Gated Calcium Channels Associated with Schizophrenia

UC San Diego UC San Diego Previously Published Works

Title A molecule-based genetic association approach implicates a range of voltage-gated calcium channels associated with schizophrenia.

Permalink https://escholarship.org/uc/item/99g7r6sz

Journal American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics, 177(4)

ISSN 1552-4841

Authors Li, Wen Fan, Chun Chieh Mäki-Marttunen, Tuomo et al.

Publication Date 2018-06-01

DOI 10.1002/ajmg.b.32634

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California Received: 1 February 2017 | Revised: 13 February 2018 | Accepted: 23 March 2018 DOI: 10.1002/ajmg.b.32634

RESEARCH ARTICLE

A molecule-based genetic association approach implicates a range of voltage-gated calcium channels associated with schizophrenia

1NORMENT, KG Jebsen Centre for Psychosis Research, Institute of Clinical Medicine, University of Oslo, Oslo, Norway

2Division of Mental Health and Addiction, Oslo University Hospital, Oslo, Norway 3Department of Neurosciences, University of California, San Diego, La Jolla, California

4Multimodal Imaging Laboratory, University of California, San Diego, La Jolla, California

5Department of Psychiatry, University of California, San Diego, La Jolla, California

6Institute of Biological Psychiatry, Mental Health Centre Sct. Hans, Mental Health Services, Copenhagen, Denmark

7The Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Copenhagen, Denmark

8Department of Cognitive Sciences, University of California, San Diego, La Jolla, California

9Department of Medical Genetics, Oslo University Hospital, Oslo, Norway

10NORMENT, KG Jebsen Centre for Psychosis Research, Department of Clinical Science, University of Bergen, Bergen, Norway

11Department of Radiology, University of California, San Diego, La Jolla, California

Correspondence Traditional genome-wide association studies (GWAS) have successfully detected genetic variants Yunpeng Wang, NORMENT, KG Jebsen associated with schizophrenia. However, only a small fraction of heritability can be explained. Centre, Building 48, Oslo University Hospital, Ullevål, Kirkeveien 166, PO Box Gene-set/pathway-based methods can overcome limitations arising from single nucleotide poly- 4956 Nydalen, 0424 Oslo, Norway. morphism (SNP)-based analysis, but most of them place constraints on size which may exclude E-mail: [email protected] highly specific and functional sets, like macromolecules. Voltage-gated calcium (Cav) channels,

Funding information belonging to macromolecules, are composed of several subunits whose encoding genes are located EU funding (PsychDPC); Research Council far away or even on different chromosomes. We combined information about such molecules with of Norway, Grant/Award Number: RCN GWAS data to investigate how functional channels associated with schizophrenia. We defined a #223273, #251134; South East Norway biologically meaningful SNP-set based on channel structure and performed an association study by Regional Health Authority, KG Jebsen Foundation, Grant/Award Number: SKGJ- using a validated method: SNP-set (sequence) kernel association test. We identified eight subtypes

MED-008 of Cav channels significantly associated with schizophrenia from a subsample of published data

(N 5 56,605), including the L-type channels (Cav1.1, Cav1.2, Cav1.3), P-/Q-type Cav2.1, N-type

Cav2.2, R-type Cav2.3, T-type Cav3.1, and Cav3.3. Only genes from Cav1.2 and Cav3.3 have been

implicated by the largest GWAS (N 5 82,315). Each subtype of Cav channels showed relatively high chip heritability, proportional to the size of its constituent gene regions. The results suggest

that abnormalities of Cav channels may play an important role in the pathophysiology of schizophrenia and these channels may represent appropriate drug targets for therapeutics. Analyzing

*Memberships of the Schizophrenia Working Group of the Psychiatric Genomics Consortium are provided in Acknowledgments section.

454 | VC 2018 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/ajmgb Am J Med Genet. 2018;177B:454–467. LI ET AL. | 455

subunit-encoding genes of a macromolecule in aggregate is a complementary way to identify more genetic variants of polygenic diseases. This study offers the potential of power for discovery the biological mechanisms of schizophrenia.

KEYWORDS channels, molecule-based GWAS, schizophrenia, SKAT, SNP-sets

1 | INTRODUCTION constraints on size from 10 to a few 100 genes (Ramanan et al., 2012). Restriction to pathways with more than 10 genes may exclude highly Schizophrenia is a highly heritable complex disease (Lichtenstein et al., specific and potentially informative functional SNP-sets, like 2009). The biological underpinnings of schizophrenia remain an enigma, macromolecules. making prevention difficult and delaying development of better treat- A macromolecule is a very large molecule created by polymeriza- ment alternatives (Van Os & Kapur, 2009). Recently, advances in tech- tion of multiple smaller subunits. Voltage-gated calcium (Cav) channels nology and the establishment of an international consortium, the that belong to macromolecules are pore-forming membrane proteins Psychiatric Genomics Consortium (PGC), have made it possible to per- involved in diverse physiological processes including depolarization of form genome-wide association studies (GWAS) involving more than a neuronal action potentials, neurotransmitter release, neuronal excitabil- hundred thousand individuals. The latest study from PGC has reported ity, and intracellular signaling (Simms & Zamponi, 2014). Before inter- 108 independent genomic regions associated with schizophrenia esting GWAS findings emerged, they have already received (Schizophrenia Working Group of the Psychiatric Genomics Consor- considerable physiological investigations in psychiatric and neurological tium, 2014). However, the variants identified can only explain a small disorders due to their importance to brain function (Catterall, 2000; fraction of the estimated heritability (Giusti-Rodríguez & Sullivan, Simms & Zamponi, 2014). Cav channels are key mediators of calcium 2013; Goldstein 2009; Ripke et al., 2013; Schizophrenia Working entry into neurons (Turner, Anderson, & Zamponi, 2011) and calcium Group of the Psychiatric Genomics Consortium, 2014), and the func- signaling is involved in major molecular hypothesis of schizophrenia tional consequences of these variants remain largely uncharacterized. such as dopamine, glutamatergic, and GABAergic hypothesis (Lidow, These problems may originate from inherent limitations of the GWAS 2003). In fact, calcium signaling dysfunction has been suggested as a methodology: The mass univariate testing approach requires an unifying pathological mechanism in schizophrenia (Lidow, 2003). Thus, extremely stringent significance threshold to control false positives, Cav channels gene variants are of large interest in relationship to schiz- thus reducing power; Genetic heterogeneity further complicate inter- ophrenia and we chose to perform the macromolecular analysis of pretation in large meta-analysis; Connecting SNP markers to the causal functional Cav channels. variants they represent is not straightforward; And, robust, efficient Recently, GWAS have identified several associated neuronal ion methods for detecting interactions among genetic variants remain channel genes (e.g., CACNA1C, CACNB2, CACNA1I, KCNB1, HCN1, elusive. CHRNA3, CHRNA5, CHRNB4) (Cross-Disorder Group of the Psychiatric Gene-based, and gene-set/pathway-based methods provide prom- Genomics Consortium, 2013; Ripke et al., 2013). In particular, associa- ising alternatives to overcome certain limitations of GWAS (Askland, tions at CACNA1C, CACNB2,andCACNA1I, which encode Cav channel

Read, O’Connell, & Moore, 2012). Typically, genetic variants within or subunits, extend previous findings implicating members of Cav channels near to a gene are aggregated and tested for associations with a dis- in schizophrenia (Hamshere et al., 2013; Ripke et al., 2013). Cav chan- ease (Liu et al., 2010). Gene-set/pathway-based analyses aggregate nels can either be monomers (one subunit), or heteromultimers (three functionally related genes, providing a potentially powerful and biologi- or four subunits). Although, these subunits physically bind together to cally oriented bridge between genotypes and phenotypes (Ramanan, form a channel, their encoding genes are located in different regions of Shen, Moore, & Saykin, 2012; Wang, Li, & Hakonarson, 2010). These a chromosome or even on different chromosomes. For example, in the methods, complementary to GWAS, have several advantages: they can Cav1.1 channel (Bannister & Beam, 2013), the a1 subunit gene CAC- reduce the number of tests performed; they may reduce the impact of NA1S, a2d subunit gene CACNA2D1, b subunit gene CACNB1,andg genetic heterogeneity across cohorts; and they can facilitate the inter- subunit gene CACNG1 are located at chromosomal bands 1q32, 7q21- pretation of findings. On the other hand, they also have limitations: q22, 17q21-q22, and 17q24, respectively (Figure 1). Due to the limita- genes typically work in concert with one another (Liu et al., 2010), thus tions of gene-based and gene set-based analysis mentioned above, it is gene-based methods cannot take into account the joint effect among possible that taking the macromolecules (Cav channels) as a joint entity genes; the organization of pathways is typically derived from experi- can explain more for the risk of schizophrenia than one single locus ments of model organisms or predicted from mathematical models so alone. uncertainties may be present (Bauer-Mehren, Furlong, & Sanz, 2009); We defined a SNP-set from single channel genes and investigated the mechanism of the pathways is rarely clear (Khatri, Sirota, & Butte, how this biologically functional unit is associated with schizophrenia, 2012); and most published gene-set/pathway analyses place using the accessible PGC schizophrenia GWAS data (N 5 56,605: 456 | LI ET AL.

