HHS Public Access Author manuscript Nat Genet. Author manuscript; available in PMC 2021 November 17. Author Manuscript Published in final edited form as: Nat Genet. 2021 June ; 53(6): 817–829. doi:10.1038/s41588-021-00857-4. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms * Corresponding authors: Niamh Mullins, niamh.mullins@mssm.edu; Ole A. Andreassen, ole.andreassen@medisin.uio.no. Author contributions Writing group: N.M., A.J.F., K.S.O’C., B.C., J.R.I.C., J.M.B., J.I.N., S. Cichon, H.J.E., E.A.S., A. McQuillin, A.D.F., R.A.O., O.A.A. PGC BD PI group: A.J.F., M.I., H.-H.W., D.C., R.A., I.A., M.A., L. Alfredsson, G. Babadjanova, L.B., B.T.B., F.B., S. Bengesser, W.H.B., D.H.R.B., M. Boehnke, A.D.B., G. Breen, V.J.C., S. Catts, A.C., N.C., U.D., D.D., T. Esko, B.E., P.F., M.F., J.M.F., M.G., E.S.G., F.S.G., M.J. Green, M.G.-S., J. Hauser, F.H., J. Hillert, K.S.H., D.M.H., C.M.H., K. Hveem, N.I., A.V.J., I.J., L.A.J., R.S.K., J.R.K., G.K., M. Landén, M. Leboyer, C.M.L., Q.S.L., J. Lissowska, C. Lochner, C. Loughland, N.G.M., C.A.M., F.M., S.L.M., A.M.M., F.J.M., I.M., P. Michie, L. Milani, P.B. Mitchell, G.M., O.M., P.B. Mortensen, B.M., B.M.-M., R.M.M., B.M.N., C.M.N., M.N., M.M.N., M.C.O’D., K.J.O., T.O., M.J.O., S.A.P., C. Pantelis, C. Pato, M.T.P., G.P.P., R.H.P., D.P., J.A.R-Q., A.R., E.Z.R., M. Author Manuscript Ribasés, M. Rietschel, S.R., G.A.R., T.S., U.S., M.S., P.R.S., T.G.S., L.J.S., R.J.S., A.S., C.S.W., J.W.S., H.S., K.S., E. Stordal, F. Streit, P.F.S., G.T., A.E.V., E.V., J.B.V., I.D.W., T.W.W., T.W., N.R.W., J-A.Z., J.M.B., J.I.N., S. Cichon, H.J.E., E.A.S., A. McQuillin, A.D.F., R.A.O., O.A.A. Bioinformatics: N.M., A.J.F., J.R.I.C., S. Børte, M.J. Gandal, M. Kim, B.M.S., L.G.S., B.S.W., H.-H.W., N.A.-R., S.E.B., B.M.B., V.E.-P., S.H., P.A.H., Y.K., M. Koromina, M. Kubo, M. Leber, P.H.L., C. Liao, L.M.O.L., T.R., P.R., P.D.S., M.S.A., C. Terao, T.E.T., S.X., H.Y., P.P.Z., S. Bengesser, G. Breen, P.F., E.S.G., Q.S.L., G.A.R., H.S., T.W., E.A.S. Clinical: O.K.D., M.I., L. Abramova, K.A., E.A., N.A.-R., A. Anjorin, A. Antoniou, J.H.B., N.B., M. Bauer, A.B., C.B.P., E.B., M.P.B., R.B., M. Brum, N.B.-K., M. Budde, W.B., M. Cairns, M. Casas, P.C., A.C.-B., D.C., P.M.C., N.D., A.D., T. Elvsåshagen, L. Forty, L. Frisén, K.G., J. Garnham, M.G.P., I.R.G., K.G.-S., J. Grove, J.G.-P., K. Ha, M. Haraldsson, M. Hautzinger, U.H., D.H., J.L. Kalman, J.L. Kennedy, S.K.-S., M. Kogevinas, T.M.K., R.K., S.A.K., J.L., H.-J.L., C. Lewis, S.L., M. Lundberg, D.J.M., W.M., D.M., L. Martinsson, M.M., P. McGuffin, H.M., V.M., C.O’D., L.O., S.P., A. Perry, A. Pfennig, E.P., J.B.P., D.Q., M.H.R., J.R.D., E.J.R., J.P.R., F.R., J.R., E.C.S., F. Senner, E. Sigurdsson, L.S., C.S., O.B.S., D.J. Smith, J.L.S., A.T.S., J.S.S., B.S., P.T., M.P.V., H.V., A.H.Y., L.Z., HUNT All-In Psychiatry, R.A., I.A., M.A., G. Babadjanova, L.B., B.T.B., F.B., S. Bengesser, D.H.R.B., A.D.B., A.C., N.C., U.D., D.D., B.E., P.F., M.F., M.G., E.S.G., F.S.G., M.J. Green, M.G.-S., J. Hauser, K.S.H., N.I., I.J., L.A.J., R.S.K., G.K., M. Landén, C.M.L., J. Lissowska, N.G.M., C.A.M., F.M., S.L.M., A.M.M., I.M., P.B. Mitchell, G.M., O.M., P.B. Mortensen, M.C.O’D., K.J.O., M.J.O., C. Pato, M.T.P., R.H.P., J.A.R.-Q., A.R., E.Z.R., M. Rietschel, T.S., T.G.S., A.S., C.S.W., J.W.S., E. Stordal, F. Streit, A.E.V., E.V., J.B.V., I.D.W., T.W.W., T.W., J.I.N., A. McQuillin, A.D.F. Genomic assays/data generation: A.J.F., M.I., E.A., M.A.E., D.A., M.B.-H., E.C.B., C.B.P., J.B.-G., M. Cairns, T.-K.C., C.C., J.C., F.S.D., F.D., S.D., A.F., J.F., N.B.F., J. Gelernter, M.G.P., P.H., S.J., Y.K., H.R.K., M. Kubo, S.E.L., C. Liao, E.M., N.W.M., J.D.M., Author Manuscript G.W.M., J.L.M., D.W.M., T.W.M., N.O’B., M. Rivera, C.S.-M., S. Sharp, C.S.H., C. Terao, C. Toma, E.-E.T., S.H.W., HUNT All-In Psychiatry, G. Breen, A.C., T. Esko, J.M.F., E.S.G., D.M.H., N.I., F.J.M., L. Milani, R.M.M., M.M.N., M. Ribasés, G.A.R., T.S., G.T., S. Cichon Obtained funding for BD samples: M.I., M. Cairns, I.N.F., L. Frisén, S.J., Y.K., J.A.K., M. Kubo, C. Lavebratt, S.L., D.M., P. McGuffin, G.W.M., J.B.P., M.H.R., J.R.D., D.J. Stein, J.S.S., C. Terao, A.H.Y., P.P.Z., M.A., L. Alfredsson, L.B., B.T.B., F.B., W.H.B., M. Boehnke, A.D.B., G. Breen, A.C., N.C., B.E., M.F., J.M.F., E.S.G., M.J. Green, M.G.-S., K.S.H., K. Hveem, N.I., I.J., L.A.J., M. Landén, M. Leboyer, N.G.M., F.J.M., P.B. Mitchell, O.M., P.B. Mortensen, B.M.N., M.N., M.M.N., M.C.O’D., T.O., M.J.O., C. Pato, M.T.P., G.P.P., M. Rietschel, G.A.R., T.S., M.S., P.R.S., T.G.S., C.S.W., J.W.S., G.T., J.B.V., T.W.W., T.W., J.M.B., J.I.N., H.J.E., R.A.O. Statistical analysis: N.M., K.S.O’C., B.C., J.R.I.C., Z.Q., T.D.A., T.B.B., S. Børte, J.B., A.W.C., O.K.D., M.J. Gandal, S.P.H., N.K., M. Kim, K.K., G.P., B.M.S., L.G.S., S. Steinberg, V.T., B.S.W., H.-H.W., V.A., S.A., S.E.B., B.M.B., A.M.D., A.L.D., V.E.-P., T.M.F., O.F., S.D.G., T.A.G., J. Grove, P.A.H., L.H., J.S.J., Y.K., M. Kubo, C. Lavebratt, M. Leber, P.H.L., S.H.M., A. Maihofer, M.M., S.A.M., S.E.M., L.M.O.L., A.F.P., T.R., P.R., D.M.R., O.B.S., C. Terao, T.E.T., J.T.R.W., W.X., J.M.K.Y., H.Y., P.P.Z., H.Z., A.D.B., G. Breen, E.S.G., F.S.G., Q.S.L., B.M.-M., C.M.N., D.P., S.R., H.S., P.F.S., T.W., N.R.W., J.M.B., E.A.S. Kevin S. O’Connell, Brandon Coombes, Jonathan R. I. Coleman, and Zhen Qiao contributed equally to this work and should be regarded as joint second authors. Data availability GWAS summary statistics are publicly available on the PGC website (https://www.med.unc.edu/pgc/results-and-downloads). Author Manuscript Individual-level data are accessible through collaborative analysis proposals to the Bipolar Disorder Working Group of the PGC (https://www.med.unc.edu/pgc/shared-methods/how-to/). This study included some publicly available datasets accessed through dbGaP (PGC bundle phs001254.v1.p1) and the Haplotype Reference Consortium reference panel v1.0 (http://www.haplotype- reference-consortium.org/home). Databases used: Drug-Gene Interaction Database DGIdb v.2 (https://www.dgidb.org); Psychoactive Drug Screening Database Ki DB (https://pdsp.unc.edu/databases/kidb.php); DrugBank 5.0 (www.drugbank.ca); LDHub (http:// ldsc.broadinstitute.org); FUMA (https://fuma.ctglab.nl). Code availability All software used is publicly available at the URLs or references cited. Consortia HUNT All-In Psychiatry Bendik S. Winsvold19,33, Eystein Stordal221,222, Gunnar Morken21,198, John-Anker Zwart18,19,33, Ole Kristian Drange21,22, Sigrid Børte17,18,19 A full list of consortium members and affiliations appears in the Supplementary Note. Competing interests Mullins et al. Page 2 Author Manuscript Genome-wide association study of over 40,000 bipolar disorder cases provides new insights into the underlying biology A full list of authors and affiliations appears at the end of the article. Abstract Bipolar disorder (BD) is a heritable mental illness with complex etiology. We performed a genome-wide association study (GWAS) of 41,917 BD cases and 371,549 controls of European ancestry, which identified 64 associated genomic loci. BD risk alleles were enriched in genes in synaptic signaling pathways and brain-expressed genes, particularly those with high specificity of expression in neurons of the prefrontal cortex and hippocampus. Significant signal enrichment was Author Manuscript found in genes encoding targets of antipsychotics, calcium channel blockers, antiepileptics, and anesthetics. Integrating eQTL data implicated 15 genes robustly linked to BD via gene expression, encoding druggable targets such as HTR6, MCHR1, DCLK3 and FURIN. Analyses of BD subtypes indicated high but imperfect genetic correlation between BD type I and II and identified additional associated loci. Together, these results advance our understanding of the biological etiology of BD, identify novel therapeutic leads, and prioritize genes for functional follow-up studies. T.E. Thorgeirsson, S. Steinberg, H. Stefansson and K. Stefansson are employed by deCODE Genetics/Amgen. Multiple additional Author Manuscript authors work for pharmaceutical or biotechnology companies in a manner directly analogous to academic co-authors and collaborators. A.H. Young has given paid lectures and advisory boards for the following companies with drugs used in affective and related disorders: Astrazenaca, Eli Lilly, Lundbeck, Sunovion, Servier, Livanova, Janssen, Allegan, Bionomics, Sumitomo Dainippon Pharma. A.H. Young was Lead Investigator for Embolden Study (Astra Zeneca), BCI Neuroplasticity study and Aripiprazole Mania Study. Also an investigator for Janssen, Lundbeck, Livanova, Compass. J. Nurnberger is an investigator for Janssen. P.F. Sullivan reports the following potentially competing financial interests: Lundbeck (advisory committee), Pfizer (Scientific Advisory Board member), and Roche (grant recipient, speaker reimbursement). G. Breen reports consultancy and speaker fees from Eli Lilly and Illumina and grant funding from Eli Lilly. M. Landén has received speaker fees from Lundbeck. O.A. Andreassen has received speaker fees from Lundbeck. J.Antoni Ramos-Quiroga was on the speakers bureau and/or acted as consultant for Eli-Lilly, Janssen-Cilag, Novartis, Shire, Lundbeck, Almirall, Braingaze, Sincrolab, and Rubió in the last 5 years. He also received travel awards (air tickets + hotel) for taking part in psychiatric meetings from Janssen-Cilag, Rubió, Shire, and Eli- Lilly. The Department of Psychiatry chaired by him received unrestricted educational and research support from the following companies in the last 5 years: Eli-Lilly, Lundbeck, Janssen- Cilag, Actelion, Shire, Ferrer, Oryzon, Roche, Psious, and Rubió. Dr. E. Vieta has received grants and served as consultant, advisor or CME speaker for the following entities: AB-Biotics, Abbott, Allergan, Angelini, AstraZeneca, Bristol-Myers Squibb, Dainippon Sumitomo Pharma, Farmindustria, Ferrer, Forest Research Institute, Gedeon Richter, Glaxo-Smith-Kline, Janssen, Lundbeck, Otsuka, Pfizer, Roche, SAGE, Sanofi-Aventis, Servier, Shire, Sunovion, Takeda, the Brain and Behaviour Foundation, the Catalan Government (AGAUR and PERIS), the Spanish Ministry of Science, Innovation, and Universities (AES and CIBERSAM), the Seventh European Author Manuscript Framework Programme and Horizon 2020, and the Stanley Medical Research Institute. T. Elvsåshagen has received speaker fees from Lundbeck. S. Kittel-Schneider received author’s and consultant honoraria from Medice Arzneimittel Pütter GmbH and Shire/Takeda. Prof. Serretti is or has been consultant/speaker for: Abbott, Abbvie, Angelini, Astra Zeneca, Clinical Data, Boheringer, Bristol Myers Squibb, Eli Lilly, GlaxoSmithKline, Innovapharma, Italfarmaco, Janssen, Lundbeck, Naurex, Pfizer, Polifarma, Sanofi, Servier. J.R. DePaulo has served as an unpaid consultant to Myriad – Neuroscience (formerly Assurex Health) in 2017 and 2019 and owns stock in CVS Health. H.R. Kranzler serves as an advisory board member for Dicerna Pharmaceuticals, is a member of the American Society of Clinical Psychopharmacology’s Alcohol Clinical Trials Initiative, which was sponsored in the past three years by AbbVie, Alkermes, Amygdala Neurosciences, Arbor Pharmaceuticals, Ethypharm, Indivior, Lilly, Lundbeck, Otsuka, and Pfizer. HRK is named as an inventor on PCT patent application #15/878,640 entitled: “Genotype-guided dosing of opioid agonists,” filed January 24, 2018. ALM and CL are employees of Indivior, Inc. AA was an employee of Indivior, Inc. at the time the work was performed. All other authors declare no financial interests or potential conflicts of interest. Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 3 Bipolar disorder (BD) is a complex mental disorder characterized by recurrent episodes of Author Manuscript (hypo)mania and depression. It is a common condition affecting an estimated 40 to 50 million people worldwide1. This, combined with the typical onset in young adulthood, an often chronic course, and increased risk of suicide2, make BD a major public health concern and a major cause of global disability1. Clinically, BD is classified into two main subtypes: bipolar I disorder, in which manic episodes typically alternate with depressive episodes, and bipolar II disorder, characterized by the occurrence of at least one hypomanic and one depressive episode3. These subtypes have a lifetime prevalence of ~1% each in the population4,5. Family and molecular genetic studies provide convincing evidence that BD is a multifactorial disorder, with genetic and environmental factors contributing to its development6. On the basis of twin and family studies, the heritability of BD is estimated at 60–85%7,8. Genome-wide association studies (GWAS)9–23 have led to valuable insights into Author Manuscript the genetic etiology of BD. The largest such study has been conducted by the Psychiatric Genomics Consortium (PGC), in which genome-wide SNP data from 29,764 BD patients and 169,118 controls were analyzed and 30 genome-wide significant loci were identified 2 (PGC2)24. SNP-based heritability (ℎSNP ) estimation using the same data suggested that common genetic variants genome-wide explain ~20% of BD’s phenotypic variance24. Polygenic risk scores generated from the results of this study explained ~4% of phenotypic variance in independent samples. Across the genome, genetic associations with BD converged on specific biological pathways including regulation of insulin secretion25,26, retrograde endocannabinoid signaling24, glutamate receptor signaling27 and calcium channel activity9. Despite this considerable progress, only a fraction of the genetic etiology of BD has been Author Manuscript identified, and the specific biological mechanisms underlying the development of the disorder are still unknown. In the present study, we report the results of the third GWAS meta-analysis of the PGC Bipolar Disorder Working Group, comprising 41,917 individuals with BD and 371,549 controls. These results confirm and expand on many previously reported findings, identify novel therapeutic leads, and prioritize genes for functional follow- up studies28,29. Thus, our results further illuminate the biological etiology of BD. Results GWAS results. We conducted a GWAS meta-analysis of 57 BD cohorts collected in Europe, North America and Australia (Supplementary Table 1), totaling 41,917 BD cases and 371,549 controls of Author Manuscript European descent (effective n = 101,962, see Online Methods). For 52 cohorts, individual- level genotype and phenotype data were shared with the PGC and cases met international consensus criteria (DSM-IV, ICD-9 or ICD-10) for lifetime BD, established using structured diagnostic interviews, clinician-administered checklists or medical record review. BD GWAS summary statistics were received for five external cohorts (iPSYCH30, deCODE genetics31, Estonian Biobank32, Trøndelag Health Study (HUNT)33 and UK Biobank34), in which most cases were ascertained using ICD codes. The GWAS meta-analysis identified 64 Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 4 independent loci associated with BD at genome-wide significance (P < 5 × 10−8) (Fig. 1, 2 Author Manuscript Table 1, and Supplementary Table 2). Using LD Score regression (LDSC)35, the ℎSNP of BD was estimated to be 18.6% (s.e. = 0.008, P = 5.1 × 10−132) on the liability scale, assuming a BD population prevalence of 2%, and 15.6% (s.e. = 0.006, P = 5.0 × 10−132) assuming a population prevalence of 1% (Supplementary Table 3). The genomic inflation factor (λGC) was 1.38 and the LD Score regression (LDSC) intercept was 1.04 (s.e. = 0.01, P = 2.5 × 10−4) (Supplementary Fig. 1). While the intercept has frequently been used as an indicator of confounding from population stratification, it can rise above 1 with increased sample size and heritability. The attenuation ratio—(LDSC intercept - 1)/(mean of association chi-square statistics - 1)—which is not subject to these limitations, was 0.06 (s.e. = 0.02), indicating that the majority of inflation of the GWAS test statistics was due to polygenicity35,36. Of the 64 genome-wide significant loci, 33 are novel discoveries (i.e. loci not overlapping with any locus previously reported as genome-wide significant for BD). Author Manuscript Novel loci include the major histocompatibility complex (MHC) and loci previously reaching genome-wide significance for other psychiatric disorders, including 10 for schizophrenia, 4 for major depression, and 3 for childhood-onset psychiatric disorders or problematic alcohol use (Table 1). Enrichment analyses. Genome-wide analyses using MAGMA37 indicated significant enrichment of BD associations in 161 genes (Supplementary Table 4) and 4 gene sets related to synaptic signaling (Supplementary Table 5). The BD association signal was enriched amongst genes expressed in different brain tissues (Supplementary Table 6), especially genes with high specificity of gene expression in neurons (both excitatory and inhibitory) versus other cell types, within cortical and subcortical brain regions in mice (Supplementary Fig. 2)38. In Author Manuscript human brain samples, signal enrichment was also observed in hippocampal pyramidal neurons and interneurons of the prefrontal cortex and hippocampus, compared with other cell types (Supplementary Fig. 2). In a gene-set analysis of the targets of individual drugs (from the Drug-Gene Interaction Database DGIdb v.239 and the Psychoactive Drug Screening Database Ki DB40), the targets of the calcium channel blockers mibefradil and nisoldipine were significantly enriched (Supplementary Table 7). Grouping drugs according to their Anatomical Therapeutic Chemical (ATC) classes41, there was significant enrichment in the targets of four broad drug classes (Supplementary Table 8): psycholeptics (drugs with a calming effect on behavior) (especially hypnotics and sedatives, antipsychotics and anxiolytics), calcium channel blockers, antiepileptics, and (general) anesthetics (Supplementary Table 8). Author Manuscript eQTL integrative analyses. We conducted a transcriptome-wide association study (TWAS) using FUSION42 and eQTL data from the PsychENCODE Consortium (1,321 brain samples)43. BD-associated alleles significantly influenced expression of 77 genes in the brain (Supplementary Table 9 and Supplementary Fig. 3). These genes encompassed 40 distinct regions. We performed TWAS fine-mapping using FOCUS44 to model the correlation among the TWAS signals and prioritize the most likely causal gene(s) in each region. Within the 90%-credible set, FOCUS Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 5 prioritized 22 genes with a posterior inclusion probability (PIP) > 0.9 (encompassing 20 Author Manuscript distinct regions) and 32 genes with a PIP > 0.7 (29 distinct regions) (Supplementary Table 10). We used summary data-based Mendelian randomization (SMR)45,46 to identify putative causal relationships between SNPs and BD via gene expression by integrating the BD GWAS results with brain eQTL summary statistics from the PsychENCODE43 Consortium and blood eQTL summary statistics from the eQTLGen Consortium (31,684 whole blood samples)47. The eQTLGen results represent the largest existing eQTL study and provide independent eQTL data. Of the 32 genes fine-mapped with PIP > 0.7, 15 were significantly associated with BD in the SMR analyses and passed the HEIDI (heterogeneity in dependent instruments) test45,46, suggesting that their effect on BD is mediated via gene expression in the brain and/or blood (Supplementary Table 11). The genes located in genome-wide significant loci are labeled in Figure 1. Other significant genes included HTR6, DCLK3, Author Manuscript HAPLN4 and PACSIN2. MHC locus. Variants within and distal to the major histocompatibility complex (MHC) locus were associated with BD at genome-wide significance. The most highly associated SNP was rs13195402, 3.2 Mb distal to any HLA gene or the complement component 4 (C4) genes (Supplementary Fig. 4). Imputation of C4 alleles using SNP data uncovered no association between the five most common structural forms of the C4A/C4B locus (BS, AL, AL-BS, AL-BL, and AL-AL) and BD, either before or after conditioning on rs13195402 (Supplementary Fig. 5). While genetically predicted C4A expression initially showed a weak association with BD, this association was non-significant after controlling for rs13195402 (Supplementary Fig. 6). Author Manuscript Polygenic risk scoring. The performance of polygenic risk scores (PRS) based on these GWAS results was assessed by excluding cohorts in turn from the meta-analysis to create independent test samples. PRS explained ~4.57% of phenotypic variance in BD on the liability scale (at GWAS P-value threshold (PT) < 0.1, BD population prevalence 2%), based on the weighted mean R2 across cohorts (Fig. 2 and Supplementary Table 12). This corresponds to a weighted mean area under the curve (AUC) of 65%. Results per cohort and per wave of recruitment to the PGC are in Supplementary Tables 12 and 13 and Supplementary Figure 7. At PT < 0.1, individuals in the top 10% of BD PRS had an odds ratio of 3.5 (95% CI 1.7–7.3) of being affected with the disorder compared with individuals in the middle decile (based on the weighted mean OR across PGC cohorts), and an odds ratio of 9.3 (95% CI 1.7–49.3) Author Manuscript compared with individuals in the lowest decile. The generalizability of PRS from this meta- analysis was examined in several non-European cohorts. PRS explained up to 2.3% and 1.9% of variance in BD in two East Asian samples, and 1.2% and 0.4% in two admixed African American samples (Fig. 2 and Supplementary Table 14). The variance explained by the PRS increased in every cohort with increasing sample size of the PGC BD European discovery sample (Supplementary Fig. 8 and Supplementary Table 14). Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 6 Genetic architecture of BD and other traits. Author Manuscript The genome-wide genetic correlation (rg) of BD with a range of diseases and traits was assessed on LD Hub48. After correction for multiple testing, BD showed significant rg with 16 traits among 255 tested from published GWAS (Supplementary Table 15). Genetic correlation was positive with all psychiatric disorders assessed, particularly schizophrenia (rg = 0.68) and major depression (rg = 0.44), and to a lesser degree anorexia, attention deficit/hyperactivity disorder, and autism spectrum disorder (rg ≈ 0.2). We found evidence of positive rg between BD and smoking initiation, cigarettes per day, problematic alcohol use, and drinks per week (Fig. 3). BD was also positively genetically correlated with measures of sleep quality (daytime sleepiness, insomnia, sleep duration) (Fig. 3). Among 514 traits measured in the general population of the UK Biobank, there was significant rg between BD and many psychiatric-relevant traits or symptoms, dissatisfaction with interpersonal relationships, poorer overall health rating, and feelings of loneliness or isolation Author Manuscript (Supplementary Table 16). Bivariate gaussian mixture models were applied to the GWAS summary statistics for BD and other complex traits using the MiXeR tool49,50 to estimate the number of variants 2 influencing each trait that explain 90% of ℎSNP and their overlap between traits. MiXeR estimated that approximately 8.6 k (s.e. = 0.2 k) variants influence BD, which is similar to the estimate for schizophrenia (9.7 k, s.e. = 0.2 k) and somewhat lower than that for major depression (12.3 k, s.e. = 0.6 k) (Supplementary Table 17 and Supplementary Fig. 9). When considering the number of shared loci as a proportion of the total polygenicity of each trait, the vast majority of loci influencing BD were also estimated to influence major depression (97%) and schizophrenia (96%) (Supplementary Table 17 and Supplementary Fig. 9). Interestingly, within these shared components, the variants that influenced both BD and Author Manuscript schizophrenia had high concordance in direction of effect (80%, s.e. = 2%), while the portion of concordant variants between BD and MDD was only 69% (s.e. = 1%) (Supplementary Table 17). Genetic and causal relationships between BD and modifiable risk factors. Ten traits associated with BD from clinical and epidemiological studies were investigated in detail for genetic and potentially causal relationships with BD via LDSC35, generalized summary statistics-based Mendelian randomization (GSMR)51, and bivariate gaussian mixture modeling49. BD has been strongly linked with sleep disturbances52, alcohol use53 and smoking54, higher educational attainment55,56, and mood instability57. Most of these traits had modest but significant genetic correlations with BD (rg = −0.05–0.35) (Fig. 3). Examining the effects of these traits on BD via GSMR, smoking initiation was associated Author Manuscript with BD, corresponding to an OR of 1.49 (95% CI 1.38–1.61) for developing the disorder (P = 1.74 × 10−22) (Fig. 3). Testing the effect of BD on the traits, BD was significantly associated with reduced likelihood of being a morning person and increased number of drinks per week (P < 1.47 × 10−3) (Fig. 3). Positive bi-directional relationships were identified between BD and longer sleep duration, problematic alcohol use, educational attainment (EA), and mood instability (Fig. 3). Notably, the instrumental variables for mood instability were selected from a GWAS conducted in the general population, excluding Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 7 individuals with psychiatric disorders58. For all of the aforementioned BD-trait Author Manuscript relationships, the effect size estimates from GSMR were consistent with those calculated using the inverse variance weighted regression method, and there was no evidence of bias from horizontal pleiotropy. Full MR results are in Supplementary Tables 18 and 19. Bivariate gaussian mixture modeling using MiXeR indicated large proportions of variants influencing both BD and all other traits tested, particularly educational attainment, where approximately 98% of variants influencing BD were estimated to also influence EA. While cigarettes per day was a trait of interest, MiXeR could not model these data due to low polygenicity and heritability, and the effect of cigarettes per day on BD was inconsistent between MR methods, suggesting a violation of MR assumptions (Supplementary Tables 18–20). BD subtypes. Author Manuscript We conducted GWAS meta-analyses of bipolar I disorder (BD I) (25,060 cases, 449,978 controls) and bipolar II disorder (BD II) (6,781 cases, 364,075 controls). The BD I analysis identified 44 genome-wide significant loci, 31 of which overlapped with genome-wide significant loci from the main BD GWAS (Table 1 and Supplementary Table 21). The remaining 13 genome-wide significant loci for BD I all had P < 4.0 × 10−5 in the main BD GWAS. One genome-wide significant locus was identified in the GWAS meta-analysis of BD II and had a P < 1.1 × 10−4 in the main GWAS of BD (Supplementary Table 21). The 2 ℎSNP estimates on the liability scale for BD I and BD II were 20.9% (s.e. = 0.009, P = 1.0 × 10−111) and 11.6% (s.e. = 0.01, P = 3.9 × 10−15), respectively, assuming a 1% population prevalence of each subtype. These heritability values are significantly different from each other (P = 2.4 × 10−25, block jackknife). The genetic correlation between BD I and BD II was 0.85 (s.e. = 0.05, P = 2.88 × 10−54), which is significantly different from 1 (P = 1.6 × Author Manuscript 10−3). The genetic correlation of BD I with schizophrenia (rg = 0.66, s.e. = 0.02) was higher than that of BD II (rg = 0.54, s.e. = 0.05), whereas major depression was more strongly genetically correlated with BD II (rg = 0.66, s.e. = 0.05) than with BD I (rg = 0.34, s.e. = 0.03) (Supplementary Table 22). Discussion In a GWAS of 41,917 BD cases, we identified 64 associated genomic loci, 33 of which are novel discoveries. With a 1.5-fold increase in effective sample size compared with the PGC2 BD GWAS, this study more than doubled the number of associated loci, representing an inflection point in the rate of risk variant discovery. We observed consistent replication of known BD loci, including 28/30 loci from the PGC2 GWAS24 and several implicated by other BD GWAS15,16,17, including a study of East Asian cases59. Author Manuscript The 33 novel loci discovered here encompass genes of expected biological relevance to BD, such as the ion channels CACNB2 and KCNB1. Amongst the 64 BD loci, 17 have previously been implicated in GWAS of schizophrenia60, and seven in GWAS of major depression61, representing the first overlap of genome-wide significant loci between the mood disorders. For these genome-wide significant loci shared across disorders, 17/17 and 5/7 of the BD index SNPs had the same direction of effect on schizophrenia and major Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 8 depression, respectively (Supplementary Table 23). More generally, 50/64 and 62/64 BD loci Author Manuscript had a consistent direction of effect on major depression and schizophrenia, respectively, considerably greater than chance (P < 1 × 10−5, binomial test). Bivariate gaussian mixture modeling estimated that across the entire genome, almost all variants influencing BD also influence schizophrenia and major depression, albeit with variable effects62. SNPs in and around the MHC locus reached genome-wide significance for BD for the first time. However, unlike in schizophrenia, we found no influence of C4 structural alleles or gene expression63. Rather the association was driven by variation outside the classical MHC locus, with the index SNP (rs13195402) being a missense variant in BTN2A1, a brain- expressed gene64 encoding a plasma membrane protein. The genetic correlation of BD with other psychiatric disorders was consistent with previous reports65,66. Our results also corroborate previous genetic and clinical evidence of associations between BD and sleep disturbances67, problematic alcohol use68, and Author Manuscript smoking69. While the genome-wide genetic correlations with these traits were modest (rg = −0.05–0.35), MiXeR estimated that, for all traits, more than 55% of trait-influencing variants also influence BD (Fig. 3). Taken together, these results point to shared biology as one possible explanation for the high prevalence of substance use in BD. However, excluding genetic variants associated with both traits, MR analyses suggested that smoking is also a putatively “causal” risk factor for BD, while BD has no effect on smoking, consistent with a previous report70. (We use the word “causal” with caution here as we consider MR an exploratory analysis to identify potentially modifiable risk factors that warrant more detailed investigations to understand their complex relationship with BD.) In contrast, MR indicated that BD had bi-directional “causal” relationships with problematic alcohol use, longer sleep duration, and mood instability. Insights into the relationship of such behavioral correlates with BD may have future impact on clinical decision making in Author Manuscript the prophylaxis or management of the disorder. Higher educational attainment has previously been associated with BD in epidemiological studies55,56, while lower educational attainment has been associated with schizophrenia and major depression71,72. Here, educational attainment had a significant positive effect on risk of BD and vice versa. Interestingly, MiXeR estimated that almost all variants that influence BD also influence educational attainment. The substantial genetic overlap observed between BD and the other phenotypes suggests that many variants likely influence multiple phenotypes, which may be differentiated by phenotype-specific effect size distributions among the shared influencing variants. The integration of eQTL data with our GWAS results yielded 15 high-confidence genes for which there was converging evidence that their association with BD is mediated via gene Author Manuscript expression. Amongst these were HTR6, encoding a serotonin receptor targeted by antipsychotics and antidepressants73, and MCHR1 (melanin-concentrating hormone receptor 1), encoding a target of the antipsychotic haloperidol73. We note that, for both of these genes, their top eQTLs have opposite directions of effect on gene expression in the brain and blood, possibly playing a role in the tissue-specific gene regulation influencing BD74. BD was associated with decreased expression of FURIN, a gene with a neurodevelopmental role that has already been the subject of functional genomics experiments in neuronal cells following its association with schizophrenia in GWAS75. The top association in our GWAS Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 9 was in the TRANK1 locus on chromosome 3, which has previously been implicated in BD12,18,59. Although BD-associated SNPs in this locus are known to regulate TRANK1 Author Manuscript expression76, our eQTL analyses support a stronger but correlated regulation of DCLK3, located 87 kb upstream of TRANK143,77. Both FURIN and DCLK3 also encode druggable proteins (although they are not targets for any current psychiatric medications)73,78. These eQTL results provide promising BD candidate genes for functional follow-up experiments29. While several of these are in genome-wide significant loci, many are not the closest gene to the index SNP, highlighting the value of probing underlying molecular mechanisms to prioritize the most likely causal genes in the loci. GWAS signals were enriched