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

Association study of polymorphisms in synaptic vesicle-associated genes, SYN2 and CPLX2, with... 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 contribution of genetic factors to the vulnerability to Schizophrenia is a severe, chronic mental illness affecting schizophrenia has been well established by family, twin, 0.5–1.5% of the general population worldwide [1]. The and adoption studies that have suggested a significant Page 1 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 Genomic or Figure 1 ganization of SYN2 and CPLX2 and locations of SNPs 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. heritability of approximately 50–70% [2]. Many studies [8,9]. The expression levels of both synapsins were signif- have attempted to identify the allelic variants that confer icantly decreased in the hippocampal tissue of schizo- susceptibility to the illness, but no single genes have been phrenic patients [10]. The levels of synapsin 2 and identified that produce a major effect on the vulnerability complexin 2 mRNA were also significantly reduced in the [3]. prefrontal cortex, cerebellum, and hippocampus of schiz- ophrenics [11-14]. Recently the synaptic hypothesis of schizophrenia has gained attention by attributing the fundamental pathol- SYN2 was mapped to chromosome 3p25 [15], and CPLX2 ogy of schizophrenia to the dysfunction of synaptic trans- is located on chromosome 5q35.3 (OMIM 605033). mission involving various molecules [4]. Synapsins, a These loci were identified as potential regions conferring family of synaptic vesicle-associated phosphoproteins, susceptibility to schizophrenia in diverse populations play a crucial role in the regulation of neurotransmission, [16-18]. Based on their localization, well-established neu- synaptogenesis, and neuronal plasticity [5]. Three human robiological roles, and expression patterns in schizo- synapsin genes have been identified (SYN1, 2, and 3; phrenic patients, we selected SYN2 and CPLX2 as OMIM 313440, 600755, and 602705) [6]. Complexin 1 candidate genes for conferring susceptibility to schizo- and complexin 2, which are encoded by CPLX1 (OMIM phrenia. In this report, we present an association study of 605032) and CPLX2 (OMIM 605033), respectively, and SYN2 and CPLX2 with schizophrenia using 12 polymor- are also called synaphins, are pre-synaptic membrane pro- phisms in the Korean population. teins that preferentially bind to syntaxin within the SNARE (soluble N-ethylmaleimide-sensitive fusion Results SYN2 polymorphisms in the schizophrenia and control attachment protein receptors) complex. These proteins are important regulators of transmitter release immedi- groups ately preceding vesicle fusion [7]. Previous studies have Of the seven polymorphisms in SYN2, rs2623873 (SYN2- demonstrated that the concentrations of synapsins and 1) is located in the promoter region, whereas the others complexins are reduced in the brains of schizophrenics are all located in the intronic regions (SYN2-2–7) (Fig. 1, Page 2 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 Table 1: Twelve polymorphisms genotyped in this study. Gene Name dbSNP rs# Region Allele Methods SYN2 SYN2-1 rs2623873 Promoter G/T Direct Sequencing SYN2 SYN2-2 rs2308169 Intron ATGCT/- Direct Sequencing SYN2 SYN2-3 rs308961 Intron T/G Direct Sequencing SYN2 SYN2-4 rs308963 Intron C/G Direct Sequencing SYN2 SYN2-5 rs308952 Intron A/G DdeI RFLP SYN2 SYN2-6 rs2279750 Intron A/C Direct Sequencing SYN2 SYN2-7 rs310762 Intron C/T Direct Sequencing CPLX2 CPLX2-1 rs2247916 Promoter G/T MaeIII RFLP CPLX2 CPLX2-2 rs2243404 5'UTR C/T Cac8I RFLP CPLX2 CPLX2-3 rs890736 Intron C/T AvaII RFLP CPLX2 CPLX2-4 rs890737 Intron C/T Direct Sequencing CPLX2 CPLX2-5 rs4390706 Intron G/A Direct Sequencing Tissue inhibitor of metalloproteinase 4 (Timp4) gene is nested within the intron of SYN2 in reverse orientation. Table 1). The genotypic distributions and allelic frequen- observed more frequently in schizophrenia than the con- cies of polymorphisms in SYN2 were determined in 113 trols (Table 4). schizophrenic patients and 114 normal healthy controls by direct sequencing or DdeI RFLP. The genotypic distri- We also investigated the association of three-way haplo- butions and allelic frequencies of polymorphisms in types formed by SYN2-1, SYN2-2, and SYN2-4 with schiz- SYN2 are shown in Table 2. The average allelic frequency ophrenia. A significant difference in the haplotype of the SNPs was 0.312. Given the equivalent frequency for frequencies between the schizophrenia and control 2 -5 the susceptible allele, the expected detection power for groups was observed (χ = 35.0, df = 7, P = 1.1 × 10 ). corr SYN2 was 0.9538 to 0.9929 under the multiplicative For the combination of SYN2-1, SYN2-2, and SYN2-4, the model with a genotype relative risk = 1.8 to 2.0 [22]. None estimated frequencies of the T-deletion-G haplotype dif- of the SNPs showed any significant deviation from Hardy- fered between the schizophrenia (0.570) and controls Weinberg equilibrium (P > 0.05). We observed no signif- groups (0.440). icant difference in the genotypic distributions and allelic frequencies between the schizophrenics and control CPLX2 polymorphisms in schizophrenia and control groups (Table 2). groups Of the five SNPs in