An association study between polymorphisms in five genes in glutamate and GABA pathway and paranoid schizophrenia

European Psychiatry 20 (2005) 45–49 http://france.elsevier.com/direct/EURPSY/

Original article

An association study between polymorphisms in five genes in glutamate and GABA pathway and paranoid schizophrenia Boyu Zhang a,b, Yanbo Yuan c, Yanbin Jia a,b, Xin Yu c, Qi Xu a,b, Yucun Shen c, Yan Shen a,b,* a

National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC), 5, Dong Dan San Tiao, Beijing 100005, China b National Center of Human Genome Research, Beijing 100176, China c Peking University Institute of Mental Health, Beijing 100083, China Received 15 June 2004; accepted 9 September 2004 Available online 13 December 2004

Abstract Dysfunctions of glutamatergic and GABAergic neurotransmission are two important hypotheses for the pathogenesis of schizophrenia. Thus, genes in the pathway are candidates for schizophrenia susceptibility. Phosphate-activated glutaminase (GLS), glutamine synthetase (GLUL), glutamic acid decarboxylase (GAD), GABA transaminase (ABAT) and succinic semialdehyde dehydrogenase (ALDH5A1) are five primary enzymes in glutamate and GABA synthetic and degradative pathway. In order to investigate the possible involvement of these genes in the development of paranoid schizophrenia, we genotyped 80 paranoid schizophrenics from northern China and 108 matched controls by polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) methods or directly sequencing of PCR product. Seven SNPs were found to be polymorphic in the population investigated. No significant differences in the genotype distributions or allele frequencies between patients and controls were found. Therefore, we conclude the polymorphisms studied in the five genes do not play major roles in pathogenesis of paranoid schizophrenia in the population investigated. © 2004 Elsevier SAS. All rights reserved. Keywords: Schizophrenia; Association study; GLS; GLUL; GAD; ABAT; ALDH5A1

1. Introduction Several models have been put forward to explain the neurochemical basis of schizophrenia. These models relate to the dopaminergic, glutamatergic, serotonergic, cholinergic or GABAergic systems. In contrast to the classic dopamine hypothesis of schizophrenia, recent theories about the neurochemical mechanisms of the disorder emphasized the potential role of amino acid neurotransmitters. Glutamate is a primary excitatory neurotransmitter in the CNS. Evidence supporting an association between glutamatergic dysfunction and schizophrenia came from pharmacological studies. NMDA antagonists are regarded as the best pharmacological models of schizophrenia, mimicking both positive and negative symptoms of the disease [15]. In addition, postmortem studies of schizophrenics have identified abnormalities of glutamate and glutaminergic receptor * Corresponding author. E-mail address: [email protected] (Y. Shen). 0924-9338/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.eurpsy.2004.09.028

density and subunit composition in the prefrontal cortex, thalamus, hippocampus and temporal lobe [7,10,13]. GABA is another principal neurotransmitter in the brain. Much evidence supported the GABA hypothesis. GABA mimetic agents can be psychotogenic, and their psychotogenic effects probably resemble schizophrenia more closely than the one of NMDA antagonists. Changes in gene expression of GABAAR subunits [1] and glutamic acid decarboxylase (GAD) [2,9,16] have been reported in postmortem studies of schizophrenia patients. These results gave us suggestions to guide research on the pathophysiology and treatment of schizophrenia. The two neurotransmitters, glutamate and GABA, are involved in one metabolic pathway in CNS. Five major enzymes involved in glutamate and GABA synthetic and degradative pathway were selected in our study. The five enzymes are phosphate-activated glutaminase (GLS) [EC3.5.1.2], glutamine synthetase (GLUL) [EC6.3.1.2], GAD [EC4.1.1.15], GABA transaminase (ABAT) [EC2.6.1.19] and succinic semialdehyde dehydrogenase (ALDH5A1)

46

B. Zhang et al. / European Psychiatry 20 (2005) 45–49

to patients group on gender, age and ethnicity. Each subject gave informed consent for the research involved in this study. Ethical approval for the study was obtained from the Ethical Committee of the National Center of Human Genome Research, Beijing.