in the gene targets of existing BD pharmacological agents, such as antipsychotics, mood stabilizers, and antiepileptics. However, enrichment was also found in the targets of calcium channel blockers used to treat hypertension and GABA- receptor targeting anesthetics (Supplementary Table 8). Calcium channel antagonists have Author Manuscript long been investigated for the treatment of BD, without becoming an established therapeutic approach, and there is evidence that some antiepileptics have calcium channel-inhibiting effects79,80. These results underscore the opportunity for repurposing some classes of drugs, particularly calcium channel antagonists, as potential BD treatments81. BD associations were enriched in gene sets involving neuronal parts and synaptic signaling. Neuronal and synaptic pathways have been described in cross-disorder GWAS of multiple psychiatric disorders including BD82–84. Dysregulation of such pathways has also been suggested by previous functional and animal studies85. Analysis of single-cell gene expression data revealed enrichment in genes with high specificity of gene expression in neurons (both excitatory and inhibitory) of many brain regions, in particular the cortex and hippocampus. These findings are similar to those reported in GWAS data of schizophrenia86 Author Manuscript and major depressive disorder38. PRS for BD explained on average 4.57% of phenotypic variance (liability scale) across European cohorts, although this varied in different waves of the BD GWAS, ranging from 6.6% in the PGC1 cohorts to 2.9% in the External biobank studies (Supplementary Fig. 7 2 and Supplementary Table 12). These results are in line with the ℎSNP of BD per wave, which ranged from 24.6% (s.e. =0.01) in PGC1 to 11.9% (s.e. = 0.01) in External studies 2 (Supplementary Table 3). Some variability in ℎSNP estimates may arise from the inclusion of cases from population biobanks, who may have more heterogeneous clinical presentations or less severe illness than BD patients ascertained via inpatient or outpatient psychiatric 2 clinics. Across the waves of clinically ascertained samples within the PGC, ℎSNP and the R2 of PRS also varied, likely reflecting clinical and genetic heterogeneity in the type of BD Author Manuscript cases ascertained; the PGC1 cohorts consisted mostly of BD I cases9, known to be the most heritable of the BD subtypes11,24, while later waves included more individuals with BD II24. 2 Overall, the ℎSNP of BD calculated from the meta-analysis summary statistics was 18% on the liability scale, a decrease of ~2% compared with the PGC2 GWAS24, which may be due 2 to the addition of cohorts with lower ℎSNP estimates and heterogeneity between cohorts 2 (Supplementary Table 3). However, despite differences in ℎSNP and R2 of PRS per wave, Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 10 the genetic correlation of BD between all waves was high (weighted mean rg = 0.94, s.e. = Author Manuscript 0.03), supporting our rationale for combining cases with different BD subtypes or ascertainment to increase power for discovery of risk variants. In Europeans, individuals in the top 10% of PRS had an OR of 3.5 for BD, compared with individuals with average PRS (middle decile), which translates into a modest absolute lifetime risk of the disorder (7% based on PRS alone). While PRS are invaluable tools in research settings, the current BD PRS lack sufficient power to separate individuals into clinically meaningful risk categories, and therefore have no clinical utility at present87,88. PRS from this European BD meta- analysis yield higher R2 values in diverse ancestry samples than PRS based on any currently available BD GWAS within the same ancestry59. However, performance still greatly lags behind that in Europeans, with ~2% variance explained in East Asian samples and substantially less in admixed African American samples, likely due to differences in allele frequencies and LD structures, consistent with previous studies89,90. There is a pressing Author Manuscript need for more and larger studies in other ancestry groups to ensure that any future clinical utility is broadly applicable. Exploiting the differences in LD structure between diverse ancestry samples will also assist in the fine-mapping of risk loci for BD. Our analyses confirmed that BD is a highly polygenic disorder, with an estimated 8.6 k 2 variants explaining 90% of its ℎSNP . Hence, many more SNPs than those identified here are expected to account for the common variant architecture underlying BD. This GWAS marks an inflection point in risk variant discovery, and we expect that, from this point forward, the addition of more samples will lead to a dramatic increase in genetic findings. Nevertheless, fewer genome-wide significant loci have been identified in BD than in a schizophrenia GWAS of comparable sample size60. This may be due to the clinical and genetic heterogeneity that exists in BD. Author Manuscript Our GWAS of subtypes BD I and BD II identified additional associated loci. Consistent with previous findings24, our analysis showed that the two subtypes were highly but imperfectly genetically correlated (rg = 0.85), and that BD I is more genetically correlated with schizophrenia, while BD II has stronger genetic correlation with major depression. The subtypes are sufficiently similar to justify joint analysis as BD, but are not identical in their genetic composition, and as such contribute to the genetic heterogeneity of BD91. We identified 13 loci passing genome-wide significance for BD I, and one for BD II, which did not reach significance in the main BD GWAS, further illustrating the partially differing genetic composition of the two subtypes. Understanding the shared and distinct genetic components of BD subtypes and symptoms requires detailed phenotyping efforts in large cohorts and is an important area for future psychiatric genetics research. Author Manuscript In summary, these new data advance our understanding of the biological etiology of BD and prioritize a set of candidate genes for functional follow-up experiments. Several lines of evidence converge on the involvement of calcium channel signaling, providing a promising avenue for future therapeutic development. Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 11 Methods Author Manuscript Sample description. The meta-analysis sample comprises 57 cohorts collected in Europe, North America and Australia, totaling 41,917 BD cases and 371,549 controls of European descent (Supplementary Table 1). The total effective n, equivalent to an equal number of cases and controls in each cohort (4*ncases*ncontrols/(ncases + ncontrols)), is 101,962. For 52 cohorts, individual-level genotype and phenotype data were shared with the PGC. Cohorts have been added to the PGC in five waves (PGC19, PGC224, PGC PsychChip, PGC3 and External Studies); all cohorts from previous PGC BD GWAS were included. The source and inclusion/exclusion criteria for cases and controls for each cohort are described in the Supplementary Note. Cases were required to meet international consensus criteria (DSM-IV, ICD-9, or ICD-10) for a lifetime diagnosis of BD, established using structured diagnostic Author Manuscript instruments from assessments by trained interviewers, clinician-administered checklists, or medical record review. In most cohorts, controls were screened for the absence of lifetime psychiatric disorders and randomly selected from the population. For five cohorts (iPSYCH30, deCODE genetics31, Estonian Biobank32, Trøndelag Health Study (HUNT)33 and UK Biobank34), GWAS summary statistics for BD were shared with the PGC. In these cohorts, BD cases were ascertained using ICD codes or self-report during a nurse interview, and the majority of controls were screened for the absence of psychiatric disorders via ICD codes. Follow-up analyses included four non-European BD case-control cohorts, two from East Asia (Japan59 and Korea92), and two admixed African American cohorts22,93, providing a total of 5,847 cases and 65,588 controls. These BD cases were ascertained using international consensus criteria (DSM-IV)22,93 through psychiatric interviews (Supplementary Note). Author Manuscript Genotyping, quality control and imputation. For 52 cohorts internal to the PGC, genotyping was performed following local protocols and genotypes were called using standard genotype calling softwares from commercial sources (Affymetrix and Illumina). Subsequently, standardized quality control, imputation and statistical analyses were performed centrally using RICOPILI (Rapid Imputation for COnsortias PIpeLIne) (version 2018_Nov_23.001)94, separately for each cohort. Briefly, the quality control parameters for retaining SNPs and subjects were: SNP missingness < 0.05 (before sample removal), subject missingness < 0.02, autosomal heterozygosity deviation (Fhet < 0.2), SNP missingness < 0.02 (after sample removal), difference in SNP missingness between cases and controls < 0.02, SNP Hardy-Weinberg equilibrium (P > 10 × 10−10 in psychiatric cases and P > 10 × 10−6 in controls). Relatedness was calculated across cohorts Author Manuscript using identity by descent and one of each pair of related individuals (pi_hat > 0.2) was excluded. Principal components (PCs) were generated using genotyped SNPs in each cohort separately using EIGENSTRAT v6.1.495. Based on visual inspection of plots of PCs for each dataset (which were all of European descent according to self-report/clinical data), we excluded samples to obtain more clearly homogeneous datasets. Genotype imputation was performed using the pre-phasing/ imputation stepwise approach implemented in Eagle v2.3.596 and Minimac397 to the Haplotype Reference Consortium (HRC) reference panel v1.098. Data on the X chromosome were available for 50 cohorts internal to the PGC and Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 12 one external cohort (HUNT), and the X chromosome was imputed to the HRC reference Author Manuscript panel in males and females separately within each cohort. The five external cohorts were processed by the collaborating research teams using comparable procedures and imputed to the HRC or a custom reference panel as appropriate. Full details of the genotyping, quality control and imputation for each of these cohorts are available in the Supplementary Note. Identical individuals between PGC cohorts and the Estonian Biobank and UK Biobank cohorts were detected using genotype-based checksums (https://personal.broadinstitute.org/ sripke/share_links/zpXkV8INxUg9bayDpLToG4g58TMtjN_PGC_SCZ_w3.0718d.76) and removed from PGC cohorts. Genome-wide association study. For PGC cohorts, GWAS were conducted within each cohort using an additive logistic regression model in PLINK v1.9099, covarying for PCs 1–5 and any others as required. Author Manuscript Association analyses of the X chromosome were conducted in males and females separately using the same procedures, with males coded as 0 or 2 for 0 or 1 copies of the reference allele. Results from males and females were then meta-analyzed within each cohort. For external cohorts, GWAS were conducted by the collaborating