
 C558488 (0.87)68 (0.12)2 (0.00)0.521044 (0.94)72 (0.06)0.870.821
4rs10814991  CCCTTT CT  
 Sz571119 (0.21)275 (0.48)177 (0.31) 513 (0.45)629 (0.55) 0.523
 C567123 (0.22)282 (0.50)162 (0.29)0.67528 (0.47)606 (0.53)0.430.989
5rs7032326  TTTCCC TC  
 Sz57295 (0.17)258 (0.45)219 (0.38) 448 (0.39)696 (0.61) 0.201
 C56586 (0.15)245 (0.43)234 (0.41)0.54417 (0.37)713 (0.63)0.270.102
6rs7860087  GGGCCC GC  
 Sz572458 (0.80)107 (0.19)7 (0.01) 1023 (0.89)121 (0.11) 0.790
 C572473 (0.83)92 (0.16)7 (0.01)0.501038 (0.91)106 (0.09)0.290.299
7rs10814995  AAACCC AC  
 Sz572310 (0.54)222 (0.39)40 (0.07) 842 (0.74)302 (0.26) 0.976
 C561278 (0.50)227 (0.40)56 (0.10)0.11783 (0.70)339 (0.30)0.040.338
8rs10491732  GGGAAA GA  
 Sz569417 (0.73)137 (0.24)15 (0.03) 971 (0.85)167 (0.15) 0.358
 C567402 (0.71)148 (0.26)17 (0.03)0.66952 (0.84)182 (0.16)0.360.455
9rs1980943  AAAGGG AG  
 Sz572183 (0.32)292 (0.51)97 (0.17) 658 (0.58)486 (0.42) 0.286
 C571153 (0.27)289 (0.51)129 (0.23)0.03595 (0.52)547 (0.48)0.010.737
10rs10814998  AAAGGG AG  
 Sz572265 (0.46)252 (0.44)55 (0.10) 782 (0.68)362 (0.32) 0.660
 C575260 (0.45)259 (0.45)56 (0.10)0.93779 (0.68)371 (0.32)0.750.463
11rs7022369  CCCGGG CG  
 Sz572432 (0.76)115 (0.20)25 (0.04) 979 (0.86)165 (0.14) 0.000009
 C566383 (0.68)156 (0.28)27 (0.05)0.01922 (0.81)210 (0.19)0.010.04
12rs2026828  AAAGGG AG  
 Sz570202 (0.35)273 (0.48)95 (0.17) 677 (0.59)463 (0.41) 0.865
 C569181 (0.32)268 (0.47)120 (0.21)0.13630 (0.55)508 (0.45)0.050.262
13rs4641119  AAACCC AC  
 Sz573431 (0.75)128 (0.22)14 (0.02) 990 (0.86)156 (0.14) 0.230
 C576384 (0.67)170 (0.30)22 (0.04)0.002938 (0.81)214 (0.19)0.001a0.559
14rs3780415  TTTCCC TC  
 Sz574429 (0.75)132 (0.23)13 (0.02) 990 (0.86)158 (0.14) 0.454
 C568419 (0.74)134 (0.24)15 (0.03)0.89972 (0.86)164 (0.14)0.640.283
15rs10974625  GGGAAA GA  
 Sz565180 (0.32)262 (0.46)123 (0.22) 622 (0.55)508 (0.45) 0.134
 C564183 (0.32)266 (0.47)115 (0.20)0.85632 (0.56)496 (0.44)0.640.309
16rs3780413  GGGCCC GC  
 Sz567289 (0.51)223 (0.39)55 (0.10) 801 (0.71)333 (0.29) 0.216
 C569299 (0.53)218 (0.38)52 (0.09)0.86816 (0.72)322 (0.28)0.570.183
17rs10974629  AAAGGG AG  
 Sz571314 (0.55)216 (0.38)41 (0.07) 844 (0.74)298 (0.26) 0.646
 C569308 (0.54)201 (0.35)60 (0.11)0.12817 (0.72)321 (0.28)0.260.002
18rs2072657  TTTGGG TG  
 Sz573282 (0.49)229 (0.40)62 (0.11) 793 (0.69)353 (0.31) 0.135
 C564303 (0.54)212 (0.38)49 (0.09)0.24818 (0.73)310 (0.27)0.080.176
19rs3087879  GGGCCC GC  
 Sz574432 (0.75)131 (0.23)11 (0.02) 995 (0.87)153 (0.13) 0.771
 C568422 (0.74)127 (0.22)19 (0.03)0.32971 (0.85)165 (0.15)0.410.018