CPLX2, rs2247916 (CPLX2-1) is located in the promoter region, rs2243404 (CPLX2-2) is We compared the LD for all possible two-way compari- sons of the SNPs in the controls (Table 3). The pairwise D' located in the 5'UTR, and the others are located in the values for the seven SNPs were consistently high, except in intronic regions (Fig. 1, Table 1). We determined the gen- one instance (SYN2-2 vs. SYN2-6; D' = 0.300, r = 0.200). otypic distributions and allelic frequencies of the SNPs in Out of the 21 possible pairs of SNPs, significant haplotype 154 schizophrenic patients and 133 normal healthy con- associations with schizophrenia were observed for 4 pairs: trols by direct sequencing or RFLP analysis. The genotypic 2 -6 SYN2-1 – SYN2-2 (χ = 27.58, df = 3, P = 4.45 × 10 ), distributions and allelic frequencies for CPLX2 SNPs are 2 -4 SYN2-2 – SYN2-4 (χ = 16.46, df = 3, P = 9.12 × 10 ), shown in Table 2. The average allelic frequency of the SYN2-2 – SYN2-7 (χ = 8.08, df = 3, P = 0.044), and SYN2- SNPs was 0.126. Given the equivalent frequency for the 3 – SYN2-4 (χ = 10.66, df = 3, P = 0.014) (Table 3). Even susceptible allele, the expected detection power for CPLX2 after the Bonferroni correction (number of haplotypes, n was 0.7445 to 0.8802 based on the multiplicative model = 21), the associations of the SYN2-1 – SYN2-2 and SYN2- with the genotype relative risk = 1.8 to 2.0 [22]. None of 2 – SYN2-4 haplotypes with schizophrenia remained sig- the five SNPs showed any significant deviations from -5 nificant (P = 9.35 × 10 and P = 0.019) (Table 3). The Hardy-Weinberg equilibrium. We observed no significant corr corr T allele-the deletion allele haplotype for the SYN2-1 – differences in genotypic distributions or allelic frequen- SYN2-2 combination and the deletion allele-the G allele cies between the schizophrenia and control groups (Table haplotype for the SYN2-2 – SYN2-4 combination were 2). Page 3 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 Table 2: Genotype distributions and allele frequencies of each polymorphism of the SYN2 and CPLX2 in the schizophrenia and control groups. Polymorphism Subjects Genotype distribution (frequency) Allele frequency a b 11 12 22 P 12 P SYN2-1 Cases (n = 113) 47 (0.416) 50 (0.442) 16 (0.142) 0.527 0.637 0.363 0.679 Controls (n = 114) 41 (0.360) 59 (0.518) 14 (0.122) 0.618 0.382 SYN2-2 Cases (n = 113) 40 (0.354) 55 (0.487) 18 (0.159) 0.758 0.597 0.403 0.438 Controls (n = 114) 36 (0.316) 56 (0.491) 22 (0.193) 0.561 0.439 SYN2-3 Cases (n = 113) 83 (0.735) 30 (0.265) 0 (0.000) 0.762 0.867 0.133 0.975 Controls (n = 114) 85 (0.746) 28 (0.246) 1 (0.009) 0.868 0.132 SYN2-4 Cases (n = 113) 49 (0.434) 45 (0.398) 19 (0.168) 0.722 0.633 0.367 0.612 Controls (n = 114) 44 (0.386) 51 (0.447) 19 (0.167) 0.610 0.390 SYN2-5 Cases (n = 113) 72 (0.637) 35 (0.310) 6 (0.053) 0.973 0.792 0.208 0.869 Controls (n = 114) 74 (0.649) 34 (0.298) 6 (0.053) 0.798 0.202 SYN2-6 Cases (n = 113) 67 (0.593) 39 (0.345) 7 (0.062) 0.786 0.765 0.235 0.622 Controls (n = 114) 63 (0.553) 44 (0.386) 7 (0.061) 0.746 0.254 SYN2-7 Cases (n = 113) 39 (0.345) 54 (0.478) 20 (0.177) 0.847 0.584 0.416 0.576 Controls (n = 114) 42 (0.368) 55 (0.482) 17 (0.149) 0.610 0.390 CPLX2-1 Cases (n = 154) 132 (0.857) 22 (0.143) 0 (0.000) 0.441 0.929 0.071 0.612 Controls (n = 133) 113 (0.850) 18 (0.135) 2 (0.015) 0.917 0.083 CPLX2-2 Cases (n = 154) 132 (0.857) 22 (0.143) 0 (0.000) 0.254 0.929 0.071 0.198 Controls (n = 133) 108 (0.812) 23 (0.173) 2 (0.015) 0.898 0.102 CPLX2-3 Cases (n = 154) 115 (0.747) 34 (0.221) 5 (0.032) 0.129 0.857 0.143 0.072 Controls (n = 133) 85 (0.639) 43 (0.323) 5 (0.038) 0.801 0.199 CPLX2-4 Cases (n = 154) 110 (0.714) 40 (0.260) 4 (0.026) 1.000 0.844 0.156 0.849 Controls (n = 133) 94 (0.707) 35 (0.263) 4 (0.030) 0.838 0.162 CPLX2-5 Cases (n = 154) 124 (0.805) 30 (0.195) 0 (0.000) 0.312 0.903 0.097 0.541 Controls (n = 133) 112 (0.842) 20 (0.150) 1 (0.008) 0.917 0.083 Fisher's exact probability tests, case vs controls (2 × 3 genotype-based analysis) Fisher's exact probability tests, case vs controls (2 × 2 allele-based analysis) Table 3: Pairwise linkage disequilibrium and haplotype association of SNPs in SYN2. SYN2-1 SYN2-2 SYN2-3 SYN2-4 SYN2-5 SYN2-6 SYN2-7 SYN2-1 0.532 0.776 0.758 0.865 0.516 0.642 0.222 0.056 0.553 0.306 0.482 0.397 -6 a SYN2-2 4.45 × 10 0.613 0.453 0.734 0.300 0.429 0.044 0.250 0.174 0.200 0.225 SYN2-3 0.246 0.632 1.000 0.997 0.480 1.000 0.097 0.038 0.433 0.097 -4 a SYN2-4 0.845 9.12 × 10 0.014 1.000 0.480 0.806 0.395 0.433 0.650 SYN2-5 0.896 0.655 0.135 0.133 0.938 0.865 0.651 0.295 SYN2-6 0.116 0.892 0.602 0.602 0.147 0.722 0.278 SYN2-7 0.646 0.044 0.155 0.392 0.404 0.0.72 2 2 Upper diagonal top: D', bottom: r in controls; Lower diagonal: P value by χ test (df = 3) P < 0.05 after Bonferroni correction Page 4 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 Table 4: Estimated haplotype frequencies of the SYN2-1 – SYN2-2 and SYN2-2 – SYN2-4 combination on the SYN2 Haplotypes Estimated frequency SYN2-1 SYN2-2 Cases Controls T Deletion 0.583 0.461 T ATGCT 0.054 0.158 G Deletion 0.014 0.100 G ATGCT 0.349 0.281 SYN2-2 SYN2-4 Cases Controls Deletion G 0.569 0.463 Deletion C 0.028 0.098 ATGCT G 0.064 0.146 ATGCT C 0.339 0.293 Table 5: Pairwise linkage disequilibrium and haplotype association of SNPs in CPLX2. CPLX2-1 CPLX2-2 