research teams using comparable procedures (Supplementary Note). To control test statistic inflation at SNPs with low minor allele frequency (MAF) in small cohorts, SNPs were retained only if cohort MAF was > 1% and minor allele count was > 10 in either cases or controls (whichever had smaller n). There was no evidence of stratification artifacts or uncontrolled inflation of test statistics in the results from any cohort (λGC = 0.97–1.05) (Supplementary Table 1). Meta-analysis of GWAS summary statistics was conducted using an inverse variance-weighted fixed effects model in METAL (version 2011-03-25)100 across 57 cohorts for the autosomes (41,917 BD cases and 371,549 controls) and 51 cohorts for the X chromosome (35,691 BD cases and Author Manuscript 96,731 controls). A genome-wide significant locus was defined as the region around a SNP with P < 5 × 10−8, with linkage disequilibrium (LD) r2 > 0.1, within a 3,000 kilobase (kb) window. Regional association plots and forest plots of the index SNP for all genome-wide significant loci are presented in Supplementary Data 1 and 2, respectively. Overlap of loci with other psychiatric disorders. Genome-wide significant loci for BD were assessed for overlap with genome-wide significant loci for other psychiatric disorders, using the largest available GWAS results for major depression61, schizophrenia60, attention deficit/hyperactivity disorder101, post- traumatic stress disorder102, lifetime anxiety disorder103, Tourette’s Syndrome104, anorexia nervosa105, alcohol use disorder or problematic alcohol use68, autism spectrum disorder106, mood disorders91 and the cross-disorder GWAS of the Psychiatric Genomics Consortium66. Author Manuscript The boundaries of the genome-wide significant loci were calculated in the original publications. Overlap of loci was calculated using bedtools v2.29.2107. Enrichment analyses. P values quantifying the degree of association of genes and gene sets with BD were calculated using MAGMA v1.0837, implemented in FUMA v1.3.6a64,108. Gene-based tests were performed for 19,576 genes (Bonferroni-corrected P-value threshold = 2.55 × 10−6). A total of 11,858 curated gene sets including at least 10 genes from MSigDB V7.0 were tested Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 13 for association with BD (Bonferroni-corrected P-value threshold = 4.22 × 10−6). Author Manuscript Competitive gene-set tests were conducted correcting for gene size, variant density and LD within and between genes. Tissue-set enrichment analyses were also performed using MAGMA implemented in FUMA, to test for enrichment of association signal in genes expressed in 54 tissue types from GTEx V8 (Bonferroni-corrected P-value threshold = 9.26 × 10−4)64,108. For single-cell enrichment analyses, publicly available single-cell RNA-seq data were compiled from five studies of the adult human and mouse brain86,109–112. The mean expression for each gene in each cell type was computed from the single-cell expression data (if not provided). For the Zeisel dataset109, we used the mean expression at level 4 (39 cell types from 19 regions for the mouse nervous system). For the Saunders dataset110, we computed the mean expression of the different classes in each of the 9 different brain regions sampled (88 cell types in total). We filtered out any genes with non-unique names, genes not Author Manuscript expressed in any cell types, non-protein coding genes, and, for mouse datasets, genes that had no expert curated 1:1 orthologs between mouse and human (Mouse Genome Informatics, The Jackson Laboratory, version 11/22/2016, http://www.informatics.jax.org/ downloads/reports/index.html#homology), resulting in 16,472 genes. Gene expression was then scaled to a total of 1 million UMIs (unique molecular identifiers) (or transcript per million (TPM)) for each cell type/tissue. Using a previously described method38, a metric of gene expression specificity was calculated by dividing the expression of each gene in each cell type by the total expression of that gene in all cell types, leading to values ranging from 0 to 1 for each gene (0 meaning that the gene is not expressed in that cell type and 1 meaning that all of the expression of the gene is in that cell type). We then selected the top 10% most specific genes for each cell type/tissue for enrichment analysis. MAGMA v1.0837 was used to test gene-set enrichment using GWAS summary statistics, covarying for gene Author Manuscript size, gene density, mean sample size for tested SNPs per gene, the inverse of the minor allele counts per gene and the log of these metrics. We excluded any SNPs with INFO score < 0.6, with MAF < 1% or with estimated odds ratio > 25 or smaller than 1/25, as well as SNPs located in the MHC region (chr6:25–34 Mb). We set a window of 35 kb upstream to 10 kb downstream of the gene coordinates to compute gene-level association statistics and used the European reference panel from the phase 3 of the 1000 Genomes Project as the reference population113. We then used MAGMA to test whether the 10% most specific genes (with an expression of at least 1 TPM or 1 UMI per million) for each cell type/tissue were associated with BD. The P-value threshold for significance was P < 9.1 × 10−3, representing a 5% false discovery rate (FDR) across datasets. Further gene-set analyses were performed restricted to genes targeted by drugs, assessing Author Manuscript individual drugs and grouping drugs with similar actions. This approach has been described previously41. Gene-level and gene-set analyses were performed in MAGMA v1.0837. Gene boundaries were defined using build 37 reference data from the NCBI, available on the MAGMA website (https://ctg.cncr.nl/software/magma), extended 35 kb upstream and 10 kb downstream to include regulatory regions outside of the transcribed region. Gene-level association statistics were defined as the aggregate of the mean and the lowest variant-level P value within the gene boundary, converted to a Z-value. Gene sets were defined comprising the targets of each drug in the Drug-Gene Interaction database DGIdb v.239 and Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 14 in the Psychoactive Drug Screening Database Ki DB40, both downloaded in June 201641. Author Manuscript Analyses were performed using competitive gene-set analyses in MAGMA. Results from the drug-set analysis were then grouped according to the Anatomical Therapeutic Chemical class of the drug41. Only drug classes with at least 10 valid drug gene sets within them were analyzed. Drug-class analysis was performed using enrichment curves. All drug gene sets were ranked by their association in the drug set analysis, and then for a given drug class an enrichment curve was drawn scoring a “hit” if the drug gene set was within the class, or a “miss” if it was outside of the class. The area under the curve was calculated, and a P-value for this calculated as the Wilcoxon Mann-Whitney test comparing drug gene sets within the class to drug gene sets outside of the class41. Multiple testing was controlled using a Bonferroni-corrected significance threshold of P < 5.60 × 10−5 for drug-set analysis and P < 7.93 × 10−4 for drug-class analysis, accounting for 893 drug-sets and 63 drug classes tested. eQTL integrative analysis. Author Manuscript A transcriptome-wide association study (TWAS) was conducted using the precomputed gene expression weights from PsychENCODE data (1,321 brain samples)43, available online with the FUSION software42. For genes with significant cis-SNP heritability (13,435 genes), FUSION software (vOct 1, 2019) was used to test whether SNPs influencing gene expression are also associated with BD (Bonferroni-corrected P-value threshold < 3.72 × 10−6). For regions including a TWAS significant gene, TWAS fine-mapping of the region was conducted using FOCUS (fine-mapping of causal gene sets, v0.6.10)44. Regions were defined using the correlation matrix of predicted effects on gene expression around TWAS significant genes44. A posterior inclusion probability (PIP) was assigned to each gene for being causal for the observed TWAS association signal. Based on the PIP of each gene and a null model, whereby no gene in the region is causal for the TWAS signal, the 90%-credible gene set for each region was computed44. Author Manuscript Summary data-based Mendelian randomization (SMR) (v1.03)45,46 was applied to further investigate putative causal relationships between SNPs and BD via gene expression. SMR was performed using eQTL summary statistics from the eQTLGen (31,684 blood samples)47 and PsychENCODE43 consortia. SMR analysis is limited to transcripts with at least one significant cis-eQTL (P < 5 × 10−8) in each dataset (15,610 in eQTLGen; 10,871 in PsychENCODE). The Bonferroni-corrected significance threshold was P < 3.20 × 10−6 and P < 4.60 × 10−6 for eQTLGen and PsychENCODE, respectively. The significance threshold for the HEIDI test (heterogeneity in dependent instruments) was PHEIDI ≥ 0.0146. While the results of TWAS and SMR indicate an association between BD and gene expression, a non- significant HEIDI test additionally indicates either a direct causal role or a pleiotropic effect of the BD-associated SNPs on gene expression. Author Manuscript Complement component 4 (C4) imputation. To investigate the major histocompatibility complex (MHC; chr6:24–34 Mb on hg19), the alleles of complement component 4 genes (C4A and C4B) were imputed in 47 PGC cohorts for which individual-level genotype data were accessible, totaling 32,749 BD cases and 53,370 controls. The imputation reference panel comprised 2,530 reference haplotypes of MHC SNPs and C4 alleles, generated using a sample of 1,265 individuals with whole- Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 15 genome sequence data, from the Genomic Psychiatry cohort114. Briefly, imputation of C4 as a multi-allelic variant was performed using Beagle v4.1115,116, using SNPs from the MHC Author Manuscript region that were also in the haplotype reference panel. Within the Beagle pipeline, the reference panel was first converted to bref format. We used the conform-gt tool to perform strand-flipping and filtering of specific SNPs for which strand remained ambiguous. Beagle was run using default parameters with two key exceptions: we used the GRCh37 PLINK recombination map, and we set the output to include genotype probability (i.e., GP field in VCF) for correct downstream probabilistic estimation of C4A and C4B joint dosages. The output consisted of dosage estimates for each of the common C4 structural haplotypes for each individual. The five most common structural forms of the C4A/C4B locus (BS, AL, AL-BS, AL-BL, and AL-AL) could be inferred with reasonably high accuracy (generally 0.70 < r2 < 1.00). The imputed C4 alleles were tested for association with BD in a joint logistic regression that included (i) terms for dosages of the five most common C4 structural Author Manuscript haplotypes (AL-BS, AL-BL, AL-AL, BS, and AL), (ii) rs13195402 genotype (top lead SNP in the MHC) and (iii) PCs as per the GWAS. The genetically regulated expression of C4A was predicted from the imputed C4 alleles using a model