Association of NPAS3 exonic variation with schizophrenia

Schizophrenia Research 120 (2010) 143–149

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Schizophrenia Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c h r e s

Association of NPAS3 exonic variation with schizophrenia Georgina Macintyre a, Tyler Alford a, Lan Xiong c, Guy A. Rouleau c, Philip G. Tibbo b,1, Diane W. Cox a,⁎ a

Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7 Department of Psychiatry and Bebensee Schizophrenia Research Unit, University of Alberta, Edmonton, Alberta, Canada, T6G 4R7 Centre of Excellence in Neuromics of University of Montreal, CHUM Research Center and the Department of Medicine, University of Montreal, Montréal, Québec, Canada, H2L 4M1 b c

a r t i c l e

i n f o

Article history: Received 21 December 2009 Received in revised form 5 April 2010 Accepted 7 April 2010 Available online 14 May 2010

Keywords: NPAS3 Genetic variation Schizophrenia Neurogenesis

a b s t r a c t Background: We previously identified the neuronal PAS3 (NPAS3) gene as a candidate gene for schizophrenia. A mother and daughter, both with schizophrenia, were carriers of a translocation, t(9;14)(q34;q13), that disrupts the NPAS3 gene. The gene is located at 14q13, a region implicated in schizophrenia and bipolar disorder in various linkage studies. NPAS3 belongs to the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor family, involved in diverse processes including the regulation of cell differentiation and circadian rhythms, and the development and function of the nervous system. Methods: The 12 exons encoding NPAS3 were sequenced in DNA from individuals with schizophrenia. NPAS3 variants were identified in exons 6 and 12, initially in 12 patients only. These two exons were then sequenced in 83 patients and 83 controls. Results and conclusion: Three common variants of NPAS3, also found in controls, showed a positive association with schizophrenia (NM_001164749: rs12434716, c.1654GNC, p = 0.009; rs10141940, c.2208CNT, p = 0.01; rs10142034, c.2262CNG, p = 0.01). The c.1654GNC variant, results in an p.Ala552Pro change and may affect NPAS3 protein function directly. Alternatively, the three SNPs may affect the splicing of NPAS3 transcripts, as they are each located within putative exonic splicing enhancer (ESE) motifs (ESEFinder). A c.726CNT variant, identified in three patients, is located in an ESE element and is predicted to reduce the function of the motif. Other variants, identified in controls, included c.2089GNA (p.Gly697Ser) and c.2097TNC. Our identification of potentially defective NPAS3 variants supports recent studies that implicate perturbations in NPAS3 pathways in impaired neurogenesis and psychosis. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Our characterization of a translocation, t(9;14)(q34:q13), in a mother and two daughters affected with schizophrenia,

⁎ Corresponding author. Department of Medical Genetics, 8-43B Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7. Tel.: + 1 780 492 7501; fax: + 1 780 492 1998. E-mail addresses: [email protected] (G. Macintyre), [email protected] (T. Alford), [email protected] (L. Xiong), [email protected] (G.A. Rouleau), [email protected] (P.G. Tibbo), [email protected] (D.W. Cox). 1 Now at: Department of Psychiatry, Dalhousie University, Rm 3030, 3rd Floor, Abbie Lane Bldg, 5909 Veterans' Memorial Lane, Halifax, Nova Scotia, Canada, B3H 2E2. 0920-9964/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2010.04.002

led to the identification of a break in the neuronal PAS3 gene, NPAS3 (Kamnasaran et al., 2003). The NPAS3 break in this family was later confirmed independently (Pickard et al, 2005). NPAS3 is located at 14q13 in a chromosomal region implicated in brain development, neurocognition and psychiatric disorders, including schizophrenia (selected references: (Arinami et al., 2005; Bailer et al., 2000; Blouin et al., 1998; Buyske et al., 2006; Chiu et al., 2002; Kamnasaran et al., 2005; Lerer et al., 2003; Lewis et al., 2003). NPAS3 belongs to the highly conserved basic helix loop helix (bHLH) protein family (reviewed in (Pickard et al., 2006)). Members contain characteristic DNA bHLH and protein dimerization, Per-Arnt-Sim (PAS: Period, Aryl hydrocarbon receptor, Single minded) domains (Crews and Fan,