CPLX2-3 CPLX2-4 CPLX2-5 CPLX2-1 0.412 0.325 0.058 0.008 0.136 0.300 0.001 0.008 -4 a CPLX2-2 9.0 × 10 0.309 0.715 0.301 0.042 0.011 0.001 CPLX2-3 0.494 0.056 0.027 0.019 0.015 0.016 CPLX2-4 0.994 0.830 0.564 0.143 0.000 CPLX2-5 1.000 0.564 0.650 0.992 2 2 Upper diagonal top: D', bottom: r in controls; Lower diagonal: P value by χ test (df = 3) P < 0.01 after Bonferroni correction Table 6: Estimated haplotype frequencies of the CPLX2-1 – CPLX2-2 combination on the CPLX2 Haplotypes Estimated frequency CPLX2-1 CPLX2-2 Cases Controls T C 0.010 0.036 T T 0.061 0.047 G C 0.919 0.862 G T 0.010 0.055 We compared LD for all possible two-way comparisons of (CPLX2-2 vs. CPLX2-4; D' = 0.715, r = 0.011). Only one the SNPs in controls (Table 5). The pairwise D' values for pair of SNPs (CPLX2-1 vs. CPLX2-2) showed a significant the five SNPs were consistently low, except in one instance haplotype association with schizophrenia (χ = 16.28, df Page 5 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 = 3, P = 0.0009), even after the Bonferroni correction (n = controvertible results have also been reported [25]. The 10, P = 0.009, Table 5). For the combination of CPLX2- altered expression levels of other presynaptic proteins, corr 1 – CPLX2-2, the G allele-the C allele haplotype was complexin 1 and complexin 2, have been reported in observed more frequently in the schizophrenia group schizophrenic patients [11-13]. Interestingly, complexin 1 than the control group (Table 6). is enriched in axosomatic regions, inhibitor neurons, and their synapses, while complexin 2 is enriched in the axo- Discussion dendritic terminals [9,26]. The differential expression of In this study, we observed significant, pairwise haplotype complexins 1 and 2 implies their involvement in the exci- associations with schizophrenia for two pairs of SNPs in tatory synapse in the hippocampus of schizophrenic SYN2 (SYN2-1 – SYN2-2 and SYN2-2 – SYN2-4; P = patients [11]. These observations suggest that abnormal corr -5 9.35 × 10 and P = 0.019, respectively) and one pair of expression of SYN2 and CPLX2 may cause the vulnerabil- corr SNPs in CPLX2 (CPLX2-1 – CPLX2-2, P = 0.009) (Table ity to schizophrenia by altering neurotransmitter release corr 3, 5). The three-way haplotype (SYN2-1, SYN2-2, and and neuroplasticity. SYN2-4) also showed a significant association with schiz- -5 = 1.1 × 10 ). The SYN2-1 and CPLX2-1 ophrenia (P Conclusion corr SNPs are located in the respective promoter regions, -98 We found significant differences in the haplotype fre- and -156. SYN2-1 was located within the GC box motif quencies in both SYN2 and CPLX2 polymorphisms and CPLX2-1 within the C/EBP motif in a database search between schizophrenia and control groups. In addition, http://www.cbrc.jp/research/db/TFSEARCH. The positive the haplotype constructed from three polymorphisms haplotype associations seem to be based on an increase of (SYN2-1, SYN2-2, and SYN2-4) showed a significant asso- LD in the schizophrenia group compared to the control ciation with schizophrenia. Our results suggest that both group because the D' values of the schizophrenia group SYN2 and CPLX2 polymorphisms may contribute suscep- were higher than those of the controls [(SYN2-1 – SYN2- tibility to schizophrenia in the Korean population. 2, 0.935 vs. 0.531 (schizophrenics vs. controls)), (SYN2-2 – SYN2-4, 0.750 vs. 0.453)] (Table 3). A similar situation Methods was also observed with the positive association of the Subjects A total of 154 unrelated Korean schizophrenia patients haplotype in CPLX2 with schizophrenia [CPLX2-1 – CPLX2-2, 0.852 vs. 0.412 (schizophrenics vs. controls)] (80 male and 74 female with a mean ± SD age of 43.8 ± (Table 5). 11.4 yr) and 133 unrelated Korean controls (64 male and 69 female; age 50.6 ± 11.7 yr) were recruited. For the Chen et al. [23] recently reported an association study of SYN2 analysis, 113 unrelated Korean schizophrenia four SNPs in SYN2 using Han Chinese samples. They patients (60 male and 53 female with a mean ± SD age of found significant associations of SNP rs795009 and a 42.2 ± 11.3 yr) and 114 unrelated Korean controls (60 haplotype constructed by the four SNPs with schizophre- male and 54 female; age 51.7 ± 10.9 yr) were participated. nia. Chen et al. [23] and our study examined two SNPs The schizophrenia patients were diagnosed using the (rs2308169 and rs308963) in common, and their geno- Diagnostic and Statistical Manual of Mental Disorders typic and allelic frequencies were similar in both studies. (DSM)-IV criteria. The control subjects were recruited after Although Chen et al. [23] did not mention the pairwise they had been designated as mentally healthy in a general haplotype association study that we performed, they did health check-up program. The average age of the controls report a significant difference in the overall four-way hap- exceeded 50 years because we tried to avoid misincorpo- lotype frequencies between schizophrenics and controls. ration of patients with late onset schizophrenia in the Since two independent studies have reported a significant control group, while it may produce statistical bias poten- haplotype association of SYN2 with schizophrenia, this tially. Written informed consent was obtained from all gene is probably involved in the pathogenesis of subjects. This