Brief report

Sex-specific association of the ST8SIAII gene with schizophreniain a Spanish population

Javier Gilabert-Juan a,b,d,e, Juan Nacher b,d,e, Julio Sanjuán c,d,e, María Dolores Moltó a,d,e,n

a Genetics Department, Faculty of Biological Sciences, University of Valencia, Spainb Neurobiology Unit and Program in Basic and Applied Neurosciences, Department of Cell Biology, University of Valencia, Spainc Psychiatric Unit, Faculty of Medicine, University of Valencia, Spaind CIBERSAM, Spaine INCLIVA, Valencia, Spain

a r t i c l e i n f o

Article history:Received 11 March 2013Received in revised form30 July 2013Accepted 2 September 2013

Keywords:SchizophreniaSexST8SIAII

a b s t r a c t

We investigated the association between ST8SIAII and schizophrenia in a sample of Spanish origin. Wefound that the G allele (P¼0.044) and the AG genotype (P¼0.040) of rs3759916 were associated infemales. The ACAG haplotype (rs3759914, rs3759915, rs3759916 and rs2305561) was associated in males(P¼0.028).

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The alpha-2,8-sialyltransferases II and IV are two enzymes thatcatalyze the transfer of polysialic acid (PSA) to the neural cell adhesionmolecule (NCAM). These enzymatic activities are crucial in neuraldevelopment, modulating the adhesive properties of NCAM, whichare involved in cell–cell and cell-extracellular matrix recognition(Rutishauser, 2008). NCAM plays important roles in neuronal migra-tion, neurite growth, axon guidance, synaptic plasticity (Maness andSchachner, 2007); regulation of circadian cues, and learning andmemory processes (Conboy et al., 2010).

Different studies in human postmortem samples and in animalmodels have suggested that alterations in PSA-NCAM expression inthe central nervous system increase the vulnerability to severalpsychiatric disorders (Barbeau et al., 1995; Gilabert-Juan et al., 2012;Varea et al., 2012). The gene coding for polysialyltransferase II(ST8SIAII) map to chromosomal region reported as susceptibility locifor schizophrenia (Maziade et al., 2005). Interestingly, several SingleNucleotide Polymorphisms (SNPs) and haplotypes of ST8SIAII havebeen associated with schizophrenia in different Asian (Arai et al.,2006; Tao et al., 2007) and Australian (McAuley et al., 2012) popula-tion samples.

To futher investigate the relationship between schizophreniaand ST8SIAII, we performed a case-control association study in aCaucasian sample of Spanish origin.

2. Material and methods

2.1. Subjects

A total of 508 unrelated patients (185 females, mean ages¼44.09713.42 years,age at onset¼29.26711.99 years; 323 males, mean ages¼37.62711.18, age at onset¼24.9079.04 years) with schizophrenia all met DSM-IV-TR criteria (AmericanPsychiatric Association, 2000) for this disease. Duration of the disease was similar inmales (13.88711.13) than in females (15.52710.83). The diagnoses for every patientwere confirmed by a consensus meeting with the treating psychiatrist and one of thepsychiatrists of our research group. Patients with drug abuse based on the clinicalinterview during assessment using the Mini-International Neuropsychiatric Interview(M.I.N.I., Sheehan et al., 1998) and clinical records were excluded from the study.Patients were on antipsychotic treatment at evaluation time. The mean daily dose was5.3 mg equivalent Risperidone, with no differences between males and females. A totalof 428 control individuals (132 females, mean age¼36.93715.28 years; 296 males,mean age¼38.10714.00) were drawn from unrelated local volunteers. Controlindividuals were screened using the M.I.N.I. (Sheehan et al., 1998) to discardpsychiatric records and drug abuse. All subjects were of Spanish descent. Nostratification has been found in the Spanish population (Laayouni et al., 2010),therefore no allelic differences due to ethnic procedure were expected. This studywas approved by the Ethical Committee of Valencia University and all subjects gavetheir written informed consent.

2.2. Genotyping

Four SNPs located in the promoter (rs3759914, rs3759915 and rs3759916) andthe coding (rs2305561) regions of ST8SIAII were analyzed. Genomic DNA was

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n Corresponding author at: Genetics Department, Faculty of Biological Sciences,University of Valencia, Spain. Tel.: þ34 963543400; fax: þ34 963543029.

E-mail address: dmolto@uv.es (M.D. Moltó).

