Association analysis of ANK3 gene variants in nordic bipolar disorder and schizophrenia caseЦcontrol samples.код для вставкиСкачать
RESEARCH ARTICLE Neuropsychiatric Genetics Association Analysis of ANK3 Gene Variants in Nordic Bipolar Disorder and Schizophrenia Case–Control Samples Martin Tesli,1,2* Pernille Koefoed,3,4 Lavinia Athanasiu,1,2,5 Morten Mattingsdal,5 Omar Gustafsson,2,6 Ingrid Agartz,1,7,8 Lars M. Rimol,8 Andrew Brown,1,9,10 Katrine V. Wirgenes,1,2 Lisa-Lena Smorr,1,2 Anna K. K€ahler,2,11,12 Thomas Werge,13 Ole Mors,14 Erling Mellerup,3,4 Erik G. J€onsson,7 Ingrid Melle,1,2 Gunnar Morken,15,16 Srdjan Djurovic,1,2,5 and Ole A. Andreassen1,2 1 Institute of Clinical Medicine, University of Oslo, Oslo, Norway Division of Mental Health and Addiction, Oslo University Hospital, Oslo, Norway 2 3 Department of Neuroscience and Pharmacology, Laboratory of Neuropsychiatry, University of Copenhagen, Copenhagen, Denmark 4 Psychiatric Centre Copenhagen, Department O, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Department of Medical Genetics, Oslo University Hospital, Oslo, Norway 5 6 deCODE Genetics, Reykjavik, Iceland 7 Department of Clinical Neuroscience, HUBIN Project, Psychiatry Section, Karolinska Institutet and Hospital, Stockholm, Sweden Department of Psychiatric Research, Diakonhjemmet Hospital, Oslo, Norway 8 9 Department of Biostatistics, University of Oslo, Oslo, Norway 10 Department of Mathematics, University of Oslo, Oslo, Norway Department of Genetics, University of North Carolina at Chapel Hill, North Carolina 27599 11 12 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 13 Research Institute of Biological Psychiatry, Mental Health Center Sct. Hans, Copenhagen University Hospital, Roskilde, Denmark Center for Psychiatric Research, Aarhus University Hospital, Aarhus, Denmark 14 15 Østmarka Psychiatric Department, St. Olavs Hospital, Trondheim, Norway 16 Institute of Neuroscience, Norwegian University of Technology and Science, Trondheim, Norway Received 29 October 2010; Accepted 16 September 2011 Genetic variants in ankyrin 3 (ANK3) have recently been shown to be associated with bipolar disorder (BD). We genotyped three ANK3 SNPs previously found to be associated with BD (rs10994336, rs1938526, and rs9804190) in a Scandinavian BD case–control sample (N ¼ 854/2,614). Due to evidence of genetic overlap between BD and schizophrenia (SZ), we also genotyped these three SNPs in a Scandinavian SZ case–control sample (N ¼ 1,073/2,919). Combining our Scandinavian samples with an Icelandic sample (N ¼ 435 BD cases, 651 SZ cases, and 11,491 healthy controls), we found rs10994336 and rs9804190 to be nominally signiﬁcantly associated with BD in this combined Nordic BD sample (N ¼ 1,289/14,105). Nominal P was 0.015/ 0.018 (ﬁxed/random effect) for rs10994336 (Bonferroni corrected P ¼ 0.044/0.053) and 0.023 for rs9804190 (Bonferroni corrected P ¼ 0.069). None of the SNPs were signiﬁcantly associated with SZ in the combined Nordic SZ case–control sample (N ¼ 1,724/14,410). These results further support that ANK3 is a Ó 2011 Wiley Periodicals, Inc. Additional Supporting Information may be found in the online version of this article. Grant sponsor: University of Oslo; Grant sponsor: Research Council of Norway; Grant numbers: 167153/V50, 163070/V50; Grant sponsor: SouthEast Norway Health Authority; Grant number: 2004123; Grant sponsor: Danish National Psychiatric Research Foundation; Grant sponsor: Lundbeck Foundation; Grant sponsor: Ivan Nielsen Foundation; Grant sponsor: Stanley Medical Research Institute; Grant sponsor: Wallenberg Foundation; Grant sponsor: HUBIN Project; Grant sponsor: Swedish