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ZDHHC8 gene may play a role in cortical volumes of patients with schizophrenia
Schizophrenia Research 145 (2013) 33–35
Contents lists available at SciVerse ScienceDirect
Schizophrenia Research journal homepage: www.elsevier.com/locate/schres
ZDHHC8 gene may play a role in cortical volumes of patients with schizophrenia Vanessa K. Ota a, b, Ary Gadelha b, c, Idaiane B. Assunção b, Marcos L. Santoro a, b, Denise M. Christofolini d, Fernanda T. Bellucco a, Airton F. Santos-Filho c, Gustavo L. Ottoni e, Diogo R. Lara e, Jair J. Mari c, Maria I. Melaragno a, Marília A.C. Smith a, Rodrigo A. Bressan b, c, Sintia I. Belangero a, b, c, Andrea P. Jackowski b, c,⁎ a Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de Sao Paulo (UNIFESP), Rua Botucatu, 740, Edifício Leitao da Cunha, 1° andar, CEP 04023-900, Sao Paulo, Brazil b Laboratório Interdisciplinar de Neurociências Clínicas (LiNC), Departamento de Psiquiatria, Universidade Federal de Sao Paulo (UNIFESP), Sao Paulo, Brazil c Departamento de Psiquiatria, Universidade Federal de Sao Paulo (UNIFESP), Sao Paulo, Brazil d Disciplina de Genética e Reproducao Humana, Departamento de Ginecologia e Obstetrícia, Faculdade de Medicina do ABC (FMABC), São Paulo, Brazil e Faculdade de Biociências, Pontifícia Universidade do Rio Grande do Sul (PUCRS), Porto Alegre, Brazil
a r t i c l e
i n f o
Article history: Received 4 July 2012 Received in revised form 11 January 2013 Accepted 14 January 2013 Available online 8 February 2013 Keywords: Schizophrenia ZDHHC8 22q11 Cortical volumes MRI
a b s t r a c t ZDHHC8 rs175174 polymorphism is located in 22q11.2 region and its role in brain volume has not been fully addressed. A total of 282 schizophrenia patients and 379 controls were genotyped. A sample of 138 patients underwent brain MRI scan. No association was found between schizophrenia and genotypes. Nevertheless, GG-genotype carriers presented gray matter volume (GMV) reduction in frontal lobe compared to A-allele carriers, and cerebellar hemispheres GMV reductions were found in G-allele carriers compared to AA-genotype. Moreover, A-allele carriers presented posterior brain GMV reductions when compared to GG-genotype. These data suggest that ZDHHC8 may play a role in cortical volumes. © 2013 Elsevier B.V. All rights reserved.
1. Introduction One of the strongest genetic risk factors identified for schizophrenia is the presence of 22q11 deletion (Levinson et al., 2011). The zinc finger DHHC domain-containing protein 8 gene (ZDHHC8) is located at 22q11.2 and encodes a putative transmembrane palmitoyltransferase enzyme (Mukai et al., 2008). Protein palmitoylation is a common protein lipid modification in the brain (Fukata and Fukata, 2010). It increases the hydrophobicity of neuronal proteins, affecting their interaction with cell membranes (el-Husseini Ael and Bredt, 2002; Drew et al., 2011). Since palmitoylation is critical for some proteins involved in
⁎ Corresponding author at: Departamento de Psiquiatria UNIFESP/EPM, Rua Pedro de Toledo 669, 3o. andar, SP, Brasil. Tel.: +55 11 55764845. E-mail addresses: [email protected] (V.K. Ota), [email protected] (A. Gadelha), [email protected] (I.B. Assunção), [email protected] (M.L. Santoro), [email protected] (D.M. Christofolini), [email protected] (F.T. Bellucco), [email protected] (A.F. Santos-Filho), [email protected] (G.L. Ottoni), [email protected] (D.R. Lara), [email protected] (J.J. Mari), [email protected] (M.I. Melaragno), [email protected] (M.A.C. Smith), [email protected] (R.A. Bressan), [email protected] (S.I. Belangero), [email protected] (A.P. Jackowski). 0920-9964/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.schres.2013.01.011
