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Positive association between SIAT8B and schizophrenia in the Chinese Han population
Schizophrenia Research 90 (2007) 108 – 114 www.elsevier.com/locate/schres
Positive association between SIAT8B and schizophrenia in the Chinese Han population Ran Tao a,b,1 , Chao Li a,b,1 , Yonglan Zheng a,b , Wei Qin a,b , Jing Zhang a,b , Xingwang Li a,b , Yifeng Xu c , Yong Yong Shi a,b , Guoyin Feng c , Lin He b,d,⁎ a
d
Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China b Bio-X Life Science Research Center, Shanghai Jiao Tong University, Shanghai 200030, China c Shanghai Institute of Mental Health, Shanghai 200030, China NHGG, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Received 6 September 2006; received in revised form 25 September 2006; accepted 27 September 2006 Available online 28 November 2006
Abstract The Sialyltransferase 8B gene (SIAT8B) is located at 15q26, a susceptibility region for both schizophrenia and bipolar disorder. The protein encoded by this gene has an important role in neural development and sialic acid synthesis on the neural cell adhesion molecule (NCAM). Previous research had indicated that the promoter region of SIAT8B is associated with schizophrenia in the Japanese population. To take this further we carried out an association study based on 643 unrelated schizophrenics and 527 unrelated healthy subjects, all Han Chinese, recruited from Shanghai. Although our results differed from those of the Japanese research, rs3759915, also located in the promoter region of SIAT8B, showed nominally significant association with schizophrenia (P = 0.0036). Moreover, haplotypes constructed from rs3759915 and another two SNPs reported in the Japanese study (rs3759914 and rs3759916, also located in promoter region of SIAT8B) which located in the same LD block were significantly associated with schizophrenia (global P = 0.0000050). Our findings indicate that SIAT8B may be a candidate susceptibility gene for schizophrenia in the Chinese Han population and may also provide further support for the potential importance of polysaccharide-synthesizing genes in the etiology of schizophrenia. © 2006 Elsevier B.V. All rights reserved. Keywords: SIAT8B; SNP; Haplotype; Association; Chinese Han population
1. Introduction Schizophrenia (OMIM 181500) is a chronic psychotic disorder with a lifetime prevalence of approximately ⁎ Corresponding author. Bio-X Center, Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China; or Shanghai Jiao Tong University, PO Box 501, Hao Ran Building, 1954 Hua Shan Road, Shanghai 200030, China. Tel./fax: +86 21 62822491. E-mail address: [email protected] (L. He). 1 Contributed equally to this work. 0920-9964/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2006.09.029
1%. The Sialyltransferase 8B (SIAT8B) gene localizes on chromosome 15q26, a novel positive region reported in a genome scan of Eastern Quebec families (Maziade et al., 2005). The protein encoded by this gene is thought to catalyze the transfer of sialic acid from CMP-sialic acid to N-linked oligosaccharides and glycoproteins. The adhesive properties of the neural cell adhesion molecule (NCAM1) are modulated by its linkage to polysialic acid (PSA). This linkage is regulated by sialyltransferases, including sialyltransferase 8B (SIAT8B) (Angata et al., 2000). PSA ligated-NCAM1 (PSA-NCAM1) is widely
R. Tao et al. / Schizophrenia Research 90 (2007) 108–114
expressed in the embryonic and neonatal brain, but expression is highly restricted in the adult central nervous system to areas such as the hippocampus and olfactory bulb, that maintain a permanent capacity for neurogenesis (Kiss and Muller, 2001; Kiss et al., 2001; Seki and Arai, 1993). It has been reported that cells cotransfected with NCAM1 and SIAT8B can support neurite outgrowth better than cells transfected with NCAM1 alone (Angata et al., 1997). Barbeau et al. observed a 20–95% reduction in the number of hilar PSA-NCAM-immunoreactive cells in the great majority of schizophrenic brains (Barbeau et al., 1995). The most conspicuous phenotype of Ncam1-deficit mice is in the olfactory bulb, where granule cells are both reduced in number and disorganized (Tomasiewicz et al., 1993). Olfactory impairments are common in schizophrenia and low olfactory bulb volumes have been reported in first-degree relatives of schizophrenics (Turetsky et al., 2003). Structural abnormalities of the olfactory system in schizophrenia may partly reflect preexisting genetic vulnerability to the illness. SIAT8B-deficient mice also show a PSA deficit
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in brain regions of neurogenesis, and this deficiency results in the misguidance of infrapyramidal mossy fibers and the formation of ectopic synapses in the hippocampus (Angata et al., 2004). Disturbances of the anatomical organization and hippocampal function have long been implicated in the etiology of schizophrenia (Harrison, 2004). All indications above mentioned implied that SIAT8B is one of candidate genes for schizophrenia. In present study, we evaluated the potential association between SIAT8B and schizophrenia by a case-control study. 2. Materials and methods 2.1. Subjects For the case-control study, we recruited 643 unrelated schizophrenics (means age 45.54, SD = 14.70) from the Shanghai Mental Center, of whom 346 were male and 297 female. The average onset age of disease was 25.06 years (SD = 10.30). All the schizophrenia patients were in-patients and had been diagnosed according to the
Table 1 Allele and genotype frequencies of the nine SNPs within SIAT8B Allele frequency (%)
rs3759916 Cases Controls rs3759915b Cases Controls rs3759914 Cases Controls rs3848153 Cases Controls rs3931230 Cases Controls rs8035191 Cases Controls rs2168351 Cases Controls rs3784727 Cases Controls rs3784724 Cases Controls a
Genotype frequency (%) a
Allele 1
Allele 2
P (df = 1)
OR (95%CI)
11
12
22
P (df = 2)a
T 850 (66.1) 677 (64.2) C 666 (51.8) 482 (45.7) T 870 (67.7) 755 (71.6) T 879 (68.4) 708 (67.2) A 876 (68.1) 691 (65.6) C 927 (72.1) 758 (71.9) G 847 (65.9) 686 (65.1) G 725 (56.4) 567 (53.8) C 816 (63.5) 701 (66.5)
C 436 (33.9) 377 (35.8) G 620 (48.2) 572 (54.3) C 416 (32.3) 299 (28.4) C 407 (31.6) 346 (32.8) C 410 (31.9) 363 (34.4) T 359 (27.9) 296 (28.1) A 439 (34.1) 368 (34.9) C 561 (43.6) 487 (46.2) G 470 (36.5) 353 (33.5)
0.36
0.92 (0.78–1.09)
1.27 (1.08–1.50)
0.039
1.21 (1.01–1.44)
0.56
1.06 (0.89–1.26)
0.20
1.12 (0.94–1.33)
0.96
1.01 (0.84–1.21)
0.70
1.04 (0.87–1.23)
0.23
1.11 (0.94–1.31)
0.13
1.14 (0.96–1.36)
TC 274 (42.6) 249 (47.2) CG 312 (48.5) 254 (48.2) TC 290 (45.1) 201 (38.1) TC 279 (43.4) 230 (43.6) AC 280 (43.5) 225 (42.7) CT 259 (40.3) 202 (38.3) GA 297 (46.2) 256 (48.6) GC 307 (47.7) 281 (53.3) CG 286 (44.5) 221 (41.9)
CC 81 (12.6) 64 (12.1) GG 154 (24.0) 159 (30.2) CC 63 (9.7) 49 (9.3) CC 64 (10.0) 58 (11.0) CC 65 (10.1) 69 (13.1) TT 50 (7.8) 47 (8.9) AA 71 (11.0) 56 (10.6) CC 127 (19.8) 103 (19.5) GG 92 (14.3) 66 (12.5)
0.27
0.0036
TT 288 (44.8) 214 (40.6) CC 177 (27.5) 114 (21.6) TT 290 (45.1) 277 (52.6) TT 300 (46.7) 239 (45.4) AA 298 (46.3) 233 (44.2) CC 334 (51.9) 278 (52.8) GG 275 (42.8) 215 (40.8) GG 209 (32.5) 143 (27.1) CC 265 (41.2) 240 (45.5)
Significant P values (<0.05) are in boldface. b Context sequence of rs3759915: CGAGGGCGGA
GGAAGGCGGC.