Medical Research Council; Grant numbers: 2006-2992, 2006-986, 2008-2167. *Correspondence to: Martin Tesli, MD, Oslo University Hospital HF, Division of Mental Health and Addiction Psychosis Research Unit/TOP, Kirkeveien 166, N-0407 Oslo, Norway. E-mail: firstname.lastname@example.org Published online 3 October 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ajmg.b.31244 969 970 susceptibility gene speciﬁc to BD and that more than one risk locus is involved. Ó 2011 Wiley Periodicals, Inc. Key words: bipolar disorder; schizophrenia; ANK3; genetic association INTRODUCTION Bipolar disorder (BD) and schizophrenia (SZ) are common, severe, and highly heritable psychiatric disorders [Smoller and Finn, 2003; Sullivan, 2005]. However, few susceptibility genes have been ﬁrmly established for these two disorders and the underlying pathophysiological mechanisms are not well understood [Nothen et al., 2010]. Findings from epidemiological [Craddock et al., 1995] and molecular genetic [Owen et al., 2009] studies suggest that these disorders have a polygenic basis, and that multiple genes, each with a small effect, interact with each other and with environmental factors to give rise to BD and SZ. Moreover, results from recent epidemiological studies [Lichtenstein et al., 2009] and genetic association studies [Moskvina et al., 2009] indicate a genetic overlap between these two disorders. Of the most promising recent ﬁndings from BD genome-wide association studies (GWAS) is ANK3. In a collaborative GWAS comprising 4,387 cases and 6,209 controls, a genome-wide signiﬁcant association between the two ANK3 SNPs rs10994336 (P ¼ 9.1 109) and rs1938526 (P ¼ 1.3 108) and BD was found [Ferreira et al., 2008]. The association between rs10994336 and BD has been replicated in a sample of 1,668 cases and 1,604 controls (P ¼ 1.7 105) [Schulze et al., 2009] as well as in a sample of 638 cases and 904 controls (P ¼ 0.042) [Scott et al., 2009]. Furthermore, the association between rs1938526 and BD (P ¼ 0.036) (N ¼ 1,001 cases and 1,033 controls) has also been replicated [Smith et al., 2009]. All the above mentioned studies consisted of European individuals, but there is also evidence from other populations that genetic variants in ANK3 are involved in the etiology of BD. In the ﬁrst GWAS on bipolar I disorder in the Han Chinese population (N ¼ 1,000 cases and 1,000 controls) one SNP nearby the ANK3 SNP rs1938526 was found to be signiﬁcantly associated with bipolar I disorder (P ¼ 6.55 105) [Lee et al., 2010]. ANK3 is expressed in the central and peripheral nervous system, where its encoded protein Ankyrin G was ﬁrst discovered in the axonal initial segments and nodes of Ranvier [Kordeli et al., 1995]. Ankyrin G has been shown to link membrane proteins to the spectrin–actin cytoskeleton, and to be important for clustering of ion channels at axon initial segments and for normal action potential ﬁring [Kordeli et al., 1995; Zhou et al., 1998; Pan et al., 2006]. These ﬁndings have led to the hypothesis that ANK3 is involved in BD pathophysiology through ion channel dysregulation [Ferreira et al., 2008]. We investigated the association between the three most signiﬁcant ANK3 loci from previous BD studies (rs10994336, rs1938526, and rs9804190) [Ferreira et al., 2008; Schulze et al., 2009] in a Scandinavian case–control sample independent of the abovementioned samples. Due to the evidence of genetic overlap between AMERICAN JOURNAL OF MEDICAL GENETICS PART B How to Cite this Article: Tesli M, Koefoed P, Athanasiu L, Mattingsdal M, Gustafsson O, Agartz I, Rimol LM, Brown A, Wirgenes KV, Smorr L-L, K€ahler AK, Werge T, Mors O, Mellerup E, J€ onsson EG, Melle I, Morken G, Djurovic S, Andreassen OA. 2011. Association Analysis of ANK3 Gene Variants in Nordic Bipolar Disorder and Schizophrenia Case–Control Samples. Am J Med Genet Part B 156:969–974. BD and SZ [Lichtenstein et al., 2009; Moskvina et al., 2009], we also tested for association between these three ANK3 SNPs and SZ. MATERIALS AND METHODS Sample Description The SCOPE bipolar disorder case–control sample. The SCOPE BD sample is based on two independent case–control samples from Norway and Denmark, all included in the Scandinavian Collaboration on Psychiatric Etiology (SCOPE) study. The total number of subjects was 854 BD cases and 2,614 controls. The Norwegian patients (N ¼ 345) had been diagnosed with bipolar I disorder (N ¼ 230), bipolar II disorder (N ¼ 96), or BD not otherwise speciﬁed (N ¼ 19), according to DSM-IV using the Structural Clinical Interview for DSM-IV (SCID) [Spitzer et al., 1992]. The Danish sample (N ¼ 509) consisted of patients with bipolar affective disorder F31 (N ¼ 423) and manic episode F30 (N ¼ 3) according to ICD-10 (http://www.who.int/classiﬁcations/icd/en), bipolar I disorder according to DSM-IV (N ¼ 1), and BD (N ¼ 15), mania with psychosis (N ¼ 1), and bipolar with psychosis (N ¼ 66) according to the OPCRIT classiﬁcation system. [McGufﬁn et al., 1991]. The SCOPE BD, SZ, and control samples are further described in Table I. The SCOPE schizophrenia case–control sample. The SZ association study was based on three independent case–control samples from Norway, Sweden and Denmark, included in the SCOPE. The Norwegian patients (N ¼ 349) had been diagnosed with SZ (N ¼ 272), schizoaffective disorder (N ¼ 59), schizophreniform disorder (N ¼ 15), and persistent delusional disorder (N ¼ 3) according to DSM-IV using the Structural Clinical Interview for DSM-IV (SCID) [Spitzer et al., 1992]. The Danish sample (N ¼ 467) consisted of patients with SZ (N ¼ 422), schizotypal personality disorder (N ¼ 4), persistent delusional disorder (N ¼ 2), and schizoaffective disorder (N ¼ 39) according to ICD-10 F20, F21, F22, and F25 using clinical interviews. The Swedish patients (N ¼ 257) had been diagnosed with SZ (N ¼ 224), schizoaffective disorder (N ¼ 25), or schizophreniform disorder (N ¼ 8), according to DSM-III-R/DSM-IV criteria using medical record reviews and clinical interviews. A total of 1,073 SZ cases and 2,919 control subjects were genotyped in this study. The Norwegian Scientiﬁc-Ethical Committees, the Norwegian Data Protection Agency, the Danish Scientiﬁc Committees, the TESLI ET AL. 971 TABLE I. Characteristics for the SCOPE BD and SZ Case–Control Samples Sample Denmark N (females, %) Mean age (SD)a Norway N (females, %) Mean age (SD)a Sweden N (females, %) Mean age (SD)a Sum BD SZ CTR Sum 509 (0.58) 53.1 (15.0) 467 (0.41) 47.8 (12.3) 2182 (0.46) 46.0 (11.8) 3,158 345 (0.58) 41.2 (13.1) 349 (0.44) 37.2 (10.7) 432 (0.50) 39.2 (10.4) 1,126 305 (0.38) 54.3 (10.4) 2,919 562 854 257 (0.38) 58.1 (15.2) 1,073 4,846 SCOPE, Scandinavian Collaboration on Psychiatric Etiology; BD, bipolar disorder; SZ, schizophrenia; CTR, controls; SD, standard deviation. a Mean age in 2010. Danish Data Protection Agency, the Ethical Committee of the Karolinska Hospital, the Stockholm Regional Ethical Committee, and the Swedish Data Inspection Board approved the study. All subjects have given written informed consent prior to inclusion into the project. Icelandic bipolar disorder and schizophrenia case–control samples. The Icelandic BD and SZ samples consisted of 435 BD cases, 651 SZ cases, and 11,491 controls. Patients and controls were recruited from all over Iceland. For 316 of the BD