Fig. 1. Important enzymes in glutamate and GABA pathway. 1.phosphate-activated glutaminase, 2.glutamine synthetase, 3.glutamic acid decarboxylase, 4.GABA transaminase, 5.succinic semialdehyde dehydrogenase

[EC1.2.1.24]. Glutamate can be synthesized from glutamine by GLS, glutamate can be converted to glutamine by GLUL, glutamate can be catalyzed to GABA by GAD, and GABA metabolized further to succinate by ABAT and ALDH5A1 (see Fig. 1). The aim of this study was to examine the relationship between the polymorphisms in the genes and the paranoid schizophrenia.

2.2. DNA analysis Each patient and control agreed to provide 5 ml venous blood for the study. Venous blood samples were collected in EDTA-lined tubes, and DNA was extracted from peripheral leukocytes according to the standard method. All SNPs were selected from a SNPs database (http://www.ncbi.nlm.nih.gov/SNP) in accordance with the following criteria: SNPs are located in the coding or untranslated regions of their genes. Each SNP was genotyped in 10 randomly selected patients and 10 randomly selected controls at first. If the SNP showed any polymorphism in either patients or controls, we chose it as a marker in further test. Polymerase chain reactions (PCRs) were conducted in 25 ul volumes containing 60 ng genomic DNA, 200 uM dNTPs, 0.2 uM each primers, 1× buffer (Yuanpinghao, Inc., Beijing, China), and 1 unit Taq DNA polymerase (Yuanpinghao, Inc.). PCR conditions were denaturation at 94 °C for 5 min followed by 35 cycles at 94 °C for 20 s, annealing for 20 s (temperature: see Table 1), 72 °C for 20 s with a final elongation step at 72 °C for 5 min. Primers and restrict enzymes were listed in Table 1 (Not including non polymorphic SNPs). Restriction digestion was performed by commercially available enzymes according to the recommendations of the manufacturer.

2. Materials and methods 2.1. Subjects Schizophrenia is a highly clinical and genetic heterogeneous disorder. Different clinical subtypes may have different genetic bases. More homogeneous clinical samples may increase the power of association studies. Therefore, we selected paranoid schizophrenia, the most common subtype of the disorder, as the object in this study. Samples of the study were all inpatients admitted by Peking University Institute of Mental Health. After structured interviews, clinical observations and comparing medical records and past history, 80 patients from northern China (mean age: 24.8 ± 5.8; mean age of onset: 21.9 ± 5.5) entered our study. They met ICD-10 criteria for schizophrenia and were categorized as paranoid subtype. The homogeneity of the sample is of benefit to our investigation. A total of 108 Chinese normal unrelated controls (mean age 25.7 ± 5.8) were recruited for genetic analysis. They were in good health and reported themselves free from current and previous psychiatry illness. The controls, living in the same area with patients were well matched

2.3. Statistical analysis Statistical differences in genotype and allele frequencies between schizophrenic and control subjects were evaluated by the v2-test at significance level of 0.05. Odds ratio (OR)

Table 1 Polymorphic SNPs and primers of PCRs and corresponding restriction enzymes Genes

Polymorphisms (amino acid change)

SNPs localization

Restriction enzymes

PCR product size and digest fragment (bp)

GLUL

rs1058111 A/G (Ser/Ser)

Exon 3

Bsr I

GLS

rs1546648 A/T

3′UTR

Seqence

249 (A: 249 G: 167 + 82) 253

rs1546647 C/A

3′UTR

Seqence

253

58

GAD67

rs769395 C/T

3′UTR

Mnl I

58

ABAT

rs1731017 A/G (Gln/Arg)

Exon 2

Ava I

rs1641022 C/A (Val/Val)

Exon 3

Rsa I

206 (T: 79 + 127 C: 79 + 44 + 83) 126 (A: 126 G: 46 + 80) 224 (A: 224 C: 150 + 74)

3′UTR

BamH I

232 (C: 232 A: 88 + 144)

56

ALDH5A1 rs1054899 C/A

Sequence means the SNPs were genotyped by directly sequencing of PCR product method.