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1999). Many aspects of brain development and physiology are controlled by proteins in this family with some members implicated in schizophrenia, including hypoxia-inducible factor-1α (HIF-1α), a key regulator of genes required for adaptation to low oxygen, such as the erythropoietin gene, EPO (Semenza, 2004). EPO has neurotrophic and synaptogenic properties and EPO receptors are up-regulated in patients with schizophrenia. This latter finding may lead to EPO therapies for patients (Ehrenreich et al., 2007). The NPAS subfamily has three other members, NPAS1, NPAS2 and NPAS4. These proteins share sequence homology only within the bHLH-PAS domains and all are expressed in neuronal tissue. Of the above, NPAS3 is most closely related to NPAS1 (Pickard et al., 2005). NPAS1 is involved in branching morphogenesis of the lung, cortical interneuron migration and differentiation, and EPO gene expression (Levesque et al., 2007; Ohsawa et al., 2005; Zhao et al., 2008b). NPAS3 has also been implicated in early lung development (Zhou et al., 2009). NPAS2 controls mammalian circadian rhythms, plays a role in cued and contextual memory in mice, (Garcia et al., 2000) and has been associated with seasonal affective disorder (Partonen et al, 2007). NPAS4 may contribute to the modulation of neuronal synapse activity and is upregulated in brain ischemia (Lin et al., 2008). Recent evidence supports our hypothesis (Kamnasaran et al., 2003) that NPAS3 is a potential candidate gene for schizophrenia. First, Npas3 knockout mice exhibit altered brain structure and biochemistry, behavioral and pharmacological responses. Hippocampal, cerebellar and cortical changes occur in the Npas3−/−mice, and reelin protein levels are reduced (Erbel-Sieler et al., 2004). Fibroblast growth factor (FGF)mediated adult hippocampal neurogenesis is implicated (Pieper et al., 2005). Behavioural tests suggest anxiety, impaired learning and social recognition deficits (Brunskill et al., 2005; Erbel-Sieler et al., 2004). Some of these changes are reminiscent of alterations seen in individuals with schizophrenia, including structural changes in the hippocampus, cerebellum and enlarged ventricles, and reduced levels of adult neural stem cells and reelin (Bilder et al., 1995; DeLisi et al., 1997; Gaser et al., 2004; Harrison and Weinberger, 2005; Lieberman et al., 1993; Reif et al., 2006; Tissir and Goffinet, 2003). Subsequent SNP studies in humans suggest the involvement of NPAS3 in neurophysiology. A large case-control study of schizophrenia and bipolar disorder, using intronic tagged SNPs, has identified risk and protective haplotypes associated with four regions of the NPAS3 gene (Pickard et al., 2008). A meta-analysis of three genome-wide association studies (GWAS) for bipolar disorder also identified an association with NPAS3 (Ferreira et al., 2008). NPAS3 intronic variants are also associated with a positive response to treatment with the anti-psychotic iloperidone in schizophrenia (Lavedan et al., 2009), with interferon beta therapy in multiple sclerosis (Byun et al., 2008) and with successful smoking cessation (Uhl et al., 2008). The NPAS3 gene is ∼ 863 kb in size and consists of 12 exons that encode at least six alternate transcripts. The full-length transcript encodes a protein containing a bHLH dimerization domain, two PAS domains, a nuclear localization signal and two adjacent polyglycine repeats in a putative transactivation domain (Kamnasaran et al., 2003; Pickard et al., 2005). We report here our case-control study to assess the contribution of genetic variation within the exons of NPAS3 to schizophre-