study was approved by the Ethics Commit- schizophrenia. tee of Kyung Hee University, Faculty of Medicine. Genomic DNA was extracted from whole blood cells Several studies have suggested that the decreased expres- using a NucleoSpin Blood kit (Macherey-Nagel, Easton, sion of synaptic genes is characteristic of schizophrenia. In PA). the hippocampus of schizophrenic patients, several stud- ies have shown a consistent pattern of decreases in presy- SNP Selection and PCR-based Genotyping naptic proteins and their encoding mRNAs, such as Since the genomic sizes of SYN2 and CPLX2 are about 187 synapsin 2, synaptophysin, and synaptosomal-associated and 89 kb, respectively, we initially intended to select protein-25 (SNAP-25) [8-10,24]. Furthermore, a reduc- common polymorphisms at intervals of approximately tion in the synapsin 2 mRNA levels was observed in the 20–50 kb from the dbSNP http://www.ncbi.nlm.nih.gov/ prefrontal cortex of schizophrenic patients [14], but SNP/. After validating the frequency of each polymor- Page 6 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 6. Kao H-T, Porton B, Hilfiker S, Stefani G, Pieribone VA, DeSalle R: phism in 24 healthy Korean individuals using direct Molecular evolution of the synapsin gene family. J Exp Zool sequencing, we selected seven common polymorphisms (Mol Dev Evo) 1999, 285:360-377. from SYN2 and five from CPLX2 for further analyses (Fig. 7. Tokumaru H, Umayahara K, Pellegrini LL, Ishizuka T, Saisu H, Betz H: SNARE complex oligomerization by synaphin/complexin is 1, Table 1). We amplified the fragments containing poly- essential for synaptic vesicle exocytosis. Cell 2001, morphisms individually and genotyped DNA samples for 104:421-432. 8. Browning MD, Dudek EM, Rapier JL, Leonard S, Freedman R: Signif- each SNP with either PCR-based restriction fragment icant reductions in synapsin but not synaptophysin specific length polymorphism (RFLP) assays or direct sequencing activity in the brains of some schizophrenics. Biol Psychiatry performed with an ABI PRISM Dye Terminator Cycle 1993, 34:529-535. 9. Harrison PJ, Eastwood SL: Preferential involvement of excita- Sequencing kit (Applied Biosystems, Foster City, CA) on tory neurons in medial temporal lobe in schizophrenia. Lan- an ABI PRISM 3100 DNA sequencer (Applied Biosys- cet 1998, 352:1669-1673. tems) (Table 1). In case of unclear sequence data, we 10. Vawter MP, Thatcher L, Usen N, Hyde TM, Kleinman JE, Freed WJ: Reduction of synapsin in the hippocampus of patients with repeated direct sequencing under various conditions until bipolar disorder and schizophrenia. Mol Psychiatry 2002, the genotype was determined correctly. 7:571-578. 11. Eastwood SL, Harrison PJ: Hippocampal synaptic pathology in schizophrenia, bipolar disorder and major depression: a Statistics study of complexin mRNAs. Mol Psychiatry 2000, 5:425-432. The deviation of the genotypic frequencies from Hardy- 12. Eastwood SL, Cotter D, Harrison PJ: Cerebellar synaptic protein expression in schizophrenia. Neuroscience 2001, 105:219-229. Weinberg equilibrium was examined using the chi-square 13. Knable MB, Barci BM, Webster MJ, Meador-Woodruff L, Torrey EF: test (df = 1). Statistical differences in the genotypic distri- Molecular abnormalities of hippocampus in severe psychia- try illness: postmortem findings from the Stanley Neuropa- butions and allelic frequencies between the schizophrenia thology Consortium. Mol Psychiatry 2004, 9:609-620. and control groups were examined using the Fisher's exact 14. Mirnics K, Middleton FA, Marquez A, Lewis DA, Levitt P: Molecular probability test. We calculated D' and r to evaluate the characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex. Neuron 2000, magnitude of linkage disequilibrium (LD) [19]. We esti- 28:53-67. mated haplotype frequencies using the EH program, ver- 15. Karlin S, Chen C, Gentles AJ, Cleary M: Associations between sion 1.14 [20]. The statistical analysis of haplotype human disease genes and overlapping gene groups and mul- tiple amino acid runs. Proc Nat Acad Sci 2002, 99:17008-17013. association was done as previously described [21]. We 16. Lewis CM, Levinson DF, Wise LH, DeLisi LE, Straub RE, Hovatta I: applied the Bonferroni correction to multiple testing Genome scan meta-analysis of schizophrenia and bipolar dis- order, Part II: Schizophrenia. Am J Hum Genet 2003, 73:34-48. based on the number of haplotypes. The significance level 17. 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Nat Genet 2000, 25:324-328. bata and Y Fukumaki participated in its design, carried out 20. Xie X, Ott J: Testing linkage disequilibrium between a disease the statistical analyses, and participated in the interpreta- gene and marker loci. Am J Hum Genet 1993, 53:1170. 21. Sham P: The analysis of allelic association. In Statistics in Human tion of data. All authors read and approved the final Genetics Edited by: Sham P. London: Arnold; 1997:145-185. manuscript. 22. Ohashi J, Tokunaga K: The power of genome-wide association studies of complex disease genes: statistical limitations of indirect approaches using SNP markers. J Hum Genet 2001, Acknowledgements 46:478-482. This work was supported by JPSS and KOSEF, and in part by a grant of the 23. Chen Q, He G, Wang XY, Chen QY, Liu XM, Gu ZZ: Positive asso- Korean Health 21 R&D Project, Ministry of Health & Welfare, Republic of ciation between synapsin II and schizophrenia. Biol Psychiatry 2004, 56:177-181. Korea. (0405-NS01-0704-0001). 