Annealing temperature (°C) 58 58

56 56

Primers

F: 5′-gaggaggggtatccatagctg-3′ R: 5′-tgcttggaaagctcctgtatg-3′ F: 5′-atgggtcaaggttgcctttag-3′ R: 5′-ttcacacatacgttttggtcac-3′ F: 5′-atgggtcaaggttgcctttag-3′ R: 5′-ttcacacatacgttttggtcac-3′ F: 5′-aggatactaatggggagggtgtg-3′ R: 5′-ccttcagcagttaccgaggag-3′ F: 5′-tatgatgggcctctgatgaag-3′ R: 5′-ctaagcttggctgtgcctttg-3′ F: 5′-ctcccacgggtgtttatttct-3′ R: 5′-gctccaactcacagcattagg-3′ F: 5′-tcagaactcatccagtccttg-3′ R: 5′-ggcagtgatcaagcctttgt-3′

B. Zhang et al. / European Psychiatry 20 (2005) 45–49

and their 95% confidence intervals (95% CI) were calculated to evaluate the effect of different alleles. These analyses were estimated by EPI info 2002 program (http://www.cdc.gov). Case–control comparisons of haplotype distributions were done with the Arlequin program (http://lgb.unige.ch/ arlequin/).

3. Results We have examined six genes in our study, including 31 SNPs (rs1058094, rs912901, rs912900, rs1058111, rs8527 and rs8762 in GLUL gene; rs1168, rs1546648, rs1546647, rs1546646 and rs10437 in GLS gene; rs1049731, rs1049733, rs769399, rs769395, rs954947, rs2270335 and rs1049730 in GAD67 gene; rs2839673, rs1556234, rs1805397, rs2210118, rs1360845 and rs1335547 in GAD65 gene; rs1731017, rs1062593, rs1641022, rs1042495 and rs737696 in ABAT gene; rs2760118 and rs1054899 in ALDH5A1 gene). Of the studied 31 SNPs, only seven SNPs (details in Table 2) were found to be polymorphic in the population investigated. GAD exists as two isoforms of 67 kDa (GAD67) and 65 kDa (GAD65) [6,11]. The former isoform is abundant in the nerve terminal and the latter in the neuronal soma and dendrites. Six SNPs studied in GAD65 gene did not show any polymorphisms in our samples. The obtained genotype distributions and allele frequencies among the patients and the controls are shown in Table 2. The genotype distributions for all the diagnostic groups and controls were in Hardy–Weinberg equilibrium. No significant differences in genotype distributions or allele frequencies were observed in the seven SNPs between the controls and patients. The two SNPs (rs1546647 and rs1546648) in GLS gene were in completely linkage disequilibrium (D’ = 1). But the two SNPs (rs1731017 and rs1641022) in ABAT gene were in weak linkage disequilibrium (D′ = 0.0729). Distributions of haplotypy frequencies of the two SNPs in ABAT gene did not show a significant association between any haplotypes and paranoid schizophrenia (v2 = 0.455 df = 3 P = 0.929).

4. Discussion The hypothesis of abnormal glutamatergic and GABAergic neurotransmission in schizophrenia suggests that genes involved in the metabolism are prime candidates for the disorder. The five enzymes we investigated play important roles in the regulation of the metabolism of glutamate and GABA. The aim of present study was to research the relationships between the five genes in glutamate and GABA pathway and the paranoid schizophrenia. GLS and GLUL are the key enzymes involved in glutamate pathway. Burbaeva et al. [4] reported that significantly changed level of GLUL was found in the prefrontal cortex of patients with schizophrenia. The result was not consistent with