patients, diagnoses were assigned according to Research Diagnostic Criteria (RDC) [Spitzer et al., 1978] through the use of the Schedule for Affective Disorders and Schizophrenia Lifetime Version (SADS-L) [Spitzer, 1977]. The remaining BD patients were recruited through a genetic study of anxiety and depression [Thorgeirsson et al., 2003] and had been characterized using the Composite International Diagnostic Interview (CIDI) [Peters and Andrews 1995; Wittchen et al., 1996]. For the Icelandic SZ patients, diagnoses were assigned according to RDC [Spitzer et al., 1978] through the use of the SADS-L [Spitzer, 1977. The 11,491 controls were recruited as a part of various genetic programs at deCODE genetics and were not screened for psychiatric disorders. Genotyping Genomic DNA was extracted from whole blood. ANK3 SNPs rs10994336, rs1938526, and rs9804190 were genotyped in the SCOPE BD and SZ case–control samples with predesigned TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. Allelic discrimination of samples was done using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) in combination with the SDS 3.2 software. For a subset of the Norwegian sample, genotype data were based on imputation of a Norwegian GWAS data set comprising patients with BD and SZ, as well as healthy controls [Athanasiu et al., 2010; Djurovic et al., 2010]. Statistical Analysis SCOPE bipolar disorder and schizophrenia case–control samples. All SNPs were tested for deviation from Hardy–Weinberg equilibrium (HWE) in the controls using PLINK (version 1.07; http://pngu.mgh.harvard.edu/purcell/plink/) [Purcell et al., 2007]. Single SNP association tests were performed in PLINK with the allelic test for each subsample and the Cochran–Mantel–Haenszel (CMH) test for the SCOPE BD and SZ samples, using case–control sample origin as the stratiﬁcation factor. The heterogeneity of the sample speciﬁc odds ratios (ORs) was evaluated with the Breslow–Day test. Icelandic bipolar disorder and schizophrenia case–control samples. Single SNP allele tests were performed to investigate for association between the three ANK3 SNPs and BD and SZ in the Icelandic samples. Additionally, genomic control adjustments were undertaken for all SNPs. Combined Nordic bipolar disorder and schizophrenia case–control samples. Association tests for ANK3 SNPs rs10994336, rs1938526, and rs9804190 in the combined Nordic BD case–control control sample (N ¼ 1,289/14,105) and SZ case–control sample (N ¼ 1,724/14,410) were performed with the meta-analysis function in PLINK. Power estimates. Statistical power was estimated with the Genetic Power Calculator (http://pngu.mgh.harvard.edu/purcell/gpc/), assuming a disease prevalence of 0.01 for BD, an additive allelic model, a 10:1 control to case ratio and a type I error rate of 0.05. We used high risk allele frequencies from the HapMap (http://hapmap. ncbi.nlm.nih.gov/) and ORs from previous studies [Ferreira et al., 2008; Schulze et al., 2009]. The results from these analyses are presented in Supplementary material Table II. RESULTS No SNPs had genotype distributions in Hardy–Weinberg disequilibrium in controls (P < 0.05). 1,022/2,634 SCOPE, Scandinavian Collaboration on Psychiatric Etiology; BD, bipolar disorder; SZ, schizophrenia; OR, odds ratio; MAF, minor allele frequency; CMH, Cochrane–Mantel–Haenszel test; F/R, ﬁxed/random effect; Q, P-value for Cochrane’s Q statistic; I, I2 heterogeneity index (0–100). 