neurodevelopment (Young et al., 2012), ZDHHC8 might participate in the brain morphology. Indeed, ZDHHC8 is highly expressed in the cortex and hippocampus (Liu et al, 2002). The A-allele of rs175174, a single nucleotide polymorphism (SNP) in ZDHHC8, was associated to schizophrenia (Liu et al., 2002). This allele was shown to increase levels of unspliced form, which is predicted to encode a truncated protein (Mukai et al., 2004). However, another study in Han-Chinese population reported that G-allele was the risk allele (Chen et al., 2004). Structural neuroimaging techniques are useful tools in understanding the biological underpinnings of schizophrenia (Perlini et al., 2012). There is evidence for gray matter reductions of anterior cingulate, frontal and temporal lobes, hippocampus/amygdala, thalamus, and insula that may be magnified over time (Shepherd et al., 2012) providing support for the neurodevelopmental hypothesis of schizophrenia. Genes related to schizophrenia and neurodevelopment have been associated with brain-based changes, mainly variations of gray matter volumes (Wang et al., 2009; Trost et al., in press; Wei et al., 2012). Furthermore, patients with 22q11.2 deletion syndrome present abnormalities in brain structure, such as global brain volumetric reduction including several cortical regions, cerebellum and hippocampus (Tan et al., 2009). To our knowledge, no studies assessed the effect of ZDHHC8 on regional gray matter volume in patients with schizophrenia.
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V.K. Ota et al. / Schizophrenia Research 145 (2013) 33–35
Considering the broad importance of palmitoylation to neuronal function, the localization of ZDHHC8 gene and the divergent results from previous studies on schizophrenia, we aimed to further clarify the role of this SNP in schizophrenia and in regional gray matter volume (GMV), using voxel-based morphometry. 2. Material and methods
This hypothesis-driven analysis was conducted using the small volume correction (SVC) method, with the purpose of constraining the total number of voxels included in the analyses. SVC-based analyses were performed by placing a sphere with a 5 mm radius centered at the local maximum, over the respective volume of interest (frontal, temporal and parietal lobes and cerebellum), with voxel clusters comprising at least 50 voxels, and reported as significant if surviving false discovery rate (FDR) correction for multiple comparisons (p b 0.05).
2.1. Subjects and ZDHHC8 genotyping The Research Ethics Committee of UNIFESP approved the research protocol and participants entered the study after signing an informed consent. A total of 282 patients (199 men and 83 women, age=36.85± 10.64) were recruited from the PROESQ at UNIFESP (N=193) and from the out-patient unit of the PUCRS (N=89). The Structured Diagnostic Interview (SCID) applied by trained psychiatrists was used to confirm the DSM-IV criteria for schizophrenia. Three hundred and seventy nine healthy subjects (213 men and 166 women, age= 41.29± 15.34) were recruited by UNIFESP (N= 257) and PUCRS (N= 122). They did not have psychiatric diseases and family history of psychotic illness, assessed through a questionnaire derived from SCID item for psychotic symptoms. All of them were genotyped using TaqMan® genotyping technology (Applied Biosystems, Foster City, CA) and a sample of 138 patients from UNIFESP was submitted to Magnetic Resonance Imaging (MRI). Hardy–Weinberg equilibrium was verified using Chi-square test. Logistic regression was performed to associate rs175174 with schizophrenia and p b 0.05 was considered significant. 