Association study of polymorphisms in synaptic vesicle-associated genes, SYN2 and CPLX2, with schizophrenia

Background The occurrence of aberrant functional connectivity in the neuronal circuit is one of the integrative theories of the etiology of schizophrenia. Previous studies have reported that the protein and mRNA levels of the synapsin 2 (SYN2) and complexin 2 (CPLX2) genes were decreased in patients with schizophrenia. Synapsin 2 and complexin 2 are involved in synaptogenesis and the modulation of neurotransmitter release. This report presents a study of the association of polymorphisms of SYN2 and CPLX2 with schizophrenia in the Korean population. Methods Six single nucleotide polymorphisms (SNPs) and one 5-bp insertion/deletion in SYN2 and five SNPs in CPLX2 were genotyped in 154 Korean patients with schizophrenia and 133 control patients using direct sequencing or restriction fragment length polymorphism analysis. An intermarker linkage disequilibrium map was constructed for each gene. Results Although there was no significant difference in the genotypic distributions and allelic frequencies of either SYN2 or CPLX2 polymorphisms between the schizophrenia and control groups, the two-way haplotype analyses revealed significant associations with the disease (P < 0.05 after Bonferroni correction). The three-way haplotype analyses also revealed a significant association of SYN2 with schizophrenia (P < 0.001 after Bonferroni correction). Conclusion These results suggest that both SYN2 and CPLX2 may confer susceptibility to schizophrenia in the Korean population.


Background
Schizophrenia is a severe, chronic mental illness affecting 0.5-1.5% of the general population worldwide [1]. The contribution of genetic factors to the vulnerability to schizophrenia has been well established by family, twin, and adoption studies that have suggested a significant heritability of approximately 50-70% [2]. Many studies have attempted to identify the allelic variants that confer susceptibility to the illness, but no single genes have been identified that produce a major effect on the vulnerability [3].
Recently the synaptic hypothesis of schizophrenia has gained attention by attributing the fundamental pathology of schizophrenia to the dysfunction of synaptic transmission involving various molecules [4]. Synapsins, a family of synaptic vesicle-associated phosphoproteins, play a crucial role in the regulation of neurotransmission, synaptogenesis, and neuronal plasticity [5]. Three human synapsin genes have been identified (SYN1, 2, and 3; OMIM 313440, 600755, and 602705) [6]. Complexin 1 and complexin 2, which are encoded by CPLX1 (OMIM 605032) and CPLX2 (OMIM 605033), respectively, and are also called synaphins, are pre-synaptic membrane proteins that preferentially bind to syntaxin within the SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptors) complex. These proteins are important regulators of transmitter release immediately preceding vesicle fusion [7]. Previous studies have demonstrated that the concentrations of synapsins and complexins are reduced in the brains of schizophrenics [8,9]. The expression levels of both synapsins were significantly decreased in the hippocampal tissue of schizophrenic patients [10]. The levels of synapsin 2 and complexin 2 mRNA were also significantly reduced in the prefrontal cortex, cerebellum, and hippocampus of schizophrenics [11][12][13][14].
SYN2 was mapped to chromosome 3p25 [15], and CPLX2 is located on chromosome 5q35.3 (OMIM 605033). These loci were identified as potential regions conferring susceptibility to schizophrenia in diverse populations [16][17][18]. Based on their localization, well-established neurobiological roles, and expression patterns in schizophrenic patients, we selected SYN2 and CPLX2 as candidate genes for conferring susceptibility to schizophrenia. In this report, we present an association study of SYN2 and CPLX2 with schizophrenia using 12 polymorphisms in the Korean population.

SYN2 polymorphisms in the schizophrenia and control groups
Of the seven polymorphisms in SYN2, rs2623873 (SYN2-1) is located in the promoter region, whereas the others are all located in the intronic regions (SYN2-2-7) (Fig. 1, Genomic organization of SYN2 and CPLX2 and locations of SNPs Figure 1 Genomic organization of SYN2 and CPLX2 and locations of SNPs. a; SYN2 spans over 140 kb and is composed of 14 exons. Seven markers are indicated with the dbSNP reference ID http://www.ncbi.nlm.nih.gov/SNP. b; CPLX2 spans over 83 kb and is composed 3 exons. Five markers are indicated with the dbSNP reference ID http://www.ncbi.nlm.nih.gov/SNP. Table 1). The genotypic distributions and allelic frequencies of polymorphisms in SYN2 were determined in 113 schizophrenic patients and 114 normal healthy controls by direct sequencing or DdeI RFLP. The genotypic distributions and allelic frequencies of polymorphisms in SYN2 are shown in Table 2. The average allelic frequency of the SNPs was 0.312. Given the equivalent frequency for the susceptible allele, the expected detection power for SYN2 was 0.9538 to 0.9929 under the multiplicative model with a genotype relative risk = 1.8 to 2.0 [22]. None of the SNPs showed any significant deviation from Hardy-Weinberg equilibrium (P > 0.05). We observed no significant difference in the genotypic distributions and allelic frequencies between the schizophrenics and control groups ( Table 2).

