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Keywords:

  • association study;
  • DAP-1 gene;
  • direct sequencing;
  • NMDA receptor;
  • polymorphism;
  • schizophrenia

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Glutamate dysfunction has been hypothesized to be involved in the pathophysiology of schizophrenia. The human homolog of Drosophila discs large protein (hDLG) and post-synaptic density-95-associated protein-1 (DAP-1) is one of the major proteins that are involved in intracellular signal transduction via N-methyl-d-aspartate receptors. In the present study 33 Japanese patients with schizophrenia were screened for mutations in the DAP-1 gene. A single nucleotide polymorphism was identified in the DAP-1 gene (1618A/G). A case–control study using a larger sample of unrelated patients and controls did not reveal a significant association between this polymorphism and schizophrenia. The results do not provide evidence that the DAP-1 gene is involved in vulnerability to schizophrenia.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Glutamate dysfunction has been proposed to be associated with the pathophysiology of schizophrenia. The most compelling evidence linking abnormal glutamatergic neurotransmission and schizophrenia is the psychotomimetic action of phencyclidine (PCP)1,2 and the therapeutic effects of N-methyl-d-aspartate (NMDA) receptor agonists in schizophrenic patients.3,4 Recently, rat synapse-associated protein 90/post-synaptic-density-95-associated protein (SAPAP)-1, also called DAP-1 (human homolog of Drosophila discs large protein (hDLG) and post-synaptic-density-95-associated protein-1)5,6 has been identified from a gene regulated by PCP in the rat brain.7 The family of proteins known as DAP is concentrated in the post-synaptic density and binds to the guanylate kinase domain of post-synaptic density 95 (PSD-95). The DAP-1 protein is specifically expressed in the brain and is colocalized with PSD-95 and NMDA receptor (NMDA-R) at the synapses in the rat brain.6 Recent findings show that there is an interaction between NMDA-R and members of membrane-associated guanylate kinase (MAGUK) proteins including PSD-95 and DAP-1. Therefore, DAP-1 may directly regulate the electrophysiological properties and the clustering of the NMDA-R.8 Thus, DAP-1 is one of the major proteins involved in intracellular signal transduction via NMDA-R. In addition, the DAP-1 gene is located on chromosome 18p11 which has been linked to schizophrenia and/or bipolar disorder.9,10 Based on this evidence, we hypothesized that the DAP-1 gene could be involved in predisposition to schizophrenia. In the present study we screened for genetic variations in the coding region of the DAP-1 gene in 33 patients with schizophrenia by direct sequencing. To investigate a possible involvement of this polymorphism to the etiology of schizophrenia, we compared frequencies of genetic variations in unrelated patients with schizophrenia and a healthy comparison group.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Subjects

The subjects for an association study consisted of 325 Japanese patients with schizophrenia meeting Diagnostic and Statistical Manual of Mental Disorders (4th edn; DSM-IV) diagnostic criteria (188 male patients, 137 female patients; mean age: 50.7 years; SD = 12.5) and 342 healthy control subjects (202 male subjects, 140 female subjects; mean age: 52.0 years; SD = 14.9). The present study was approved by the Ethics Committee of Kobe University Graduate School of Medicine. All subjects were recruited from the Hyogo area around Kobe city and gave written informed consent to participate.

Polymerase chain reaction

The sequence of the DAP-1 gene cDNA was based on AB000277.5 The genomic structure of the DAP-1 gene was determined through basic local alignment search tool (BLAST) searches of the sequence NT-011069. The gene consists of 11 exons and all coding exons and exon–intron junctions were amplified by polymerase chain reaction (PCR). We used 15 pairs of primers that were designed to amplify all exons and exon–intron junctions of the DAP-1 gene.

Direct sequencing of polymerase chain reaction products

For mutation detection, we screened a sample of 33 patients with schizophrenia. Each PCR product was purified with a Microcon Centrifugal Filter Device (Millipore; Bedford, MA, USA). The sequencing reaction was performed with the ABI PRISM DNA sequencing kit (BigDye Terminator Cycle Sequencing Ready Reaction; Applied Biosystems, Tokyo, Japan). The labeled products were purified and were analyzed with an automated capillary sequencer (ABI PRISM 310; Applied Biosystems).