24. Thompson PM, Egbufoama S, Vawter MP: SNAP-25 reduction in the hippocampus of patients with schizophrenia. Prog Neu- References ropsychopharmacol Biol Psychiatry 2003, 27:411-417. 1. Lewis DA, Lieberman JA: Catching up on schizophrenia: natural 25. Imai C, Sugai T, Iritani S, Niizato K, Nakamura R, Makifuchi T: A history and neurobiology. Neuron 2002, 28:325-334. quantitative study on the expression of synapsin II and N- 2. Owen MJ, O'Donovan MC, Gottesman II: Schizophrenia. In Psychi- ethylmaleimide-sensitive fusion protein in schizophrenic atric Genetics & Genomics Edited by: McGuffin P, Owen MJ, Gottesman patients. NeuroSci Lett 2001, 305:185-188. II. Oxford; 2002:247-266. 26. Takahashi S, Yamamoto H, Matsuda Z, Ogawa M, Yagyu K, Taniguchi 3. Owen MJ, Williams NM, O'Donovan MC: The molecular genetics T: Identification of two highly homologous presynaptic pro- of schizophrenia: new findings promise new insights. Mol teins distinctly localized at the dendritic and somatic Psychiatry 2004, 9:14-27. synapses. FEBS Lett 1995, 368:455-460. 4. Frankle WG, Lerma J, Laruelle M: The synaptic hypothesis of schizophrenia. Neuron 2003, 39:205-215. 5. Hilfiker S, Pieribone VA, Czernik AJ, Kao H-T, Augustine GJ, Green- gard P: Synapsins as regulators of neurotransmitter release. Phil Trans R Soc Lond B 1999, 354:269-279. Page 7 of 7 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral and Brain Functions Springer Journals

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

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Copyright © 2005 by Lee et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
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Abstract

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 contribution of genetic factors to the vulnerability to Schizophrenia is a severe, chronic mental illness affecting schizophrenia has been well established by family, twin, 0.5–1.5% of the general population worldwide [1]. The and adoption studies that have suggested a significant Page 1 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 Genomic or Figure 1 ganization of SYN2 and CPLX2 and locations of SNPs 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. heritability of approximately 50–70% [2]. Many studies [8,9]. The expression levels of both synapsins were signif- have attempted to identify the allelic variants that confer icantly decreased in the hippocampal tissue of schizo- susceptibility to the illness, but no single genes have been phrenic patients [10]. The levels of synapsin 2 and identified that produce a major effect on the vulnerability complexin 2 mRNA were also significantly reduced in the [3]. prefrontal cortex, cerebellum, and hippocampus of schiz- ophrenics [11-14]. Recently the synaptic hypothesis of schizophrenia has gained attention by attributing the fundamental pathol- SYN2 was mapped to chromosome 3p25 [15], and CPLX2 ogy of schizophrenia to the dysfunction of synaptic trans- is located on chromosome 5q35.3 (OMIM 605033). mission involving various molecules [4]. Synapsins, a These loci were identified as potential regions conferring family of synaptic vesicle-associated phosphoproteins, susceptibility to schizophrenia in diverse populations play a crucial role in the regulation of neurotransmission, [16-18]. Based on their localization, well-established neu- synaptogenesis, and neuronal plasticity [5]. Three human robiological roles, and expression patterns in schizo- synapsin genes have been identified (SYN1, 2, and 3; phrenic patients, we selected SYN2 and CPLX2 as OMIM 313440, 600755, and 602705) [6]. Complexin 1 candidate genes for conferring susceptibility to schizo- and complexin 2, which are encoded by CPLX1 (OMIM phrenia. In this report, we present an association study of 605032) and CPLX2 (OMIM 605033), respectively, and SYN2 and CPLX2 with schizophrenia using 12 polymor- are also called synaphins, are pre-synaptic membrane pro- phisms in the Korean population. teins that preferentially bind to syntaxin within the SNARE (soluble N-ethylmaleimide-sensitive fusion Results SYN2 polymorphisms in the schizophrenia and control attachment protein receptors) complex. These proteins are important regulators of transmitter release immedi- groups ately preceding vesicle fusion [7]. Previous studies have Of the seven polymorphisms in SYN2, rs2623873 (SYN2- demonstrated that the concentrations of synapsins and 1) is located in the promoter region, whereas the others complexins are reduced in the brains of schizophrenics are all located in the intronic regions (SYN2-2–7) (Fig. 1, Page 2 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 Table 1: Twelve polymorphisms genotyped in this study. Gene Name dbSNP rs# Region Allele Methods SYN2 SYN2-1 rs2623873 Promoter G/T Direct Sequencing SYN2 SYN2-2 rs2308169 Intron ATGCT/- Direct Sequencing SYN2 SYN2-3 rs308961 Intron T/G Direct Sequencing SYN2 SYN2-4 rs308963 Intron C/G Direct Sequencing SYN2 SYN2-5 rs308952 Intron A/G DdeI RFLP SYN2 SYN2-6 rs2279750 Intron A/C Direct Sequencing SYN2 SYN2-7 rs310762 Intron C/T Direct Sequencing CPLX2 CPLX2-1 rs2247916 Promoter G/T MaeIII RFLP CPLX2 CPLX2-2 rs2243404 