2.2. Imaging processing and analysis Brain MRI was obtained from 138 subjects with schizophrenia from UNIFESP (93 men, age= 35.55± 10.29; 45 women, age = 38.49 ± 10.84) using a 1.5T scanner [Magnetom Sonata (Maestro Class) Siemens AG, Medical Solutions, Erlangen, Germany] with an eight-channel head coil. Two conventional sequences were performed to exclude structural lesions: a) axial T2-weighted FLAIR (fluid-attenuated inversion recovery) in a plane parallel to the anterior commissure–posterior commissure (AC-PC) line [TR (repetition time) = 8500 ms, TE (echo time) = 107 ms, IT (inversion time) = 2500 ms, slice thickness= 5.0 mm, slice interval= 1.5 mm, FOV (field of view) = 240 mm, matrix size= 256 × 256, NEX= 1] and b) sagittal T1-gradient echo volumetric acquisition for multiplanar reconstruction (TR = 2000 ms, TE= 3.42 ms, flip angle=15°, FOV= 245 mm, 1.0 mm slice thickness with no gaps, totaling 160 slices per slab, matrix size= 256 × 256, NEX = 1). Scans of low image quality were excluded. T1-weighted images were processed using VBM5 toolbox implemented in SPM5 software (Wellcome Department of Imaging Neuroscience, London, England; http://www.fil. ion.ucl.ac.uk/spm), running in Matlab® 7.0 (MathworksTM, Natick, MA), to normalize, segment, smooth and analyze all images according to previously described protocols (Ashburner and Friston, 2005). The resulting images were smoothed using a 10-mm-FWHM isotropic Gaussian kernel. Smoothed modulated gray matter images were analyzed through a factorial analysis of covariance (ANCOVA) design using intracranial volume as a covariate. ANCOVA was computed to detect differences in gray matter volume between groups (AA, AG and GG-genotype). Total intracranial volume (TIV) was included as nuisance covariate. TIV was calculated as the sum of GM, White Matter and Cerebrospinal Fluid (CSF) volumes, derived from SPM5 toolbox's HMRF segmentations. The VBM analysis performed in this study was hypothesis-driven fashion, searching for voxel clusters in specific brain regions hypothesized a priori taking in consideration brain regions involved in the pathophysiology of schizophrenia (frontal, temporal and parietal lobes and cerebellum).
3. Results 3.1. ZDHHC8 and schizophrenia rs175174 genotype distributions did not deviate from that predicted by Hardy–Weinberg equilibrium (controls: p = 0.20; cases: p = 0.66). Minor G-allele frequencies were 0.4393 and 0.4344 in controls and patients, respectively. No association between genotypes and schizophrenia was found after correcting for gender, age and ethnicity, assuming a codominant model (p= 0.395). Also, after grouping minor G-allele carriers (AG or GG-genotypes carriers) and comparing to AA-genotype carriers, no association was found (p= 0.341; OR= 1.190; 95%CI = 0.832–1.703).
3.2. MRI Patients submitted to a MRI scan were divided into three groups according to rs175174 genotypes: AA (N = 52), AG (N = 60) and GG (N = 26). There were significant associations between the rs175174 polymorphism and frontal, cerebellar and parietal lobe gray matter volumes. According to the ANCOVA of global GMV in the three groupcomparisons, the right inferior frontal gyrus GMV of the GG genotype group was significantly reduced in comparison to the AA genotype group. The same reduction was observed when AA and AG-genotype carriers were grouped and compared with subjects with GG-genotype carriers. AG-genotype carriers presented a significant reduction of the GMV of the right cerebellar hemisphere in comparison to AA-genotype carriers. This finding remained significant when subjects with AG and GG were included in the same group and compared with AA-carriers. Also, subjects with the AA-genotype also presented a reduction in the parietal lobe in comparison to subjects with GG and AG-genotypes. For details, see Table 1.
Table 1 Areas of significantly reduced gray matter volume according to ZDHHC8 rs175174 genotype. Comparison
Brain region
AA > GG Right inferior frontal gyrus AA + AG > GG Right inferior frontal gyrus AA > AG Right cerebellar hemisphere AA > AG + GG Right cerebellar hemisphere GG > AA
Right supramarginal gyrus (inferior parietal lobule) GG > AA + AG Right supramarginal gyrus (inferior parietal lobule) AG + GG > AA Left inferior parietal lobule a
Peak Number of MNI coordinates Z-score voxelsa (x,y,z) 46 45 23 41 21 40 55
14 14 12 14 −68 −
3.57 3.50 3.85
678 54 72
−68 −
3.35
100
−47 30
3.89
242
55 −47 30
3.68
142
39 −66 38
3.49
54
Total number of contiguous voxels in each regions that surpassed in the initial cutoff of Z > 2.83.