nia in a Northern and Western European population. We identified NPAS3 variants that might encode defective NPAS3 protein or alter transcriptional processing of NPAS3 RNA. 2. Methods 2.1. Patient ascertainment All patients were diagnosed with schizophrenia according to DSM III/IV criteria. Blood samples were collected in Alberta from 83 patients of European ancestry (cohort 1). Samples were not collected from family members. Controls were of Northern and Western European origin and collected locally (n = 51), or from a neurologically normal control panel (Caucasian; National Institute of Neurological Disorders and Stroke [NINDS], www.ninds.nih.gov/; NDPT023; n = 32), and were matched for sex (females = 25; males = 58). Ethics approval was obtained from the University of Alberta Health Research Ethics Board. Cohort 2 comprised 41 DNA samples, some of unknown ethnicity, collected previously as part of an independent schizophrenia research study (http://www. synapse2disease.ca/en_publications.html). 2.2. Sequence analysis Genomic DNA was prepared from peripheral blood leukocytes (Qiagen FlexiGene DNA Kit, QIAGEN, Ontario). Primers flanking each of the twelve exons were designed using Primer 3 (http://primer3.sourceforge.net/) and used for PCR (primers and PCR conditions are described in supplementary Table S1). PCR fragments were either treated with ExoSAPIT (GE Healthcare, Baie d'Urfe, Canada), or purified after electrophoresis through 1% agarose using the QIAquick gel extraction kit (QIAGEN). DNA sequencing was carried out using a BigDye® Terminator v3.1 Ready Reaction Mix and a 3130 × l Genetic Analyzer (both from Applied Biosystems, California). NPAS3 cDNA, GenBank accession no. NM_001164749, was used as the reference sequence; +1 is the A in the ATG translation initiation codon. Initially, the twelve exons of the NPAS3 gene were sequenced in DNA samples from twelve patients in cohort 2, and in control DNA samples from two healthy volunteers, to identify exons containing NPAS3 variants unique to the patient group. We identified NPAS3 variants in exons 6 and 12 only. We then sequenced exons 6 and 12 in the DNA from the 83 patients in cohort 1 and in 83 control DNAs. We also sequenced exons 6 and 12 in a further 29 patients from cohort 2. Subsequently, we sequenced the complete NPAS3 coding sequence in 16 of the patients in cohort 1. No additional coding sequence variants were identified. Typical DNA sequencing electropherograms for previously unreported variants are included in supplementary data (Fig. S1). 2.3. Statistics Chi-square and Fisher's exact tests were used, as appropriate, to estimate the significance of association. p-values of less than 0.05 were considered significant. All SNPs were in Hardy– Weinberg equilibrium. Samples from cohort 2 were used in the initial sequence screening, and only cohort 1 was used for statistical analysis.

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2.4. NPAS3 missense variants: predicted effect on NPAS3 protein function Identification of NPAS3 protein sequences from other species, for alignment, was performed using NCBI-BLAST and the human NPAS3 933 amino acid sequence (GenBank accession no. Q8IXF0). Eleven orthologous proteins were identified and aligned using Clustal W (www.ebi.ac.uk/ clustalw/; Fig. 1). The alignment produced by the Clustal W analysis was also submitted to SIFT (Sorting Intolerant from Tolerant; http://sift.jcvi.org/), for prediction of the effect of each non-synonymous NPAS3 variant upon protein function. SIFT uses conservation of protein sequence across species and characteristics of the amino acid substitution to assign a tolerance score for each variant; b0.05 predicts a functional deficit (Kumar et al., 2009). Q8IXF0 was also submitted to the PMUT server (http://mmb2.pcb.ub.es:8080/PMut/), and analysed using the program's default settings. PMUT employs a neural network approach to predict the pathological character of genetic variants (Ferrer-Costa et al., 2005).

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2.5. NPAS3 variants: predicted impact on splicing Splice site enhancer motifs attract components of the splicing machinery to exonic sites to encourage splicing at weak exon/intron junction sites. ESE Finder predicts potential splice enhancer motifs within cDNAs, and assigns each a score, above or below a calculated threshold for each splicing factor (Splicing Factor arginine/serine 1/Alternate Splicing Factor, SF2/ASF; Splicing factor arginine/serine 2, SC35; Splicing factor aRginine/serine rich 5, SRp40; Splicing factor arginine/serine rich 6, SRp55). NPAS3 cDNA sequence (NM_001164749) was submitted to ESEFinder (3.0; http://rulai.cshl.edu/cgi-bin/tools/ESE3/ esefinder.cgi?process=home) to identify ESE motifs within the coding sequence. NPAS3 variant cDNA sequences were also submitted to identify putative changes within splicing enhancer motifs. This analysis included both synonymous and nonsynonymous variants. The strength of each splice donor (5’) and acceptor (3’) sites was calculated using Splice Site Score (http://rulai.cshl.edu/cgi-bin/New_Alt_Exon_Db/score.pl).

Fig. 1. NPAS3 amino acid sequence alignment. ClustalW was used to align eleven orthologous sequences to the human NPAS3 sequence. NPAS3 variants (shaded) were identified in each of the two regions shown: A, residues 547–624, and B, residues 689–850. The sequences shown form part of the C-terminal transactivation domain. Full length NPAS3 is 933amino acids in length. Regions of secondary structure (2o) are shown; E — beta-turn, H — helix. NPAS3 nuclear localization signal and missense variants are identified.