47

that of Gluck et al. [8]. The activity of GLS in the dorsolateral prefrontal cortex in the schizophrenic patients is greater than the one in the controls [8]. But no evidence supports the effect of the two genes on susceptibility to paranoid schizophrenia in our study. GAD, ABAT, and ALDH5A1 belong to the GABA pathway. GAD is one of the most important enzymes in GABA pathway. Many studies reported that the GAD activity or gene expression was abnormal in schizophrenics [2,3,8,16]. But the results from investigations on genetic variants in GAD gene did not validate that the gene plays a major role in the predisposition to schizophrenia and unipolar depression [5,12]. In our test, the SNPs studied in GAD65 gene did not show any polymorphism, and only one SNP in GAD67 gene was polymorphic in our samples. No association was observed between the polymorphism and the paranoid schizophrenia. ABAT and ALDH5A1 locate in the downstream of the GABA pathway. The activity of the two enzymes did not show any obvious difference between patients and controls [8,14]. Neither genotype distributions nor allele frequencies show any difference in our study. As schizophrenia is such a complex trait disease, hunting for schizophrenia susceptibility genes is a challenging task. Although the genes we investigated are expected to be important in development of schizophrenia, our data provided no direct evidence supporting the involvement of the genes in pathogenesis of paranoid schizophrenia. When the low frequency allele of each polymorphism is assumed to be the risk, the sample size in the current study has a power of 90% with 5% significance level to detect an OR of 2.25 for rs1058111, 1.95 for rs1546648, 1.95 for rs1546647, 1.96 for rs769395, 2.00 for rs1731017, 1.96 for rs1641022 and 2.03 for rs1054899, respectively. Although we did not find evidence supporting the involvement of the five genes studied in pathogenesis of schizophrenia, these genes are still important in psychopharmacology. Larger samples for these markers are needed in future study. And as one or two markers excluded in our study in each gene are very unlikely to cover its whole structure, the involvement of other polymorphisms in the same gene can not be excluded on susceptibility to schizophrenia. Systematic investigations on additional markers may be necessary. In conclusion, our data suggests that these polymorphisms studied in five genes may not play major roles in the pathogenesis of paranoid schizophrenia. As the schizophrenia is a complex trait disease, further studies to analyze combined effect of multiple genes on the susceptibility to schizophrenia should be performed. Acknowledgements This project was supported by the grants from the National High Technology Research and Development Program of China (Grant No. 2001AA221071), the National Key Technology Research and Development Program of China (Grant No. 2002BA711A07).

48

Table 2 Alleles and genotypes distribution of seven SNPs

GLS 2q32–q34

GAD 2q31

ABAT 16p13.2

ALDH5A 1 6p22.2–p22.3

Polymorphism

rs1058111 G/A Case Control rs1546648 A/T Case Control rs1546647 C/A Case Control rs769395 C/T Case Control rs1641022 C/A Case Control rs1731017 G/A Case Control rs1054899 A/C Case Control

Genotype and frequency

GG 58 (0.725) 83 (0.769) AA 38 (0.475) 48 (0.444) CC 38 (0.475) 48 (0.444) CC 14 (0.175) 15 (0.139) CC 11 (0.138) 16 (0.148) GG 23 (0.288) 25 (0.231) AA 4 (0.05) 3 (0.028)

AA 2 (0.025) 1 (0.009) TT 12 (0.15) 10 (0.093) AA 13 (0.163) 10 (0.093) TT 28 (0.35) 32 (0.296) AA 27 (0.337) 43 (0.398) AA 23 (0.288) 30 (0.278) CC 58 (0.725) 73 (0.676)

GA 20 (0.25) 24 (0.222) AT 30 (0.375) 50 (0.463) CA 29 (0.363) 50 (0.463) CT 38 (0.475) 61 (0.565) CA 42 (0.525) 49 (0.454) GA 34 (0.425) 53 (0.491) AC 18 (0.225) 32 (0.296)

v2 (P) df = 2

Hardy–Weinberg equilibrium v2 (P)

v2 = 0.981 P = 0.612

v2 = 0.031 P = 0.861 v2 = 0.263 P = 0.608

v2 = 2.224 P = 0.329

v2 = 2.085 P = 0.149 v2 = 0.348 P = 0.555

v2 = 3.0334 P = 0.2194

v2 = 3.090 P = 0.079 v2 = 0.348 P = 0.555

v2 = 1.508 P = 0.471

v2 = 0.032 P = 0.858 v2 = 2.707 P = 0.100

v2 = 0.973 P = 0.615

v2 = 0.703 P = 0.402 v2 = 0.111 P = 0.739

v2 = 1.0095 P = 0.6037

v2 = 1.800 P = 0.180 v2 = 0.029 P = 0.865

v2 = 1.647 P = 0.439

v2 = 2.404 P = 0.121 v2 = 0.052 P = 0.820

Allele

G 136 190 A 106 146 C 105 146 C 66 91 C 64 81 G 80 103 A 26 38

A 24 26 T 54 70 A 55 70 T 94 125 A 96 135 A 80 113 C 134 178

v2 (P) df = 1

v2 = 0.70 (P = 0.403) OR = 0.78 95% CI: 0.41–1.47 v2 = 0.07 (P = 0.784) OR = 0.94 95% CI: 0.60–1.49 v2 = 0.16 (P = 0.689) OR = 0.92 95% CI: 0.58–1.44 v2 = 0.03 (P = 0.864) OR = 0.96 95% CI: 0.62–1.49 v2 = 0.24 (P = 0.622) OR = 1.11 95% CI: 0.71–1.73 v2 = 0.20 (P = 0.657) OR = 1.10 95% CI: 0.71–1.69 v2 = 0.12 (P = 0.732) OR = 0.91 95% CI: 0.51–1.62