0.016
0.035
0.81
0.28
0.69
0.72
0.10
0.31
110
R. Tao et al. / Schizophrenia Research 90 (2007) 108–114
criteria of the Diagnostic and Statistical Manual of Mental Disorder, Third Revised Edition (DSM-III-R). Diagnosis and review of psychiatric case records were independently checked and verified by two senior psychiatrists. 527 unrelated healthy subjects (280 males and 247 females with a mean age of 35.34 years, SD = 8.71) screened for absence of major mental illness were recruited from the same geographic region as case subjects. These were recruited from blood donors and were interviewed to establish that relatives within the third degree were also free from psychoses. Written informed consents, reviewed and approved by the Shanghai Ethical Committee of Human Genetic Resources, were obtained from all participating subjects after the nature of study had been fully explained. All subjects were Han Chinese in origin. 2.2. Single nucleotide polymorphism genotyping To replicate positive results of Arai et al., rs3759916 (-1126T > C; Arai et al., 2006), rs3759915 (-907G > C; Arai et al., 2006) and rs3759914 (-851T > C; Arai et al., 2006) in promoter region were recruited. In addition, according to Chinese population data of Hapmap (www. hapmap.org) and our pre-processing experiment (genotyped candidate SNPs in 48 random individuals of our samples population), we also selected other six common SNPs (rs3848153, rs3931230, rs8035191, rs2168351, rs3784727 and rs3784724) (Minor Allele Frequency, MAF > 0.2) to cover the region of SIAT8B. Genomic DNA was isolated from peripheral blood samples of the subjects using the phenol-chloroform method. Three SNPs in the promoter region were genotyped by direct DNA sequencing. Polymerase chain reaction (PCR) amplification was carried out in a final volume of 15 μl, containing 10 ng genomic DNA, 0.2 μM of each primer, 2.5 mM MgCl2, 2 mM deoxynucleoside triphosphate (dNTP), 1.5 μl of 10 × buffer (Qiagen, Basel, Switzerland), and 1 U HotStar Taq polymerase (Qiagen, Basel, Switzerland). Thermocycling was performed on a Gene Amp PCR system 9700 (Applied Biosystem, Foster City, CA) according to a modified touchdown program, with an initial denaturation at 95 °C for 5 min, followed by 44 cycles of denaturation at 95 °C for 30 s and extension at 72 °C for 45 s. The annealing temperature was decreased from 63 °C by 0.5 °C per cycle for a total of 14 cycles, followed by 30 cycles at the final annealing temperature of 56 °C and final extension of 10 min at 72 °C. The PCR products for sequencing were incubated with 0.1 U shrimp alkaline phosphatase (Roche, Basel, Switzerland) at 37 °C for 45 min, followed by heat
inactivation at 85 °C for 20 min. The treated PCR products were then sequenced using an ABI Prism BigDye Terminator Cycle Sequencing Kit, version 3.1 (Applied Biosystems) on an ABI Prism 3100 sequencer (Applied Biosystems). Six SNPs were genotyped by Single Nucleotide Primer Extension (SNuPE). To amplify the region under investigation PCR was carried out according to the above protocol. The PCR products were treated with Exonuclease I and Shrimp Alkaline Phosphatase (Exo/ SAP) according to the manufacturer's protocol. SNuPE reaction was then performed according to the protocol of MegaBACE SNuPE Genotyping Kit. After a final cleanup (SAP), the SNuPE reaction products were electrophorized on MegaBACE 1000 instruments (Amersham Biosciences) and analyzed using Genetic profiler software (Amersham Biosciences). To ensure that the genotypes obtained were valid, all genotypes were called blind to their case status in the genotyping process and regenotyping was performed on 48 random DNA samples for each of the nine SNPs. Details of the nine marker and the primer sequences are listed in Appendix A. 2.3. Statistical analysis Deviations from Hardy–Weinberg equilibrium were computed on an online calculator (http://www.kursus.kvl. dk/shares/vetgen/_Popgen/genetik/applets/kitest.htm). The software CLUMP 2.3 implementing a Monte Carlo simulation strategy was used to compare the discrepancies of allele, genotype, and haplotype frequencies between case and control subject samples using 100,000 simulations (Sham and Curtis, 1995). The odds ratio (OR) with 95% confidence intervals (CI) was also calculated online (http://www.hutchon.net/ConfidOR.htm). QVALUE version 1.0 was use for the correction of P value (measures the minimum false discovery rate that is incurred when calling that test significant) (Storey and Tibshirani, 2003). The pairwise linkage disequilibrium (LD), measured by standardized D', was estimated on 2LD software (Zapata et al., 2001). The program COCAPHASE was used to test haplotypes associated with schizophrenia and to perform 1000 permutation tests for estimating statistical significance. Power calculations for our sample size performed using the G⁎Power program (Erdfelder et al., 1996). In this study, the P values were two tailed and significance was set at P < 0.05. 3. Results Genotypic distributions of all nine SNPs in patients and control subjects were consistent with Hardy–
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Weinberg equilibrium (χ2 < 2). Between the 643 patients and 527 control subjects, the allele frequencies of two SNPs (rs3759915 and rs3759914) showed statistically significant difference (Table 1). After applying QVALUE correction tests, rs3759915 (P = 0.0036, OR = 1.27, 95%CI = 1.08–1.50, q-value = 0.022) still had significant allelic associations with schizophrenia and rs3759914 no significant difference (P = 0.039, OR = 1.21, 95% CI = 1.01–1.44, q-value = 0.122). The frequency of the C allele of rs3759915 was greater in patients (51.8%) than in control subjects (45.7%). At the genotype level, the difference of the CC genotype frequency of rs3759915 between patients and control subjects was also significant (CC vs. CG + GG, 27.5% vs. 21.6%, P = 0.020, df = 1, OR = 1.38, 95%CI = 1.05–1.80). After calculating LD for all pairs of SNP markers (expressed in D' and r2, Fig. 1) in cases and controls, strong LD (D' > 0.6) was observed among two groups of SNP markers (rs3759916, rs3759915, rs3759914 and