5'UTR C/T Cac8I RFLP CPLX2 CPLX2-3 rs890736 Intron C/T AvaII RFLP CPLX2 CPLX2-4 rs890737 Intron C/T Direct Sequencing CPLX2 CPLX2-5 rs4390706 Intron G/A Direct Sequencing Tissue inhibitor of metalloproteinase 4 (Timp4) gene is nested within the intron of SYN2 in reverse orientation. Table 1). The genotypic distributions and allelic frequen- observed more frequently in schizophrenia than the con- cies of polymorphisms in SYN2 were determined in 113 trols (Table 4). schizophrenic patients and 114 normal healthy controls by direct sequencing or DdeI RFLP. The genotypic distri- We also investigated the association of three-way haplo- butions and allelic frequencies of polymorphisms in types formed by SYN2-1, SYN2-2, and SYN2-4 with schiz- SYN2 are shown in Table 2. The average allelic frequency ophrenia. A significant difference in the haplotype of the SNPs was 0.312. Given the equivalent frequency for frequencies between the schizophrenia and control 2 -5 the susceptible allele, the expected detection power for groups was observed (χ = 35.0, df = 7, P = 1.1 × 10 ). corr SYN2 was 0.9538 to 0.9929 under the multiplicative For the combination of SYN2-1, SYN2-2, and SYN2-4, the model with a genotype relative risk = 1.8 to 2.0 [22]. None estimated frequencies of the T-deletion-G haplotype dif- of the SNPs showed any significant deviation from Hardy- fered between the schizophrenia (0.570) and controls Weinberg equilibrium (P > 0.05). We observed no signif- groups (0.440). icant difference in the genotypic distributions and allelic frequencies between the schizophrenics and control CPLX2 polymorphisms in schizophrenia and control groups (Table 2). groups Of the five SNPs in CPLX2, rs2247916 (CPLX2-1) is located in the promoter region, rs2243404 (CPLX2-2) is We compared the LD for all possible two-way compari- sons of the SNPs in the controls (Table 3). The pairwise D' located in the 5'UTR, and the others are located in the values for the seven SNPs were consistently high, except in intronic regions (Fig. 1, Table 1). We determined the gen- one instance (SYN2-2 vs. SYN2-6; D' = 0.300, r = 0.200). otypic distributions and allelic frequencies of the SNPs in Out of the 21 possible pairs of SNPs, significant haplotype 154 schizophrenic patients and 133 normal healthy con- associations with schizophrenia were observed for 4 pairs: trols by direct sequencing or RFLP analysis. The genotypic 2 -6 SYN2-1 – SYN2-2 (χ = 27.58, df = 3, P = 4.45 × 10 ), distributions and allelic frequencies for CPLX2 SNPs are 2 -4 SYN2-2 – SYN2-4 (χ = 16.46, df = 3, P = 9.12 × 10 ), shown in Table 2. The average allelic frequency of the SYN2-2 – SYN2-7 (χ = 8.08, df = 3, P = 0.044), and SYN2- SNPs was 0.126. Given the equivalent frequency for the 3 – SYN2-4 (χ = 10.66, df = 3, P = 0.014) (Table 3). Even susceptible allele, the expected detection power for CPLX2 after the Bonferroni correction (number of haplotypes, n was 0.7445 to 0.8802 based on the multiplicative model = 21), the associations of the SYN2-1 – SYN2-2 and SYN2- with the genotype relative risk = 1.8 to 2.0 [22]. None of 2 – SYN2-4 haplotypes with schizophrenia remained sig- the five SNPs showed any significant deviations from -5 nificant (P = 9.35 × 10 and P = 0.019) (Table 3). The Hardy-Weinberg equilibrium. We observed no significant corr corr T allele-the deletion allele haplotype for the SYN2-1 – differences in genotypic distributions or allelic frequen- SYN2-2 combination and the deletion allele-the G allele cies between the schizophrenia and control groups (Table haplotype for the SYN2-2 – SYN2-4 combination were 2). Page 3 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 Table 2: Genotype distributions and allele frequencies of each polymorphism of the SYN2 and CPLX2 in the schizophrenia and control groups. Polymorphism Subjects Genotype distribution (frequency) Allele frequency a b 11 12 22 P 12 P SYN2-1 Cases (n = 113) 47 (0.416) 50 (0.442) 16 (0.142) 0.527 0.637 0.363 0.679 Controls (n = 114) 41 (0.360) 59 (0.518) 14 (0.122) 0.618 0.382 SYN2-2 Cases (n = 113) 40 (0.354) 55 (0.487) 18 (0.159) 0.758 0.597 0.403 0.438 Controls (n = 114) 36 (0.316) 56 (0.491) 22 (0.193) 0.561 0.439 SYN2-3 Cases (n = 113) 83 (0.735) 30 (0.265) 0 (0.000) 0.762 0.867 0.133 0.975 Controls (n = 114) 85 (0.746) 28 (0.246) 1 (0.009) 0.868 0.132 SYN2-4 Cases (n = 113) 49 (0.434) 45 (0.398) 19 (0.168) 0.722 0.633 0.367 0.612 Controls (n = 114) 44 (0.386) 51 (0.447) 19 (0.167) 0.610 0.390 SYN2-5 Cases (n = 113) 72 (0.637) 35 (0.310) 6 (0.053) 0.973 0.792 0.208 0.869 Controls (n = 114) 74 (0.649) 34 (0.298) 6 (0.053) 0.798 0.202 SYN2-6 Cases (n = 113) 67 (0.593) 39 (0.345) 7 (0.062) 0.786 0.765 0.235 0.622 Controls (n = 114) 63 (0.553) 44 (0.386) 7 (0.061) 0.746 0.254 SYN2-7 Cases (n = 113) 39 (0.345) 54 (0.478) 20 (0.177) 0.847 0.584 0.416 0.576 Controls (n = 114) 42 (0.368) 55 (0.482) 17 (0.149) 0.610 0.390 CPLX2-1 Cases (n = 154) 132 (0.857) 22 (0.143) 0 (0.000) 0.441 0.929 0.071 0.612 Controls (n = 133) 113 (0.850) 18 (0.135) 2 (0.015) 0.917 0.083 CPLX2-2 Cases (n = 154) 132 (0.857) 22 (0.143) 0 (0.000) 0.254 0.929 0.071 0.198 Controls (n = 133) 108 (0.812) 23 (0.173) 2 (0.015) 0.898 0.102 CPLX2-3 Cases (n = 154) 115 (0.747) 34 (0.221) 5 (0.032) 0.129 0.857 0.143 0.072 Controls (n = 133) 85 (0.639) 43 (0.323) 5 (0.038) 0.801 0.199 