CACNA1H, CACNA1I, CACNA1S encode the a1 subunits; CACNA2D1,

CACNA2D2, CACNA2D3, CACNA2D4 encode the a2d subunits; CACNB1, CACNB2, CACNB3, CACNB4 encode the b subunits; and CACNG1, CACNG2, CACNG3, CACNG4, CACNG5, CACNG6, CACNG7, CACNG8 encode the g subunits. We only analyzed genes located on the autosomes, so the gene CACNA1F on the X-chromosome was excluded.

2.2 | Genotype data

Due to IRB restrictions from some substudies in PGC, we used the largest accessible PGC schizophrenia data which contains 36 case-control substudies (N 5 56,605; 25,629 cases and 30,976 controls compared

TABLE 1 Gene-level test result from discovery and validation stages

Type of Combined encoding Stage1 Stage2 dataset FIGURE 1 Molecular organization of voltage-gated calcium channels Gene name subunit BH_SKAT P BH_SKAT P BH_SKAT P and chromosome locations of their subunit-coding genes. Most Cav channels are multi-subunit structure (containing three or four subu- CACNA1A a1A 4.66E-01 3.80E-01 2.29E-01 nits, a , b, a d,withorwithoutg subunits), but T-type Ca channels 1 2 v CACNA1B a1B 7.15E-01 1.75E-01 1.75E-01 only have the a subunit. In one specific channel, the subunits are 1 a a a physically bound together, but their encoding genes are localized far CACNA1C a1C 2.24E-04 2.42E-12 3.07E-18 apart or even on different chromosomes. Nine autosomal genes (CAC- CACNA1D a1D 5.85E-01 7.63E-01 5.53E-01 NA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1G, CAC- a CACNA1E a1E 4.48E-01 7.49E-02 8.85E-03 NA1H, CACNA1I, CACNA1S)encodea1 subunit (connected by red a a lines), four genes (CACNB1, CACNB2, CACNB3, CACNB4)encodeb CACNA1G a1G 8.94E-03 1.75E-01 3.41E-03 subunits (connected by blue lines), four genes (CACNA2D1, CAC- CACNA1H a1H 5.60E-01 8.16E-01 3.29E-01 NA2D2, CACNA2D3, CACNA2D4) encode a2d subunit (connected by a a a green lines), and eight genes (CACNG1, CACNG2, CACNG3, CACNG4, CACNA1I a1I 3.75E-04 2.32E-04 9.88E-09 CACNG5, CACNG6, CACNG7, CACNG8)encodeg subunit (connected CACNA1S a 2.13E-01 2.64E-01 1.26E-01 by gray lines). The numbers 1, 2, 3, 7, 9, 10, 12, 16, 17, 19, and 22 1S represent chromosome numbers [Color figure can be viewed at CACNA2D1 a2d1 5.85E-01 8.42E-02 1.26E-01 wileyonlinelibrary.com] CACNA2D2 a2d2 5.60E-01 7.49E-02 1.94E-01

25,629 cases and 30,976 controls) divided into a discovery and a repli- CACNA2D3 a2d3 4.48E-01 8.42E-02 8.01E-02 cation sample. We applied the SNP-set (sequence) kernel association CACNA2D4 a2d4 4.48E-01 1.60E-01 7.15E-02 test (SKAT) (Wu et al., 2010) and identified significant associations in CACNB1 b1 7.15E-01 1.75E-01 1.53E-01 eight subtypes of Cav channels (Cav1.1, Cav1.2, Cav1.3, Cav2.1, Cav2.2, a a CACNB2 b2 3.55E-02 6.73E-02 2.41E-05 Cav2.3, Cav3.1, and Cav3.3). In contrast, only genes (CACNA1C, CACNB3 b 4.66E-01 1.92E-01 1.60E-01 CACNB2,andCACNA1I) from two subtypes were implicated by the 3 original GWAS despite its larger sample (N 5 82,315). These findings CACNB4 b4 6.68E-01 1.86E-01 1.75E-01 show the potential of the macromolecule approach to identify the pos- CACNG1 g1 6.48E-01 1.75E-01 1.53E-01 sible etiology of diseases, and suggest that abnormalities of Cav chan- CACNG2 g2 4.48E-01 1.75E-01 2.91E-01 nels may play an important role in the pathophysiology of CACNG3 g3 4.66E-01 1.60E-01 1.26E-01 schizophrenia.

CACNG4 g4 8.47E-01 7.49E-02 1.26E-01

2 | MATERIALS AND METHODS CACNG5 g5 4.66E-01 1.87E-01 1.26E-01

CACNG6 g6 4.66E-01 1.75E-01 4.98E-01

2.1 | Cav genes CACNG7 g7 5.75E-01 1.75E-01 1.26E-01

A total of 26 genes encoding subunits of Cav channels can be classified CACNG8 g8 4.66E-01 2.65E-01 1.75E-01 into four groups (Table 1) according to the types of subunits they *p-Value <.05 after correction. Stage1: discovery phase; Stage 2: valida- encode (Catterall, 2000; Simms & Zamponi, 2014). Genes CACNA1A, tion phase; BH: Benjamini Hochberg; SKAT: SNP-set (sequence) kernel CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, association test. LI ET AL. | 457 to 52 sub-studies and N 5 82,315 in the primary study) (Schizophrenia TABLE 2 Channel-level test results Working Group of the Psychiatric Genomics Consortium, 2014). Qual- Combined ity control and imputation were performed by the PGC Statistical Anal- Channel Subunits Stage1 Stage2 datasets P P P ysis Group for each dataset separately. Briefly, SNP meets with name combination BH_SKAT BH_SKAT BH_SKAT following conditions were retained: SNP missingness <0.05, SNP Cav1.1 a1S b1 a2d1 g1 4.63E-01 2.78E-02* 3.54E-02* Hardy–Weinberg equilibrium p > 1 3 1026 in controls or p > 1 3 a1C b1 a2d1 9.56E-04* 5.85E-12* 1.51E-16* a1C b1 a2d2 5.09E-05* 8.42E-14* 1.62E-19* 10210 in cases. Samples with missing rate >0.05 were removed. After a1C b2 a2d1 6.21E-05* 8.87E-13* 1.31E-20* quality control, the remaining genotypes were imputed using a1C b2 a2d1 g1 6.21E-05* 8.05E-13* 1.16E-20* SHAPEIT2/IMPUTE2 (Delaneau, Marchini, & Consortium, 2014; Howie a1C b2 a2d1 g2 5.70E-05* 6.13E-13* 1.13E-20* a1C b2 a2d1 g3 6.21E-05* 4.66E-13* 5.56E-21* et al., 2012) based on the full 1000 Genomes Project dataset (Schizo- a1C b2 a2d1 g4 6.93E-05* 6.03E-13* 1.07E-20* phrenia Working Group of the Psychiatric Genomics Consortium, a1C b2 a2d1 g5 6.21E-05* 6.93E-13* 6.59E-21* a b a d g 6.21E-05* 7.04E-13* 1.31E-20* 2014). To evaluate the replicability of our analysis, we selected out the 1C 2 2 1 6 a1C b2 a2d1 g7 6.21E-05* 8.05E-13* 1.13E-20* data used in the first phase of PGC (PGC1) as a discovery sample a1C b2 a2d1 g8 6.21E-05* 8.36E-13* 1.13E-20* (10,616 cases and 10,315 controls), and used the rest as replication Cav1.2 a1C b2 a2d2 6.66E-06* 8.72E-14* 1.75E-22* sample (15,013 cases and 20,661 controls). In addition, we also used a1C b2 a2d2 g1 6.66E-06* 8.42E-14* 1.64E-22* combined samples from both discovery and replication stages. We first a1C b2 a2d2 g2 6.66E-06* 8.42E-14* 1.61E-22* 6.66E-06* 8.42E-14* 1.16E-22* merged the best-guessed genotype data (imputation information score a1C b2 a2d2 g3 a1C b2 a2d2 g4 7.22E-06* 8.42E-14* 1.61E-22* > > 0.8 and minor allele frequency 0.05) across 36 substudies, and a1C b2 a2d2 g5 6.66E-06* 8.42E-14* 1.16E-22* then, performed the second round of quality controls using parameters a1C b2 a2d2 g6 6.66E-06* 8.42E-14* 1.64E-22* a b a d g 6.66E-06* 8.42E-14* 1.61E-22* SNP missingness <0.05 and minor allele frequency >0.05. To control 1C 2 2 2 7 a1C b2 a2d2 g8 6.66E-06* 8.42E-14* 1.61E-22* the impact of population stratification on our analysis, we computed a1C b2 a2d3 3.72E-05* 2.65E-12* 2.67E-20* the first 20 principal components based on the merged and quality con- a1C b2 a2d4 6.66E-06* 1.49E-13* 1.16E-22* a1C b3 a2d1 9.49E-04* 5.85E-12* 1.51E-16* trolled genotype data by using the program EigenSoft (Price et al., a1C b3 a2d2 5.09E-05* 8.42E-14* 1.62E-19*