V.K. Ota et al. / Schizophrenia Research 145 (2013) 33–35
4. Discussion Considering the broad importance of protein palmitoylation in neurons and the genetic and functional data, ZDHHC8 seems to be a strong candidate for schizophrenia and its phenotypes (Drew et al., 2011). Therefore, in the current study, we investigated the association of ZDHHC8 rs175174 with schizophrenia and cortical volumes. The frequencies of the minor allele-G were 0.4393 and 0.4344 in control and patient groups, respectively, similar to European populations (Faul et al., 2005; Glaser et al., 2005), but lower than Asian populations (Chen et al., 2004; Otani et al., 2005; Saito et al., 2005). Probably, these differences were due to our admixed sample, which presented a higher European ancestry component than African and Native-American/Asian ancestries. Comparing patients and controls, no significant association between rs175174 genotypes and schizophrenia was found, corroborating a recent meta-analysis study, which included both European and Asian populations (Xu et al., 2010). The role of ZDHHC8 in brain structure in schizophrenia is still unknown. Significant cortical reductions have been reported in patients with 22q11.2 deletion syndrome and schizophrenia, which are consistent with those from studies involving schizophrenia samples from the general population (Chow et al., 2011). The gene encoding ZDHHC8 is expressed in both hippocampus and cortex (Mukai et al., 2004). In our study, GG-genotype carriers presented GMV reduction in frontal lobe compared to A-allele carriers, and cerebellar hemisphere GMV reductions were found in G-alelle carriers compared to AA-genotype. Moreover, A-allele carriers presented posterior brain GMV reductions when compared to GG-genotype. Some limitations of this study may include the small sample size, and the lack of MRI evaluation in a healthy control group, which could interrogate the effect of rs175174 genotypes in the general population, not only under pathological condition. However, regarding MRI, considering large effects (effect size= 0.40) and using a F-test with α = 0.05 and N = 138, the power of this analysis was >99% (http://www. psycho.uni-duesseldorf.de/aap/projects/gpower/). In addition, all inconsistent findings reported for rs175174 may be due either to the different ethnic origins of the various populations or attributed to a closely linked genetic locus, and not to ZDHHC8 itself. Although the association of rs175174 and schizophrenia may be controversial, it seems to have a role in cortical structure. Given our results and the ZDHHC8 role in palmitoylation, it is plausible to suggest further investigation of this potential relationship. Role of funding source Funding for this study was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2007/58736-1 and 2011/00030-1, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Brazil), which had no role in the study design, in the data collection and analysis, in the writing of the paper, or in the decision to submit the paper for publication.
Contributors Authors S.I. Belangero, F.T.S. Bellucco, D.M. Christofolini, M.I. Melaragno and M.A.C. Smith designed the study and wrote the protocol. Authors A. Gadelha, A.F. Santos-Filho, G.L. Ottoni and D.R. Lara selected and interviewed patients and controls. Authors V.K. Ota and A.P. Jackowski undertook the statistical analysis. Authors V.K. Ota, S.I. Belangero and A.P. Jackowski managed the literature searches and analyses and wrote the first draft of the manuscript. Authors A.P. Jackowski, I.B. Assunção and V.K. Ota provided the data acquisition. Authors A. Gadelha, M.L. Santoro, S.I. Belangero, D.R. Lara, R. Bressan, M.I. Melaragno, J.J. Mari, and M.A.C. Smith revised the manuscript critically for important intellectual content. All authors contributed to and have approved the final manuscript.
Conflict of interest The authors declare that they have no competing interests.