0 0.70/0.78 (1/1) 1.02/1.02 0.21 34.5 T/C 1,673/14,123 1.05 (0.92–1.20) 0.50 (1) 0.22 0.23 T/C 651/11,489 0.99 (0.87–1.13) 0.88 (1) 0.21 0 rs9804190 T/C 0.91/0.91 (1/1) 1.01/1.01 0.74 0.46/0.46 (1/1) 1.06/1.06 0.94 T/C G/A 1,720/14,320 1,683/14,324 1.08 (0.83–1.39) 1.03 (0.81–1.31) 0.32 (0.96) 0.56 (1) 0.050 0.058 0.060 0.055 T/C G/A 651/11,473 629/11,429 1.05 (0.86–1.29) 0.99 (0.81–1.22) 0.073 0.95 (1) 0.068 0.64 (1) 1,069/2,847 rs1938526 T/C 1,054/2,895 rs10994336 SZ sample G/A 0 0.88/0.88 0.68 (0.069/0.069) 0.023/0.023 T/C 1,183/13,843 0.84 (0.70–0.99) 0.050 (0.15) 0.22 0.19 T/C 434/11,489 0.18 (0.54) 0.90 (0.78–1.05) 0.21 749/2,354 rs9804190 T/C 1.11/1.11 0.60 (0.71/0.71) 0.24/0.24 G/A 1,226/14,012 1.23 (0.93–1.61) 0.32 (0.96) 0.058 0.070 G/A 433/11,473 1.04 (0.83–1.30) 0.072 0.75 (1) 806/2,542 rs1938526 T/C 839/2,596 rs10994336 G/A 0.050 0.071 T/C 421/11,429 0.068 0.30 (0.90) 1.13 (0.90–1.41) OR (95%CI) corrected) MAF allele cases/controls BD sample Sample SNP 0 5.05 1.25/1.24 0.35 0.015/0.018 T/C 1,260/14,025 0.012 (0.036) 1.45 (1.09–1.90) OR (95%CI) corrected) affected unaffected allele controls Allelic P (Bonferroni MAF MAF Minor/major Total number cases/ (Bonferroni CMH P Minor/ major Total number (0.044/0.053) Q OR F/R corrected) allele cases/controls P F/R (Bonferroni major Total number Minor/ Combined Nordic Icelandic The main ﬁndings in this study were signiﬁcant associations between ANK3 SNPs rs10994336 (Bonferroni corrected) and rs9804190 (nominally signiﬁcant) and BD in our combined Nordic sample. These SNPs have previously been found to be associated with BD in three studies comprising individuals of European ancestry [Ferreira et al., 2008; Schulze et al., 2009; Scott et al., 2009]. We did not ﬁnd evidence for association between rs10994336, rs1938526, or rs9804190 and SZ, which indicates that these three ANK3 SNPs are involved speciﬁcally in the pathophysiology of BD, or at least in neurobiological processes more prevalent in the BD spectrum than in the SZ spectrum in the proposed continuum of severe psychiatric disorders [Craddock et al., 2009]. ANK3 being a speciﬁc risk gene for BD is further supported by the ﬁndings from a recent meta-analysis [Liu et al., 2010] combining and comparing results from the aforementioned GWAS on BD (N ¼ 4,387 BD cases and 6,209 controls) [Ferreira et al., 2008] with a GWAS on Major Depressive Disorder (MDD) (N ¼ 1,695 MDD cases and 1761 controls) [Sullivan et al., 2009], where no evidence for ANK3 involvement in MDD was detected. There is increasing evidence of interesting molecular and neurobiological mechanisms related to ANK3 in BD. Ion channelopathy has been proposed as one potential mechanism [Ferreira et al., 2008; Askland et al., 2009]. This hypothesis is based on the ﬁndings from animal studies showing that a dysfunction in ankyrin G could lead to abnormal clustering of potassium channels and voltage-gated sodium channels at axon initial segments and cause abnormal action potential ﬁring [Zhou et al., 1998; Pan et al., 2006]. Further support for this hypothesis comes from GWA studies ﬁnding signiﬁcant association between L-type voltage-gated calcium channel gene (CACNA1C), a gene involved in calcium channel regulation, and BD [Ferreira et al., 2008; Sklar et al., 2008] and from a recent animal study showing the importance of ankyrin G for SCOPE DISCUSSION TABLE II. Association Analyses of ANK3 SNPs in Nordic Bipolar Disorder and Schizophrenia Case–Control Samples None of the three SNPs were signiﬁcantly associated with BD or SZ in the Scandinavian subsamples, but rs10994336 was nominally signiﬁcantly associated with BD in the Icelandic BD sample (nominal P ¼ 0.012). Rs9804190 attained a nominal P-value of 0.05 for association with BD in the Icelandic sample. When combining our results from the Scandinavian samples with the results from the Icelandic samples, we found