Analysis of polymorphic restriction enzyme sites

In order to analyze the A/G single nucleotide polymorphism (SNP) in exon 4, we prepared a sequence of 404 base pairs (bp) by PCR amplification using the forward primer 5′-AGATGCTTGGAGAGGTTGAT-3′ and the reverse primer 5′-CAACTATGTGTGTTGACAGC-3′. The PCR products were digested with 2 U Acc-II (Takara, Otsu, Japan) at 37°C for 8 h. The A/A genotype gives two bands of 318 and 86 bp. In the G/G genotype, the enzyme further digests the 318 bp band into 269 and 49 bp fragments. The PCR products of each digestion were analyzed by separation on 2.0% agarose gel electrophoresis. The bands were visualized by ethidium bromide staining and ultraviolet transillumination.

Statistical methods

The association of the DAP-1 SNP was tested using a χ2 test. Probability differences of P < 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

An SNP was detected in the coding sequence. It was located at exon 4 of the DAP-1 gene, at cDNA nucleotide position 1618 (A/G), which also altered an Acc-II restriction site. However, this SNP does not show an amino acid substitution. This SNP is the same one that has been open to the public in the National Center for Biotechnology Information (NCBI) SNP database (rs3745051) and the Japanese SNP (JSNP) database (IMS-JST096473) recently.11 No other mutations were found in the coding region of the DAP-1 gene. We then carried out a genetic case–control association study based on this SNP in the patients with schizophrenia and healthy control subjects. The genotype distributions in the two groups did not deviate from Hardy–Weinberg equilibrium. The genotype and allele frequencies in the patients with schizophrenia did not differ from those in the comparison group (Table 1).

Table 1. Genotype and allele frequencies of the 1618A/G polymorphism of the DAP-1 gene
 Schizophrenia (n = 325)Controls (n = 342)
  1. No significant differences were found between the patients with schizophrenia and the comparison group in either the genotype distribution (χ2 = 2.606, d.f. = 2, P = 0.272) or the allele frecuencies (χ2 = 2.451, d.f. = 1, P = 0.117).

  2. DAP-1, human homolog of Drosophila discs large protein and post-synaptic-density-95-associated protein-1.

Genotype
A/A 47 (14.5%) 41 (12.0%)
A/G161 (49.5%)158 (46.2%)
G/G117 (36.0%)143 (41.8%)
Allele
A255 (39.2%)240 (35.1%)
G395 (60.8%)444 (64.9%)

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The DAP-1 protein is a member of the MAGUK proteins. The MAGUK proteins have recently been shown to have several functions in the modulation of NMDA-R, including regulation of ion channel function, synaptic targeting and coupling to downstream signaling pathways and cytoskeletal components.8 Therefore, a defect in the MAGUK proteins may be associated with hypofunction of the NMDA-R-mediated glutamatergic system in schizophrenia. The present results do not provide evidence for the involvement of the DAP-1 gene in schizophrenia. It is possible that other members of MAGUK proteins are involved in the etiology of schizophrenia.

The DAP-1 gene is located on chromosome 18p11, which has been linked not only to schizophrenia but also to bipolar disorder.9,10 Berrettini hypothesizes that schizophrenic and bipolar syndromes share some genetic risk factors and chromosome 18p11 is one of four genomic regions of overlap for bipolar and schizophrenic susceptibility loci.12 Although this SNP was not associated with schizophrenia, there is a possibility of an association with bipolar disorder. The present results indicate that 1618A/G polymorphism and other polymorphisms, which are in linkage disequilibrium with this SNP, are not associated with schizophrenia. However, it is still possible that there are some genetic markers, which are associated with schizophrenia, in the DAP-1 gene. We would need further studies to find genetic variation in non-coding regions of the DAP-1 gene, such as a promoter region, which might affect the DAP-1 gene expression.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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