Molecular Psychiatry
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March 2001, Volume 6, Number 2, Pages 186-192
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Original Research Article
Structure and polymorphisms of the human metabotropic glutamate receptor type 2 gene (GRM2): analysis of association with schizophrenia
A Joo1, H Shibata1, H Ninomiya2, H Kawasaki2, N Tashiro2 and Y Fukumaki1

1Division of Disease Genes, Institute of Genetic Information, Kyushu University, Japan

2Department of Neuropsychiatry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Correspondence to: Y Fukumaki MD PhD, Division of Disease Genes, Institute of Genetic Information, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: yfukumak@gen.kyushu-u.ac.jp

Abstract

Metabotropic glutamate receptors (mGluRs) belong to the class of GTP-binding protein coupled receptors and consist of eight different subtypes. The subtype 2 metabotropic glutamate receptor (mGluR2) gene (GRM2) is one of the possible candidate genes for schizophrenia. Phencyclidine (PCP)-induced increase in glutamate efflux and schizophrenia-like behavioral abnormalities were reduced by pretreatment of the mGluRII agonist LY354740 in rats and its effects are mediated via mGluR2. To evaluate involvement of the mGluR2 gene in the pathogenesis of schizophrenia, we isolated the human mGluR2 gene and determined the transcription initiation site, the entire nucleotide sequence and the chromosomal localization. The hmGluR2 gene spans 13 kb with six exons, including one non-coding exon. The gene was mapped to chromosome 3 p12-p11 by Radiation Hybrid Panel analysis. We screened polymorphisms in the coding exons of the mGluR2 gene, using the SSCP procedure. The thirteen polymorphisms identified included ten missense, one silent mutation and two one-base substitutions in the 5'-untranslated region. We genotyped 213 Japanese schizophrenics and 220 controls to study the association of polymorphisms in the mGluR2 gene with schizophrenia. As we found no statistically significant differences in allele frequencies of each polymorphism, these polymorphisms apparently do not play a major role in schizophrenia. Molecular Psychiatry (2001) 6, 186-192.

Keywords

metabotropic glutamate receptor type 2 gene; RH mapping; polymorphisms; SSCP; schizophrenia

Introduction

Glutamate is a major excitatory neurotransmitter in the central nervous system. There are two types of glutamate receptors, ionotropic glutamate receptors (iGluRs) including NMDA and non-NMDA subtypes which form ligand-gated cation channel complexes, and metabotropic glutamate receptors (mGluRs) which couple to GTP-binding proteins. Metabotropic glutamate receptors (mGluRs) belong to the class of seven-transmembrane domain receptors and eight distinct subtypes have been reported (mGluR1-8). These subtypes are classified into three groups. Group I (mGluR1 and 5) activates phospholipase C and stimulates metabolism of phosphoinositide, while group II (mGluR2 and 3) and group III (mGluR4, 6, 7, and 8) inhibit the activities of adenylyl cyclase and suppress production of cyclic AMP (cAMP). mGluRs modulate a variety of intracellular signal transduction pathways and are involved in excitatory neuronal transmissions, synaptic plasticity, neural cell death, learning and memory.1,2,3,4,5

Although many theories of pathogenesis of schizophrenia are available,6 possible involvement of dysfunction of the glutamate neurotransmitter system in the pathogenesis of schizophrenia attracts much attention.7,8 Phencyclidine (PCP), an uncompetitive antagonist of the NMDA receptor, induces both positive and negative psychotic symptoms in normal individuals which are similar to findings in schizophrenics, and also exacerbate preexisting symptoms in schizophrenics.9 The NMDA receptor is critical in guiding axons to their targets10 and is involved in synaptic pruning in development,11 suggesting that dysfunction of this receptor may lead to schizophrenia which is considered to have a neuro-developmental component. Transgenic mice with reduced levels of expression of NMDAR1, an obligate subunit of NMDA receptors, showed behavioral abnormalities, including increased motor activity and stereotypy and deficits in social and sexual interactions. These behavioral alterations are similar to those observed in pharmacologically-induced animal models of schizophrenia and can be ameliorated by treatment with haloperidol or clozapine.12 These results suggest NMDA receptor dysfunction in schizophrenia, however an apparent NMDA receptor abnormality might be due to a primary problem in another receptor subtype that interacts with the NMDA receptor. Metabotropic glutamate receptors are important modulators of the glutamate system because this type of receptor can regulate functions of ionotropic glutamate receptors.13 Particularly, mGluR2 and mGluR3 genes are of great interest as possible candidate genes for schizophrenia. PCP-induced increase in glutamate efflux and schizophrenia-like behavioral abnormality were reduced by pretreatment of the mGluRII agonist LY354740 in rats and the effects of LY354740 are mediated not via mGluR3 but via the mGluR2 receptor.14,15,16 Thus, the mGluRII genes may be related to the pathogenesis of schizophrenia and targeting mGluRII may provide a therapeutic strategy for treatment of PCP-induced psychiatric disorders and schizophrenia.

