Abstract

Schizophrenia is an idiopathic mental disorder with a heritable component and a substantial public health impact. We conducted a multi-stage genome-wide association study (GWAS) for schizophrenia beginning with a Swedish national sample (5,00 cases and 6,243 controls) followed by meta-analysis with previous schizophrenia GWAS (8,832 cases and 2,067 controls) and finally by replication of SNPs in 68 genomic regions in independent samples (7,43 cases, 9,762 controls and 58 parent-offspring trios). We identified 22 loci associated at genome-wide significance; 3 of these are new, and was previously implicated in bipolar disorder. Examination of candidate genes at these loci suggests the involvement of neuronal calcium signaling. We estimate that 8,300 independent, mostly common SNPs (95% credible interval of 6,300-0,200 SNPs) contribute to risk for schizophrenia and that these collectively account for at least 32% of the variance in liability. Common genetic variation has an important role in the etiology of schizophrenia, and larger studies will allow more detailed understanding of this disorder. significance RESULTS Results for Swedish samples We analyzed genome-wide SNP data in 5,001 schizophrenia cases and 6,243 controls from a population-based sampling frame in Sweden (N = 11,244; There are only a handful of robust reported genetic associations for schizophrenia. Genome-wide linkage studies so far have been inconclusive 9 , and no compelling mendelian variants have been identified 2 ADVANCE ONLINE PUBLICATION Nature GeNetics A r t i c l e s Project Phase 1 reference panel, we obtained allelic dosages for 9,871,789 high-quality polymorphic SNPs. Given that this imputation panel is based on >800 chromosomes from individuals of European ancestry and includes the detail afforded by genome sequencing, we anticipated increased power in finding and describing association signals. Indeed, we observed 10,201 SNPs and 187 genomic regions with association P < 1 × 10 −5 using 1000 Genomes Project imputation compared with 1,594 SNPs and 133 genomic regions for HapMap 3 imputation (counts include only one region from the MHC). The resulting genomic inflation factor (λ GC ) was 1.075, and λ 1000 (refs. 19-21) was 1.013. Quantile-quantile and Manhattan plots are shown in Results for Swedish and PGC samples We reanalyzed schizophrenia data from the Psychiatric Genomics Consortium (PGC) using 1000 Genomes Project imputation (8,832 cases and 12,067 controls, excluding Swedish samples) We then conducted a meta-analysis of the Swedish and independent PGC schizophrenia samples using the same quality control, imputation and analysis pipeline. This GWAS meta-analysis of 13,833 schizophrenia cases and 18,310 controls (Table 1) afforded power to detect genotypic relative risks of 1.10-1.14 for reference allele frequencies of 0.15-0.85 (power = 0.8; α = 5 × 10 −8 , log-additive model). We evaluated the comparability of the Swedish and PGC studies using sign tests: of 608 SNPs selected from the PGC results with association P < 0.0001 and in approximate linkage equilibrium, 62.6% had logistic regression β coefficients with the same sign in the Swedish results, an observation highly inconsistent with the null hypothesis of no association (P = 2.2 × 10 −10 ). λ GC was 1.186, and λ 1000 was 1.012, values consistent with a polygenic pattern of association but not with gross inflation due to technical artifacts 20 . Manhattan and quantilequantile plots are shown in We used risk score profiling GWAS often omit the X chromosome. This omission is problematic, as the X chromosome is approximately as large as chromosome 8 and is enriched for genes important in brain development. Using a previously described approach, we imputed X-chromosome SNPs using the 1000 Genomes Project reference panel Most GWAS-associated variants were found outside of proteincoding regions Results from Swedish, PGC and replication samples We then obtained association results for SNPs in 194 genomic regions in 6 independent samples for a total sample size of over 21,000 cases and 38,000 controls ( © 2013 Nature America, Inc. All rights reserved. Nature GeNetics ADVANCE ONLINE PUBLICATION 3 A r t i c l e s replication genotypes were sought were identified using LD clumping, defined by LD (r 2 > 0.5) and a minimum association P of <1 × 10 −5 in the Swedish and PGC meta-analysis. Only one MHC SNP was included. The Swedish and PGC meta-analysis and replication results were highly concordant, with 76.3% of the logistic regression β coefficients having the same direction of effect (sign test P = 1.5 × 10 −17 ). Indeed, of the top 100 SNPs in the Sweden and PGC metaanalysis, 90% had the same sign in the replication results. This result strongly suggests that many more loci will achieve genome-wide significance with further increases in sample size. The combined results in which SNPs at 24 loci reached genomewide significance is shown