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Acknowledgements This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Brazil) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil). We would like to thank the patients, their families, and the psychiatrists for their participation in this study.
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Contents lists available at SciVerse ScienceDirect
Schizophrenia Research journal homepage: www.elsevier.com/locate/schres
ZDHHC8 gene may play a role in cortical volumes of patients with schizophrenia Vanessa K. Ota a, b, Ary Gadelha b, c, Idaiane B. Assunção b, Marcos L. Santoro a, b, Denise M. Christofolini d, Fernanda T. Bellucco a, Airton F. Santos-Filho c, Gustavo L. Ottoni e, Diogo R. Lara e, Jair J. Mari c, Maria I. Melaragno a, Marília A.C. Smith a, Rodrigo A. Bressan b, c, Sintia I. Belangero a, b, c, Andrea P. Jackowski b, c,⁎ a Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de Sao Paulo (UNIFESP), Rua Botucatu, 740, Edifício Leitao da Cunha, 1° andar, CEP 04023-900, Sao Paulo, Brazil b Laboratório Interdisciplinar de Neurociências Clínicas (LiNC), Departamento de Psiquiatria, Universidade Federal de Sao Paulo (UNIFESP), Sao Paulo, Brazil c Departamento de Psiquiatria, Universidade Federal de Sao Paulo (UNIFESP), Sao Paulo, Brazil d Disciplina de Genética e Reproducao Humana, Departamento de Ginecologia e Obstetrícia, Faculdade de Medicina do ABC (FMABC), São Paulo, Brazil e Faculdade de Biociências, Pontifícia Universidade do Rio Grande do Sul (PUCRS), Porto Alegre, Brazil
a r t i c l e
i n f o
Article history: Received 4 July 2012 Received in revised form 11 January 2013 Accepted 14 January 2013 Available online 8 February 2013 Keywords: Schizophrenia ZDHHC8 22q11 Cortical volumes MRI
a b s t r a c t ZDHHC8 rs175174 polymorphism is located in 22q11.2 region and its role in brain volume has not been fully addressed. A total of 282 schizophrenia patients and 379 controls were genotyped. A sample of 138 patients underwent brain MRI scan. No association was found between schizophrenia and genotypes. Nevertheless, GG-genotype carriers presented gray matter volume (GMV) reduction in frontal lobe compared to A-allele carriers, and cerebellar hemispheres GMV reductions were found in G-allele carriers compared to AA-genotype. Moreover, A-allele carriers presented posterior brain GMV reductions when compared to GG-genotype. These data suggest that ZDHHC8 may play a role in cortical volumes. © 2013 Elsevier B.V. All rights reserved.
1. Introduction One of the strongest genetic risk factors identified for schizophrenia is the presence of 22q11 deletion (Levinson et al., 2011). The zinc finger DHHC domain-containing protein 8 gene (ZDHHC8) is located at 22q11.2 and encodes a putative transmembrane palmitoyltransferase enzyme (Mukai et al., 2008). Protein palmitoylation is a common protein lipid modification in the brain (Fukata and Fukata, 2010). It increases the hydrophobicity of neuronal proteins, affecting their interaction with cell membranes (el-Husseini Ael and Bredt, 2002; Drew et al., 2011). Since palmitoylation is critical for some proteins involved in
⁎ Corresponding author at: Departamento de Psiquiatria UNIFESP/EPM, Rua Pedro de Toledo 669, 3o. andar, SP, Brasil. Tel.: +55 11 55764845. E-mail addresses: [email protected] (V.K. Ota), [email protected] (A. Gadelha), [email protected] (I.B. Assunção), [email protected] (M.L. Santoro), [email protected] (D.M. Christofolini), [email protected] (F.T. Bellucco), [email protected] (A.F. Santos-Filho), [email protected] (G.L. Ottoni), [email protected] (D.R. Lara), [email protected] (J.J. Mari), [email protected] (M.I. Melaragno), [email protected] (M.A.C. Smith), [email protected] (R.A. Bressan), [email protected] (S.I. Belangero), [email protected] (A.P. Jackowski). 0920-9964/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.schres.2013.01.011