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rs3848153, rs3931230, rs8035191, rs2168351). rs3759916, rs3759915 and rs3759914 were therefore designated as Group I, and rs3848153, rs3931230, rs8035191 and rs2168351 as Group II. In both Groups, we analyzed the haplotypes which are observed with certainty in at least one subject and only listed the common haplotypes (frequency > 3% in either cases or controls). In Appendix B, we list the haplotype frequency estimation for different marker groups and the comparison of the frequencies between the cases and controls in detail. In Group I and II, haplotypes with probabilities greater than 3% accounted for the majority of haplotype diversity (>92%). We constructed haplotypes in a sliding window fashion on both LD blocks (Appendix B). Haplotype analysis in Group II revealed that there are no differences in frequencies of haplotypes between cases and controls (Global P = 0.474; df = 2). In Group I, each window showed that overall frequencies were significantly different between cases and controls. The most significant
Fig. 1. A: Genomic structure and locations of polymorphic sites in the SIAT8B. SIAT8B spans over 78.58 kb and includes 6 exons. Nine markers are indicated with their Single Nucleotide Polymorphism Database reference identification number. B: Estimation of linkage disequilibrium (D' and r 2 value) between the nine SNPs. Blank squares means low linkage disequilibrium (LD). C: Estimated individual Haplotype frequencies of block 1. Block 1 spanned rs3759916, rs3759915 and rs3759914 (Group I). After 1000 permutations, the Global P of block 1 (Group I) is equal to 0.0010.
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windows spanned rs3759916, rs3759915 and rs3759914, giving Global P = 0.0000050 (df = 3; P = 0.0010 after 1000 permutation) (Fig. 1C). Several two- and threemarker haplotypes showed statistically significant difference between cases and controls (Appendix B). The haplotype (rs3759916/rs3759915/rs3759914, C/G/T) was found to be strongly associated with control subjects because of its significantly increased frequency in controls rather than cases (P = 0.0000021; OR = 0.42, 95%CI = 0.29–0.60) (Fig. 1C). In addition, the other haplotype (rs3759916/rs3759915/rs3759914, T/G/T) was also overrepresented in controls, but the significance was marginal (P = 0.038; OR = 0.80, 95%CI = 0.64–1.00). In power calculations using the G⁎Power program, we found that the sample size had > 90% power for detecting a significant association (alpha < 0.05) when an effect size index of 0.1 (corresponding to a “weak” gene effect) was used. 4. Discussion Arai et al. reported that rs3759916 and rs3759914, located in the promoter region of SIAT8B showed nominally significant association with schizophrenia using 188 schizophrenics and 156 controls recruited from the Japanese population (allelic associations, P = 0.014 and P = 0.007, respectively). Furthermore they constructed a risk haplotype for schizophrenia in the LD block including the two SNPs, and also provided in vitro evidence for the risk haplotype (Arai et al., 2006). In our study, we followed up the work of Arai et al. by testing nine SNPs within the SIAT8B region and tested haplotypes for association with schizophrenia in a large, fully independent, rigorously diagnosed sample of Han Chinese. Besides three SNPs (rs3759916, rs3759915 and rs3759914) tested by Arai et al. in the promoter region, another six common SNPs (MAF > 0.2) were genotyped to cover SIAT8B. After applying the QVALUE correction test, we still observed a significant difference in allele frequency of rs3759915 (P = 0.0036, OR = 1.27, 95%CI = 1.08–1.50, q-value = 0.022) as well as genotype frequency (CC vs. CG + GG, 27.5% vs. 21.6%, P = 0.020, df = 1, OR = 1.38, 95%CI = 1.05–1.80) between cases and controls. rs3759915 was not observed significantly association with schizophrenia in the work of Arai et al. But the SNP located in the same LD block as rs3759916 and rs3759914. According to our data, the higher frequency of the C allele of rs3759915 in cases than in controls implied that the C allele could be a risk allele for schizophrenia and the G allele might be a protective allele. However, we did not replicate the positive association found by Arai et al. with regard to rs3759916 and
rs3759914 in our samples. Furthermore, the allele frequencies of the three SNPs (rs3759916, rs3759915 and rs3759914) were different in the two population samples. Since our association study was based on a distinct ethnic population, these discrepancies might result from the allelic heterogeneity that occurs in complex diseases and the sample sizes involved in the two investigations. As far as is known, different haplotype background can result in variations in the same allele in different racial groups and the predisposing SNPs may be different among various populations. To further increase the statistical power for association with the disease, we used haplotypes constructed by contiguous SNPs in Group I and Group II, according to LD structure. In Group II (rs3848153, rs3931230, rs8035191 and rs2168351), we did not observed evidence for association with schizophrenia (Global P = 0.474; df = 2). In Group I, three-marker haplotypes covering rs3759916, rs3759915 and rs3759914 were significantly associated with schizophrenia (Global P = 0.0000050; df = 3; P = 0.0010 after 1000 permutation). The haplotypes, T/G/T and C/G/T, was overrepresented in controls (Fig. 1C) and both included the same haplotype of -/G/T covering rs3759915 and rs3759914, which had higher frequencies in controls than in cases (26.6% in controls and 18.5% in cases; P = 0.0000011) (Appendix B). Therefore, the haplotype -/G/T covering rs3759915 and rs3759914 might be protective a factor for schizophrenia. Conversely, the complementary haplotype -/C/C ought to show a significantly higher trend in patients than in controls. Although we observed the expected significance of -/C/ C in our samples (P = 0.00020; OR = 4.04, 95% CI = 1.80–9.07), we ignored it because of its low frequency (< 0.01) in controls. Arai et al. in Japanese sampled population constructed haplotypes covering rs3759916, rs3759915, rs3759914 and two other contiguous SNPs in promoter region of SIAT8B and the most significant window was a three-SNP window spanning rs3759916, rs3759915 and rs3759914 (Global P = 0.0026). Though we also observed significant frequencies difference between cases and controls in the same window (rs3759916/rs3759915/ rs3759914, Global P = 0.0000050), the frequency of each haplotype in our sampled population was totally different with Arai et al. reported. These different distributions of haplotypes, especially in control group, suggested that our sampled Chinese population has different haplotype background with Japanese population in this region. The protein encoded by SIAT8B is an important modulator for fusion of PSA ligated-NCAM1 which is