in Of these 22 associated loci Themes We highlight four themes from these results (see also Supplementary Table 2). First, these results implicate calcium signaling in the etiology of schizophrenia. As in previous studies of bipolar disorder and schizophrenia 17,27,28 , we replicated genome-wide significant association for a SNP in CACNA1C (encoding Ca v 1.2; P = 5.2 × 10 −12 at the intronic SNP rs1006737). Intriguingly, we identified a genome-wide significant association with schizophrenia in CACNB2 (P = 1.3 × 10 −10 at the intronic SNP rs17691888), which encodes the β 2 subunit of L-type calcium channels (Ca v β2). This locus was previously found to be significant when considering five psychiatric disorders as affected A gene-set test showed enrichment of smaller P values in genes encoding calcium channel subunits In L-type calcium channels, the α 1c subunit forms the transmembrane pore and directly interacts with the intracellular β 2 subunit 32 . The β 2 subunit also antagonizes an endoplasmic reticulum retention motif on the α 1c subunit to facilitate transport to the plasma membrane The genetics and biology of calcium channels have been the subject of considerable investigation, owing to their importance in fundamental neuronal processes and human diseases. L-type voltage-gated calcium channels are involved in learning, memory and synaptic plasticity, and Cacna1c knockout mice show notable deficits in long-term potentiation © 2013 Nature America, Inc. All rights reserved. ADVANCE ONLINE PUBLICATION Nature GeNetics A r t i c l e s mendelian calcium channelopathies can have episodic phenomena, perhaps reminiscent of the episodic nature of psychotic disordersexamples of such episodic phenotypes and the underlying genes include intermittent hypoglycemia and hypocalcemia in Timothy syndrome (CACNA1C), episodic ataxia (CACNA1A and CACNB4), migraine (CACNA1A), epilepsy (CACNA1H and CACNB4), periodic paralysis (CACNA1S) and malignant hyperthermia (CACNA1S and CACNA2D1) 32,40 . Our GWAS for schizophrenia suggests candidate genes involved in calcium channels. A calcium channel functional complex has also been suggested as a mechanism in the etiology of bipolar disorder and autism. These results suggest hypotheses for clinical translation. Multiple approved medications act at calcium channels, including some antipsychotics (for example, pimozide) along with adjuvants for treatment non-response for schizophrenia and bipolar disorder (for example, the calcium channel blockers verapamil and nifedipine). It is A r t i c l e s possible that drugs that act on the protein products of CACNA1C and CACNB2 for a different therapeutic indication could be repurposed for the treatment of schizophrenia. For example, there has been at least one clinical trial of the efficacy of isradipine in bipolar disorder (an approved antihypertensive acting at the protein product of CACNA1C; R. Perlis, personal communication). In addition, given © 2013 Nature America, Inc. All rights reserved. ADVANCE ONLINE PUBLICATION Nature GeNetics A r t i c l e s that many approved antipsychotics increase the cardiac QT interval, genetic variation in calcium channel genes might be used to identify individuals at higher risk of sudden cardiac death Third, multiple genomic lines of evidence support a role for MIR137 in the etiology of schizophrenia. We provide additional support for a common variant association located upstream of the MIR137 transcript (P = 1.7 × 10 −12 ; The SNP with the strongest association with schizophrenia (rs1198588) is 39 kb upstream of MIR137 and might regulate the transcription of MIR137. However, this has not been proven experimentally, and there is another candidate gene in the region. rs1198588 is in an LD block that includes DPYD (169 kb upstream of rs1198588), and rs1198588 is a significant local expression quantitative trait locus (eQTL) for DPYD. We note that DPYD also contains a predicted miR-137 target site. An exome sequencing study reported two putative functional de novo variants in DPYD in cases with schizophrenia 11 . Fourth, 13 of the 22 regions listed in Genetic architecture There has been considerable debate about the genetic architecture of schizophrenia. We estimated the proportion of variance in liability to schizophrenia explained by SNPs using GCTA (Genomewide Complex Trait Analysis) The Bayesian framework used by ABPA also allows simultaneous estimation of the number of independent SNP loci that contribute to risk for schizophrenia. Here we assume that the number of genomewide significant SNP associations and the amount of variance they explain in the Sweden and PGC meta-analysis results only partly reflect the underlying genetic architecture of schizophrenia, owing to inadequate sample size. Using 1000 Genomes Project imputation for Swedish and PGC samples and assuming a population risk of 0.01, we estimated that 8,300 independent SNPs contribute to the genetic basis