We report here the complete structure of the human mGluR2 gene (GRM2) and the chromosomal localization. We found genetic variations in the coding exons of the gene and results are given of a case-control study done to explore the association between polymorphisms in the mGuR2 gene and schizophrenia.

Materials and methods

Isolation and sequencing of the human mGluR2 gene

A 281-bp fragment (position 146-426) was amplified as a probe from genomic human DNA using a primer pair, 5'-ACTGTGGTCCTGTCAATGAG-3' and 5'-ACCGCCAATAACACCAGTGA-3'. The PCR products were subcloned into pBluescriptSK+ and sequenced. The clones containing the mGluR2 gene were isolated from a human genomic library (Clontech, Palo Alto, CA, USA) by standard hybridization techniques under high-stringency conditions with the probe labeled with [alpha-32P]dCTP.17 For DNA sequencing we used an ABI Prism Dye Terminator Cycle Sequencing Kit and an ABI Prism Dye Primer Cycle Sequencing Kit (Perkin-Elmer, Norwalk, CT, USA) on an ABI Prism 377 DNA sequencer (Perkin-Elmer).

Determination of the transcription initiation site

The 5' end of mGluR2 cDNA was determined, using the Cap Site cDNA of human brain (Nippon Gene, Tokyo, Japan). The first PCR was done in 25-mul reactions containing 10 pmol of each primer, 1RC and TGP1, 2.5 mM of MgCl2, 200 muM of each dNTP and 1.25 units of AmpliTaq Gold (Perkin-Elmer). An initial denaturing step of 10 min at 95°C was followed by 35 cycles of 95°C for 20 s, 60°C for 20 s and 72°C for 20 s and a final step of 72°C for 5 min. For nested PCR we used primers 2RC and TGP2, under the same conditions except that cycle number was 10. Sequences of primers are 1RC (5'-CAAGGTACGCCACAGCGTATG 3'), TGP1 (5'-CAAGAAGAGAGATGCGAAGG-3'), 2RC (5'-GTACGCCACAGCGTATGATGC-3') and TGP2 (5'-AGGGAGAAGCAGGGAGGA-3').

Radiation hybrid (RH) maps

The GENEBRIDGE 4 radiation hybrid panel (Research Genetics, Huntsville, AL, USA) was used to determine chromosomal localization of the mGluR2 gene. Sequence-tagged site (STS) markers were assayed by PCR, performed in 10-mul reactions containing 25 ng of DNA, 10 pmol of primers which were used for genomic library screening, 2.5 mM of MgCl2, 200 muM of each dNTP and 0.5 units of Taq DNA polymerase (Promega, Madison, WI, USA). Optimized PCR conditions were denatured at 95°C for 5 min, followed by 30 cycles of 95°C for 1 min and 55°C for 30 s and 72°C for 30 s and extending at 72°C for 5 min. PCR products were examined on 1.2% agarose gels stained with ethidium bromide. We also did Southern hybridization with the probe labeled with [alpha-32P]dCTP, using standard techniques. The result of analysis with a list of the highly-linked markers on the map was obtained by submitting the marker data to the RH server.

Human subjects

Blood samples were obtained with informed written consent from unrelated Japanese individuals. The schizophrenic group was recruited from patients of hospitals around Fukuoka and Oita areas and included 213 patients with schizophrenia (mean age 51; 43.2% female). All patients fulfilled the DSM-IV criteria for schizophrenia. The normal control group was recruited from the Fukuoka area and consisted of 220 individuals (mean age 48; 41.3% female). This study was approved by the Ethics Committee of Kyushu University, Faculty of Medicine.