2006). Since, some Cav genes are close together in genomic position a1C b4 a2d1 3.50E-03* 7.39E-11* 8.50E-15* a b a d 7.72E-04* 4.67E-11* 6.26E-15* (e.g., CACNG6, CACNG7,andCACNG8), it is possible that some SNPs 1C 4 2 2 a1D b3 a2d1 5.71E-01 6.08E-02 8.46E-02 may be assigned to more than one genes. To avoid such undesired a1D b3 a2d2 5.71E-01 1.85E-01 3.93E-01 bias, we annotated SNPs to the closest gene (GENCODEv1.9) based a1D b3 a2d3 3.17E-01 5.79E-02 4.26E-02* on genomic positions that were derived from the human genome Cav1.3 a1D b3 a2d4 4.39E-01 3.18E-01 1.21E-01 assembly build hg19 (Supporting Information Table S8). Then based on a1D b4 a2d1 6.41E-01 4.63E-02* 6.17E-02 a1D b4 a2d2 6.78E-01 1.22E-01 2.00E-01 the SNPs list, the genotypes of the 25 Cav genes were extracted. a1D b4 a2d3 4.07E-01 4.36E-02* 3.54E-02* Ca channels can either be monomers (only the a subunit), or het- v 1 a1D b4 a2d4 5.71E-01 1.72E-01 9.55E-02 eromultimers (three subunits a1, b, a2d;orfoursubunitsa1, b, a2d, g). a1A b1 a2d1 4.99E-01 4.00E-02* 4.42E-02* a1A b4 a2d1 5.71E-01 3.28E-02* 3.72E-02* Great diversity of Cav channels allows them to fulfill highly specialized roles in specific neuronal subtypes (Simms & Zamponi, 2014). Thus, for Cav2.1 a1A b4 a2d2 5.71E-01 7.90E-02 1.02E-01 a b a d 3.49E-01 3.27E-02* 2.13E-02* each a1 subunit (principal subunit for classifying subtypes of Cav chan- 1A 4 2 3 a1A b4 a2d4 4.69E-01 1.13E-01 4.99E-02* nels), co-assembly of a variety of ancillary subunits (b, a2d, g)exists a1B b1 a2d1 6.41E-01 2.34E-02* 3.92E-02* (Table 2). In some Cav channels, the ancillary subunit types are not a1B b1 a2d2 7.14E-01 3.28E-02* 1.02E-01 completely known. So for channel-level association analysis, we test all a1B b1 a2d3 3.58E-01 2.34E-02* 2.16E-02* a1B b3 a2d1 6.41E-01 2.34E-02* 3.92E-02* of the possible combinations based on the current literatures (Buraei &

Yang, 2010; Catterall, 1996; Davies et al., 2010; Hofmann, Flockerzi, Cav2.2 a1B b3 a2d2 6.92E-01 3.28E-02* 1.02E-01 a b a d 3.58E-01 2.34E-02* 2.16E-02* Kahl, & Wegener, 2014; Schlick, Flucher, & Obermair, 2010). According 1B 3 2 3 a1B b4 a2d1 6.90E-01 2.15E-02* 3.51E-02* to different subunit gene combinations (three or four genes per set), a1B b4 a2d2 7.66E-01 3.97E-02* 8.89E-02 a b a d 4.37E-01 2.15E-02* 1.92E-02* genotypes of the genes consisting of a Cav channel were concatenated. 1B 4 2 3 a b a d 4.25E-01 5.15E-03* 1.47E-03* Therefore, each SNP-set is corresponding to one functional channel 1E 1 2 1 that exists in nature. Cav2.3 a1E b2 a2d1 4.53E-02* 4.25E-04* 2.30E-07*

a1E b3 a2d1 4.25E-01 5.15E-03* 1.47E-03* a1E b4 a2d1 4.82E-01 5.15E-03* 1.56E-03* 2.3 | SNP-set (sequence) kernel association test

Cav3.1 a1G 2.23E-03* 1.28E-01 1.05E-03* SKAT was used to test for association between a set of genetic var- Ca 3.2 a 4.82E-01 8.16E-01 3.08E-01 iants and dichotomous or quantitative phenotypes. It uses the logistic v 1H kernel-machine regression modeling framework. SKAT aggregates indi- Cav3.3 a1I 7.31E-05* 3.85E-05* 1.64E-09* vidual score test statistics of SNPs in a SNP-set and computes SNP-set *p-Value <0.05 after corrections. Stage1: discovery phase; Stage 2: validation level p-values. SKAT can be used for common or/and rare variants phase; BH: Benjamini Hochberg; SKAT: sequencing kernel association test. 458 | LI ET AL.

(Ionita-Laza et al., 2013; Wu et al., 2010, 2011). In the current study, schizophrenia candidates (Schizophrenia Working Group of the Psychi- we focus on the common variants in line with the PGC schizophrenia atric Genomics Consortium, 2014). study and used SKAT version 1.07 (Wu et al., 2010). The linear kernel with beta (p, 1.25), where p is the minor allele frequency of a SNP, was 3.2 | Association of Cav channels with schizophrenia used. In our analysis, we carefully selected the cohort indicators and (macromolecule level) the first six principal components as covariates after comparing results Macromolecule-level testing in the discovery cohort identified hetero- including different number of principal components (3, 6, and 10) (Sup- mers Ca 1.2 (all possible subunits combinations), Ca 2.3 (a b a d ), porting Information Table S1). At the same time, to overcome the issue v v 1E 2 2 1 and monomers Ca 3.1 (a )andCa3.3 (a ) as associated (corrected of the large number of degrees of freedom, SKAT employs a test that v 1G v 1I p < .05). All of them except Cav3.1 (a1G) were replicated in the separate adaptively estimates the degrees of freedom by accounting for correla- samples by SKAT analysis (Table 2). In the combined sample, hetero- tion (LD) among the SNPs (Wu et al., 2010). In this study, a SNP-set mers Cav1.1 (a1S b1 a2d1 g1); Cav1.2 (all possible subunits combina- can be a collection of SNPs from a gene or several genes consisting of tions); Cav1.3 (a1D b3 a2d3, a1D b4 a2d3); Cav2.1 (a1A b1 a2d1, a1A b4 a heteromeric channel. The Benjamini Hochberg (BH) procedure was a2d1, a1A b4 a2d3, a1A b4 a2d4); Cav2.2 (a1B b1 a2d1, a1B b1 a2d3, a1B used to correct for multiple comparisons both in the Tables 1 and 2 b3 a2d1, a1B b3 a2d3, a1B b4 a2d1, a1B b4 a2d3); and Cav2.3 (a1E b1 (Hochberg & Benjamini, 1990; Wu et al., 2011). a2d1, a1E b2 a2d1, a1E b3 a2d1, a1E b4 a2d1), and monomers Cav3.1

(a1G)andCav3.3 (a1I) associate with the risk of schizophrenia (cor- | 2.4 Estimate schizophrenia heritability contributed rected p < .05) (Table 2). by Cav channels SNPs