neurodevelopment (Young et al., 2012), ZDHHC8 might participate in the brain morphology. Indeed, ZDHHC8 is highly expressed in the cortex and hippocampus (Liu et al, 2002). The A-allele of rs175174, a single nucleotide polymorphism (SNP) in ZDHHC8, was associated to schizophrenia (Liu et al., 2002). This allele was shown to increase levels of unspliced form, which is predicted to encode a truncated protein (Mukai et al., 2004). However, another study in Han-Chinese population reported that G-allele was the risk allele (Chen et al., 2004). Structural neuroimaging techniques are useful tools in understanding the biological underpinnings of schizophrenia (Perlini et al., 2012). There is evidence for gray matter reductions of anterior cingulate, frontal and temporal lobes, hippocampus/amygdala, thalamus, and insula that may be magnified over time (Shepherd et al., 2012) providing support for the neurodevelopmental hypothesis of schizophrenia. Genes related to schizophrenia and neurodevelopment have been associated with brain-based changes, mainly variations of gray matter volumes (Wang et al., 2009; Trost et al., in press; Wei et al., 2012). Furthermore, patients with 22q11.2 deletion syndrome present abnormalities in brain structure, such as global brain volumetric reduction including several cortical regions, cerebellum and hippocampus (Tan et al., 2009). To our knowledge, no studies assessed the effect of ZDHHC8 on regional gray matter volume in patients with schizophrenia.
34
V.K. Ota et al. / Schizophrenia Research 145 (2013) 33–35
Considering the broad importance of palmitoylation to neuronal function, the localization of ZDHHC8 gene and the divergent results from previous studies on schizophrenia, we aimed to further clarify the role of this SNP in schizophrenia and in regional gray matter volume (GMV), using voxel-based morphometry. 2. Material and methods
This hypothesis-driven analysis was conducted using the small volume correction (SVC) method, with the purpose of constraining the total number of voxels included in the analyses. SVC-based analyses were performed by placing a sphere with a 5 mm radius centered at the local maximum, over the respective volume of interest (frontal, temporal and parietal lobes and cerebellum), with voxel clusters comprising at least 50 voxels, and reported as significant if surviving false discovery rate (FDR) correction for multiple comparisons (p b 0.05).
2.1. Subjects and ZDHHC8 genotyping The Research Ethics Committee of UNIFESP approved the research protocol and participants entered the study after signing an informed consent. A total of 282 patients (199 men and 83 women, age=36.85± 10.64) were recruited from the PROESQ at UNIFESP (N=193) and from the out-patient unit of the PUCRS (N=89). The Structured Diagnostic Interview (SCID) applied by trained psychiatrists was used to confirm the DSM-IV criteria for schizophrenia. Three hundred and seventy nine healthy subjects (213 men and 166 women, age= 41.29± 15.34) were recruited by UNIFESP (N= 257) and PUCRS (N= 122). They did not have psychiatric diseases and family history of psychotic illness, assessed through a questionnaire derived from SCID item for psychotic symptoms. All of them were genotyped using TaqMan® genotyping technology (Applied Biosystems, Foster City, CA) and a sample of 138 patients from UNIFESP was submitted to Magnetic Resonance Imaging (MRI). Hardy–Weinberg equilibrium was verified using Chi-square test. Logistic regression was performed to associate rs175174 with schizophrenia and p b 0.05 was considered significant. 