Psychiatry Research 210 (2013) 1293–1295

isolated from the peripheral blood using the PUREGENEs DNA isolation kit (GentraSystems, MN, USA). SNP genotyping was performed through the iPLEX assay on theMassARRAY platform (Sequenom, Santiago de Compostela, Spain). Exclusioncriteria during quality control of the genotyping procedure were the following:(i) genotyping call rate lower than 99%; (ii) deviations from Hardy–WeinbergEquilibrium (HWE) in the control sample (Po0.05).

2.3. Statistical analysis

For each demographic condition, mean7SD was determined and the resultingvalues were then subjected to unpaired Student's t test statistical analysis, using theIBM SPSS statistics software (version 19).

QUANTO software v.1.2.4 (http://hydra.usc.edu/gxe/) was used to calculate thestatistical power of our sample. It was 54% to detect a risk allele over rare allelefrequencies (0.05–0.50) and assuming an odds ratio of 1.5 with 95% confidenceintervals. We also set the prevalence of schizophrenia at 1%.

The HWE of all the SNPs was assessed by applying a χ2 test implemented withSNPator software (http://www.snpator.org/). Differences in allelic and genotypicfrequencies between patients and controls were evaluated with a χ2 test via SNPator.Pair-wise marker linkage disequilibrium (LD) was analyzed using the programHaploview 4.1 (Barrett et al., 2005). It showed one Block (rs3759914, rs3759915and rs3759916) with D′40.8 between markers. Haplotypes were constructed andtheir frequencies compared between cases and controls using the SNPator package.Haplotype frequencies were estimated through a retrospective likelihood algorithm.Bonferroni multiple test correction was applied at each level of analysis taking intoaccount the number of SNPs studied (in the allelic and genotypic analyses) or thenumber of haplotypes.

3. Results

No significant differences in the allelic or genotypic frequenciesbetween cases and controls were detected. Sample grouped by sexshowed some positive results (Table 1). A significant associationbetween the G allele of rs3759916 and the disease in the femalesample was found (χ2¼6.514, P¼0.011; corrected P¼0.044).Among 132 control females none of them has this allele, whilenine of the 185 females with schizophrenia are G carriers. In themale subgroup, a significant association was detected with the Gallele of the rs2305561, but it did not overcome the multiple testcorrection (χ2¼4.201, P¼0.04; corrected P¼0.16). Concerning thegenotypic analyses, the AG genotype of rs3759916 was associate tothe disease in female (χ2¼6.609, P¼0.01; corrected P¼0.04),although this result should be treated with caution because isbased on small number of females. As can be seen in Table 1, therewere no individuals with the GG genotype in our sample. In male,a positive association was observed with the GG genotype ofrs2305561, but it was not significant after Bonferroni correction(χ2¼3.966, P¼0.046; corrected P¼0.18).

Haplotype was constructed with the four SNPs and no sig-nificant differences between cases and controls were found.However, the GCAG haplotype was associated to the disease infemales (Table 1), but it lost the statistical significance after

Table 1Allelic, genotypic and haplotypic frequencies of the ST8SIAII polymorphisms grouped by sex.

Polymorphisms Allele counts (frequency) P value P valuea Genotype counts (frequency) P valueb P valuea

Females rs3759916 A G 0.011 0.044 AA AG GG 0.01 0.04Schizophrenia 361(0.98) 9(0.02) 176(0.95) 9(0.05) 0(0.0)Control 264(1.0) 0(0.0) 132(1.0) 0(0.0) 0(0.0)

rs3759915 C G 0.33 1 CC CG GG 0.23 0.92Schizophrenia 17(0.05) 353(0.95) 2(0.01) 13(0.07) 170(0.92)Control 8(0.03) 254(0.97) 0(0.0) 8(0.06) 123(0.94)

rs3759914 A G 0.23 0.92 AA AG GG 0.23 0.92Schizophrenia 366(0.99) 2(0.005) 182(0.99) 2(0.01) 0(0.0)Control 264(1.0) 0(0.0) 132(1.0) 0(0.0) 0(0.0)

rs2305561 C G 0.85 1 CC CG GG 0.21 0.84Schizophrenia 55(0.15) 311(0.85) 5(0.03) 45(0.25) 133(0.73)Control 38(0.15) 224(0.85) 1(0.007) 36(0.27) 94(0.72)