Channels significantly associated with schizophrenia (Table 2; Support- 3.3 | Chip heritability of Cav channels ing Information Table S6) were selected. For each subtype of Ca chan- v We estimate that 0.0567% (s.e. 0.0391%), 0.5051% (s.e. 0.1172%), nel, all of the auxiliary subunit ( , , ) genes contributing to a b a2d g 0.2453% (s.e. 0.0946%), 0.1788% (s.e. 0.0708%), 0.2578% (s.e. significant association with schizophrenia were grouped with each a1 0.0929%), 0.176% (s.e. 0.0658%), 0.0272% (s.e. 0.0316%), and gene. The following gene lists Cav1.1 (CACNA1S, CACNA2D1, CACNB1, 0.0569% (s.e. 0.0464%) of the variance in schizophrenia can be CACNG1); Cav1.2 (CACNA1C, CACNA2D1, CACNA2D2, CACNA2D3, explained by Cav1.1, Cav1.2, Cav1.3, Cav2.1, Cav2.2, Cav2.3, Cav3.1, CACNA2D4, CACNB1, CACNB2, CACNB3, CACNB4, CACNG1, CACNG2, and Cav3.3 SNPs, respectively (Figure 2a). The Cav1.2 account for the CACNG3, CACNG4, CACNG5, CACNG6, CACNG7, CACNG8); Cav1.3 largest amount of chip heritability (0.5051%, s.e. 0.1172%) and the

(CACNA1D, CACNA2D3, CACNB3, CACNB4); Cav2.1 (CACNA1A, CAC- Cav3.1 account for the least (0.0272%, s.e. 0.0316%). However, after NA2D1, CACNA2D3, CACNA2D4, CACNB1, CACNB4); Cav2.2 (CAC- accounting for the number of SNPs included in each Cav subtype, NA1B, CACNA2D1, CACNA2D3, CACNB1, CACNB3, CACNB4); Ca 2.3 v Cav3.1 and Cav3.3 show largest fold enrichment (39.83 and 36.51, (CACNA1E, CACNA2D1, CACNB1, CACNB2, CACNB3, CACNB4); Ca 3.1 v respectively) (Figure 2b). All tested subtypes of Cav channels show – (CACNA1G); Cav3.3 (CACNA1I)wereusedtoextractgenotype pheno- more than sixfold enrichment. The variance explained by each subtype type data for estimating chip heritability by using the linear mixed of Cav channels is proportional to its number of SNPs (Supporting method BOLT-REML (Loh et al., 2015). The level of enrichment for Information Figure S1). This is in line with the previous discovery that association with schizophrenia was represented by the ratio of propor- the larger the genomic region, the higher the proportion of chip herit- tion of chip heritability (from each subtype of channel) in total heritabil- ability that can be accounted for (Yang et al., 2011). ity (33%) (Ripke et al., 2013) to the proportion of their SNPs in all SNPs (9423850 variants, minor allele frequency >0.05) from the 1000 3.4 | Robustness of the channel-based association Genomes Project. Cav channels that are significantly associated with schizophrenia reported by SKAT were also identified by another program MAGMA 3 | RESULTS (de Leeuw, Mooij, Heskes, & Posthuma, 2015) (Supporting Information Tables S4 and S5). However, MAGMA identified fewer channels at the 3.1 | Association of Cav genes with schizophrenia discovery stage compared with SKAT (Table 2; Supporting Information (gene level) Table S5). But for the largest European dataset (49 substudies), Two genes, CACNA1C and CACNA1I significantly associate with schizo- MAGMA reports similar results with SKAT. phrenia in the discovery cohort (corrected p < .05) and in the replication cohort (corrected p < .05) both according to the SKAT) method 4 | DISCUSSION (Table 1) and univariate analysis (Supporting Information Table S2). Within the combined sample (56,605 subjects) a further three genes In the current study, we applied a macromolecule approach to a sub- were identified by the SKAT analysis: CACNA1E, CACNA1G,and sample of published schizophrenia GWAS (N 5 56,605) and identified

CACNB2. CACNA1C, CACNA1I,andCACNB2 were previously reported, eight subtypes of Cav channels associated with schizophrenia, including whereas CACNA1E and CACNA1G have not been reported as the L-type Cav channels (Cav1.1, Cav1.2, Cav1.3), P-/Q-type Cav2.1, LI ET AL. | 459

FIGURE 2 Estimates of the schizophrenia variance explained by SNPs from each subtype of Cav channels. (a) Chip heritability of each significant subtype of Cav channel, (b) fold enrichment of each significant subtype of Cav channel in schizophrenia. The fold enrichment is the ratio of the proportion of chip heritability (from each significant subtype of channel) in total heritability (33%) to the proportion of their SNPs in all SNPs (9,423,850 variants, minor allele frequency >0.05) from 1000 Genomes Projects [Color figure can be viewed at wileyonlinelibrary.com]

N-type Cav2.2, R-type Cav2.3, T-type channels (Cav3.1, Cav3.3). Only on different chromosomes, the encoding subunits are physically bind- genes (CACNA1C, CACNB2,andCACNA1I)fromCav1.2 and Cav3.3 ing together in one functional unit to deal with flow of calcium ions. were implicated in the primary PGC analysis, which was based on a This macromolecule-based approach is different from grouping genes larger sample (N 5 82,315) (Schizophrenia Working Group of the based on their functional catalogs or pathways since their products Psychiatric Genomics Consortium, 2014). In addition, we used another (proteins) interact directly or indirectly and they could not form a published statistical tool MAGMA to confirm our analysis. The results unique functional macromolecule. Our approach combining biological are highly consistent, although the two programs are based on different priors with GWAS data identified eight subtypes of Cav channels asso- assumptions and statistical models. It demonstrates that analyzing ciated with the risk of schizophrenia. It is possible that the associations macromolecule subunit genes in aggregate is a complementary way to of whole channels with schizophrenia may be due to a highly associ- identify more genetic variants of schizophrenia compare to the tradi- ated component gene. This is likely the case for Cav1.2, where a few tional GWAS that treating each SNP separately. possible subunit combinations (e.g., Cav1.2: a1C b1 a2d2 that encoded The macromolecule subunits physically bind together to achieve by genes CACNA1C, CACNB1,andCACNA2D2)showtheirsignificance their cellular functions, thus perturbations of any of their subunits may thanks to the a1 subunit gene CACNA1C (Table 1; Supporting Informa- contribute to disease pathogenesis. In previous, GWAS of schizophre- tion Table S7), although most of the others are not. The significant nia, only a handful of channel subunits were implicated, perhaps due to associations of the other heteromultimeric channels may be not due to the limited power of the massive univariate tests (Lichtenstein et al., a single significant gene. For example, during the discovery and replica-

2009; Ripke et al., 2013; Schizophrenia Working Group of the Psychi- tion stages, the Cav2.3 channel (subunits encoded by CACNA1E, atric Genomics Consortium, 2014). To the best of our knowledge, only CACNB2,andCACNA2D1) was discovered and replicated by SKAT but Askland et al. (2012) have performed an association analysis of ion none of their composing genes was identified at the gene-level test. channels with schizophrenia, but the gene sets defined in their study is The univariate analysis (minP SNP represents channel) could not iden- a mixture of subunit-encoding genes from many ionic species and does tify this channel in small samples (discovery and replication stages), but not therefore correspond to a macromolecule existing in nature. In the combined sample could confirm this finding when applying a addition, it was tested in a much smaller sample. To test whether each macromolecule-based approach (Supporting Information Table S3). functional Cav channel is associated with schizophrenia or not, we com- None of the channels Cav1.1, Cav1.3, Cav2.1, and Cav2.2 subunit genes posed specific gene set based on molecular structures of Cav channels was identified in gene-level testing, but the channels show significant (Buraei & Yang, 2010; Catterall, 1996; Davies et al., 2010; Schlick et al., association with schizophrenia in the combined sample. These results 2010; Simms & Zamponi, 2014). For each channel (macromolecule- indicate that subunit genes can collectively associate with disease sus- based analysis), although the containing genes locate far away or even ceptibility, even if individual genes do not exhibit significant 460 | LI ET AL. association. It seems that analyzing channel SNPs as a set can capture are used for hypertension, have been examined in clinical trials in schiz- the joint effect of multiple variants located on different chromosomes. ophrenia (Lencz & Malhotra, 2015). Revisiting the effect of existing