R. Tao et al. / Schizophrenia Research 90 (2007) 108–114
indispensable for normal neurogenesis (Kiss and Muller, 2001; Kiss et al., 2001). Its expression level is modest in the adult heart, brain, and thymus. Previous studies have implied that the mRNA levels of SIAT8B are strictly controlled for normal neuronal development and maintenance of proper neuronal function, because either overproduction or insufficiency of PSA can disturb the integrity of this system (Angata et al., 2004; Barbeau et al., 1995; Brocco et al., 2003). In our association study, the haplotype constructed by three SNPs (Group I) showed a protective trend due to the over-representation of the C/G/T haplotype in controls (P = 0.0000021). In addition, one of the three SNPs, rs3759915, revealed a normally significant difference between cases and controls even after a QVALUE correction test (P = 0.0036, qvalue = 0.022). The schizophrenia associated haplotype block, located in the promoter region of SIAT8B included several putative transcriptional binding sites (CCAAT, MZF1, CREB, GATA, TATA and SP1) (Arai et al., 2006). The significant allele and haplotype might functionally affect the expression level of Sialyltransferase 8B in some specific gene backgrounds and increase the risk for schizophrenia. Allele-specific or haplotype-specific in vitro or in vivo experiments should routinely be performed to test the prediction. On the other hand, unlike conditions involving single gene defects, the genetic contributions to common diseases (including schizophrenia) are generally considered to be related to susceptibility loci, influencing but not determining overall disease risk. Common disease susceptibility alleles should not be considered disease genes because although necessary, they are not sufficient to cause diseases. A straightforward hypothesis, namely the common disease/common variant (CD/CV) hypothesis has been proposed. This hypothesis states that, “the genetic risk for common diseases will often be due to disease-producing alleles found at relatively high frequencies (> 1%)” (Becker, 2004). Although the C allele of rs3759915 was considerably more common in cases than in controls, the molecular variants may be components in complex multi-component networks that contribute in additive ways to the ultimate disease phenotype. In summary, our study provides supportive evidence that SIAT8B may be a potential susceptibility gene for schizophrenia and polysaccharide dysregulation may involve in the etiology of schizophrenia. Due to the complex patterns of association results in complex disorders, additional studies in other ethnic groups are needed to further examine the role of SIAT8B. In addition, further exploration of NCAM1 associated signaling is warranted.
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Acknowledgements We would sincerely thank all the subjects for their participation in this study and all the medical staff involved in specimen collecting. This work was supported by grants from the National 973 and 863 Programs, the National Natural Science Foundation of China and the Key grant Project of the Chinese Ministry of Education (NO.10414), PRC. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. schres.2006.09.029. References Angata, K., Nakayama, J., Fredette, B., Chong, K., Ranscht, B., Fukuda, M., 1997. Human STX polysialyltransferase forms the embryonic form of the neural cell adhesion molecule. Tissuespecific expression, neurite outgrowth, and chromosomal localization in comparison with another polysialyltransferase, PST. J. Biol. Chem. 272 (11), 7182–7190. Angata, K., Suzuki, M., McAuliffe, J., Ding, Y., Hindsgaul, O., Fukuda, M., 2000. Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct alpha 2,8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III. J. Biol. Chem. 275 (24), 18594–18601. Angata, K., Long, J.M., Bukalo, O., Lee, W., Dityatev, A., WynshawBoris, A., Schachner, M., Fukuda, M., Marth, J.D., 2004. Sialyltransferase ST8Sia-II assembles a subset of polysialic acid that directs hippocampal axonal targeting and promotes fear behavior. J. Biol. Chem. 279 (31), 32603–32613. Arai, M., Yamada, K., Toyota, T., Obata, N., Haga, S., Yoshida, Y., Nakamura, K., Minabe, Y., Ujike, H., Sora, I., Ikeda, K., Mori, N., Yoshikawa, T., Itokawa, M., 2006. Association between polymorphisms in the promoter region of the sialyltransferase 8B (SIAT8B) gene and schizophrenia. Biol. Psychiatry 59 (7), 652–659. Barbeau, D., Liang, J.J., Robitalille, Y., Quirion, R., Srivastava, L.K., 1995. Decreased expression of the embryonic form of the neural cell adhesion molecule in schizophrenic brains. Proc. Natl. Acad. Sci. U. S. A. 92 (7), 2785–2789. Becker, K.G., 2004. The common variants/multiple disease hypothesis of common complex genetic disorders. Med. Hypotheses 62 (2), 309–317. Brocco, M., Pollevick, G.D., Frasch, A.C., 2003. Differential regulation of polysialyltransferase expression during hippocampus development: implications for neuronal survival. J. Neurosci. Res. 74 (5), 744–753. Erdfelder, E., Faul, F., Buchner, A., 1996. GPOWER: a general power analysis program. Behav. Res. Meth. Instrum. Comput. 28 (1), 1–11. Harrison, P.J., 2004. The hippocampus in schizophrenia: a review of the neuropathological evidence and its pathophysiological implications. Psychopharmacology (Berl) 174 (1), 151–162. Kiss, J.Z., Muller, D., 2001. Contribution of the neural cell adhesion molecule to neuronal and synaptic plasticity. Rev. Neurosci. 12 (4), 297–310.