CPLX2 polymorphisms in schizophrenia and control groups
Of the five SNPs in CPLX2, rs2247916 (CPLX2-1) is located in the promoter region, rs2243404 (CPLX2-2) is located in the 5'UTR, and the others are located in the intronic regions ( Fig. 1, Table 1). We determined the genotypic distributions and allelic frequencies of the SNPs in 154 schizophrenic patients and 133 normal healthy controls by direct sequencing or RFLP analysis. The genotypic distributions and allelic frequencies for CPLX2 SNPs are shown in Table 2. The average allelic frequency of the SNPs was 0.126. Given the equivalent frequency for the susceptible allele, the expected detection power for CPLX2 was 0.7445 to 0.8802 based on the multiplicative model with the genotype relative risk = 1.8 to 2.0 [22]. None of the five SNPs showed any significant deviations from Hardy-Weinberg equilibrium. We observed no significant differences in genotypic distributions or allelic frequencies between the schizophrenia and control groups ( Table  2).

Gene
Name dbSNP rs# Region Allele Methods Intron G/A Direct Sequencing a Tissue inhibitor of metalloproteinase 4 (Timp4) gene is nested within the intron of SYN2 in reverse orientation.     = 3, P = 0.0009), even after the Bonferroni correction (n = 10, P corr = 0.009, Table 5). For the combination of CPLX2-1 -CPLX2-2, the G allele-the C allele haplotype was observed more frequently in the schizophrenia group than the control group (Table 6).
Chen et al. [23] recently reported an association study of four SNPs in SYN2 using Han Chinese samples. They found significant associations of SNP rs795009 and a haplotype constructed by the four SNPs with schizophrenia. Chen et al. [23] and our study examined two SNPs (rs2308169 and rs308963) in common, and their genotypic and allelic frequencies were similar in both studies. Although Chen et al. [23] did not mention the pairwise haplotype association study that we performed, they did report a significant difference in the overall four-way haplotype frequencies between schizophrenics and controls.
Since two independent studies have reported a significant haplotype association of SYN2 with schizophrenia, this gene is probably involved in the pathogenesis of schizophrenia.
Several studies have suggested that the decreased expression of synaptic genes is characteristic of schizophrenia. In the hippocampus of schizophrenic patients, several studies have shown a consistent pattern of decreases in presynaptic proteins and their encoding mRNAs, such as synapsin 2, synaptophysin, and synaptosomal-associated protein-25 (SNAP-25) [8][9][10]24]. Furthermore, a reduction in the synapsin 2 mRNA levels was observed in the prefrontal cortex of schizophrenic patients [14], but controvertible results have also been reported [25]. The altered expression levels of other presynaptic proteins, complexin 1 and complexin 2, have been reported in schizophrenic patients [11][12][13]. Interestingly, complexin 1 is enriched in axosomatic regions, inhibitor neurons, and their synapses, while complexin 2 is enriched in the axodendritic terminals [9,26]. The differential expression of complexins 1 and 2 implies their involvement in the excitatory synapse in the hippocampus of schizophrenic patients [11]. These observations suggest that abnormal expression of SYN2 and CPLX2 may cause the vulnerability to schizophrenia by altering neurotransmitter release and neuroplasticity.

Conclusion
We found significant differences in the haplotype frequencies in both SYN2 and CPLX2 polymorphisms between schizophrenia and control groups. In addition, the haplotype constructed from three polymorphisms (SYN2-1, SYN2-2, and SYN2-4) showed a significant association with schizophrenia. Our results suggest that both SYN2 and CPLX2 polymorphisms may contribute susceptibility to schizophrenia in the Korean population.

SNP Selection and PCR-based Genotyping
Since the genomic sizes of SYN2 and CPLX2 are about 187 and 89 kb, respectively, we initially intended to select common polymorphisms at intervals of approximately 20-50 kb from the dbSNP http://www.ncbi.nlm.nih.gov/ SNP/. After validating the frequency of each polymor-phism in 24 healthy Korean individuals using direct sequencing, we selected seven common polymorphisms from SYN2 and five from CPLX2 for further analyses (Fig.  1, Table 1). We amplified the fragments containing polymorphisms individually and genotyped DNA samples for each SNP with either PCR-based restriction fragment length polymorphism (RFLP) assays or direct sequencing performed with an ABI PRISM ® Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) on an ABI PRISM ® 3100 DNA sequencer (Applied Biosystems) ( Table 1). In case of unclear sequence data, we repeated direct sequencing under various conditions until the genotype was determined correctly.

Statistics
The deviation of the genotypic frequencies from Hardy-Weinberg equilibrium was examined using the chi-square test (df = 1). Statistical differences in the genotypic distributions and allelic frequencies between the schizophrenia and control groups were examined using the Fisher's exact probability test. We calculated D' and r 2 to evaluate the magnitude of linkage disequilibrium (LD) [19]. We estimated haplotype frequencies using the EH program, version 1.14 [20]. The statistical analysis of haplotype association was done as previously described [21]. We applied the Bonferroni correction to multiple testing based on the number of haplotypes. The significance level for all the statistical tests was 0.05.