Thus, genetic variants with weak or moderate effects could be identi- agents on Cav channels or designing new drugs could be a high priority fied when we combined them together based on biological knowledge for new schizophrenia treatment development. of the macromolecule. The genetic association test of macromolecules may also suggest

We also observed enrichment of heritability in significant Cav candidates for nonadditive interactions (epistasis) and improve poly- channels SNPs for schizophrenia and it may point to a major role of the genic predictions. In addition, while we only considered Cav channels, inherited genetic variants in the risk of schizophrenia. These eight sub- future work could consider other types of channels, such as potassium types of Cav channels may provide more knowledge about the pathol- channels, sodium channels, and proton channels as interesting suscepti- ogy of schizophrenia. Cav channels are the primary mediators of bility candidates for schizophrenia and other psychiatric disorders. depolarization-induced calcium entry into neurons (Simms & Zamponi, The present findings illustrate the power of the macromolecule- 2014). Calcium-dependent processes such as neurotransmitter release, based approach applied to schizophrenia, which identified eight sub- neuronal gene transcription, and activation of calcium-dependent types of Cav channels associated with the disorder. The results high- enzymes are of critical importance to brain function (Clapham, 2007; light the combined role of different aspects of calcium signaling in

Simms & Zamponi, 2014). L-type Cav channels (Cav1.1, Cav1.2, Cav1.3) schizophrenia pathophysiology, and suggest several new potential drug are involved in learning, memory, and synaptic plasticity (Moosmang targets for development of novel therapeutics. et al., 2005; White et al., 2008; Woodside, Borroni, Hammonds, &

Teyler, 2004). Mutations in CACNA1C, the gene encoding the a1 subu- ACKNOWLEDGEMENTS nit of Ca 1.2, are responsible for Timothy syndrome, a multisystem dis- v This work was supported by the EU funding (PsychDPC); Research order including cognitive impairment and autism spectrum disorder Council of Norway [RCN #223273, #251134]; South East Norway (Splawski et al., 2004, 2005). SNPs located in CACNA1C are linked to Regional Health Authority; and KG Jebsen Foundation [SKGJ-MED- development of schizophrenia, bipolar disorder and depression (Dao 008]. et al., 2010; Green et al., 2010; He et al., 2014). Data from mice and humans suggest an involvement of Cav1.3 channels in neurophysiologi- SCHIZOPHRENIA WORKING GROUP OF THE cal functions, in particular in the dopaminergic system (Simms & PSYCHIATRIC GENOMICS CONSORTIUM Zamponi, 2014), which is involved in the pathology of schizophrenia Stephan Ripke1,2, Benjamin M. Neale1,2,3,4, Aiden Corvin5, James T. R. (Brisch et al., 2014). Although, in humans, mutations in Cav1.1 have 6 1 6,7 1,2,4 been linked to hypokalemic periodic paralysis (Ptaček et al., 1994) and Walters , Kai-How Farh , Peter A. Holmans ,PhilLee , Brendan 1,2 8,9 1,3 malignant hyperthermia (Monnier, Procaccio, Stieglitz, & Lunardi, 1997), Bulik-Sullivan , David A. Collier ,HailiangHuang ,TuneH. 3,10,11 12,13,14 15,16,17 18 a pathway analysis for a set of calcium channel genes implicated CAC- Pers , Ingrid Agartz ,EsbenAgerbo , Margot Albus , 19 20,21 22 NA1S (Cav1.1 channel a1 subunit gene) as one of the 20 gene regions Madeline Alexander , Farooq Amin , Silviu A. Bacanu ,Martin 23 2 24,25 associated in the five psychiatric disorder meta-analysis (Cross-Disorder Begemann , Richard A Belliveau Jr ,JuditBene , Sarah E. Bergen 2,26 2 22 27 Group of the Psychiatric Genomics Consortium, 2013). P-/Q-type chan- , Elizabeth Bevilacqua , Tim B Bigdeli , Donald W. Black , Richard 28 29 30,31,32 nel Cav2.1 and N-type channel Cav2.2 play a role in neurotransmitter Bruggeman , Nancy G. Buccola , Randy L. Buckner , William 33 34 35,36 39,120,170 release at the presynaptic terminal and in neuronal integration in many Byerley ,WiepkeCahn ,GuiqingCai , Murray J. Cairns , 37 38 39,40 neuronal types (Williams et al., 1992). R-type channel Cav2.3 is strongly Dominique Campion , Rita M. Cantor , Vaughan J. Carr ,Noa expressed in cortex, hippocampus, striatum, amygdala, and interpedun- Carrera6, Stanley V. Catts39,41, Kimberly D. Chambert2, Raymond C. K. 42 43 43,44 45 cular nucleus (Parajuli et al., 2012). The T-type channels (Cav3.1, Chan , Ronald Y. L. Chen , Eric Y. H. Chen ,WeiCheng ,EricF. 46 47 48 Cav3.3) appear to play important roles in regulating neuronal excitability C. Cheung , Siow Ann Chong , C. Robert Cloninger , David (Simms & Zamponi, 2014). Although, there is no direct evidence associ- Cohen49, Nadine Cohen50, Paul Cormican5, Nick Craddock6,7, 210 51 52,53 ating Cav2.1, Cav2.2, Cav2.3, and Cav3.1 with schizophrenia, due to Benedicto Crespo-Facorro , James J. Crowley ,DavidCurtis , their strong expression and wide distribution in the human brain, these Michael Davidson54, Kenneth L. Davis36, Franziska Degenhardt55,56, 57 128,129, 17,58,59 four subtypes of Cav channels are likely involved in some aspects of Jurgen Del Favero , Lynn E. DeLisi Ditte Demontis , schizophrenia pathology. A recent study of rare variants in schizophre- Dimitris Dikeos60,TimothyDinan61,SrdjanDjurovic14,62, Gary Dono- 5,63 36 64,65 66 nia demonstrated that a gene set containing 26 Cav genes yielded a hoe , Elodie Drapeau , Jubao Duan , Frank Dudbridge , Naser large odds ratio of 8.4 (Purcell et al., 2014). Given the central role of Durmishi67, Peter Eichhammer68, Johan Eriksson69,70,71,Valentina 6 72 73,74,75,76 Cav channels in regulating neurotransmitter release and neuronal gene Escott-Price , Laurent Essioux , Ayman H. Fanous ,Martilias transcription, the identified channels may represent convenient drug S. Farrell51, Josef Frank77, Lude Franke78,RobertFreedman79, Nelson targets for novel therapeutics. Designing drugs for specific channels by B. Freimer80,MarionFriedl81, Joseph I. Friedman36,Menachem 1,2,4,82 2 6 targeting a1 subunit, or designing more universal drugs for some chan- Fromer ,GiulioGenovese, Lyudmila Georgieva , Elliot S. nels by targeting shared ancillary subunits can improve efficiency of Gershon209,InaGiegling81,83, Paola Giusti-Rodríguez51, Stephanie God- 84 1,3 85 86 treatments. There are some Cav channels blockers in clinical use. A few ard ,JacquelineI.Goldstein ,VeraGolimbet , Srihari Gopal ,Jacob 87 88 23 L-type Cav channel antagonists such as verapamil and nifedipine, which Gratten , Lieuwe de Haan , Christian Hammer ,MarianL. LI ET AL. | 461