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Positive association between SIAT8B and schizophrenia in the Chinese Han population Ran Tao a,b,1 , Chao Li a,b,1 , Yonglan Zheng a,b , Wei Qin a,b , Jing Zhang a,b , Xingwang Li a,b , Yifeng Xu c , Yong Yong Shi a,b , Guoyin Feng c , Lin He b,d,⁎ a
d
Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China b Bio-X Life Science Research Center, Shanghai Jiao Tong University, Shanghai 200030, China c Shanghai Institute of Mental Health, Shanghai 200030, China NHGG, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Received 6 September 2006; received in revised form 25 September 2006; accepted 27 September 2006 Available online 28 November 2006
Abstract The Sialyltransferase 8B gene (SIAT8B) is located at 15q26, a susceptibility region for both schizophrenia and bipolar disorder. The protein encoded by this gene has an important role in neural development and sialic acid synthesis on the neural cell adhesion molecule (NCAM). Previous research had indicated that the promoter region of SIAT8B is associated with schizophrenia in the Japanese population. To take this further we carried out an association study based on 643 unrelated schizophrenics and 527 unrelated healthy subjects, all Han Chinese, recruited from Shanghai. Although our results differed from those of the Japanese research, rs3759915, also located in the promoter region of SIAT8B, showed nominally significant association with schizophrenia (P = 0.0036). Moreover, haplotypes constructed from rs3759915 and another two SNPs reported in the Japanese study (rs3759914 and rs3759916, also located in promoter region of SIAT8B) which located in the same LD block were significantly associated with schizophrenia (global P = 0.0000050). Our findings indicate that SIAT8B may be a candidate susceptibility gene for schizophrenia in the Chinese Han population and may also provide further support for the potential importance of polysaccharide-synthesizing genes in the etiology of schizophrenia. © 2006 Elsevier B.V. All rights reserved. Keywords: SIAT8B; SNP; Haplotype; Association; Chinese Han population
1. Introduction Schizophrenia (OMIM 181500) is a chronic psychotic disorder with a lifetime prevalence of approximately ⁎ Corresponding author. Bio-X Center, Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China; or Shanghai Jiao Tong University, PO Box 501, Hao Ran Building, 1954 Hua Shan Road, Shanghai 200030, China. Tel./fax: +86 21 62822491. E-mail address: [email protected] (L. He). 1 Contributed equally to this work. 0920-9964/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2006.09.029
1%. The Sialyltransferase 8B (SIAT8B) gene localizes on chromosome 15q26, a novel positive region reported in a genome scan of Eastern Quebec families (Maziade et al., 2005). The protein encoded by this gene is thought to catalyze the transfer of sialic acid from CMP-sialic acid to N-linked oligosaccharides and glycoproteins. The adhesive properties of the neural cell adhesion molecule (NCAM1) are modulated by its linkage to polysialic acid (PSA). This linkage is regulated by sialyltransferases, including sialyltransferase 8B (SIAT8B) (Angata et al., 2000). PSA ligated-NCAM1 (PSA-NCAM1) is widely
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expressed in the embryonic and neonatal brain, but expression is highly restricted in the adult central nervous system to areas such as the hippocampus and olfactory bulb, that maintain a permanent capacity for neurogenesis (Kiss and Muller, 2001; Kiss et al., 2001; Seki and Arai, 1993). It has been reported that cells cotransfected with NCAM1 and SIAT8B can support neurite outgrowth better than cells transfected with NCAM1 alone (Angata et al., 1997). Barbeau et al. observed a 20–95% reduction in the number of hilar PSA-NCAM-immunoreactive cells in the great majority of schizophrenic brains (Barbeau et al., 1995). The most conspicuous phenotype of Ncam1-deficit mice is in the olfactory bulb, where granule cells are both reduced in number and disorganized (Tomasiewicz et al., 1993). Olfactory impairments are common in schizophrenia and low olfactory bulb volumes have been reported in first-degree relatives of schizophrenics (Turetsky et al., 2003). Structural abnormalities of the olfactory system in schizophrenia may partly reflect preexisting genetic vulnerability to the illness. SIAT8B-deficient mice also show a PSA deficit
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in brain regions of neurogenesis, and this deficiency results in the misguidance of infrapyramidal mossy fibers and the formation of ectopic synapses in the hippocampus (Angata et al., 2004). Disturbances of the anatomical organization and hippocampal function have long been implicated in the etiology of schizophrenia (Harrison, 2004). All indications above mentioned implied that SIAT8B is one of candidate genes for schizophrenia. In present study, we evaluated the potential association between SIAT8B and schizophrenia by a case-control study. 2. Materials and methods 2.1. Subjects For the case-control study, we recruited 643 unrelated schizophrenics (means age 45.54, SD = 14.70) from the Shanghai Mental Center, of whom 346 were male and 297 female. The average onset age of disease was 25.06 years (SD = 10.30). All the schizophrenia patients were in-patients and had been diagnosed according to the
Table 1 Allele and genotype frequencies of the nine SNPs within SIAT8B Allele frequency (%)
rs3759916 Cases Controls rs3759915b Cases Controls rs3759914 Cases Controls rs3848153 Cases Controls rs3931230 Cases Controls rs8035191 Cases Controls rs2168351 Cases Controls rs3784727 Cases Controls rs3784724 Cases Controls a
Genotype frequency (%) a
Allele 1
Allele 2
P (df = 1)
OR (95%CI)
11
12
22
P (df = 2)a
T 850 (66.1) 677 (64.2) C 666 (51.8) 482 (45.7) T 870 (67.7) 755 (71.6) T 879 (68.4) 708 (67.2) A 876 (68.1) 691 (65.6) C 927 (72.1) 758 (71.9) G 847 (65.9) 686 (65.1) G 725 (56.4) 567 (53.8) C 816 (63.5) 701 (66.5)
C 436 (33.9) 377 (35.8) G 620 (48.2) 572 (54.3) C 416 (32.3) 299 (28.4) C 407 (31.6) 346 (32.8) C 410 (31.9) 363 (34.4) T 359 (27.9) 296 (28.1) A 439 (34.1) 368 (34.9) C 561 (43.6) 487 (46.2) G 470 (36.5) 353 (33.5)
0.36
0.92 (0.78–1.09)
1.27 (1.08–1.50)
0.039
1.21 (1.01–1.44)
0.56
1.06 (0.89–1.26)
0.20
1.12 (0.94–1.33)
0.96
1.01 (0.84–1.21)
0.70
1.04 (0.87–1.23)
0.23
1.11 (0.94–1.31)
0.13
1.14 (0.96–1.36)
TC 274 (42.6) 249 (47.2) CG 312 (48.5) 254 (48.2) TC 290 (45.1) 201 (38.1) TC 279 (43.4) 230 (43.6) AC 280 (43.5) 225 (42.7) CT 259 (40.3) 202 (38.3) GA 297 (46.2) 256 (48.6) GC 307 (47.7) 281 (53.3) CG 286 (44.5) 221 (41.9)
CC 81 (12.6) 64 (12.1) GG 154 (24.0) 159 (30.2) CC 63 (9.7) 49 (9.3) CC 64 (10.0) 58 (11.0) CC 65 (10.1) 69 (13.1) TT 50 (7.8) 47 (8.9) AA 71 (11.0) 56 (10.6) CC 127 (19.8) 103 (19.5) GG 92 (14.3) 66 (12.5)
0.27
0.0036
TT 288 (44.8) 214 (40.6) CC 177 (27.5) 114 (21.6) TT 290 (45.1) 277 (52.6) TT 300 (46.7) 239 (45.4) AA 298 (46.3) 233 (44.2) CC 334 (51.9) 278 (52.8) GG 275 (42.8) 215 (40.8) GG 209 (32.5) 143 (27.1) CC 265 (41.2) 240 (45.5)
Significant P values (<0.05) are in boldface. b Context sequence of rs3759915: CGAGGGCGGA
GGAAGGCGGC.