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3. Results 3.1. NPAS3 variants showing a positive association with schizophrenia We identified seven NPAS3 variants, one in exon 6 and six in exon 12 (cohort 1; Table 1). The exon 6 variant, c.726CNT, a synonymous change, was observed only in the patient group and has not been previously reported. Three of six variants detected in exon 12 have been reported in the SNP database, dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), rs12434716 (c.1654GNC), rs10141940 (c.2208CNT) and rs10142034 (c.2262CNG). The three variants form an invariant haplotype in most individuals. However, in one patient and one control, c.2208T and c.2262G were found in the absence of c.1654C. The allele frequency of the c.1654C variant in the patient group is 19.9%, significantly higher (p = 0.014) than in the control group (9.6%), and higher than reported in the HapMap dataset for CEU (CEPH, Utah residents with ancestry from northern and western Europe; http://hapmap.ncbi.nlm.nih.gov/; 13.8%). Four patients were homozygous for the three variants. No homozygotes were observed in controls (Table 1). c.2089GNA (p.Gly697Ser) was found in three healthy controls, in a heterozygous state (Table 1). The genotype and allele frequencies were not calculated for additional variants, c.1500GNC and c.2097 TNC, that were observed in the control group, in single individuals and in a heterozygous state. The NPAS3 variant, c.1857CNG (p.Ser619Arg), was observed in a healthy control. However, this sample was excluded from the case control study due to uncertain ethnicity. Cohort 2 comprised DNA from 41 probands, previously collected for a separate family trio study of schizophrenia. Ethnic heterogeneity was noted for this group and matched

controls were unavailable. This cohort was not used in the case control study. We used this cohort only to identify exons that showed variation in patients, for sequencing in cohort 1. We subsequently sequenced exon 6 and 12 in the remaining samples in cohort 2. The c.726CNT variant was observed in three of the 41 patients. The three SNP haplotype was also present. For c.1654GNC, frequencies were calculated for genotype (G/G 68.3%, G/C 29.3%, C/C 2.4%) and for each allele, G, 82.9% and A, 15.8%. One patient was homozygous for the variant haplotype, another carried the c.1654GNC variant in the absence of c.2208CNT and c.2262CNG. A rare change, c.2171CNT, p.Ala724Val, was identified in a single patient. 3.2. NPAS3 variants are predicted to affect NPAS3 protein function Currently, a limited number of sequences are available that contain the full coding sequence for NPAS3. Eleven nonhuman NPAS3 orthologues were identified using the human sequence in a BLAST search (Fig. 1). Of the five missense variants identified, two are predicted to be deleterious, using SIFT, p.Gly699Arg (control) and p.Ala724Val (patient; Table 2). Using the human NPAS3 protein sequence and the default alignment produced in PMUT, p.Gly699Arg was the only variant predicted to be pathological (Table 2). The terms ‘deleterious’ and ‘pathological’ refer to changes from the query sequence, gain- or loss-of-function cannot be inferred. 3.3. NPAS3 variants are predicted to affect RNA splicing The identification of NPAS3 variants in exons 6 and 12 prompted the examination of these exons for splicing regulatory elements. The probable efficiency of splicing was determined using Splice Site Finder. The 5’ and 3’ splice sites for the splicing of exons 5 and 6 were predicted to be efficient

Table 1 NPAS3 exonic variants identified in cohort 1. Variant c.726CNT

rs12434716 c.1654GNC p.Ala552Pro

c.2089GNA p.Gly697Ser

rs10141940 c.2208CNT

rs10142034 c.2262CNG

Population

Sample size

Schizophrenia

83

Control

83

Schizophrenia

83

Control

83

Schizophrenia

83

Control

83

Schizophrenia

83

Control

83

Schizophrenia

83

Control

83

Genotype C/C 81 (97.5%) 83 (100%) G/G 54 (65.1%) 67 (80.7%) G/G 83 (100%) 80 (96.4%) C/C 53 (63.9%) 66 (79.5%) C/C 53 (63.9%) 66 (79.5%)

p C/T 2 (2.4%) 0 (0%) G/C 25 (30.1%) 16 (19.3%) G/A 0 (0%) 3 (3.6%) C/T 26 (31.3%) 17 (20.5%) C/G 26 (31.3%) 17 (20.5%)