Hamshere6, Mark Hansen89, Thomas Hansen17,90, Vahram Haroutu- Andreassen14,133, Douglas H. R. Blackwood132,ElviraBramon190,Joseph nian36,91,92, Annette M. Hartmann81, Frans A. Henskens39,93,94,Stefan D. Buxbaum35,36,91,191, Anders D. Børglum17,58,59,138,Sven Herms55,56,95, Joel N. Hirschhorn3,11,96, Per Hoffmann55,56,95, Andrea Cichon55,56,95,192, Ariel Darvasi193, Enrico Domenici194, Hannelore Hofman55,56, Mads V. Hollegaard97, David M. Hougaard97,Masashi Ehrenreich23,Tonu~ Esko3,11,96,135,PabloV.Gejman64,65,MichaelGill5, Ikeda98,IngeJoa99,AntonioJulia100,ReneS.Kahn 34, Luba Kalayd- Hugh Gurling53, Christina M. Hultman26, Nakao Iwata98,AssenV. jieva101,102, Sena Karachanak-Yankova103, Juha Karjalainen78,David Jablensky39,102,186,195,ErikG.Jonsson€ 12,14, Kenneth S. Kendler196, Kavanagh6, Matthew C. Keller104,BrianJ.Kelly120,JamesL.Ken- George Kirov6,JoKnight105,106,107,ToddLencz197,198,199, Douglas F. nedy105,106,107, Andrey Khrunin108, Yunjung Kim51, Janis Klovins109, Levinson19, Qingqin S. Li86, Jianjun Liu188,200,AnilK.Malhotra197,198,199, James A. Knowles110, Bettina Konte81, Vaidutis Kucinskas111,Zita Steven A. McCarroll2,96, Andrew McQuillin53, Jennifer L. Moran2,Preben Ausrele Kucinskiene111, Hana Kuzelova-Ptackova112, Anna K. Kähler26, B. Mortensen15,16,17,BryanJ.Mowry87,201,MarkusM.Nothen€ 55,56,Roel Claudine Laurent19,113,JimmyLeeCheeKeong47,114,S.HongLee87, A. Ophoff38,80,34, Michael J. Owen6,7,AarnoPalotie2,4,161,CarlosN. Sophie E. Legge6, Bernard Lerer115, Miaoxin Li43,44,116 Tao Li117, Kung- Pato110, Tracey L. Petryshen2,128,202, Danielle Posthuma203,204,205,Mar- Yee Liang118, Jeffrey Lieberman119, Svetlana Limborska108,CarmelM. cella Rietschel77,BrienP.Riley196,DanRujescu81,83,PakC.Sham43,44,116 Loughland39,120, Jan Lubinski121,JoukoLonnqvist€ 122,MilanMacekJr112, Pamela Sklar82,91,165, David St Clair206, Daniel R. Weinberger178,207,Jens Patrik K. E. Magnusson26,BrionS.Maher123,WolfgangMaier124,Jac- R. Wendland166,ThomasWerge17,90,208,MarkJ.Daly1,2,3, Patrick F. Sul- ques Mallet125,SaraMarsal100, Manuel Mattheisen17,58,59,126, Morten livan26,51,160 &MichaelC.O’Donovan6,7 Mattingsdal14,127,RobertW.McCarley128,129, Colm McDonald130, 1Analytic and Translational Genetics Unit, Massachusetts General Andrew M. McIntosh131,132, Sandra Meier77, Carin J. Meijer88,Bela Hospital, Boston, Massachusetts Melegh24,25,IngridMelle14,133, Raquelle I. Mesholam-Gately128,134, 2Stanley Center for Psychiatric Research, Broad Institute of MIT Andres Metspalu135,PatriciaT.Michie39,136, Lili Milani135, Vihra Mila- and Harvard, Cambridge, Massachusetts nova137, Younes Mokrab8, Derek W. Morris5,63,OleMors17,58,138,Kieran 3Medical and Population Genetics Program, Broad Institute of MIT C. Murphy139, Robin M. Murray140, Inez Myin-Germeys141, Bertram and Harvard, Cambridge, Massachusetts Muller-Myhsok€ 142,143,144,MariNelis135,IgorNenadic145,DeborahA. 4Psychiatric and Neurodevelopmental Genetics Unit, Massachu- Nertney146,GeraldNestadt147,KristinK.Nicodemus148, Liene Nikitina- setts General Hospital, Boston, Massachusetts Zake109, Laura Nisenbaum149, Annelie Nordin150, Eadbhard O’Calla- 5Neuropsychiatric Genetics Research Group, Department of Psy- ghan151,ColmO’Dushlaine2, F. Anthony O’Neill152,Sang-YunOh153, chiatry, Trinity College Dublin, Dublin, Ireland Ann Olincy79,LineOlsen17,90,JimVanOs141,154, Psychosis Endopheno- 6MRC Centre for Neuropsychiatric Genetics and Genomics, Insti- types International Consortium155, Christos Pantelis39,156,GeorgeN. tute of Psychological Medicine and Clinical Neurosciences, School of Papadimitriou60,SergiPapiol23, Elena Parkhomenko36,MicheleT. Medicine, Cardiff University, Cardiff, United Kingdom Pato110, Tiina Paunio157,158, Milica Pejovic-Milovancevic159,DianaO. 7National Centre for Mental Health, Cardiff University, Cardiff, Perkins160, Olli Pietiläinen158,161,JonathanPimm53, Andrew J. Pockling- United Kingdom ton6,JohnPowell140,AlkesPrice3,162,AnnE.Pulver147, Shaun M. Pur- 8Eli Lilly and Company Limited, Erl Wood Manor, Windlesham, cell82,DigbyQuested163,HenrikB.Rasmussen17,90, Abraham Surrey, United Kingdom. Reichenberg36,MarkA.Reimers164, Alexander L. Richards6, Joshua L. 9Social, Genetic and Developmental Psychiatry Centre, Institute of Roffman30,32,PanosRoussos82,165, Douglas M. Ruderfer6,82,VeikkoSal- Psychiatry, King’s College London, London, United Kingdom omaa71, Alan R. Sanders64,65,UlrichSchall39,120, Christian R. Schubert166, 10Department of Systems Biology, Center for Biological Sequence Thomas G. Schulze77,167,SibylleG.Schwab168,EdwardM.Scolnick2, Analysis, Technical University of Denmark, Kongens Lyngby, Denmark Rodney J. Scott39,169,170, Larry J. Seidman128,134, Jianxin Shi171,Engilbert 11Division of Endocrinology and Center for Basic and Translational Sigurdsson172, Teimuraz Silagadze173,JeremyM.Silverman36,174,Kang Obesity Research, Boston Children’s Hospital, Boston, Massachusetts Sim47,PetrSlominsky108,JordanW.Smoller2,4, Hon-Cheong So43,Chris 12Psychiatry Section, Department of Clinical Neuroscience, Karo- C. A. Spencer175,EliA.Stahl3,82, Hreinn Stefansson176,StacyStein- linska Institutet, Stockholm, Sweden. berg176, Elisabeth Stogmann177, Richard E. Straub178,EricStreng- 13Department of Psychiatry, Diakonhjemmet Hospital, Oslo, Norway man179,34, Jana Strohmaier77, T. Scott Stroup119,Mythily 14NORMENT, KG Jebsen Centre for Psychosis Research, Institute Subramaniam47, Jaana Suvisaari122, Dragan M. Svrakic48,JinP.Szatkie- of Clinical Medicine, University of Oslo, Oslo, Norway wicz51,ErikSoderman€ 12, Srinivas Thirumalai180, Draga Toncheva103, 15Centre for Integrative Register-based Research, CIRRAU, Aarhus Paul A. Tooney39,120,170, Sarah Tosato181, Juha Veijola182,183,JohnWad- University, Aarhus, Denmark dington184,DermotWalsh185,DaiWang86, Qiang Wang117,BradleyT. 16National Centre for Register-based Research, Aarhus University, Webb22,MarkWeiser54, Dieter B. Wildenauer186, Nigel M. Williams6, Aarhus, Denmark Stephanie Williams51, Stephanie H. Witt77,AaronR.Wolen164,EmilyH. 17The Lundbeck Foundation Initiative for Integrative Psychiatric M. Wong43, Brandon K. Wormley22, Jing Qin Wu39,170,HualinSimon Research, iPSYCH, Aarhus, Denmark Xi187,ClementC.Zai105,106, Xuebin Zheng188, Fritz Zimprich177,Naomi 18State Mental Hospital, Haar, Germany R. Wray87, Kari Stefansson176, Peter M. Visscher87,WellcomeTrust 19Department of Psychiatry and Behavioral Sciences, Stanford Case-Control Consortium 2189,RolfAdolfsson150,OleA. University, Stanford, California 462 | LI ET AL.