0.016
0.035
0.81
0.28
0.69
0.72
0.10
0.31
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criteria of the Diagnostic and Statistical Manual of Mental Disorder, Third Revised Edition (DSM-III-R). Diagnosis and review of psychiatric case records were independently checked and verified by two senior psychiatrists. 527 unrelated healthy subjects (280 males and 247 females with a mean age of 35.34 years, SD = 8.71) screened for absence of major mental illness were recruited from the same geographic region as case subjects. These were recruited from blood donors and were interviewed to establish that relatives within the third degree were also free from psychoses. Written informed consents, reviewed and approved by the Shanghai Ethical Committee of Human Genetic Resources, were obtained from all participating subjects after the nature of study had been fully explained. All subjects were Han Chinese in origin. 2.2. Single nucleotide polymorphism genotyping To replicate positive results of Arai et al., rs3759916 (-1126T > C; Arai et al., 2006), rs3759915 (-907G > C; Arai et al., 2006) and rs3759914 (-851T > C; Arai et al., 2006) in promoter region were recruited. In addition, according to Chinese population data of Hapmap (www. hapmap.org) and our pre-processing experiment (genotyped candidate SNPs in 48 random individuals of our samples population), we also selected other six common SNPs (rs3848153, rs3931230, rs8035191, rs2168351, rs3784727 and rs3784724) (Minor Allele Frequency, MAF > 0.2) to cover the region of SIAT8B. Genomic DNA was isolated from peripheral blood samples of the subjects using the phenol-chloroform method. Three SNPs in the promoter region were genotyped by direct DNA sequencing. Polymerase chain reaction (PCR) amplification was carried out in a final volume of 15 μl, containing 10 ng genomic DNA, 0.2 μM of each primer, 2.5 mM MgCl2, 2 mM deoxynucleoside triphosphate (dNTP), 1.5 μl of 10 × buffer (Qiagen, Basel, Switzerland), and 1 U HotStar Taq polymerase (Qiagen, Basel, Switzerland). Thermocycling was performed on a Gene Amp PCR system 9700 (Applied Biosystem, Foster City, CA) according to a modified touchdown program, with an initial denaturation at 95 °C for 5 min, followed by 44 cycles of denaturation at 95 °C for 30 s and extension at 72 °C for 45 s. The annealing temperature was decreased from 63 °C by 0.5 °C per cycle for a total of 14 cycles, followed by 30 cycles at the final annealing temperature of 56 °C and final extension of 10 min at 72 °C. The PCR products for sequencing were incubated with 0.1 U shrimp alkaline phosphatase (Roche, Basel, Switzerland) at 37 °C for 45 min, followed by heat
inactivation at 85 °C for 20 min. The treated PCR products were then sequenced using an ABI Prism BigDye Terminator Cycle Sequencing Kit, version 3.1 (Applied Biosystems) on an ABI Prism 3100 sequencer (Applied Biosystems). Six SNPs were genotyped by Single Nucleotide Primer Extension (SNuPE). To amplify the region under investigation PCR was carried out according to the above protocol. The PCR products were treated with Exonuclease I and Shrimp Alkaline Phosphatase (Exo/ SAP) according to the manufacturer's protocol. SNuPE reaction was then performed according to the protocol of MegaBACE SNuPE Genotyping Kit. After a final cleanup (SAP), the SNuPE reaction products were electrophorized on MegaBACE 1000 instruments (Amersham Biosciences) and analyzed using Genetic profiler software (Amersham Biosciences). To ensure that the genotypes obtained were valid, all genotypes were called blind to their case status in the genotyping process and regenotyping was performed on 48 random DNA samples for each of the nine SNPs. Details of the nine marker and the primer sequences are listed in Appendix A. 2.3. Statistical analysis Deviations from Hardy–Weinberg equilibrium were computed on an online calculator (http://www.kursus.kvl. dk/shares/vetgen/_Popgen/genetik/applets/kitest.htm). The software CLUMP 2.3 implementing a Monte Carlo simulation strategy was used to compare the discrepancies of allele, genotype, and haplotype frequencies between case and control subject samples using 100,000 simulations (Sham and Curtis, 1995). The odds ratio (OR) with 95% confidence intervals (CI) was also calculated online (http://www.hutchon.net/ConfidOR.htm). QVALUE version 1.0 was use for the correction of P value (measures the minimum false discovery rate that is incurred when calling that test significant) (Storey and Tibshirani, 2003). The pairwise linkage disequilibrium (LD), measured by standardized D', was estimated on 2LD software (Zapata et al., 2001). The program COCAPHASE was used to test haplotypes associated with schizophrenia and to perform 1000 permutation tests for estimating statistical significance. Power calculations for our sample size performed using the G⁎Power program (Erdfelder et al., 1996). In this study, the P values were two tailed and significance was set at P < 0.05. 3. Results Genotypic distributions of all nine SNPs in patients and control subjects were consistent with Hardy–
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Weinberg equilibrium (χ2 < 2). Between the 643 patients and 527 control subjects, the allele frequencies of two SNPs (rs3759915 and rs3759914) showed statistically significant difference (Table 1). After applying QVALUE correction tests, rs3759915 (P = 0.0036, OR = 1.27, 95%CI = 1.08–1.50, q-value = 0.022) still had significant allelic associations with schizophrenia and rs3759914 no significant difference (P = 0.039, OR = 1.21, 95% CI = 1.01–1.44, q-value = 0.122). The frequency of the C allele of rs3759915 was greater in patients (51.8%) than in control subjects (45.7%). At the genotype level, the difference of the CC genotype frequency of rs3759915 between patients and control subjects was also significant (CC vs. CG + GG, 27.5% vs. 21.6%, P = 0.020, df = 1, OR = 1.38, 95%CI = 1.05–1.80). After calculating LD for all pairs of SNP markers (expressed in D' and r2, Fig. 1) in cases and controls, strong LD (D' > 0.6) was observed among two groups of SNP markers (rs3759916, rs3759915, rs3759914 and