The SNPs were genotyped by the TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA). Product information on the TaqMan SNP genotyping assays used in this study is listed in Supplementary Table II. The TaqMan reaction was performed in a final volume of 3 µl consisting of 2.5 ng genomic DNA and Universal Master Mix (Eurogentc, Seraing, Belgium). Genotyping was performed with the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Because the SNPs potentially associated with schizophrenia were in the haplotype blocks that include exon 2, resequencing of SLC1A1 exon 2 was performed by direct sequencing with the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). One-third (1,152) of the samples were genotyped twice for 5 SNPs using TaqMan genotyping (Applied Biosystems), and genotype concordance was 99.5% for rs10814995, 99.4% for rs1980943, 99.8% for rs7022369, 99.7% for rs10758629, 99.9% for rs4641119, respectively. The average missing genotype rate was 1.2% (0.2–1.6%).

Determination of the Boundaries of the CNV and Genotype

The boundaries of the copy number variation (CNV) region where rs7022369 is located were determined by directly sequencing the genomic DNA around rs7022369. This region was amplified by LA Taq (Takara, Kyoto, Japan) with the primers 5′-AAGATGGAATTGGGGAGGAT and 5′-CGGACGGCTTAAGTGTCAAC, and this produced a product of approximately 14 kb. The CNV was genotyped by the size of the PCR products with the primers 5′-TTAATGCCAGTGTTGCATGAG (common 5′-primer, the primer A in Fig. 1), 5′-GCCCTGGTGTGTGATATTCC (deletion 3′-primer, the primer C in Fig. 1) and 5′-CATTTGCAAAAGTCTCTTTACCTT (wild-type 3′-primer, the primer B in Fig. 1). The 283 and 219 bp PCR product indicated the deletion type and the normal wild-type, respectively.

Real-Time Quantitative PCR for SLC1A1 Expression in Brains

Total RNA was isolated from human brain tissue (BA9) with an SV Total RNA Isolation System (Promega, Madison, WI). SLC1A1 expression was quantified by real-time quantitative polymerase chain reaction (PCR) with a TaqMan Gene Expression Assay and an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) as per the manufacturer's instructions. Primers and probes were purchased from Applied Biosystems (Assay ID: Hs00179051_m1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and measurement of the threshold cycle (Ct) was performed in triplicate. Data were collected and analyzed with Sequence Detector Software (SDS) version 2.1 (Applied Biosystems) and the standard curve method. Relative gene expression was calculated as the ratio of SLC1A1 to the internal control (GAPDH), and the mean of the three replicate measures was assigned to each individual.

Statistical Analysis

Allelic and genotypic associations were evaluated by Fisher's exact test and the Cochran–Armitage trend test, respectively. The detection power with this sample size was greater than 0.95 assuming an allelic relative risk of 1.23 and risk allele frequencies from 0.2 to 0.8 according to the Genetic Power Calculator in the total subjects [Purcell et al., 2003]. Deviation from the Hardy–Weinberg equilibrium (HWE) was evaluated by the chi-squared test. Linkage disequilibrium and haplotype frequencies/associations were evaluated with the Haploview program (http://www.broad.mit.edu/mpg/haploview/). In this study, we evaluated 19 SNPs for allelic associations with schizophrenia in the screening population, and subsequently genotyped SNPs with P < 0.05 at the screening step to confirm the association in the replication population. Corrected P-values were calculated with the Bonferroni method for SNP association analysis and with the use of 100,000 permutation as implemented in the Haploview program for haplotype association analysis.