previously described63. Predicted C4A expression was tested for association with BD in a joint logistic regression that included (i) predicted C4A expression, (ii) rs13195402 genotype (top lead SNP in the MHC) and (iii) PCs as per the GWAS. Polygenic risk scoring. PRS from our GWAS meta-analysis were tested for association with BD in individual cohorts, using a discovery GWAS where the target cohort was left out of the meta-analysis. Briefly, the GWAS results from each discovery GWAS were pruned for LD using the P- value informed clumping method in PLINK v1.9099 (r2 0.1 within a 500-kb window) based on the LD structure of the HRC reference panel98. Subsets of SNPs were selected from the Author Manuscript results below nine increasingly liberal P-value thresholds (PT) (5 × 10−8, 1 × 10−4, 1 × 10−3, 0.01, 0.05, 0.1, 0.2, 0.5, 1). Sets of alleles, weighted by their log odds ratios from the discovery GWAS, were summed into PRS for each individual in the target datasets, using PLINK v1.90 implemented via RICOPILI94,99. PRS were tested for association with BD in the target dataset using logistic regression, covarying for PCs as per the GWAS in each cohort. PRS were tested in the external cohorts by the collaborating research teams using comparable procedures. The variance explained by the PRS (R2) was converted to the liability scale to account for the proportion of cases in each target dataset, using a BD population prevalence of 2% and 1%117. The weighted average R2 values were calculated using the effective n for each cohort. The odds ratios for BD for individuals in the top decile of PRS compared with those in the lowest decile and middle decile were calculated in the 52 Author Manuscript datasets internal to the PGC. To assess cross-ancestry performance, PRS generated from the meta-analysis results were tested for association with BD using similar methods in a Japanese sample59, a Korean sample92, and two admixed African American samples. Full details of the QC, imputation, and analysis of these samples are in the Supplementary Note. Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 16 LD score regression. Author Manuscript 2 LD Score regression (LDSC)35 was used to estimate the ℎSNP of BD from GWAS summary 2 statistics. ℎSNP was converted to the liability scale, using a lifetime BD prevalence of 2% and 1%. LDSC bivariate genetic correlations attributable to genome-wide SNPs (rg) were estimated with 255 human diseases and traits from published GWAS and 514 GWAS of phenotypes in the UK Biobank from LD Hub48. Adjusting for the number of traits tested, the Bonferroni-corrected P-value thresholds were P < 1.96 × 10−4 and P < 9.73 × 10−5, respectively. MiXeR. We applied causal mixture models49,118 to the GWAS summary statistics, using MiXeR v1.3. MiXeR provides univariate estimates of the proportion of non-null SNPs Author Manuscript (“polygenicity”) and the variance of effect sizes of non-null SNPs (“discoverability”) in each phenotype. For each SNP, i, univariate MiXeR models its additive genetic effect of allele substitution, βi, as a point-normal mixture, βi = 1 − π1 N 0, 0 + π1N 0, σβ2 , where π1 represents the proportion of non-null SNPs (`polygenicity`) and σβ2 represents variance of effect sizes of non-null SNPs (`discoverability`). Then, for each SNP, j, MiXeR incorporates LD information and allele frequencies for M = 9,997,231 SNPs extracted from 1000 Genomes Phase 3 data to estimate the expected probability distribution of the signed test statistic, zj = δj + ϵj = N ∑i Hirijβi + ϵj, where N is sample size, Hi indicates heterozygosity of i-th SNP, rij indicates allelic correlation between i-th and j-th SNPs, and ϵj N 0, σ02 is the residual variance. Further, the three parameters, π1, σβ2, σ02, are fitted by direct maximization of the likelihood function. The optimization is based on a set of approximately Author Manuscript 600,000 SNPs, obtained by selecting a random set of 2,000,000 SNPs with minor allele frequency of 5% or higher, followed by LD pruning procedure at LD r2 = 0.8 threshold. The random SNP selection and full optimization procedure are repeated 20 times to obtain mean and standard errors of model parameters. The log-likelihood figures show individual curves for each of the 20 runs, each shifted vertically so that best log-likelihood point is shown at zero ordinate. The total number of trait influencing variants is estimated as Mπ1, where M = 9,997,231 gives the number of SNPs in the reference panel. MiXeR Venn diagrams report the effective number of influencing variants, ηMπ_1, where η is a fixed number, η = 0.319, which gives the fraction of influencing variants contributing to 90% of trait’s heritability (with rationale for this adjustment being that the remaining 68.1% of influencing variants are small and Author Manuscript cumulatively explain only 10% of trait’s heritability). Phenotypic variance explained on 1 average by an influencing genetic variant is calculated as Hσβ2, where H = ∑ H = 0.2075 M i i is the average heterozygosity across SNPs in the reference panel. Under the assumptions of 2 the MiXeR model, SNP-heritability is then calculated as ℎSNP = Mπ1 × Hσβ2. In the cross-trait analysis, MiXeR models additive genetic effects as a mixture of four components, representing null SNPs in both traits (π0); SNPs with a specific effect on the Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 17 first and on the second trait (π1 and π2, respectively); and SNPs with non-zero effect on both traits (π12). In the last component, MiXeR models variance-covariance matrix as Author Manuscript σ12 ρ12σ1σ2 Σ12 = where ρ12 indicates correlation of effect sizes within the shared ρ12σ1σ2 σ22 component, and σ12 and σ22 correspond to the discoverability parameter estimated in the univariate analysis of the two traits. These components are then plotted in Venn diagrams. After fitting parameters of the model, the Dice coefficient of polygenic overlap is then 2π12 ρ12π12 calculated as π1 + 2π12 + π2 , and genetic correlation is calculated as rg = . π1 + π12 π2 + π12 Fraction of influencing variants with concordant effect direction is calculated as twice the multivariate normal CDF at point (0, 0) for the bivariate normal distribution with zero mean and variance-covariance matrix Σ12. All code is available online (https://github.com/ precimed/mixer). Author Manuscript Mendelian randomization. We selected 17 traits associated with BD in clinical or epidemiological studies for Mendelian randomization (MR) to dissect their relationship with BD (Supplementary Note). Bi-directional generalized summary statistics-based MR (GSMR)51 analyses were performed between BD and the traits of interest using GWAS summary statistics, implemented in GCTA software (v1.93.1f beta). The instrumental variables (IVs) were selected by a clumping procedure internal to the GSMR software with parameters: --gwas- thresh 5e-8 --clump-r2 0.01. Traits with less than 10 IVs available were excluded from the GSMR analyses to avoid conducting underpowered tests51, resulting in 10 traits tested (Bonferroni-corrected P-value threshold < 2.5 × 10−3). The HEIDI-outlier test (heterogeneity in dependent instruments) was applied to test for horizontal pleiotropy Author Manuscript (PHEIDI < 0.01)51. For comparison, the MR analyses were also performed using the inverse variance weighted regression method, implemented via the TwoSampleMR R package, using the IVs selected by GSMR119,120. To further investigate horizontal pleiotropy, the MR Egger intercept test was conducted using the TwoSampleMR package119,120 and MR-PRESSO software was used to perform the Global Test and Distortion Test121. BD subtypes. GWAS meta-analyses were conducted for BD I (25,060 cases, 449,978 controls from 55 cohorts, effective n = 64,802) and BD II (6,781 cases, 364,075 controls from 31 cohorts, effective n = 22,560) (Supplementary Table 1) using the same procedures described for the main GWAS. BD subtypes were defined based on international consensus criteria (DSM-IV, Author Manuscript ICD-9, or ICD-10), established using structured diagnostic instruments from assessments by trained interviewers, clinician-administered checklists or medical record review. In the external biobank cohorts, BD subtypes were defined using ICD codes (Supplementary 2 Note). LDSC35 was used to estimate the ℎSNP of each subtype, and the genetic correlation 2 between the subtypes. The difference between the LDSC ℎSNP estimates for BD I and BD II was tested for deviation from 0 using the block jackknife122. The LDSC genetic correlation Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 18 (rg) was tested for difference from 1 by calculating a chi-square statistic corresponding to the estimated rg as ((rg − 1)/ SE)2. Author Manuscript Supplementary Material Refer to Web version on PubMed Central for supplementary material. Authors Niamh Mullins1,2,236,*, Andreas J. Forstner3,4,5,236, Kevin S. O’Connell6,7, Brandon Coombes8, Jonathan R. I. Coleman9,10, Zhen Qiao11, Thomas D. Als12,13,14, Tim B. Bigdeli15,16, Sigrid Børte17,18,19, Julien Bryois20, Alexander W. Charney2, Ole Kristian Drange21,22, Michael J. Gandal23, Saskia P. Hagenaars9,10, Masashi Ikeda24, Nolan Kamitaki25,26, Minsoo Kim23, Kristi Krebs27, Georgia Author Manuscript Panagiotaropoulou28, Brian M. Schilder1,29,30,31, Laura G. Sloofman1, Stacy Steinberg32, Vassily Trubetskoy28, Bendik S. Winsvold19,33, Hong-Hee Won34, Liliya Abramova35, Kristina Adorjan36,37, Esben Agerbo14,38,39, Mariam Al Eissa40, Diego Albani41, Ney Alliey-Rodriguez42,43, Adebayo Anjorin44, Verneri Antilla45, Anastasia Antoniou46, Swapnil Awasthi28, Ji Hyun Baek47, Marie Bækvad-Hansen14,48, Nicholas Bass40, Michael Bauer49, Eva C. Beins3, Sarah E. Bergen20, Armin Birner50, Carsten Bøcker Pedersen14,38,39, Erlend Bøen51, Marco P. Boks52, Rosa Bosch53,54,55,56, Murielle Brum57, Ben M. Brumpton19, Nathalie Brunkhorst- Kanaan57, Monika Budde36, Jonas Bybjerg-Grauholm14,48, William Byerley58, Murray Cairns59, Miquel Casas53,54,55,56, Pablo Cervantes60, Toni-Kim Clarke61, Cristiana Cruceanu60,62, Alfredo Cuellar-Barboza63,64, Julie Cunningham65, David Curtis66,67, Piotr M. Czerski68, Anders M. Dale69, Nina Dalkner50, Friederike S. David3, Franziska Degenhardt3,70, Srdjan Djurovic71,72, Amanda L. Dobbyn1,2, Author Manuscript Athanassios Douzenis46, Torbjørn Elvsåshagen18,73,74, Valentina Escott-Price75, I. Nicol Ferrier76, Alessia Fiorentino40, Tatiana M. Foroud77, Liz Forty75, Josef Frank78, Oleksandr Frei6,18, Nelson B. Freimer23,79, Louise Frisén80, Katrin Gade36,81, Julie Garnham82, Joel Gelernter83,84,85, Marianne Giørtz Pedersen14,38,39, Ian R. Gizer86, Scott D. Gordon87, Katherine Gordon-Smith88, Tiffany A. Greenwood89, Jakob Grove12,13,14,90, José Guzman-Parra91, Kyooseob Ha92, Magnus Haraldsson93, Martin Hautzinger94, Urs Heilbronner36, Dennis Hellgren20, Stefan Herms3,95,96, Per Hoffmann3,95,96, Peter A. Holmans75, Laura Huckins1,2, Stéphane Jamain97,98, Jessica S. Johnson1,2, Janos L. Kalman36,37,99, Yoichiro Kamatani100,101, James L. Kennedy102,103,104,105, Sarah Kittel- Schneider57,106, James A. Knowles107,108, Manolis Kogevinas109, Maria Koromina110, Thorsten