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rs3848153, rs3931230, rs8035191, rs2168351). rs3759916, rs3759915 and rs3759914 were therefore designated as Group I, and rs3848153, rs3931230, rs8035191 and rs2168351 as Group II. In both Groups, we analyzed the haplotypes which are observed with certainty in at least one subject and only listed the common haplotypes (frequency > 3% in either cases or controls). In Appendix B, we list the haplotype frequency estimation for different marker groups and the comparison of the frequencies between the cases and controls in detail. In Group I and II, haplotypes with probabilities greater than 3% accounted for the majority of haplotype diversity (>92%). We constructed haplotypes in a sliding window fashion on both LD blocks (Appendix B). Haplotype analysis in Group II revealed that there are no differences in frequencies of haplotypes between cases and controls (Global P = 0.474; df = 2). In Group I, each window showed that overall frequencies were significantly different between cases and controls. The most significant
Fig. 1. A: Genomic structure and locations of polymorphic sites in the SIAT8B. SIAT8B spans over 78.58 kb and includes 6 exons. Nine markers are indicated with their Single Nucleotide Polymorphism Database reference identification number. B: Estimation of linkage disequilibrium (D' and r 2 value) between the nine SNPs. Blank squares means low linkage disequilibrium (LD). C: Estimated individual Haplotype frequencies of block 1. Block 1 spanned rs3759916, rs3759915 and rs3759914 (Group I). After 1000 permutations, the Global P of block 1 (Group I) is equal to 0.0010.
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windows spanned rs3759916, rs3759915 and rs3759914, giving Global P = 0.0000050 (df = 3; P = 0.0010 after 1000 permutation) (Fig. 1C). Several two- and threemarker haplotypes showed statistically significant difference between cases and controls (Appendix B). The haplotype (rs3759916/rs3759915/rs3759914, C/G/T) was found to be strongly associated with control subjects because of its significantly increased frequency in controls rather than cases (P = 0.0000021; OR = 0.42, 95%CI = 0.29–0.60) (Fig. 1C). In addition, the other haplotype (rs3759916/rs3759915/rs3759914, T/G/T) was also overrepresented in controls, but the significance was marginal (P = 0.038; OR = 0.80, 95%CI = 0.64–1.00). In power calculations using the G⁎Power program, we found that the sample size had > 90% power for detecting a significant association (alpha < 0.05) when an effect size index of 0.1 (corresponding to a “weak” gene effect) was used. 4. Discussion Arai et al. reported that rs3759916 and rs3759914, located in the promoter region of SIAT8B showed nominally significant association with schizophrenia using 188 schizophrenics and 156 controls recruited from the Japanese population (allelic associations, P = 0.014 and P = 0.007, respectively). Furthermore they constructed a risk haplotype for schizophrenia in the LD block including the two SNPs, and also provided in vitro evidence for the risk haplotype (Arai et al., 2006). In our study, we followed up the work of Arai et al. by testing nine SNPs within the SIAT8B region and tested haplotypes for association with schizophrenia in a large, fully independent, rigorously diagnosed sample of Han Chinese. Besides three SNPs (rs3759916, rs3759915 and rs3759914) tested by Arai et al. in the promoter region, another six common SNPs (MAF > 0.2) were genotyped to cover SIAT8B. After applying the QVALUE correction test, we still observed a significant difference in allele frequency of rs3759915 (P = 0.0036, OR = 1.27, 95%CI = 1.08–1.50, q-value = 0.022) as well as genotype frequency (CC vs. CG + GG, 27.5% vs. 21.6%, P = 0.020, df = 1, OR = 1.38, 95%CI = 1.05–1.80) between cases and controls. rs3759915 was not observed significantly association with schizophrenia in the work of Arai et al. But the SNP located in the same LD block as rs3759916 and rs3759914. According to our data, the higher frequency of the C allele of rs3759915 in cases than in controls implied that the C allele could be a risk allele for schizophrenia and the G allele might be a protective allele. However, we did not replicate the positive association found by Arai et al. with regard to rs3759916 and
rs3759914 in our samples. Furthermore, the allele frequencies of the three SNPs (rs3759916, rs3759915 and rs3759914) were different in the two population samples. Since our association study was based on a distinct ethnic population, these discrepancies might result from the allelic heterogeneity that occurs in complex diseases and the sample sizes involved in the two investigations. As far as is known, different haplotype background can result in variations in the same allele in different racial groups and the predisposing SNPs may be different among various populations. To further increase the statistical power for association with the disease, we used haplotypes constructed by contiguous SNPs in Group I and Group II, according to LD structure. In Group II (rs3848153, rs3931230, rs8035191 and rs2168351), we did not observed evidence for association with schizophrenia (Global P = 0.474; df = 2). In Group I, three-marker haplotypes covering rs3759916, rs3759915 and rs3759914 were significantly associated with schizophrenia (Global P = 0.0000050; df = 3; P = 0.0010 after 1000 permutation). The haplotypes, T/G/T and C/G/T, was overrepresented in controls (Fig. 1C) and both included the same haplotype of -/G/T covering rs3759915 and rs3759914, which had higher frequencies in controls than in cases (26.6% in controls and 18.5% in cases; P = 0.0000011) (Appendix B). Therefore, the haplotype -/G/T covering rs3759915 and rs3759914 might be protective a factor for schizophrenia. Conversely, the complementary haplotype -/C/C ought to show a significantly higher trend in patients than in controls. Although we observed the expected significance of -/C/ C in our samples (P = 0.00020; OR = 4.04, 95% CI = 1.80–9.07), we ignored it because of its low frequency (< 0.01) in controls. Arai et al. in Japanese sampled population constructed haplotypes covering rs3759916, rs3759915, rs3759914 and two other contiguous SNPs in promoter region of SIAT8B and the most significant window was a three-SNP window spanning rs3759916, rs3759915 and rs3759914 (Global P = 0.0026). Though we also observed significant frequencies difference between cases and controls in the same window (rs3759916/rs3759915/ rs3759914, Global P = 0.0000050), the frequency of each haplotype in our sampled population was totally different with Arai et al. reported. These different distributions of haplotypes, especially in control group, suggested that our sampled Chinese population has different haplotype background with Japanese population in this region. The protein encoded by SIAT8B is an important modulator for fusion of PSA ligated-NCAM1 which is