Single-strand conformation polymorphism (SSCP) analysis18

About 5 ng of human genomic DNA prepared from white blood cells, by the standard method, was amplified with appropriate oligonucleotides (Table 1). These primers were designed to amplify all coding regions of the mGluR2 gene as eleven PCR fragments, e2-1~e6 (Figure 1). Pre-amplification PCR was performed in 10 mul reaction mixture containing 10 pmol of each primer, 2.5 mM of MgCl2 250 muM of each dNTP and 0.25 units of AmpliTaq Gold. An initial denaturing step of 10 min at 95°C was followed by 35 cycles of 94°C for 30 s and 60°C for 30 s with a final extension of 60°C for 10 min. PCR cycling was done using a Gene Amp PCR System 2400 (Perkin-Elmer). PCR products were separated by agarose gel electrophoresis, and the agarose block containing the PCR fragment was excised by laser blade and purified by the Ultrafree-DA (Millipore, Bedford, MA, USA). End labeling of primers was carried out in a 0.5-mul reaction mixture containing 0.11 MBq of [gamma-33P]ATP, 0.5 pmol primers and 0.25 units of T4 polynucleotide kinase (Nippon Gene). The reaction condition was at 37°C for 30 min and at 70°C for 10 min. Labelling PCR was done in 5-mul reactions containing purified fragments as templates, radioisotope-labeled primers, 2.5 mM of MgCl2, 200 muM of each dNTP and 0.75 units of Taq DNA polymerase. The PCR condition was at 94°C for 3 min, followed by 20 cycles of 94°C for 30 s and 60°C for 2 min with a final extension of 72°C for 5 min.

Gel electrophoresis for SSCP was carried out under two conditions for each fragment. The condition was 5% polyacrylamide gel (49:1, acrylamide: bisacrylamide ratio) containing either 0.5 ´ Tris-borate-EDTA buffer and 5% glycerol, or 1 ´ Tris-MES-EDTA buffer pH 6.8. Gels were dried onto filter paper, and autoradiography was done in the presence of an intensifying screen. The shifted bands were excised from the dried gel, re-amplified and sequenced to identify mutations. The primer sequence and the size of each amplified fragment are shown in Table 1.

Statistical analysis

Statistical differences in genotype and allele frequencies between schizophrenics and controls were evaluated using chi2 test and the Fisher's exact probability test at a significant level of 0.05. Linkage disequilibrium was quantified with the EH program.19

Results

Isolation of the human mGluR2 gene

Approximately 8.0 ´ 105 clones of a human genomic DNA library were screened with a 32P-labelled probe derived from human genomic DNA. Four hybridization-positive clones were identified and subjected to restriction mapping. These maps revealed that two of the four independent clones overlapped and they spanned over a 13-kb genomic region containing the entire mGluR2 gene (GRM2). The Bam HI and Hin dIII fragments of two representative clones named ph10 and ph11 were subcloned and the entire nucleotide sequence covering the human mGluR2 gene was determined.

Structural organization of the human mGluR2 gene

We determined the complete genomic structure, including the 5'-untranslated region of the mGluR2 gene. The mGluR2 gene consists of six exons, including one non-coding exon. The nucleotide sequences of the mGluR2 gene and 3658 bp of the 5'-flanking and 386 bp of the 3'-flanking regions have been submitted to the GenBankÔ/EMBL Data Bank and are available under accession number AB045011. The sequence of all the exon/intron boundaries has consensus splicing signals conforming to the GT/AG rule (Table 2).

To determine the transcription initiation site of the mGluR2 gene, PCR was performed using a Cap Site human brain cDNA library. We determined the transcription initiation site located 1573 bp upstream from the translation initiation site. The 5'-flanking region contains six consensus GC boxes between 650 and 380 bp upstream of the transcription initiation of the gene. The 569-bp region of the 5' end of the sequence obtained in this study perfectly matched the sequence from 727 to 1295 bp of cDNA encoding putative secreted protein (SIG11). SIG11 consists of 313 amino acids and the mRNA is 1295 bp in length. The distance between the translation stop codon of the SIG11 gene and the transcription start site of the mGluR2 gene is only 3.4 kb and there is no open reading frame in this region, indicating that the SIG11 and mGluR2 genes are adjacent. The polyadenylation site was deduced from the sequence of EST57801.20 The 3'-untranslated region of the mGluR2 gene consists of 495 nucleotides, and a consensus polyadenylation signal (AATAAA) was found 476 bp downstream of the stop codon.