B. Zhang et al. / European Psychiatry 20 (2005) 45–49

Gene and chromosome location GLUL 1q31

B. Zhang et al. / European Psychiatry 20 (2005) 45–49

References [1]

[2]

[3]

[4]

[5]

[6]

[7]

Akbarian S, Huntsman MM, Kim JJ, Tafazzoli A, Potkin SG, Bunney Jr. WE, et al. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls. Cereb Cortex 1995;5:550–60. Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, Bunney Jr. WE, et al. Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry 1995;52:258–66. Bird ED, Spokes EG, Barnes J, MacKay AV, Iversen LL, Shepherd M. Increased brain dopamine and reduced glutamic acid decarboxylase and choline acetyl transferase activity in schizophrenia and related psychoses. Lancet 1977;2:1157–8. Burbaeva GSH, Boksha IS, Turishcheva MS, Vorobyeva EA, Savushkina OK, Tereshkina EB. Glutamine synthetase and glutamate dehydrogenase in the prefrontal cortex of patients with schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 2003;27:675–80. De Luca V, Muglia P, Masellis M, Jane Dalton E, Wong GW, Kennedy JL. Polymorphisms in glutamate decarboxylase genes: analysis in schizophrenia. Psychiatr Genet 2004;14:39–42. Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, Tobin AJ. Two genes encode distinct glutamate decarboxylases. Neuron 1991;7:91– 100. Gao XM, Sakai K, Roberts RC, Conley RR, Dean B, Tamminga CA. Ionotropic glutamate receptors and expression of N-methyl-Daspartate receptor subunits in subregions of human hippocampus: effects of schizophrenia. Am J Psychiatry 2000;157:1141–9.

[8]

[9]

[10]

[11]

[12]

[13] [14]

[15] [16]

49

Gluck MR, Thomas RG, Davis KL, Haroutunian V. Implications for altered glutamate and GABA metabolism in the dorsolateral prefrontal cortex of aged schizophrenic patients. Am J Psychiatry 2002;159: 1165–73. Hashimoto T, Volk DW, Eggan SM, Mirnics K, Pierri JN, Sun Z, et al. Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia. J Neurosci 2003;23: 6315–26. Ibrahim HM, Hogg Jr. AJ, Healy DJ, Haroutunian V, Davis KL, Meador-Woodruff JH. Ionotropic glutamate receptor binding and subunit mRNA expression in thalamic nuclei in schizophrenia. Am J Psychiatry 2000;157:1811–23. Kobayashi Y, Kaufman DL, Tobin AJ. Glutamic acid decarboxylase cDNA: nucleotide sequence encoding an enzymatically active fusion protein. J Neurosci 1987;7:2768–72. Lappalainen J, Sanacora G, Kranzler HR, Malison R, Hibbard ES, Price LH, et al. Mutation screen of the glutamate decarboxylase67 gene and haplotype association to unipolar depression. Am J Med Genet 2004;124B:81–6. Meador-Woodruff JH, Healy DJ. Glutamate receptor expression in schizophrenic brain. Brain Res Brain Res Rev 2000;31:288–94. Sherif F, Eriksson L, Oreland L. Gamma-aminobutyrate aminotransferase activity in brains of schizophrenic patients. J Neural Transm Gen Sect 1992;90:231–40. Tamminga CA. Schizophrenia and glutamatergic transmission. Crit Rev Neurobiol 1998;12:21–36. Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA. Decreased glutamic acid decarboxylase67 messenger RNA expression in a subset of prefrontal cortical gamma-aminobutyric acid neurons in subjects with schizophrenia. Arch Gen Psychiatry 2000;57:237–45.