Differences in SLC1A1 expression as determined by real-time quantitative PCR were analyzed by the Wilcoxon test with JMP software version 8 (SAS Institute, Cary, NC), and P < 0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  8. Supporting Information

The genotype and allele distributions of the 19 tagSNPs in the screening population are shown in Table I. Four SNPs (rs10814995, rs1980943, rs7022369, and rs4641119) showed nominally significant allelic association with schizophrenia. Among them, the genotype distribution of rs7022369 deviated significantly from the HWE in both patient and control groups (Table I). Because SNP rs7022369 is located in the CNV region (Database of Genomic Variants, http://projects.tcag.ca/variation/ variation_33067, 10284, and 2785, http://projects.tcag.ca/variation/), we determined the boundary of the CNV region (Fig. 1) and developed a method to identify the CNV by PCR. The CNV was deleted between 4516798 and 4526818 (NCBI ref: NT 008413.18; Fig. 1d) with an allele frequency of 2%. The CNV was not significantly associated with schizophrenia (Table II). When individuals with the CNV were excluded, the genotype distribution of rs7022369 did not deviate from HWE in the control subjects (Table II). Therefore, we excluded individuals with the CNV in the following analysis for this SNP. Among four SNPs with nominally significant association in the screening subjects, rs7022369 was associated with schizophrenia in an independent case–control population even after Bonferroni correction (allelic nominal P-value = 0.001; allelic corrected P-value = 0.004 in the same direction as in the screening subjects; Table II). The genotype distribution of rs7022369 did not deviate significantly from HWE in the replication and total samples when individuals with the CNV were excluded (Table II). The data in the combined populations revealed significant allelic associations of rs7022369 (nominal allelic P = 5 × 10−5, allelic OR = 1.30, 95% CI: 1.14–1.47) and rs4641119 (nominal allelic P = 5 × 10−4, allelic OR = 1.24, 95% CI: 1.10–1.41; Table II). Haplotype analysis with rs7022369 and rs4641119 showed that the haplotype frequency of the C of rs7022369 and A of rs4641119 was significantly higher in the schizophrenia group (0.84) than the control group (0.80; permutation P = 1.0 × 10−3).

Table II. Genotypic and Allelic Distributions of the SLC1A1 Gene Polymorphisms in the Replication and Combined Populations
SNP no.dbSNP ID/populationSubjectsnGenotype count (frequency)PgenotypicAllele count (frequency)PallelicAllelic OR (95% CI)HWE P
  1. NV region: chromosome 4516798–4526818 (NCBI ref:NT 008413.18); Pgenotype, Cochran–Armitage trend test; Palleric, Fisher's exact test.