20Department of Psychiatry and Behavioral Sciences, Atlanta Vet- 47Institute of Mental Health, Singapore, Singapore erans Affairs Medical Center, Atlanta, Georgia 48Department of Psychiatry, Washington University, St. Louis, 21Department of Psychiatry and Behavioral Sciences, Emory Uni- Missouri versity, Atlanta Georgia 49Department of Child and Adolescent Psychiatry, Assistance Pub- 22Virginia Institute for Psychiatric and Behavioral Genetics, Depart- lique Hopitaux de Paris, Pierre and Marie Curie Faculty of Medicine ment of Psychiatry, Virginia Commonwealth University, Richmond, and Institute for Intelligent Systems and Robotics, Paris, France Virginia 50 Blue Note Biosciences, Princeton, New Jersey 23Clinical Neuroscience, Max Planck Institute of Experimental 51Department of Genetics, University of North Carolina, Chapel Medicine, Gottingen,€ Germany Hill, North Carolina 24Department of Medical Genetics, University of Pecs, Pecs, 52Department of Psychological Medicine, Queen Mary University Hungary of London, London, United Kingdom 25Szentagothai Research Center, University of Pecs, Pecs, Hungary 53Molecular Psychiatry Laboratory, Division of Psychiatry, Univer- 26Department of Medical Epidemiology and Biostatistics, Karolin- sity College London, London, United Kingdom ska Institutet, Stockholm, Sweden 54Sheba Medical Center, Tel Hashomer, Israel 27Department of Psychiatry, University of Iowa Carver College of 55Department of Genomics, Life and Brain Center, Bonn, Germany Medicine, Iowa City, Iowa 56Institute of Human Genetics, University of Bonn, Bonn, Germany 28Department of Psychiatry, University Medical Center Groningen, 57Applied Molecular Genomics Unit, VIB Department of Molecular University of Groningen, Groningen, The Netherlands Genetics, University of Antwerp, Antwerp, Belgium 29School of Nursing, Louisiana State University Health Sciences 58Centre for Integrative Sequencing, iSEQ, Aarhus University, Aar- Center, New Orleans, Louisiana hus, Denmark 30Athinoula A. Martinos Center, Massachusetts General Hospital, 59Department of Biomedicine, Aarhus University, Aarhus, Boston, Massachusetts Denmark 31Center for Brain Science, Harvard University, Cambridge, 60First Department of Psychiatry, University of Athens Medical Massachusetts School, Athens, Greece 32Department of Psychiatry, Massachusetts General Hospital, Bos- 61Department of Psychiatry, University College Cork, Co. Cork, ton, Massachusetts Ireland 33Department of Psychiatry, University of California at San Fran- 62Department of Medical Genetics, Oslo University Hospital, Oslo, cisco, San Francisco, California Norway 34Department of Psychiatry, University Medical Center Utrecht, 63Cognitive Genetics and Therapy Group, School of Psychology Rudolf Magnus Institute of Neuroscience, Utrecht, The Netherlands and Discipline of Biochemistry, National University of Ireland Galway, 35Department of Human Genetics, Icahn School of Medicine at Co. Galway, Ireland Mount Sinai, New York, New York 64Department of Psychiatry and Behavioral Neuroscience, Univer- 36Department of Psychiatry, Icahn School of Medicine at Mount sity of Chicago, Chicago, Illinois Sinai, New York, New York 65Department of Psychiatry and Behavioral Sciences, NorthShore 37 Faculty of Medicine, Centre Hospitalier du Rouvray, INSERM University HealthSystem, Evanston, Illinois U1079, Rouen, France 66Department of Non-Communicable Disease Epidemiology, Lon- 38Department of Human Genetics, David Geffen School of Medi- don School of Hygiene and Tropical Medicine, London, United cine, University of California, Los Angeles, California Kingdom 39Schizophrenia Research Institute, Sydney, Australia 67Department of Child and Adolescent Psychiatry, University Clinic 40School of Psychiatry, University of New South Wales, Sydney, of Psychiatry, Skopje, Republic of Macedonia Australia 68Department of Psychiatry, University of Regensburg, Regens- 41Royal Brisbane and Women’s Hospital, University of Queens- burg, Germany land, Brisbane, St Lucia, Australia 69Department of General Practice, Helsinki University Central 42Institute of Psychology, Chinese Academy of Science, Beijing, Hospital, University of Helsinki, Helsinki, Finland China 70Folkhälsan Research Center, Helsinki, Finland, Biomedicum Hel- 43Department of Psychiatry, Li Ka Shing Faculty of Medicine, The sinki 1, Haartmaninkatu 8, Helsinki, Finland University of Hong Kong, Hong Kong, China 71National Institute for Health and Welfare, Helsinki, Finland 44State Key Laboratory for Brain and Cognitive Sciences, Li Ka 72Translational Technologies and Bioinformatics, Pharma Research Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, and Early Development, F. Hoffman-La Roche, Basel, Switzerland China 73Department of Psychiatry, Georgetown University School of 45Department of Computer Science, University of North Carolina, Medicine, Washington, DC Chapel Hill, North Carolina 74Department of Psychiatry, Keck School of Medicine of the Uni- 46Castle Peak Hospital, Hong Kong, China versity of Southern California, Los Angeles, California LI ET AL. | 463

75Department of Psychiatry, Virginia Commonwealth University 101Centre for Medical Research, The University of Western Aus- School of Medicine, Richmond, Virginia tralia, Perth, Australia 76Mental Health Service Line, Washington VA Medical Center, 102The Perkins Institute for Medical Research, The University of Washington, DC Western Australia, Perth, Australia 77Department of Genetic Epidemiology in Psychiatry, Central Insti- 103Department of Medical Genetics, Medical University, Sofia, tute of Mental Health, Medical Faculty Mannheim, University of Hei- Bulgaria delberg, Heidelberg, Germany 104Department of Psychology, University of Colorado Boulder, 78Department of Genetics, University of Groningen, University Boulder, Colorado Medical Centre Groningen, Groningen, The Netherlands 105Campbell Family Mental Health Research Institute, Centre for 79Department of Psychiatry, University of Colorado Denver, Addiction and Mental Health, Toronto, Ontario, Canada Aurora, Colorado 106Department of Psychiatry, University of Toronto, Toronto, 80Center for Neurobehavioral Genetics, Semel Institute for Neuro- Ontario, Canada science and Human Behavior, University of California, Los Angeles, 107Institute of Medical Science, University of Toronto, Toronto, California Ontario, Canada 81Department of Psychiatry, University of Halle, Halle, Germany 108Institute of Molecular Genetics, Russian Academy of Sciences, 82Division of Psychiatric Genomics, Department of Psychiatry, Moscow, Russia Icahn School of Medicine at Mount Sinai, New York, New York 109Latvian Biomedical Research and Study Centre, Riga, Latvia 83Department of Psychiatry, University of Munich, Munich, 110Department of Psychiatry and Zilkha Neurogenetics Institute, Germany Keck School of Medicine at University of Southern California, Los 84Departments of Psychiatry and Human and Molecular Genetics, Angeles, California INSERM, Institut de Myologie, Hopital^ de la Pitiè-Salpetrière,^ Paris, 111Faculty of Medicine, Vilnius University, Vilnius, Lithuania France 112 Department of Biology and Medical Genetics, 2nd Fac- 85Mental Health Research Centre, Russian Academy of Medical ulty of Medicine and University Hospital Motol, Prague, Czech Sciences, Moscow, Russia Republic 86Neuroscience Therapeutic Area, Janssen Research and Develop- 113 Department of Child and Adolescent Psychiatry, Pierre and ment, Raritan, New Jersey Marie Curie Faculty of Medicine, Paris, France 87Queensland Brain Institute, The University of Queensland, Bris- 114Duke-NUS Graduate Medical School, Singapore, Singapore bane, Queensland, Australia 115Department of Psychiatry, Hadassah-Hebrew University Medi- 88Department of Psychiatry, Academic Medical Centre University cal Center, Jerusalem, Israel of Amsterdam, Amsterdam, The Netherlands 116Centre for Genomic Sciences, The University of Hong Kong, 89Illumina, La Jolla, California, California Hong Kong, China 90Mental Health Services Copenhagen, Institute of Biological Psy- 117Mental Health Centre and Psychiatric Laboratory, West China chiatry, Mental Health Centre Sct. Hans, Copenhagen, Denmark Hospital, Sichuan University, Chengdu, China 91Friedman Brain Institute, Icahn School of Medicine at Mount 118Department of Biostatistics, Johns Hopkins University Bloom- Sinai, New York, New York berg School of Public Health, Baltimore, Maryland 92J. J. Peters VA Medical Center, Bronx, New York, New York 119Department of Psychiatry, Columbia University, New York, 93Priority Research Centre for Health Behaviour, University of New York Newcastle, Newcastle, Australia 120Priority Centre for Translational Neuroscience and Mental 94School of Electrical Engineering and Computer Science, Univer- Health, University of Newcastle, Newcastle, Australia sity of Newcastle, Newcastle, Australia 121Department of Genetics and Pathology, International Heredi- 95Division of Medical Genetics, Department of Biomedicine, Uni- tary Cancer Center, Pomeranian Medical University in Szczecin, Szcze- versity of Basel, Basel, Switzerland cin, Poland 96Department of Genetics, Harvard Medical School, Boston, 122Department of Mental Health and Substance Abuse Services; Massachusetts National Institute for Health and Welfare, Helsinki, Finland 97Section of Neonatal Screening and Hormones, Department of 123Department of Mental Health, Bloomberg School of Public Clinical Biochemistry, Immunology and Genetics, Statens Serum Insti- Health, Johns Hopkins University, Baltimore, Maryland tut, Copenhagen, Denmark 124Department of Psychiatry, University of Bonn, Bonn, Germany 98Department of Psychiatry, Fujita Health University School of 125Centre National de la Recherche Scientifique, Laboratoire de Medicine, Toyoake, Aichi, Japan Gen etique Moleculaire de la Neurotransmission et des Processus Neu- 99Department of Psychiatry, Regional Centre for Clinical Research rodeg en eratifs, Hopital^ de la PitieSalp etrière,^ Paris, France in Psychosis, Stavanger University Hospital, Stavanger, Norway 126Department of Genomics Mathematics, University of Bonn, 100Rheumatology Research Group, Vall d’Hebron Research Insti- Bonn, Germany tute, Barcelona, 08035, Spain 127Research Unit, Sørlandet Hospital, Kristiansand, Norway 464 | LI ET AL.