Chromosomal localization of the human mGluR2 gene

The human mGluR2 gene had already been assigned to chromosome 3.21 We mapped the mGluR2 gene, using radiation hybrid (RH) panel. The STS marker is the 281-bp fragment amplified from exon 2 of the mGluR2 gene. The gene is located between microsatellite markers D3S2372 and D3S1271. The marker D3S2372 was mapped at the 3p12, and the D3S1271 was at the 3p11-3q11. Thus we concluded that the human mGluR2 gene is located at chromosome 3 p12-p11. The previous linkage studies of schizophrenia show no positive results regarding this region.22

Nucleotide changes revealed by comparison between sequences of the gene and cDNA of mGluR2

Compared with the reported cDNA sequence,21 we found eleven substitutions in the mGluR2 gene (Table 3). Five are missense mutations, L12P, V210E, P496A, K748N, F776L, and six are silent mutations, G116, A211, E373, P490, L583, L777.

Polymorphisms in the human mGluR2 gene revealed by SSCP analysis

Ninety unrelated schizophrenia patients were screened for polymorphisms in the coding exons by SSCP analysis (Figure 1). We found polymorphisms in four regions; exons 2-1, 3-2, 4-1 and 5. In these regions additional SSCP analysis was made on 123 schizophrenics and 220 healthy controls. In total, thirteen different polymorphisms were detected, all of which were single base pair substitutions (Figures 1 and 2). Out of the eleven substitutions (found by comparison of genomic and cDNA sequences) only one, L12P, was detected by SSCP analysis. Genotype and allele frequencies of each polymorphism in schizophrenics and controls are given in Table 4. Most subjects had only one nucleotide substitution, but three had two different polymorphisms. One patient had a -34Aright arrowC and R465L, and another patient had L12P and A842V. One control individual was heterozygous for R264C and homozygous for G306, indicating that R264C and G306 are located on the same chromosome. We used the EH program19 to evaluate linkage disequilibrium between -34Aright arrowC and R465L; L12P and A842V. The D' value was 1.0 for each combination, indicating that -34Aright arrowC, R465L, and L12P, A842V are on the same chromosomes, respectively.

Since frequencies of -34Aright arrowC and N439S are higher compared with other polymorphisms and are also higher in schizophrenics than normal subjects, we performed statistical analysis to evaluate association of each polymorphism with schizophrenia. Our data revealed no significant differences in the -34Aright arrowC allele frequencies (Fisher exact test P = 0.4701, chi2 = 0.644, df = 1) between schizophrenics and controls. No differences in the N439S allele frequencies (Fisher exact test P = 0.4993, chi2 = 0.572, df = 1) were observed between the two groups. There is also no association of the other variants detected in this study with schizophrenia (data not shown).

Discussion

Among eight subtypes of the mGluR family, subtypes 2 and 3 are classified into the group II according to amino acid sequence similarities, agonist selectivities and the intracellular transduction mechanisms. The sequence of the mouse mGluR3 gene was reported.23 Although the mouse mGluR3 gene spans over 95 kb, the human mGluR2 and mouse mGluR3 genes have six exons of a comparable size.23

Compared with the reported cDNA sequence,21 eleven single nucleotide changes of the mGluR2 gene were detected (Table 3). Genomic clones of the mGluR2 gene studied here were derived from the normal whole placenta of a Caucasian, whereas mRNA source for the cDNA clone was not mentioned in the report.21 We identified only one mutation, 35Tright arrowC causing L12P of the eleven substitutions revealed by comparison of the genomic DNA and cDNA sequences as a polymorphism in Japanese. Interestingly, except for 35Tright arrowC all nucleotides of our genomic clone, which are different from cDNA sequences of human mGluR2,21 are the same as those of the rat counterpart, suggesting that these nucleotide differences between our genomic clone and the cDNA clone of human mGluR221 could be due to rare base substitutions.