7rs10814995  AAAGGG AG   
ScreeningSz572310 (0.54)222 (0.39)40 (0.07) 842 (0.74)302 (0.26)  0.976
 C561278 (0.50)227 (0.40)56 (0.10)0.04783 (0.70)339 (0.30)0.04 0.338
ReplicationSz1,324738 (0.56)494 (0.37)92 (0.07) 1970 (0.74)678 (0.26)  0.453
 C1,323680 (0.51)540 (0.41)103 (0.08)0.031900 (0.72)746 (0.28)0.02 0.769
CombinedSz1,8961048 (0.55)716 (0.38)132 (0.07) 2812 (0.74)980 (0.26)  0.520
 C1,884958 (0.51)767 (0.41)159 (0.08)0.0042683 (0.71)1085 (0.29)0.0041.16 (1.05–1.28)0.754
9rs1980943  AAAGGG AG   
ScreeningSz572183 (0.32)292 (0.51)97 (0.17) 658 (0.58)486 (0.42)  0.29
 C571153 (0.27)289 (0.51)129 (0.23)0.03595 (0.52)547 (0.48)0.01 0.74
ReplicationSz1,337432 (0.32)638 (0.48)267 (0.20) 1502 (0.56)1172 (0.44)  0.26
 C1,304389 (0.30)639 (0.49)276 (0.21)0.371417 (0.54)1191 (0.46)0.09 0.65
CombinedSz1,909615 (0.32)930 (0.49)364 (0.19) 2160 (0.57)1658 (0.43)  0.71
 C1,875542 (0.29)928 (0.49)405 (0.22)0.042012 (0.54)1738 (0.46)0.011.13 (1.03–1.23)0.83
11rs7022369  CCCGGG CG   
ScreeningSz551416 (0.75)115 (0.21)20 (0.04) 947 (0.86)155 (0.14)  0.001
 C541364 (0.67)156 (0.29)21 (0.04)0.01884 (0.82)198 (0.18)0.01 0.41
ReplicationSz1,275937 (0.73)312 (0.24)26 (0.02) 2186 (0.86)364 (0.14)  0.996
 C1,271870 (0.68)359 (0.28)42 (0.03)0.0092099 (0.83)443 (0.17)0.001 0.508
CombinedSz1,8261353 (0.74)427 (0.23)46 (0.03) 3133 (0.86)519 (0.14)  0.08
 C1,8121234 (0.68)515 (0.28)63 (0.03)6.8 × 10−52983 (0.82)641 (0.18)5 × 10−51.30 (1.14–1.47)0.309
rs7022369  C delG deldel del      
Individuals with the CNVSz8979 (0.89)6 (0.07)4 (0.04)      
 C8776 (0.87)8 (0.09)3 (0.03)      
CNV  2 Copies1 Copy0 Copy Without CNVWith CNV   
(Combined population)Sz1,9151826 (0.95)85 (0.04)4 (0.00) 3737 (0.98)93 (0.02)  0.006
 C1,8991812 (0.95)84 (0.04)3 (0.00)0.933708 (0.98)90 (0.02)0.88 0.055
13rs4641119  AAACCC AC   
ScreeningSz573431 (0.75)128 (0.22)14 (0.02) 990 (0.86)156 (0.14)  0.23
 C576384 (0.67)170 (0.30)22 (0.04)0.001938 (0.81)214 (0.19)0.001 0.56
ReplicationSz1,342983 (0.73)325 (0.24)34 (0.03) 2291 (0.85)393 (0.15)  0.25
 C1,341927 (0.69)382 (0.28)32 (0.02)0.022236 (0.83)446 (0.17)0.02 0.32
CombinedSz1,9151414 (0.74)453 (0.24)48 (0.03) 3281 (0.86)549 (0.14)  0.11
 C1,9171311 (0.68)552 (0.29)54 (0.03)5.9 × 10−43174 (0.83)660 (0.17)5 × 10−41.24 (1.10–1.41)0.65

Because the SNPs associated with schizophrenia are in the haplotype blocks that include exon 2, we resequenced exon 2 in 32 randomly selected patients. However, we did not identify any nonsynonymous mutations. Therefore, we suspected that the SNPs associated with schizophrenia found in the present study were markers regulating SLC1A1 expression. We explored the association of rs7022369 and rs4641119 with SLC1A1 expression in the postmortem prefrontal cortex of 43 individuals with schizophrenia and 11 control subjects. SLC1A1 expression was higher in brains homozygous for the major C allele of rs7022369 or the major A allele of rs4641119 than brains with the other genotypes (P = 0.003 and P = 0.02, respectively, Wilcoxon test; Fig. 2). This association was particularly obvious in the patient group (P = 0.01 at rs7022369 and P = 0.12 at rs4641119, Wilcoxon test). However, we should take into account the fact that the number of control brain samples was small. The effects on gene expression of sample pH, postmortem interval, sex, or age at death were not significant (data not shown). SLC1A1 expression was not significantly different between the patient and control groups (P = 0.17, Wilcoxon test).