rs10994336 and rs9804190 to be nominally signiﬁcantly associated with BD in the combined Nordic case–control sample. Nominal P values were 0.015/0.018 (ﬁxed/random effect) for rs10994336 and 0.023 for rs9804190; the respective P values after Bonferroni correction were 0.044/0.53 and 0.069. For both these SNPs (rs10994336 and rs9804190), the risk allele was overrepresented among patients in the total BD sample, as well as in all of the BD subsamples (Table II and Supplementary material Table I). None of the SNPs were signiﬁcantly associated with SZ in any of the samples (Table II and Supplementary material Table I). There was no evidence of heterogeneity for the population-based ORs amongst the different SCOPE subsamples, as indicated by the Breslow–Day test (Supplementary material Table I). More detailed results are presented in Table II and in Supplementary material Table I. AMERICAN JOURNAL OF MEDICAL GENETICS PART B I 972 TESLI ET AL. maintaining appropriate axo-dendritic polarity in vivo [Sobotzik et al., 2009]. Interestingly, another recent animal study showed that ANK3 was downregulated in the mouse brain in response to lithium [McQuillin et al., 2007]. This suggests a potential for drug development based on ANK3 pathophysiology. However, what effect ANK3 SNPs rs10994336 and rs9804190 might have on the function of the expressed protein ankyrin G is unknown. According to the Ensembl database (http://www. ensembl.org/), these two SNPs are located in intronic regions of ANK3. If rs10994336 and rs9804190 are directly affecting the ankyrin G protein, gene regulation affecting expression is a tentative mechanism [Quinn et al., 2010]. It is also possible that these two SNPs have no functional relevance for BD pathophysiology, and that their association with BD merely reﬂects a high linkage disequilibrium (LD) with the real risk allele. The two SNPs attaining most signiﬁcant associations in this study, rs10994336 and rs9804190, are independent markers in our SCOPE sample (R2 ¼ 0.00). As hypothesized in a previous study, this suggests allelic heterogeneity in the involvement of ANK3 in BD [Schulze et al., 2009]. In conclusion, the present results are in accordance with former ﬁndings, suggesting that ANK3 is a BD susceptibility gene, and that ion channelopathy may be involved in BD pathophysiology. Future studies should investigate how ANK3 genetic variants are related to neurobiological endophenotypes as well as clinical subphenotypes in bipolar spectrum disorders. ACKNOWLEDGMENTS We thank patients and controls for their participation in the study, and the health professionals who facilitated our work. We also thank Thomas D. Bjella for assistance with the database, and Bente Bennike, Knut-Erik Gylder, and Lars Hansson for molecular genetic technical assistance. The study was supported by grants to the TOP study group from the University of Oslo, the Research Council of Norway (#167153/V50, #163070/V50), the SouthEast Norway Health Authority (#2004123), the Danish National Psychiatric Research Foundation, the Lundbeck Foundation, the Ivan Nielsen Foundation, the Stanley Medical Research Institute, the Wallenberg Foundation, the HUBIN Project, and the Swedish Medical Research Council (2006-2992, 2006-986, 2008-2167). We would like to thank Dr. Hreinn Stefansson at deCODE Genetics for providing information from replication samples. REFERENCES Askland K, Read C, Moore J. 2009. Pathways-based analyses of wholegenome association study data in bipolar disorder reveal genes mediating ion channel activity and synaptic neurotransmission. Hum Genet 125(1):63–79. 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