Allele frequencies of -34Aright arrowC and 1316Aright arrowG were higher in schizophrenics than in controls, however, there were no statistically significant differences in these frequencies. These results do not support the possibility that polymorphisms in the coding regions of the mGluR2 gene play a major role in the pathogenesis of schizophrenia. Polymorphisms in the promoter region of the mGluR2 gene might be associated with schizophrenia, although the previous expression study revealed no abnormality of mRNA expression of mGluR2 as well as other subtypes in the postmortem thalamus specimens from elderly schizophrenic patients.24

Regarding functional consequences of fourteen missense mutations, ten mutations are located in extracellular amino-terminal domains. The conservation of Arg78, Ser159, and Thr182 in all members of the mGluR family, which correspond to Arg61, Ser145 and Thr168 of mGluR2, respectively, indicates that these amino acids may be fundamental recognition motifs for binding of agonists.25 Although the polymorphisms found in this study are not located in the three important positions mentioned above, R264C may influence receptor function by affecting the ligand binding ability because of the substitution of basic to uncharged amino acids at the conserved site between at least six members of the mGluR family.21,26 As Glu210, Ala496, Asn748 and Leu776 were observed in mouse mGluR2, the substitutions, V210E, P496A, K748N and F776L may have no functional effect. This is also the case with K748N and F776L, although these amino acid substitutions are located in the cytosol domain between the fifth and sixth transmembrane domains, and in the sixth transmembrane domain, respectively. One amino acid substitution, V796M in the seventh transmembrane domain, may affect cellular-dispositions of the receptor as it is located in conserved sites between human and rat mGluR2. Two single base substitutions in the untranslated region might have effects on expression levels of mGluR2. To better understand effects of these base substitutions on receptor activities, a functional analysis is in progress.

In this study, thirteen polymorphisms were detected by SSCP analysis, indicating that frequency of single nucleotide polymorphisms (SNPs) is about 1 SNP/214 bp in the coding exons of the mGluR2 gene. This is much higher than would be predicted for the average human nucleotide diversity level of approximately 0.1%, and fourfold less in coding exons.27,28 These observations suggest a lesser degree of structural constraint on fundamental functions in mGluR2 which may enhance diversities of modulation of glutamatergic transmission. Since mGluR2 is involved in various aspects of the function of the central nervous system, the polymorphisms reported here may be a useful tool to clarify the involvement of the functional diversities of the receptor in neuropsychiatric disorders.

Acknowledgements

We are grateful to all the medical staff who were involved in collecting specimens. We also thank Dr K Hayashi, Kyushu University for technical advice and Ms M Ohara for language assistance. This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas (c) Medical Genome Science from the Ministry of Education, Science, Sports and Culture, Japan.

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Figures

Figure 1 Structural organization of the human mGluR2 gene. Overlapping phage clones, ph 10 and ph 11, containing the human mGluR2 gene are shown. The probe used for genomic library screening is indicated by a closed box. Open boxes indicate 5' and 3' non-coding exons and shaded boxes indicate five coding exons. The translation start site is indicated by ATG and the translation termination site is indicated by TGA. The region in which the sequence was determined spans from 3658 bp upstream of the transcription initiation site (arrow a) to 386 bp downstream of the polyadenylation site (arrow b) of the mGluR2 gene.

Figure 2 Schematic representation of the PCR fragments used for SSCP analysis and polymorphisms. Boxes indicate five coding exons and eleven vertical lines indicate the PCR fragments for SSCP analysis. Locations of thirteen polymorphisms revealed by the SSCP procedure are indicated by arrows. Dotted lines indicate intron on an arbitrary scale.

Tables

Table 1 PCR primers used to amplify coding regions of the mGluR2 gene

Table 2 Size of exons and introns and nucleotide sequences of exon-intron boundaries

Table 3 Polymorphisms of the mGluR2 gene revealed by comparison of genomic DNA with mRNA sequences (genome right arrow mRNA)

Table 4 Genotype and allele frequencies in schizophrenics and controls

Received 14 August 2000; revised 11 October 2000; accepted 16 October 2000
March 2001, Volume 6, Number 2, Pages 186-192
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