2.2. Imaging processing and analysis Brain MRI was obtained from 138 subjects with schizophrenia from UNIFESP (93 men, age= 35.55± 10.29; 45 women, age = 38.49 ± 10.84) using a 1.5T scanner [Magnetom Sonata (Maestro Class) Siemens AG, Medical Solutions, Erlangen, Germany] with an eight-channel head coil. Two conventional sequences were performed to exclude structural lesions: a) axial T2-weighted FLAIR (fluid-attenuated inversion recovery) in a plane parallel to the anterior commissure–posterior commissure (AC-PC) line [TR (repetition time) = 8500 ms, TE (echo time) = 107 ms, IT (inversion time) = 2500 ms, slice thickness= 5.0 mm, slice interval= 1.5 mm, FOV (field of view) = 240 mm, matrix size= 256 × 256, NEX= 1] and b) sagittal T1-gradient echo volumetric acquisition for multiplanar reconstruction (TR = 2000 ms, TE= 3.42 ms, flip angle=15°, FOV= 245 mm, 1.0 mm slice thickness with no gaps, totaling 160 slices per slab, matrix size= 256 × 256, NEX = 1). Scans of low image quality were excluded. T1-weighted images were processed using VBM5 toolbox implemented in SPM5 software (Wellcome Department of Imaging Neuroscience, London, England; http://www.fil. ion.ucl.ac.uk/spm), running in Matlab® 7.0 (MathworksTM, Natick, MA), to normalize, segment, smooth and analyze all images according to previously described protocols (Ashburner and Friston, 2005). The resulting images were smoothed using a 10-mm-FWHM isotropic Gaussian kernel. Smoothed modulated gray matter images were analyzed through a factorial analysis of covariance (ANCOVA) design using intracranial volume as a covariate. ANCOVA was computed to detect differences in gray matter volume between groups (AA, AG and GG-genotype). Total intracranial volume (TIV) was included as nuisance covariate. TIV was calculated as the sum of GM, White Matter and Cerebrospinal Fluid (CSF) volumes, derived from SPM5 toolbox's HMRF segmentations. The VBM analysis performed in this study was hypothesis-driven fashion, searching for voxel clusters in specific brain regions hypothesized a priori taking in consideration brain regions involved in the pathophysiology of schizophrenia (frontal, temporal and parietal lobes and cerebellum).
3. Results 3.1. ZDHHC8 and schizophrenia rs175174 genotype distributions did not deviate from that predicted by Hardy–Weinberg equilibrium (controls: p = 0.20; cases: p = 0.66). Minor G-allele frequencies were 0.4393 and 0.4344 in controls and patients, respectively. No association between genotypes and schizophrenia was found after correcting for gender, age and ethnicity, assuming a codominant model (p= 0.395). Also, after grouping minor G-allele carriers (AG or GG-genotypes carriers) and comparing to AA-genotype carriers, no association was found (p= 0.341; OR= 1.190; 95%CI = 0.832–1.703).
3.2. MRI Patients submitted to a MRI scan were divided into three groups according to rs175174 genotypes: AA (N = 52), AG (N = 60) and GG (N = 26). There were significant associations between the rs175174 polymorphism and frontal, cerebellar and parietal lobe gray matter volumes. According to the ANCOVA of global GMV in the three groupcomparisons, the right inferior frontal gyrus GMV of the GG genotype group was significantly reduced in comparison to the AA genotype group. The same reduction was observed when AA and AG-genotype carriers were grouped and compared with subjects with GG-genotype carriers. AG-genotype carriers presented a significant reduction of the GMV of the right cerebellar hemisphere in comparison to AA-genotype carriers. This finding remained significant when subjects with AG and GG were included in the same group and compared with AA-carriers. Also, subjects with the AA-genotype also presented a reduction in the parietal lobe in comparison to subjects with GG and AG-genotypes. For details, see Table 1.
Table 1 Areas of significantly reduced gray matter volume according to ZDHHC8 rs175174 genotype. Comparison
Brain region
AA > GG Right inferior frontal gyrus AA + AG > GG Right inferior frontal gyrus AA > AG Right cerebellar hemisphere AA > AG + GG Right cerebellar hemisphere GG > AA
Right supramarginal gyrus (inferior parietal lobule) GG > AA + AG Right supramarginal gyrus (inferior parietal lobule) AG + GG > AA Left inferior parietal lobule a
Peak Number of MNI coordinates Z-score voxelsa (x,y,z) 46 45 23 41 21 40 55
14 14 12 14 −68 −
3.57 3.50 3.85
678 54 72
−68 −
3.35
100
−47 30
3.89
242
55 −47 30
3.68
142
39 −66 38
3.49
54
Total number of contiguous voxels in each regions that surpassed in the initial cutoff of Z > 2.83.