Males rs3759916 A G 0.66 1 AA AG GG 0.66 1Schizophrenia 640(0.99) 6(0.01) 317(0.98) 6(0.02) 0(0.0)Control 585(0.99) 7(0.01) 289(0.98) 7(0.02) 0(0.0)

rs3759915 C G 0.13 0.52 CC CG GG 0.26 1Schizophrenia 45(0.07) 601(0.93) 2(0.006) 41(0.13) 280(0.86)Control 29(0.05) 561(0.95) 0(0.0) 29(0.1) 266(0.99)

rs3759914 A G 0.14 0.56 AA AG GG 0.14 0.56Schizophrenia 642(1.0) 0(0.0) 321(1.0) 0(0.0) 0(0.0)Control 590(0.99) 2(0.003) 294(0.99) 2(0.007) 0(0.0)

rs2305561 C G 0.04 0.16 CC CG GG 0.046 0.18Schizophrenia 85(0.13) 561(0.87) 2(0.006) 81(0.25) 240(0.74)Control 102(0.17) 486(0.83) 5(0.02) 92(0.31) 197(0.67)

Haplotypes rs3759916 rs3759915 rs3759914 rs2305561 Controlfrequency

Schizophreniafrequency

P value P valuea

Females G C A G 0% 1.89% 0.025 0.15

Males A C A G 0.92% 3.56% 0.0047 0.028

a P value after Bonferroni correction.b Genotype P value with the dominant model of inheritance.

J. Gilabert-Juan et al. / Psychiatry Research 210 (2013) 1293–12951294

Bonferroni correction (χ2¼5.018, P¼0.025; corrected P¼0.15). Inmales, the ACAG haplotype was associated with the diseaseretaining the significant association after multiple test corrections(χ2¼8.007, P¼0.0047, corrected P¼0.028). Its frequency was3.56% and 0.92% in cases and controls respectively (Table 1).

4. Discussion

In this study we tried to replicate in a European populationsample positive results found between ST8SIAII and schizophreniain two Asian populations (Arai et al., 2006; Tao et al., 2007). Thesestudies found association with SNPs located in the promoterregion of ST8SIAII (rs3759914, rs3759915 and rs3759916), whichsuggests that dysregulation of ST8SIAII expression may increasethe risk to suffer schizophrenia. We did not replicate these resultsin our whole sample. Differences in the allelic frequenciesbetween the different population samples might explain theseresults. In fact, the alleles associated with schizophrenia in each ofthe two Asian samples, show lower frequencies in our Spanishcohort and in other population samples of European ancestry(http://www.ncbi.nlm.nih.gov/SNP/). Alternatively, allelic hetero-geneity that characterizes complex disease could also explainthese discrepancies. However we found interesting sex-specificassociations to schizophrenia in ST8SIAII. In females, the resultssuggested that the G allele of rs3759916 might be a risk factor forschizophrenia in the Spanish population. It would be interesting toreplicate this result in other populations, since this polymorphismis located 200 base-pairs downstream from a binding sequence ofa glucocorticoid receptor gene, a transcriptional factor implicatedin sexual features.

Regarding to rs2305561, we observed that the G allele and theGG genotype were more frequent in cases than in controls inmales, although the association was lost after the Bonferroni testcorrection. Nevertheless this polymorphism might be a real riskfactor. It is located in the coding region and each allele hasdifferent efficiency of NCAM polysialylation (Isomura et al.,2011). Therefore chain length and quantity of NCAM polysialyla-tion could vary depending on the rs2305561 genotype affecting itsbiological function.

The haplotype analysis indicated a risk haplotype ACAG inmales of the Spanish population (P¼0.028). Interestingly, the riskhaplotype reported in the Japanese sampled population (Arai et al.,2006) share the same alleles of rs3759916, rs3759915 andrs3759914, although its frequency is higher than in our sample.

A limitation of this study is that the sample size becamereduced when it was grouped by sex decreasing the power ofthe analysis. In any case, it might be expected that ST8SIAIIcontributes differently to schizophrenia in the two sexes becauseits expression is controlled by estrogens (Tan et al., 2009). There-fore estrogens regulate the PSA-NCAM expression. In addition,PSA-NCAM expression has been associated with the gonadotropin-releasing hormone (Parkash and Kaur, 2005), one of the mostimportant molecules in the reproductive life in vertebrates. There-fore PSA-NCAM may have different impact in brain developmentand maintenance in each sex. Schizophrenia occurs 1.4 times morefrequently in males than females and the course of the disease isalso different between the two sexes (McGrath et al., 2008). Allthese data shows a scenario where neural genes, such as ST8SIAII,regulated by sexual hormones may contribute differently to the

vulnerability to schizophrenia in males and females. Our studyshowed interesting sex-specific associations between ST8SIAII andschizophrenia.

Acknowledgments

Spanish Ministry of Science and Innovation (MICINN-FEDER)BFU2009-12284/BFI, MICINN-PIM2010ERN-00577/NEUCONNECTin the frame of ERA-NET NEURON”, Generalitat ValencianaACOMP/2012/229 to JN. Spanish Ministry of Health (FIS) PI10/01399 to MDM and JS.

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