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indispensable for normal neurogenesis (Kiss and Muller, 2001; Kiss et al., 2001). Its expression level is modest in the adult heart, brain, and thymus. Previous studies have implied that the mRNA levels of SIAT8B are strictly controlled for normal neuronal development and maintenance of proper neuronal function, because either overproduction or insufficiency of PSA can disturb the integrity of this system (Angata et al., 2004; Barbeau et al., 1995; Brocco et al., 2003). In our association study, the haplotype constructed by three SNPs (Group I) showed a protective trend due to the over-representation of the C/G/T haplotype in controls (P = 0.0000021). In addition, one of the three SNPs, rs3759915, revealed a normally significant difference between cases and controls even after a QVALUE correction test (P = 0.0036, qvalue = 0.022). The schizophrenia associated haplotype block, located in the promoter region of SIAT8B included several putative transcriptional binding sites (CCAAT, MZF1, CREB, GATA, TATA and SP1) (Arai et al., 2006). The significant allele and haplotype might functionally affect the expression level of Sialyltransferase 8B in some specific gene backgrounds and increase the risk for schizophrenia. Allele-specific or haplotype-specific in vitro or in vivo experiments should routinely be performed to test the prediction. On the other hand, unlike conditions involving single gene defects, the genetic contributions to common diseases (including schizophrenia) are generally considered to be related to susceptibility loci, influencing but not determining overall disease risk. Common disease susceptibility alleles should not be considered disease genes because although necessary, they are not sufficient to cause diseases. A straightforward hypothesis, namely the common disease/common variant (CD/CV) hypothesis has been proposed. This hypothesis states that, “the genetic risk for common diseases will often be due to disease-producing alleles found at relatively high frequencies (> 1%)” (Becker, 2004). Although the C allele of rs3759915 was considerably more common in cases than in controls, the molecular variants may be components in complex multi-component networks that contribute in additive ways to the ultimate disease phenotype. In summary, our study provides supportive evidence that SIAT8B may be a potential susceptibility gene for schizophrenia and polysaccharide dysregulation may involve in the etiology of schizophrenia. Due to the complex patterns of association results in complex disorders, additional studies in other ethnic groups are needed to further examine the role of SIAT8B. In addition, further exploration of NCAM1 associated signaling is warranted.
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Acknowledgements We would sincerely thank all the subjects for their participation in this study and all the medical staff involved in specimen collecting. This work was supported by grants from the National 973 and 863 Programs, the National Natural Science Foundation of China and the Key grant Project of the Chinese Ministry of Education (NO.10414), PRC. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. schres.2006.09.029. References Angata, K., Nakayama, J., Fredette, B., Chong, K., Ranscht, B., Fukuda, M., 1997. Human STX polysialyltransferase forms the embryonic form of the neural cell adhesion molecule. Tissuespecific expression, neurite outgrowth, and chromosomal localization in comparison with another polysialyltransferase, PST. J. Biol. Chem. 272 (11), 7182–7190. Angata, K., Suzuki, M., McAuliffe, J., Ding, Y., Hindsgaul, O., Fukuda, M., 2000. Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct alpha 2,8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III. J. Biol. Chem. 275 (24), 18594–18601. Angata, K., Long, J.M., Bukalo, O., Lee, W., Dityatev, A., WynshawBoris, A., Schachner, M., Fukuda, M., Marth, J.D., 2004. Sialyltransferase ST8Sia-II assembles a subset of polysialic acid that directs hippocampal axonal targeting and promotes fear behavior. J. Biol. Chem. 279 (31), 32603–32613. Arai, M., Yamada, K., Toyota, T., Obata, N., Haga, S., Yoshida, Y., Nakamura, K., Minabe, Y., Ujike, H., Sora, I., Ikeda, K., Mori, N., Yoshikawa, T., Itokawa, M., 2006. Association between polymorphisms in the promoter region of the sialyltransferase 8B (SIAT8B) gene and schizophrenia. Biol. Psychiatry 59 (7), 652–659. Barbeau, D., Liang, J.J., Robitalille, Y., Quirion, R., Srivastava, L.K., 1995. Decreased expression of the embryonic form of the neural cell adhesion molecule in schizophrenic brains. Proc. Natl. Acad. Sci. U. S. A. 92 (7), 2785–2789. Becker, K.G., 2004. The common variants/multiple disease hypothesis of common complex genetic disorders. Med. Hypotheses 62 (2), 309–317. Brocco, M., Pollevick, G.D., Frasch, A.C., 2003. Differential regulation of polysialyltransferase expression during hippocampus development: implications for neuronal survival. J. Neurosci. Res. 74 (5), 744–753. Erdfelder, E., Faul, F., Buchner, A., 1996. GPOWER: a general power analysis program. Behav. Res. Meth. Instrum. Comput. 28 (1), 1–11. Harrison, P.J., 2004. The hippocampus in schizophrenia: a review of the neuropathological evidence and its pathophysiological implications. Psychopharmacology (Berl) 174 (1), 151–162. Kiss, J.Z., Muller, D., 2001. Contribution of the neural cell adhesion molecule to neuronal and synaptic plasticity. Rev. Neurosci. 12 (4), 297–310.
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