M. Kranz57, Henry R. Kranzler111,112, Michiaki Kubo113, Author Manuscript Ralph Kupka114,115,116, Steven A. Kushner117, Catharina Lavebratt118,119, Jacob Lawrence120, Markus Leber121, Heon-Jeong Lee122, Phil H. Lee123, Shawn E. Levy124, Catrin Lewis75, Calwing Liao125,126, Susanne Lucae62, Martin Lundberg118,119, Donald J. MacIntyre127, Sigurdur H. Magnusson32, Wolfgang Maier128, Adam Maihofer89, Dolores Malaspina1,2, Eirini Maratou129, Lina Martinsson80, Manuel Mattheisen12,13,14,106,130, Steven A. McCarroll25,26, Nathaniel W. McGregor131, Peter McGuffin9, James D. McKay132, Helena Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 19 Medeiros108, Sarah E. Medland87, Vincent Millischer118,119, Grant W. Montgomery11, Jennifer L. Moran25,133, Derek W. Morris134, Thomas W. Author Manuscript Mühleisen4,95, Niamh O’Brien40, Claire O’Donovan82, Loes M. Olde Loohuis23,79, Lilijana Oruc135, Sergi Papiol36,37, Antonio F. Pardiñas75, Amy Perry88, Andrea Pfennig49, Evgenia Porichi46, James B. Potash136, Digby Quested137,138, Towfique Raj1,29,30,31, Mark H. Rapaport139, J. Raymond DePaulo136, Eline J. Regeer140, John P. Rice141, Fabio Rivas91, Margarita Rivera142,143, Julian Roth106, Panos Roussos1,2,29, Douglas M. Ruderfer144, Cristina Sánchez-Mora53,54,56,145, Eva C. Schulte36,37, Fanny Senner36,37, Sally Sharp40, Paul D. Shilling89, Engilbert Sigurdsson93,146, Lea Sirignano78, Claire Slaney82, Olav B. Smeland6,7, Daniel J. Smith147, Janet L. Sobell148, Christine Søholm Hansen14,48, Maria Soler Artigas53,54,56,145, Anne T. Spijker149, Dan J. Stein150, John S. Strauss102, Beata Świątkowska151, Chikashi Terao101, Thorgeir E. Thorgeirsson32, Claudio Toma152,153,154, Paul Tooney59, Evangelia-Eirini Tsermpini110, Marquis P. Author Manuscript Vawter155, Helmut Vedder156, James T. R. Walters75, Stephanie H. Witt78, Simon Xi157, Wei Xu158, Jessica Mei Kay Yang75, Allan H. Young159,160, Hannah Young1, Peter P. Zandi136, Hang Zhou83,84, Lea Zillich78, HUNT All-In Psychiatry161, Rolf Adolfsson162, Ingrid Agartz51,130,163, Martin Alda82,164, Lars Alfredsson165, Gulja Babadjanova166, Lena Backlund118,119, Bernhard T. Baune167,168,169, Frank Bellivier170,171, Susanne Bengesser50, Wade H. Berrettini172, Douglas H. R. Blackwood61, Michael Boehnke173, Anders D. Børglum14,174,175, Gerome Breen9,10, Vaughan J. Carr176, Stanley Catts177, Aiden Corvin178, Nicholas Craddock75, Udo Dannlowski167, Dimitris Dikeos179, Tõnu Esko26,27,180,181, Bruno Etain170,171, Panagiotis Ferentinos9,46, Mark Frye64, Janice M. Fullerton152,153, Micha Gawlik106, Elliot S. Gershon42,182, Fernando S. Goes136, Melissa J. Green152,176, Maria Grigoroiu-Serbanescu183, Joanna Hauser68, Frans Henskens59, Jan Hillert80, Author Manuscript Kyung Sue Hong47, David M. Hougaard14,48, Christina M. Hultman20, Kristian Hveem19,184, Nakao Iwata24, Assen V. Jablensky185, Ian Jones75, Lisa A. Jones88, René S. Kahn2,52, John R. Kelsoe89, George Kirov75, Mikael Landén20,186, Marion Leboyer97,98,187, Cathryn M. Lewis9,10,188, Qingqin S. Li189, Jolanta Lissowska190, Christine Lochner191, Carmel Loughland59, Nicholas G. Martin87,192, Carol A. Mathews193, Fermin Mayoral91, Susan L. McElroy194, Andrew M. McIntosh127,195, Francis J. McMahon196, Ingrid Melle6,197, Patricia Michie59, Lili Milani27, Philip B. Mitchell176, Gunnar Morken21,198, Ole Mors14,199, Preben Bo Mortensen12,14,38,39, Bryan Mowry177, Bertram Müller-Myhsok62,200,201, Richard M. Myers124, Benjamin M. Neale25,45,180, Caroline M. Nievergelt89,202, Merete Nordentoft14,203, Markus M. Nöthen3, Michael C. O’Donovan75, Ketil J. Oedegaard204,205, Tomas Olsson206, Michael J. Owen75, Sara A. Paciga207, Chris Pantelis208, Carlos Pato108, Michele T. Author Manuscript Pato108, George P. Patrinos110,209,210, Roy H. Perlis211,212, Danielle Posthuma213,214, Josep Antoni Ramos-Quiroga53,54,55,56, Andreas Reif57, Eva Z. Reininghaus50, Marta Ribasés53,54,56,145, Marcella Rietschel78, Stephan Ripke25,28,45, Guy A. Rouleau126,215, Takeo Saito24, Ulrich Schall59, Martin Schalling118,119, Peter R. Schofield152,153, Thomas G. Schulze36,78,81,136,216, Laura J. Scott173, Rodney J. Scott59, Alessandro Serretti217, Cynthia Shannon Weickert152,176,218, Jordan W. Smoller25,133,219, Hreinn Stefansson32, Kari Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 20 Stefansson32,220, Eystein Stordal221,222, Fabian Streit78, Patrick F. Sullivan20,223,224, Gustavo Turecki225, Arne E. Vaaler226, Eduard Vieta227, John B. Author Manuscript Vincent102, Irwin D. Waldman228, Thomas W. Weickert152,176,218, Thomas Werge14,229,230,231, Naomi R. Wray11,232, John-Anker Zwart18,19,33, Joanna M. Biernacka8,64, John I. Nurnberger233, Sven Cichon3,4,95,96, Howard J. Edenberg77,234, Eli A. Stahl1,2,180,237, Andrew McQuillin40,237, Arianna Di Florio75,224,237, Roel A. Ophoff23,79,117,235,237, Ole A. Andreassen6,7,237,* Affiliations 1Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 2Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 3Institute of Human Genetics, University of Bonn, School of Medicine and University Hospital Bonn, Bonn, Germany. 4Institute of Neuroscience and Medicine (INM-1), Research Centre Jülich, Author Manuscript Jülich, Germany. 5Centre for Human Genetics, University of Marburg, Marburg, Germany. 6Division of Mental Health and Addiction, Oslo University Hospital, Oslo, Norway. 7NORMENT, University of Oslo, Oslo, Norway. 8Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA. 9Social, Genetic and Developmental Psychiatry Centre, King’s College London, London, UK. 10NIHR Maudsley BRC, King’s College London, London, UK. 11Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia. 12iSEQ, Center for Integrative Sequencing, Aarhus University, Aarhus, Denmark. 13Department of Biomedicine - Human Genetics, Aarhus University, Aarhus, Denmark. 14iPSYCH, The Lundbeck Foundation Initiative for Integrative Psychiatric Research, Denmark. 15Department of Psychiatry and Behavioral Sciences, SUNY Downstate Health Sciences University, Brooklyn, NY, USA. 16VA NY Harbor Healthcare System, Author Manuscript Brooklyn, NY, USA. 17Research and Communication Unit for Musculoskeletal Health, Division of Clinical Neuroscience, Oslo University Hospital, Ullevål, Oslo, Norway. 18Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 19K. G. Jebsen Center for Genetic Epidemiology, Department of Public Health and Nursing, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway. 20Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. 21Department of Mental Health, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology (NTNU), Trondheim, Norway. 22Department of Østmarka, Division of Mental Health Care, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway. 23Department of Psychiatry and Biobehavioral Science, Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, Author Manuscript Los Angeles, CA, USA. 24Department of Psychiatry, Fujita Health University School of Medicine, Toyoake, Japan. 25Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA. 26Department of Genetics, Harvard Medical School, Boston, MA, USA. 27Estonian Genome Center, Institute of Genomics, University of Tartu, Tartu, Estonia. 28Department of Psychiatry and Psychotherapy, Charité - Universitätsmedizin, Berlin, Germany. 29Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 30Ronald M. Loeb Center for Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 21 Author Manuscript Alzheimer’s Disease, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 31Estelle and Daniel Maggin Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 32deCODE Genetics / Amgen, Reykjavik, Iceland. 33Department of Research, Innovation and Education, Division of Clinical Neuroscience, Oslo University Hospital, Oslo, Norway. 34Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, Samsung Medical Center, Seoul, South Korea. 35Russian Academy of Medical Sciences, Mental Health Research Center, Moscow, Russian Federation. 36Institute of Psychiatric Phenomics and Genomics (IPPG), University Hospital, LMU Munich, Munich, Germany. 37Department of Psychiatry and Psychotherapy, University Hospital, LMU Munich, Munich, Germany. 38National Centre for Register-Based Research, Aarhus University, Aarhus, Denmark. 39Centre for Integrated Register- Author Manuscript based Research, Aarhus University, Aarhus, Denmark. 40Division of Psychiatry, University College London, London, UK. 41Department of Neuroscience, Istituto Di Ricerche Farmacologiche Mario Negri IRCCS, Milano, Italy. 42Department of Psychiatry and Behavioral Neuroscience, University of Chicago, Chicago, IL, USA. 43Northwestern University, Chicago, IL, USA. 44Psychiatry, Berkshire Healthcare NHS Foundation Trust, Bracknell, UK. 45Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA. 46National and Kapodistrian University of Athens, 2nd Department of Psychiatry, Attikon General Hospital, Athens, Greece. 47Department of Psychiatry, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, South Korea. 48Center for Neonatal Screening, Department for Congenital Disorders, Statens Serum Institut, Copenhagen, Denmark. 49Department of Psychiatry and Psychotherapy, University Author Manuscript Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. 50Medical University of Graz, Department of Psychiatry and Psychotherapeutic Medicine, Graz, Austria. 51Department of Psychiatric Research, Diakonhjemmet Hospital, Oslo, Norway. 52Psychiatry, Brain Center UMC Utrecht, Utrecht, The Netherlands. 53Instituto de Salud Carlos III, Biomedical Network Research Centre on Mental Health (CIBERSAM), Madrid, Spain. 54Department of Psychiatry, Hospital Universitari Vall d´Hebron, Barcelona, Spain. 55Department of Psychiatry and Forensic Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain. 56Psychiatric Genetics Unit, Group of Psychiatry Mental Health and Addictions, Vall d´Hebron Research Institut (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. 57Department of Psychiatry, Psychosomatic Medicine and Psychotherapy, University Hospital Frankfurt, Frankfurt am Main, Germany. 58Psychiatry, University Author Manuscript of California San Francisco, San Francisco, CA, USA. 59University of Newcastle, Newcastle, NSW, Australia. 60Department of Psychiatry, Mood Disorders Program, McGill University Health Center, Montreal, QC, Canada. 61Division of Psychiatry, University of Edinburgh, Edinburgh, UK. 62Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Munich, Germany. 63Department of Psychiatry, Universidad Autonoma de Nuevo Leon, Monterrey, Mexico. 64Department of Psychiatry and Psychology, Mayo Clinic, Rochester, MN, USA. 65Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 22 Author Manuscript USA. 66Centre for Psychiatry, Queen Mary University of London, London, UK. 67UCL Genetics Institute, University College London, London, UK. 68Department of Psychiatry, Laboratory of Psychiatric Genetics, Poznan University of Medical Sciences, Poznan, Poland. 69Center for Multimodal Imaging and Genetics, Departments of Neurosciences, Radiology, and Psychiatry, University of California, San Diego, CA, USA. 70Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital Essen, University of Duisburg-Essen, Duisburg, Germany. 