T/T 0 (0%) 0 (0%) C/C 4 (4.8%) 0 (0%) A/A 0 (0%) 0 (0%) T/T 4 (4.8%) 0 (0%) G/G 4 (4.8%) 0 (0%)

0.5

0.025

0.25

0.026

0.026

Allele C 164 (98.8%) 166 (100%) G 133 (80.1%) 150 (90.4%) G 166 (100%) 163 (98.2%) C 132 (79.5%) 149 (89.7%) C 132 (79.5%) 149 (89.7%)

p T 2 (1.2%) 0 (0%) C 33 (19.9%) 16 (9.6%) A 0 (0%) 3 (1.8%) T 34 (20.5%) 17 (10.2%) G 34 (20.5%) 17 (10.2%)

0.5

0.009

0.25

0.01

0.01

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Table 2 NPAS3 variants identified in all groups: summary of predicted effects. Predicted functional change Variant

Amino acid change Functional domain Protein function

Splicing protein binding capacity

SIFT (b 0.05 deleterious) PMut (N0.5 pathological) Control group only c.1500GNC c.1857CNG c.2089GNA c.2097 TNC

Synonymous p.Ser619Arg p.Gly697Ser p.Gly699Arg

– Transactivation Transactivation Transactivation

– Benign (0.24) Benign (0.35) Deleterious (0.02)

– Neutral (0.49) Neutral (0.34) Pathological (0.69)

Reduced Reduced No change Increased — creates a new site

Patient group only c.726CNT c.2171CNT

Synonymous p.Ala724Val

– Transactivation

– Deleterious (0.04)

– Neutral (0.32)

Severe reduction Severe reduction

Control and patient groups c.1654GNC (rs12434716) p.Ala552Pro c.2208CNT (rs10141940) Synonymous c.2262CNG (rs10142034) Synonymous

Transactivation – –

Benign (0.42) – –

Neutral (0.29) – –

Increased — creates a new site Reduced Severe reduction

and scored higher than average (5’, 9.5; 3’, 9.7; average scores, 5’ = 8.1, 3’ = 7.9), while lower than average scores were obtained for exons 11 and 12 (5’, 4.9; 3’,6.4). Exonic splicing factor motifs were identified using ESE Finder. NPAS3 nucleotide variants that alter the splicing factor binding scores to above or below the threshold values are included in supplementary Table S2. Two variants, c.2097TNC (control group) and c.1654GNC (control and patient groups), may create new sites. The most severe reductions in potential binding capacity are noted for the NPAS3 variants found in patients only, c.726CNT, c.2171CNT, or in both groups, c.2262CNG (Table 2 and S2).

4. Discussion This study has identified variants within the NPAS3 coding sequence associated with schizophrenia. Several of the variants are also found in the control population and are predicted to affect NPAS3 transcript processing or NPAS3 protein function. These findings support the currently favoured theory that complex diseases, such as schizophrenia, are the result of interactions between particular combinations of rare and/or common variants, in one or more genes, and the environment (Burmeister et al., 2008). The observed three SNP haplotype may have a significant impact on NPAS3 function, as all three SNPs are predicted to affect NPAS3 RNA splicing. Functional testing of the three SNPs separately, and in combination, will be required to elucidate whether their influence on RNA splicing is positive or negative. In addition, the first SNP in this haplotype encodes p.Ala552Pro. The alanine residue is found in the most common form of human NPAS3, with an allele frequency of 90.4%. Alanine is found only in the human NPAS3 sequence and may confer a change in NPAS3 function, important only in humans. However, using predictions programs, such as SIFT, this substitution of a proline residue is predicted to be benign, as proline is found at this position in the frog and many of the mammalian sequences examined. Four different triplet codons can encode proline, the CCG triplet codon generated in this instance is also used at this position in the mouse