V.K. Ota et al. / Schizophrenia Research 145 (2013) 33–35
4. Discussion Considering the broad importance of protein palmitoylation in neurons and the genetic and functional data, ZDHHC8 seems to be a strong candidate for schizophrenia and its phenotypes (Drew et al., 2011). Therefore, in the current study, we investigated the association of ZDHHC8 rs175174 with schizophrenia and cortical volumes. The frequencies of the minor allele-G were 0.4393 and 0.4344 in control and patient groups, respectively, similar to European populations (Faul et al., 2005; Glaser et al., 2005), but lower than Asian populations (Chen et al., 2004; Otani et al., 2005; Saito et al., 2005). Probably, these differences were due to our admixed sample, which presented a higher European ancestry component than African and Native-American/Asian ancestries. Comparing patients and controls, no significant association between rs175174 genotypes and schizophrenia was found, corroborating a recent meta-analysis study, which included both European and Asian populations (Xu et al., 2010). The role of ZDHHC8 in brain structure in schizophrenia is still unknown. Significant cortical reductions have been reported in patients with 22q11.2 deletion syndrome and schizophrenia, which are consistent with those from studies involving schizophrenia samples from the general population (Chow et al., 2011). The gene encoding ZDHHC8 is expressed in both hippocampus and cortex (Mukai et al., 2004). In our study, GG-genotype carriers presented GMV reduction in frontal lobe compared to A-allele carriers, and cerebellar hemisphere GMV reductions were found in G-alelle carriers compared to AA-genotype. Moreover, A-allele carriers presented posterior brain GMV reductions when compared to GG-genotype. Some limitations of this study may include the small sample size, and the lack of MRI evaluation in a healthy control group, which could interrogate the effect of rs175174 genotypes in the general population, not only under pathological condition. However, regarding MRI, considering large effects (effect size= 0.40) and using a F-test with α = 0.05 and N = 138, the power of this analysis was >99% (http://www. psycho.uni-duesseldorf.de/aap/projects/gpower/). In addition, all inconsistent findings reported for rs175174 may be due either to the different ethnic origins of the various populations or attributed to a closely linked genetic locus, and not to ZDHHC8 itself. Although the association of rs175174 and schizophrenia may be controversial, it seems to have a role in cortical structure. Given our results and the ZDHHC8 role in palmitoylation, it is plausible to suggest further investigation of this potential relationship. Role of funding source Funding for this study was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2007/58736-1 and 2011/00030-1, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Brazil), which had no role in the study design, in the data collection and analysis, in the writing of the paper, or in the decision to submit the paper for publication.
Contributors Authors S.I. Belangero, F.T.S. Bellucco, D.M. Christofolini, M.I. Melaragno and M.A.C. Smith designed the study and wrote the protocol. Authors A. Gadelha, A.F. Santos-Filho, G.L. Ottoni and D.R. Lara selected and interviewed patients and controls. Authors V.K. Ota and A.P. Jackowski undertook the statistical analysis. Authors V.K. Ota, S.I. Belangero and A.P. Jackowski managed the literature searches and analyses and wrote the first draft of the manuscript. Authors A.P. Jackowski, I.B. Assunção and V.K. Ota provided the data acquisition. Authors A. Gadelha, M.L. Santoro, S.I. Belangero, D.R. Lara, R. Bressan, M.I. Melaragno, J.J. Mari, and M.A.C. Smith revised the manuscript critically for important intellectual content. All authors contributed to and have approved the final manuscript.
Conflict of interest The authors declare that they have no competing interests.
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Acknowledgements This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Brazil) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil). We would like to thank the patients, their families, and the psychiatrists for their participation in this study.
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