CPLX2-4 Cases (n = 154) 110 (0.714) 40 (0.260) 4 (0.026) 1.000 0.844 0.156 0.849 Controls (n = 133) 94 (0.707) 35 (0.263) 4 (0.030) 0.838 0.162 CPLX2-5 Cases (n = 154) 124 (0.805) 30 (0.195) 0 (0.000) 0.312 0.903 0.097 0.541 Controls (n = 133) 112 (0.842) 20 (0.150) 1 (0.008) 0.917 0.083 Fisher's exact probability tests, case vs controls (2 × 3 genotype-based analysis) Fisher's exact probability tests, case vs controls (2 × 2 allele-based analysis) Table 3: Pairwise linkage disequilibrium and haplotype association of SNPs in SYN2. SYN2-1 SYN2-2 SYN2-3 SYN2-4 SYN2-5 SYN2-6 SYN2-7 SYN2-1 0.532 0.776 0.758 0.865 0.516 0.642 0.222 0.056 0.553 0.306 0.482 0.397 -6 a SYN2-2 4.45 × 10 0.613 0.453 0.734 0.300 0.429 0.044 0.250 0.174 0.200 0.225 SYN2-3 0.246 0.632 1.000 0.997 0.480 1.000 0.097 0.038 0.433 0.097 -4 a SYN2-4 0.845 9.12 × 10 0.014 1.000 0.480 0.806 0.395 0.433 0.650 SYN2-5 0.896 0.655 0.135 0.133 0.938 0.865 0.651 0.295 SYN2-6 0.116 0.892 0.602 0.602 0.147 0.722 0.278 SYN2-7 0.646 0.044 0.155 0.392 0.404 0.0.72 2 2 Upper diagonal top: D', bottom: r in controls; Lower diagonal: P value by χ test (df = 3) P < 0.05 after Bonferroni correction Page 4 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 Table 4: Estimated haplotype frequencies of the SYN2-1 – SYN2-2 and SYN2-2 – SYN2-4 combination on the SYN2 Haplotypes Estimated frequency SYN2-1 SYN2-2 Cases Controls T Deletion 0.583 0.461 T ATGCT 0.054 0.158 G Deletion 0.014 0.100 G ATGCT 0.349 0.281 SYN2-2 SYN2-4 Cases Controls Deletion G 0.569 0.463 Deletion C 0.028 0.098 ATGCT G 0.064 0.146 ATGCT C 0.339 0.293 Table 5: Pairwise linkage disequilibrium and haplotype association of SNPs in CPLX2. CPLX2-1 CPLX2-2 CPLX2-3 CPLX2-4 CPLX2-5 CPLX2-1 0.412 0.325 0.058 0.008 0.136 0.300 0.001 0.008 -4 a CPLX2-2 9.0 × 10 0.309 0.715 0.301 0.042 0.011 0.001 CPLX2-3 0.494 0.056 0.027 0.019 0.015 0.016 CPLX2-4 0.994 0.830 0.564 0.143 0.000 CPLX2-5 1.000 0.564 0.650 0.992 2 2 Upper diagonal top: D', bottom: r in controls; Lower diagonal: P value by χ test (df = 3) P < 0.01 after Bonferroni correction Table 6: Estimated haplotype frequencies of the CPLX2-1 – CPLX2-2 combination on the CPLX2 Haplotypes Estimated frequency CPLX2-1 CPLX2-2 Cases Controls T C 0.010 0.036 T T 0.061 0.047 G C 0.919 0.862 G T 0.010 0.055 We compared LD for all possible two-way comparisons of (CPLX2-2 vs. CPLX2-4; D' = 0.715, r = 0.011). Only one the SNPs in controls (Table 5). The pairwise D' values for pair of SNPs (CPLX2-1 vs. CPLX2-2) showed a significant the five SNPs were consistently low, except in one instance haplotype association with schizophrenia (χ = 16.28, df Page 5 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 = 3, P = 0.0009), even after the Bonferroni correction (n = controvertible results have also been reported [25]. The 10, P = 0.009, Table 5). For the combination of CPLX2- altered expression levels of other presynaptic proteins, corr 1 – CPLX2-2, the G allele-the C allele haplotype was complexin 1 and complexin 2, have been reported in observed more frequently in the schizophrenia group schizophrenic patients [11-13]. Interestingly, complexin 1 than the control group (Table 6). is enriched in axosomatic regions, inhibitor neurons, and their synapses, while complexin 2 is enriched in the axo- Discussion dendritic terminals [9,26]. The differential expression of In this study, we observed significant, pairwise haplotype complexins 1 and 2 implies their involvement in the exci- associations with schizophrenia for two pairs of SNPs in tatory synapse in the hippocampus of schizophrenic SYN2 (SYN2-1 – SYN2-2 and SYN2-2 – SYN2-4; P = patients [11]. These observations suggest that abnormal corr -5 9.35 × 10 and P = 0.019, respectively) and one pair of expression of SYN2 and CPLX2 may cause the vulnerabil- corr SNPs in CPLX2 (CPLX2-1 – CPLX2-2, P = 0.009) (Table ity to schizophrenia by altering neurotransmitter release corr 3, 5). The three-way haplotype (SYN2-1, SYN2-2, and and neuroplasticity. SYN2-4) also showed a significant association with schiz- -5 = 1.1 × 10 ). The SYN2-1 and CPLX2-1 ophrenia (P Conclusion corr SNPs are located in the respective promoter regions, -98 We found significant differences in the haplotype fre- and -156. SYN2-1 was located within the GC box motif quencies in both SYN2 and CPLX2 polymorphisms and CPLX2-1 within the C/EBP motif in a database search between schizophrenia and control groups. In addition, http://www.cbrc.jp/research/db/TFSEARCH. The positive the haplotype constructed from three polymorphisms haplotype associations seem to be based on an increase of (SYN2-1, SYN2-2, and SYN2-4) showed a significant asso- LD in the schizophrenia group compared to the control ciation with schizophrenia. Our results suggest that both group because the D' values of the schizophrenia group SYN2 and CPLX2 polymorphisms may contribute suscep- were higher than those of the controls [(SYN2-1 – SYN2- tibility to schizophrenia in the Korean population. 2, 0.935 vs. 0.531 (schizophrenics vs. controls)), (SYN2-2 – SYN2-4, 0.750 vs. 0.453)] (Table 3). A similar situation Methods was also observed with the positive association of the Subjects A total of 154 unrelated Korean schizophrenia patients haplotype in CPLX2 with schizophrenia [CPLX2-1 – CPLX2-2, 0.852 vs. 0.412 (schizophrenics vs. controls)] (80 male and 74 female with a mean ± SD age of 43.8 ± (Table 5). 