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Figure 2. Expression of SLC1A1 in postmortem brains classified according to the single nucleotide polymorphism rs10758629 and rs4641119 genotype. Expression of SLC1A1 was normalized to that of glyceraldehyde-3-phosphate dehydrogenase. a: The difference in expression between the TT genotype and AA genotype in rs10758629 is significant (Wilcoxon test, P = 0.003). AA genotype, n = 7; TA genotype, n = 30; TT genotype, n = 52. b: The difference in expression between the AA genotype and CC genotype in rs4641119 is significant (Wilcoxon test, P = 0.02). CC genotype, n = 7; AC genotype, n = 28; AA genotype, n = 52. The horizontal line indicates the mean.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  8. Supporting Information

The present study identified the association between SNPs near exon 2 of the SLC1A1 gene and schizophrenia. These findings need to be replicated in other populations before accepting them. Because the OR of rs7022369 for association with schizophrenia was only 1.30 (95% CI: 1.14–1.47), more than 1,500 patients and an equal number of controls need to be examined to exceed 80% power in replication studies.

In the present study, we did not provide evidence that the SNPs examined directly cause the association with schizophrenia and/or the association of SLC1A1 expression in the prefrontal cortex. A survey of 193 neuropathologically normal human brain samples (Myers et al., 2007) showed the location of a potential cis-acting region regulating SLC1A1 expression within the 15 kb between rs1980943 and rs10758629, as calculated with PLINK [Purcell et al., 2007], where rs7022369 is located. The calculated lowest allelic P-value of 0.006 was at rs10814997, which is in complete linkage disequilibrium with rs1980943 (according to the HapMap data, r2 = 1 in the Japanese population). An association between rs1980943 and schizophrenia was suggested in the present study (nominal allelic P = 0.01, Table II). Thus, the cis-acting region regulating SLC1A1 is likely to be located in the first intronic region, although its exact position requires further investigation.

Decreases in EAAT3 have been observed in the striatum of schizophrenics [McCullumsmith and Meador-Woodruff, 2002; Nudmamud-Thanoi et al., 2007]. Preclinical studies have demonstrated that chronic treatment with clozapine or haloperidol can downregulate EAAT3 in the infralimbic cortex and hippocampal CA2 [Schmitt et al., 2003]. Therefore, EAAT3 expression is influenced by antipsychotic treatments, but it is difficult to distinguish between the cause and effect on the basis of postmortem brain studies. In the model of diminished glutamate activity in schizophrenia, potential therapeutic effects on some symptom dimensions is expected by glutamate re-uptake inhibitors, such as EAAT3 antagonist, which could increase the synaptic availability of glutamate and increase glutamatergic action at the postsynaptic neuron [Miyamoto et al., 2005]. In the present study, the risk genotype was associated with increased SLC1A1 expression levels in the prefrontal cortex. On the basis of these findings, we speculated that individuals with a tendency toward increased EAAT3 expression are susceptible to schizophrenia. Higher EAAT3 may be linked to lower synaptic availability of glutamate or more direct mechanism(s) leading to improper functioning of NMDA receptors in some cases. Because different regulation of EAAT3 among brain regions is likely and the associations between SNPs and SLC1A1 expression were not analyzed in regions other than the prefrontal cortex, further studies regarding the same are required. Furthermore, in our findings, the relationship between SNPs and SLC1A1 expression in the prefrontal cortex was observed more obviously in the patient group than the control group. Therefore, the possibility remains that the association between SNPs and SLC1A1 expression reflected antipsychotic treatment responses.

The polymorphisms in SLC1A1 have been reported to be associated with obsessive-compulsive disorder [Arnold et al., 2006; Dickel et al., 2006; Grados and Wilcox, 2007; Stewart et al., 2007]. More recently, a SLC1A1 haplotype was reported to be associated with obsessive-compulsive symptoms induced by atypical antipsychotics [Kwon et al., 2009]. These polymorphisms that were associated with obsessive-compulsive disorder or other symptoms span from introns 2 to 6 of the SLC1A1 gene, and they are not in linkage disequilibrium with SNPs identified as associated with schizophrenia in the present study (Fig. 1).

In conclusion, our findings provide evidence that the SLC1A1 gene might be involved in susceptibility to schizophrenia. Further studies on the involvement of the SLC1A1 gene in the pathophysiology of schizophrenia and confirmation of the present association in other populations are necessary.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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ajmg_31249_sm_SupplTable.doc71KSupplementary Table

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