of schizophrenia and that these SNPs account for 50% of the variance in liability to schizophrenia (95% credible intervals of 6,300-10,200 for the number of SNPs and 0.45-0.54 for total variance explained). We stress that these estimates must be interpreted in the context of the assumptions of ABPA and the strengths and weaknesses of the input data. Additional analyses (data not shown) indicate that most of the signal was derived from SNPs with allele frequencies of >0.1; low-frequency imputed SNPs were not generally inferred to be associated with schizophrenia. ABPA estimates of the genetic architecture of schizophrenia are compared with those for four biomedical diseases We previously estimated the heritability of schizophrenia in Sweden to be 0.64 (95% confidence interval of 0.617-0.675) using a national pedigree sample of 9 million individuals 5 , and a Danish national pedigree study of 2.6 million individuals reported a similar estimate (0.67, 95% confidence interval of 0.65-0.71) DISCUSSION These results provide deeper insight into the genetic architecture of schizophrenia than ever before achieved. We find support for 22 common variant loci (13 new) that highlight biological hypotheses for further evaluation. Some findings have immediate translational relevance. Larger studies are highly likely to uncover more common variant associations, as argued elsewhere Common variation is an important (and perhaps predominant) genetic contributor to risk for schizophrenia. We estimated that 6,300-10,200 independent and mostly common SNPs contribute to the etiology of schizophrenia. As one gene or structural element could contain multiple independent associations, the number of genes ultimately determined to harbor causal variation for schizophrenia will be smaller, and we expect that these genes will implicate one or more biological pathways fundamental to disease risk. Moreover, these thousands of independent loci seem to account for a considerable fraction of the heritability of schizophrenia. It is possible that the commonly used phrase 'missing heritability' lacks precision. Indeed, if thousands of SNPs underlie schizophrenia, a statistical model containing a handful of SNPs is unlikely to account for more than a small fraction of the heritability 71 . Our results imply that the genetic architecture of schizophrenia is not dominated by uncommon variation. However, a balanced plan of attack should include well-powered searches for rare, private or de novo genetic variation of strong effect, given that such variants are probably more tractable to current molecular methods. Power calculations are a fundamental component of the design of genetic studies. However, relatively extensive knowledge of genetic architecture is essential for power calculations to have maximum usefulness in study planning. We used the ABPA estimates of the posterior distribution of genotypic relative risks Thus, for the first time, there is a clear path to increased knowledge of the etiology of schizophrenia through the application of standard, off-the-shelf genomic technologies for elucidating the effects of common variation. We suggest that a relatively thorough enumeration of the genomic loci conferring risk for schizophrenia (the 'parts list') should be a priority for the field 8 . Identifying all loci would surely be an exercise in diminishing returns. However, we propose a goal for the field: the identification of the top 2,000 loci (for example) might be sufficient to confidently and clearly identify the biological processes that mediate risk and protection for schizophrenia. Achievement of this goal would provide a strong empirical impetus for targeted biological and genetic research into the precise molecular basis of risk for schizophrenia, stratification of at-risk populations (for example, psychotic prodrome) and appropriate cellular measures for the evaluation of novel therapeutics. As indicated by our findings, greater knowledge of the genetic basis of schizophrenia can converge on increasingly specific neurobiological hypotheses that can be prioritized for subsequent investigation. URLs. Results can be downloaded from the PGC website at http://pgc. unc.edu/ and visualized using Ricopili at http://www.broadinstitute. org/mpg/ricopili. Genotype data are available upon application from the National Institute of Mental Health (NIMH) Genetics Repository at https://www.nimhgenetics.org/. The JAG website is at http://ctglab.nl/ software. METhODS Methods and any associated references are available in the online version of the paper. © 2013 Nature America, Inc. All rights reserved. 8 ADVANCE ONLINE PUBLICATION Nature GeNetics A r t i c l e s AUTHOR CONTRIBUTIONS COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. voltage-dependent calcium channels mediate different aspects of acquisition and retention of a spatial memory task. Neurobiol. Learn. Mem. 81, 105-114 (2004 Genes Dev. 25, 1915-1927