11.4 yr) and 133 unrelated Korean controls (64 male and 69 female; age 50.6 ± 11.7 yr) were recruited. For the Chen et al. [23] recently reported an association study of SYN2 analysis, 113 unrelated Korean schizophrenia four SNPs in SYN2 using Han Chinese samples. They patients (60 male and 53 female with a mean ± SD age of found significant associations of SNP rs795009 and a 42.2 ± 11.3 yr) and 114 unrelated Korean controls (60 haplotype constructed by the four SNPs with schizophre- male and 54 female; age 51.7 ± 10.9 yr) were participated. nia. Chen et al. [23] and our study examined two SNPs The schizophrenia patients were diagnosed using the (rs2308169 and rs308963) in common, and their geno- Diagnostic and Statistical Manual of Mental Disorders typic and allelic frequencies were similar in both studies. (DSM)-IV criteria. The control subjects were recruited after Although Chen et al. [23] did not mention the pairwise they had been designated as mentally healthy in a general haplotype association study that we performed, they did health check-up program. The average age of the controls report a significant difference in the overall four-way hap- exceeded 50 years because we tried to avoid misincorpo- lotype frequencies between schizophrenics and controls. ration of patients with late onset schizophrenia in the Since two independent studies have reported a significant control group, while it may produce statistical bias poten- haplotype association of SYN2 with schizophrenia, this tially. Written informed consent was obtained from all gene is probably involved in the pathogenesis of subjects. This study was approved by the Ethics Commit- schizophrenia. tee of Kyung Hee University, Faculty of Medicine. Genomic DNA was extracted from whole blood cells Several studies have suggested that the decreased expres- using a NucleoSpin Blood kit (Macherey-Nagel, Easton, sion of synaptic genes is characteristic of schizophrenia. In PA). the hippocampus of schizophrenic patients, several stud- ies have shown a consistent pattern of decreases in presy- SNP Selection and PCR-based Genotyping naptic proteins and their encoding mRNAs, such as Since the genomic sizes of SYN2 and CPLX2 are about 187 synapsin 2, synaptophysin, and synaptosomal-associated and 89 kb, respectively, we initially intended to select protein-25 (SNAP-25) [8-10,24]. Furthermore, a reduc- common polymorphisms at intervals of approximately tion in the synapsin 2 mRNA levels was observed in the 20–50 kb from the dbSNP http://www.ncbi.nlm.nih.gov/ prefrontal cortex of schizophrenic patients [14], but SNP/. After validating the frequency of each polymor- Page 6 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2005, 1:15 http://www.behavioralandbrainfunctions.com/content/1/1/15 6. Kao H-T, Porton B, Hilfiker S, Stefani G, Pieribone VA, DeSalle R: phism in 24 healthy Korean individuals using direct Molecular evolution of the synapsin gene family. J Exp Zool sequencing, we selected seven common polymorphisms (Mol Dev Evo) 1999, 285:360-377. from SYN2 and five from CPLX2 for further analyses (Fig. 7. Tokumaru H, Umayahara K, Pellegrini LL, Ishizuka T, Saisu H, Betz H: SNARE complex oligomerization by synaphin/complexin is 1, Table 1). We amplified the fragments containing poly- essential for synaptic vesicle exocytosis. Cell 2001, morphisms individually and genotyped DNA samples for 104:421-432. 8. Browning MD, Dudek EM, Rapier JL, Leonard S, Freedman R: Signif- each SNP with either PCR-based restriction fragment icant reductions in synapsin but not synaptophysin specific length polymorphism (RFLP) assays or direct sequencing activity in the brains of some schizophrenics. Biol Psychiatry performed with an ABI PRISM Dye Terminator Cycle 1993, 34:529-535. 9. Harrison PJ, Eastwood SL: Preferential involvement of excita- Sequencing kit (Applied Biosystems, Foster City, CA) on tory neurons in medial temporal lobe in schizophrenia. Lan- an ABI PRISM 3100 DNA sequencer (Applied Biosys- cet 1998, 352:1669-1673. tems) (Table 1). In case of unclear sequence data, we 10. Vawter MP, Thatcher L, Usen N, Hyde TM, Kleinman JE, Freed WJ: Reduction of synapsin in the hippocampus of patients with repeated direct sequencing under various conditions until bipolar disorder and schizophrenia. Mol Psychiatry 2002, the genotype was determined correctly. 7:571-578. 11. Eastwood SL, Harrison PJ: Hippocampal synaptic pathology in schizophrenia, bipolar disorder and major depression: a Statistics study of complexin mRNAs. Mol Psychiatry 2000, 5:425-432. The deviation of the genotypic frequencies from Hardy- 12. Eastwood SL, Cotter D, Harrison PJ: Cerebellar synaptic protein expression in schizophrenia. Neuroscience 2001, 105:219-229. Weinberg equilibrium was examined using the chi-square 13. Knable MB, Barci BM, Webster MJ, Meador-Woodruff L, Torrey EF: test (df = 1). Statistical differences in the genotypic distri- Molecular abnormalities of hippocampus in severe psychia- try illness: postmortem findings from the Stanley Neuropa- butions and allelic frequencies between the schizophrenia thology Consortium. Mol Psychiatry 2004, 9:609-620. and control groups were examined using the Fisher's exact 14. 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