sequence. The CCG is the least used codon for proline in human and mouse genomes (www.kazusa.or.jp./codon/). The N-termini of the HLH-PAS proteins are highly conserved, and are likely to be tightly constrained due to their roles in DNA binding and dimerization. The C-termini of related HLH-PAS proteins encode transactivation domains, responsible for interactions with other co-transactivating proteins (Fedele et al., 2002; Kewley et al., 2004). These regions are more variable in length and sequence and this may contribute to increased diversity in transcriptional control. In NPAS3, the C-terminus is biased towards alanine, glycine, proline and histidine, and contains two distinct polyglycine (polyG) tracts (Fig. 1). PolyG tracts control the spacing of important functional domains (Elanko et al., 2001), and alteration of the tract length by one or two glycine residues can alter function (Brito et al., 2005; Brockschmidt et al., 2007; Harvey et al., 2007). In addition, polyG tracts may also directly interact with other proteins (Baldwin and Inoue, 2006; Inoue and Keegstra, 2003). NPAS1 and NPAS3 are closely related and share a high homology in their N-terminal amino acid sequence, but are markedly different at their C-termini. In mammals, a recent expansion of two NPAS3specific polyglycine tracts in the C-terminus has occurred. In addition, the second polyG tract has expanded in humans, potentially creating a unique domain(s) and altering protein function (Fig. 1). p.Gly699Arg is predicted to affect protein function. However, this change may be advantageous and confer some protection against schizophrenia, as it is only found in the three individuals in the control group. Alternatively, the change may be a predisposing risk factor, not found in our patient cohorts. The predominant impact of the NPAS3 variants identified in individuals with schizophrenia is predicted to be on NPAS3 transcript processing (Supplementary Table S2). Splice Site Finder predicted the exon 11 and 12 splice junction to be weak, and splice factor binding at ESEs in exon 12 may be essential to enable splicing. This splicing event may be particularly vulnerable to some of the ESE changes identified in exon 12. We previously described two NPAS3 transcripts; a 3.4 kb cDNA transcript that does not include exon 2 of the NPAS3 gene and encodes a 901 amino acid isoform of NPAS3,

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and a smaller transcript of 2.5 kb that encodes a 153 amino acid protein containing only the HLH-PAS region of NPAS3. This putative protein is encoded by exons 2 through 6 (Kamnasaran et al., 2003). Four additional transcripts are reported by Ensembl (www.ensembl.org). The production of all known NPAS3 transcripts may be affected by one or more of the variants identified in exons 6 and 12, resulting in changes in the availability of the various NPAS3 isoforms. New NPAS3 isoforms may also be produced using cryptic splice sites. The detection of NPAS3 variation in patients supports a neurodevelopmental origin for schizophrenia (Lewis and Levitt, 2002). Impaired adult neurogenesis is implicated in many human conditions, including learning deficits, depression, and schizophrenia (Eisch et al., 2008; Reif et al., 2007). Adult neurogenesis could be stimulated in the Npas3−/−mice by electroconvulsive therapy (Pieper et al., 2005), and approaches to stimulate dormant neural cell precursor populations may prove to be a promising route for future therapies for schizophrenia (Balu and Lucki, 2008; Pieper et al., 2005; Zhao et al., 2008a). Further studies are required to elucidate the regulatory role of NPAS3 in neurodevelopment and neurophysiology, and to understand how genetic variation in the NPAS3 gene affects brain development, psychosis, addiction and drug response. Role of funding source Funding for this project was obtained from the University Hospital Foundation, University of Alberta, Canada and the Canadian Psychiatric Research Foundation (CPRF)/CIHR R&D/Astra Zeneca (YCP-82209). These agencies had no further role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. Contributors Drs. Cox, Tibbo and Macintyre designed the study protocol. Dr. Tibbo diagnosed patients and organized blood collections in Alberta. T. Alford optimized the DNA analysis and performed initial bioinformatics studies. Drs. Rouleau and Xiong contributed DNA samples collected from individuals diagnosed with schizophrenia. Dr. Macintyre performed final statistical and bioinformatics analyses, and wrote the first draft of the manuscript. Drs. Macintyre and Cox edited the manuscript. All authors contributed to, and approved, the final manuscript. Conflict of interest None. Acknowledgements We express our appreciation to all of the study participants. We thank Lisa Prat Davies and Darren Bugbee (DB) for DNA sample preparation and data collation; Susan Kenney for sequencing (TAGC: The Applied Genomics Centre, University of Alberta); DB for assistance with figure preparation. This study was funded by the University Hospital Foundation, Edmonton, and Canadian Psychiatry Research Foundation (CPRF)/Canadian Institutes for Health Research (CIHR) R&D/Astra Zeneca.

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