128Department of Psychiatry, Harvard Medical School, Boston, 156Melbourne Neuropsychiatry Centre, University of Melbourne & Massachusetts Melbourne Health, Melbourne, Australia 129VA Boston Health Care System, Brockton, Massachusetts 157Department of Psychiatry, University of Helsinki, Helsinki, Finland 130Department of Psychiatry, National University of Ireland Gal- 158Public Health Genomics Unit, National Institute for Health and way, Co. Galway, Ireland Welfare, Helsinki, Finland 131Centre for Cognitive Ageing and Cognitive Epidemiology, Uni- 159Medical Faculty, University of Belgrade, Belgrade, Serbia versity of Edinburgh, Edinburgh, United Kingdom 160Department of Psychiatry, University of North Carolina, Chapel 132Division of Psychiatry, University of Edinburgh, Edinburgh, Hill, North Carolina United Kingdom 161Institute for Molecular Medicine Finland, FIMM, University of 133Division of Mental Health and Addiction, Oslo University Hos- Helsinki, Helsinki, Finland pital, Oslo, Norway 162Department of Epidemiology, Harvard School of Public Health, 134 Division of the Beth Israel Deaconess Medical Center, Massa- Boston, Massachusetts chusetts Mental Health Center Public Psychiatry, Boston, 163Department of Psychiatry, University of Oxford, Oxford, United Massachusetts Kingdom 135Estonian Genome Center, University of Tartu, Tartu, Estonia 164Virginia Institute for Psychiatric and Behavioral Genetics, Vir- 136School of Psychology, University of Newcastle, Newcastle, ginia Commonwealth University, Richmond, Virginia Australia 165Institute for Multiscale Biology, Icahn School of Medicine at 137First Psychiatric Clinic, Medical University, Sofia, Bulgaria Mount Sinai, New York, New York 138Department P, Aarhus University Hospital, Risskov, Denmark 166PharmaTherapeutics Clinical Research, Pfizer Worldwide 139Department of Psychiatry, Royal College of Surgeons in Ireland, Research and Development, Cambridge, Massachusetts Dublin 2, Ireland 167Department of Psychiatry and Psychotherapy, University of 140King’s College London, London, United Kingdom Gottingen, Gottingen,€ Germany 141Maastricht University Medical Centre, South Limburg Mental 168Psychiatry and Psychotherapy Clinic, University of Erlangen, Health Research and Teaching Network, EURON, Maastricht, The Erlangen, Germany Netherlands 169Hunter New England Health Service, Newcastle, Australia 142Institute of Translational Medicine, University of Liverpool, Liv- 170School of Biomedical Sciences and Pharmacy, University of erpool, United Kingdom Newcastle, Callaghan, Australia 143Max Planck Institute of Psychiatry, Munich, Germany 171Division of Cancer Epidemiology and Genetics, National Cancer 144Munich Cluster for Systems Neurology (SyNergy), Munich, Institute, Bethesda, Maryland Germany 172University of Iceland, Landspitali, National University Hospital, 145Department of Psychiatry and Psychotherapy, Jena University Reykjavik, Iceland Hospital, Jena, Germany 173Department of Psychiatry and Drug Addiction, Tbilisi State 146Department of Psychiatry, Queensland Brain Institute and Medical University (TSMU), Tbilisi, Georgia Queensland Centre for Mental Health Research, University of Queens- 174Research and Development, Bronx Veterans Affairs Medical land, Brisbane, Queensland, St Lucia, Australia Center, New York, New York 147Department of Psychiatry and Behavioral Sciences, Johns Hop- 175Wellcome Trust Centre for Human Genetics, Oxford, United kins University School of Medicine, Baltimore, Maryland Kingdom 148Department of Psychiatry, Trinity College Dublin, Dublin 2, 176deCODE Genetics, Reykjavik, Iceland Ireland 177Department of Clinical Neurology, Medical University of 149Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Vienna, Wien, Austria Indiana 178Lieber Institute for Brain Development, Baltimore, Maryland 150Department of Clinical Sciences, Psychiatry, Umeå University, 179Department of Medical Genetics, University Medical Centre Umeå, Sweden Utrecht, Universiteitsweg 100, Utrecht, The Netherlands 151DETECT Early Intervention Service for Psychosis, Blackrock, 180Berkshire Healthcare NHS Foundation Trust, Bracknell, United Co. Dublin, Ireland Kingdom 152Centre for Public Health, Institute of Clinical Sciences, Queen’s 181Section of Psychiatry, University of Verona, Verona, Italy University Belfast, Belfast, United Kingdom 182Department of Psychiatry, University of Oulu, Oulu, Finland 153Lawrence Berkeley National Laboratory, University of California 183University Hospital of Oulu, Oulu, Finland at Berkeley, Berkeley, California 184Molecular and Cellular Therapeutics, Royal College of Surgeons 154Institute of Psychiatry, King’s College London, London, United in Ireland, Dublin 2, Ireland Kingdom 185Health Research Board, Dublin 2, Ireland 155A list of authors and affiliations appear in the Supplementary 186School of Psychiatry and Clinical Neurosciences, The University Information of Western Australia, Perth, Australia LI ET AL. | 465

187Computational Sciences CoE, Pfizer Worldwide Research and COMPLIANCE WITH ETHICAL STANDARDS Development, Cambridge, Massachusetts Ethical approval 188Human Genetics, Genome Institute of Singapore, A*STAR, Sin- All procedures performed in studies involving human participants were gapore, Singapore 189 in accordance with the ethical standards of the institutional and/or A list of authors and affiliations appear in the Supplementary Information national research committee and with the 1964 Helsinki declaration 190University College London, London, United Kingdom and its later amendments or comparable ethical standards. 191Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York Data availability 192Institute of Neuroscience and Medicine (INM-1), Research Cen- Schizophrenia genotype data from the Psychiatric Genomics Consor- ter Juelich, Juelich, Germany tium can be accessed by following the consortium’s data policies: 193Department of Genetics, The Hebrew University of Jerusalem, https://www.med.unc.edu/pgc/shared-methods Jerusalem, Israel 194Neuroscience Discovery and Translational Area, Pharma CONFLICT OF INTEREST Research and Early Development, F. Hoffman-La Roche, Basel, Switzerland The authors declare that they have no conflict of interest. 195Centre for Clinical Research in Neuropsychiatry, School of Psychiatry and Clinical Neurosciences, The University of ORCID Western Australia, Medical Research Foundation Building, Perth, Wen Li http://orcid.org/0000-0003-1964-0844 Australia Ole A. Andreassen http://orcid.org/0000-0002-4461-3568 196Departments of Psychiatry and Human and Molecular Genetics, Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Com- REFERENCES monwealth University, Richmond, Virginia Askland, K., Read, C., O’Connell, C., & Moore, J. H. (2012). Ion channels 197The Feinstein Institute for Medical Research, Manhasset, New and schizophrenia: A gene set-based analytic approach to GWAS York data for biological hypothesis testing. Human Genetics, 131(3), 198The Hofstra NS-LIJ School of Medicine, Hempstead, New York 373–391. 199 The Zucker Hillside Hospital, Glen Oaks, New York Bannister, R. 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