71Department of Medical Genetics, Oslo University Hospital Ullevål, Oslo, Norway. 72NORMENT, Department of Clinical Science, University of Bergen, Bergen, Norway. 73Department of Neurology, Oslo University Hospital, Oslo, Norway. 74NORMENT, KG Jebsen Centre for Psychosis Research, Oslo University Hospital, Oslo, Norway. 75Medical Research Council Author Manuscript Centre for Neuropsychiatric Genetics and Genomics, Division of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, UK. 76Academic Psychiatry, Newcastle University, Newcastle upon Tyne, UK. 77Department of Medical and Molecular Genetics, Indiana University, Indianapolis, IN, USA. 78Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 79Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, Los Angeles, CA, USA. 80Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden. 81Department of Psychiatry and Psychotherapy, University Medical Center Göttingen, Göttingen, Germany. 82Department of Psychiatry, Dalhousie University, Halifax, NS, Canada. 83Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA. Author Manuscript 84Veterans Affairs Connecticut Healthcare System, West Haven, CT, USA. 85Departments of Genetics and Neuroscience, Yale University School of Medicine, New Haven, CT, USA. 86Department of Psychological Sciences, University of Missouri, Columbia, MO, USA. 87Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia. 88Psychological Medicine, University of Worcester, Worcester, UK. 89Department of Psychiatry, University of California San Diego, La Jolla, CA, USA. 90Bioinformatics Research Centre, Aarhus University, Aarhus, Denmark. 91Mental Health Department, University Regional Hospital, Biomedicine Institute (IBIMA), Málaga, Spain. 92Department of Psychiatry, Seoul National University College of Medicine, Seoul, South Korea. 93Landspitali University Hospital, Reykjavik, Iceland. 94Department of Psychology, Eberhard Karls Universität Tübingen, Tubingen, Germany. Author Manuscript 95Department of Biomedicine, University of Basel, Basel, Switzerland. 96Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland. 97Neuropsychiatrie Translationnelle, Inserm U955, Créteil, France. 98Faculté de Santé, Université Paris Est, Créteil, France. 99International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich, Germany. 100Laboratory of Complex Trait Genomics, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan. 101Laboratory for Statistical and Translational Genetics, RIKEN Center for Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 23 Author Manuscript Integrative Medical Sciences, Yokohama, Japan. 102Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada. 103Neurogenetics Section, Centre for Addiction and Mental Health, Toronto, ON, Canada. 104Department of Psychiatry, University of Toronto, Toronto, ON, Canada. 105Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada. 106Department of Psychiatry, Psychosomatics and Psychotherapy, Center of Mental Health, University Hospital Würzburg, Würzburg, Germany. 107Cell Biology, SUNY Downstate Medical Center College of Medicine, Brooklyn, NY, USA. 108Institute for Genomic Health, SUNY Downstate Medical Center College of Medicine, Brooklyn, NY, USA. 109ISGlobal, Barcelona, Spain. 110University of Patras, School of Health Sciences, Department of Pharmacy, Laboratory of Pharmacogenomics and Individualized Therapy, Patras, Greece. 111Mental Illness Research, Education and Author Manuscript Clinical Center, Crescenz VAMC, Philadelphia, PA, USA. 112Center for Studies of Addiction, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. 113RIKEN Center for Integrative Medical Sciences, Yokohama, Japan. 114Psychiatry, Altrecht, Utrecht, The Netherlands. 115Psychiatry, GGZ inGeest, Amsterdam, The Netherlands. 116Psychiatry, VU medisch centrum, Amsterdam, The Netherlands. 117Department of Psychiatry, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. 118Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden. 119Center for Molecular Medicine, Karolinska University Hospital, Stockholm, Sweden. 120Psychiatry, North East London NHS Foundation Trust, Ilford, UK. 121Clinic for Psychiatry and Psychotherapy, University Hospital Cologne, Cologne, Germany. 122Department of Psychiatry, Korea University College of Medicine, Seoul, South Korea. 123Psychiatric Author Manuscript and Neurodevelopmental Genetics Unit, Center for Genomic Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. 124HudsonAlpha Institute for Biotechnology, Huntsville, AL, USA. 125Department of Human Genetics, McGill University, Montréal, QC, Canada. 126Montreal Neurological Institute and Hospital, McGill University, Montréal, QC, Canada. 127Division of Psychiatry, Centre for Clinical Brain Sciences, The University of Edinburgh, Edinburgh, UK. 128Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany. 129National and Kapodistrian University of Athens, Medical School, Clinical Biochemistry Laboratory, Attikon General Hospital, Athens, Greece. 130Department of Clinical Neuroscience, Centre for Psychiatry Research, Karolinska Institutet, Stockholm, Sweden. 131Systems Genetics Working Group, Department of Genetics, Stellenbosch University, Stellenbosch, South Author Manuscript Africa. 132Genetic Cancer Susceptibility Group, International Agency for Research on Cancer, Lyon, France. 133Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA. 134Centre for Neuroimaging and Cognitive Genomics (NICOG), National University of Ireland Galway, Galway, Ireland. 135Medical faculty, University Sarajevo School of Science and Technology, Sarajevo, Bosnia and Herzegovina. 136Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 137Oxford Health NHS Foundation Trust, Warneford Hospital, Oxford, UK. 138Department of Psychiatry, Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 24 Author Manuscript University of Oxford, Warneford Hospital, Oxford, UK. 139Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA, USA. 140Outpatient Clinic for Bipolar Disorder, Altrecht, Utrecht, The Netherlands. 141Department of Psychiatry, Washington University in Saint Louis, Saint Louis, MO, USA. 142Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, Granada, Spain. 143Institute of Neurosciences, Biomedical Research Center (CIBM), University of Granada, Granada, Spain. 144Medicine, Psychiatry, Biomedical Informatics, Vanderbilt University Medical Center, Nashville, TN, USA. 145Department of Genetics, Microbiology and Statistics, Faculty of Biology, Universitat de Barcelona, Barcelona, Catalonia, Spain. 146Faculty of Medicine, Department of Psychiatry, School of Health Sciences, University of Iceland, Reykjavik, Iceland. 147Institute of Health and Wellbeing, University of Author Manuscript Glasgow, Glasgow, UK. 148Psychiatry and the Behavioral Sciences, University of Southern California, Los Angeles, CA, USA. 149Mood Disorders, PsyQ, Rotterdam, The Netherlands. 150SAMRC Unit on Risk and Resilience in Mental Disorders, Department of Psychiatry and Neuroscience Institute, University of Cape Town, Cape Town, South Africa. 151Department of Environmental Epidemiology, Nofer Institute of Occupational Medicine, Lodz, Poland. 152Neuroscience Research Australia, Sydney, NSW, Australia. 153School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia. 154Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid and CSIC, Madrid, Spain. 155Department of Psychiatry and Human Behavior, School of Medicine, University of California, Irvine, Irvine, CA, USA. 156Psychiatry, Psychiatrisches Zentrum Nordbaden, Wiesloch, Germany. 157Computational Sciences Center of Emphasis, Pfizer Global Author Manuscript Research and Development, Cambridge, MA, USA. 158Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada. 159Department of Psychological Medicine, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK. 160South London and Maudsley NHS Foundation Trust, Bethlem Royal Hospital, Monks Orchard Road, Beckenham, Kent, UK. 161A list of consortium authors appears at the end of the paper. 162Department of Clinical Sciences, Psychiatry, Umeå University Medical Faculty, Umeå, Sweden. 163NORMENT, KG Jebsen Centre for Psychosis Research, Division of Mental Health and Addiction, Institute of Clinical Medicine and Diakonhjemmet Hospital, University of Oslo, Oslo, Norway. 164National Institute of Mental Health, Klecany, Czech Republic. 165Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 166Institute of Pulmonology, Russian State Medical University, Author Manuscript Moscow, Russian Federation. 167Department of Psychiatry, University of Münster, Münster, Germany. 168Department of Psychiatry, Melbourne Medical School, The University of Melbourne, Melbourne, VIC, Australia. 169The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, Australia. 170Université de Paris, INSERM, Optimisation Thérapeutique en Neuropsychopharmacologie, UMRS-1144, Paris, France. 171APHP Nord, DMU Neurosciences, GHU Saint Louis-Lariboisière-Fernand Widal, Département de Psychiatrie et de Médecine Addictologique, Paris, France. 172Psychiatry, University Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 25 Author Manuscript of Pennsylvania, Philadelphia, PA, USA. 173Center for Statistical Genetics and Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA. 174Department of Biomedicine and the iSEQ Center, Aarhus University, Aarhus, Denmark. 175Center for Genomics and Personalized Medicine, CGPM, Aarhus, Denmark. 176School of Psychiatry, University of New South Wales, Sydney, NSW, Australia. 177University of Queensland, Brisbane, QLD, Australia. 178Neuropsychiatric Genetics Research Group, Department of Psychiatry and Trinity Translational Medicine Institute, Trinity College Dublin, Dublin, Ireland. 179National and Kapodistrian University of Athens, 1st Department of Psychiatry, Eginition Hospital, Athens, Greece. 180Medical and Population Genetics, Broad Institute, Cambridge, MA, USA. 181Division of Endocrinology, Children’s Hospital Boston, Boston, MA, USA. 182Department of Human Genetics, University of Chicago, Author Manuscript Chicago, IL, USA. 183Biometric Psychiatric Genetics Research Unit, Alexandru Obregia Clinical Psychiatric Hospital, Bucharest, Romania. 184HUNT Research Center, Department of Public Health and Nursing, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway. 185University of Western Australia, Nedlands, WA, Australia. 186Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden. 187Department of Psychiatry and Addiction Medicine, Assistance Publique - Hôpitaux de Paris, Paris, France. 188Department of Medical and Molecular Genetics, King’s College London, London, UK. 189Neuroscience Therapeutic Area, Janssen Research and Development, LLC, Titusville, NJ, USA. 190Cancer Epidemiology and Prevention, M. Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland. 191SA MRC Unit on Risk and Resilience in Mental Disorders, Department of Author Manuscript Psychiatry, Stellenbosch University, Stellenbosch, South Africa. 192School of Psychology, The University of Queensland, Brisbane, QLD, Australia. 193Department of Psychiatry and Genetics Institute, University of Florida, Gainesville, FL, USA. 194Research Institute, Lindner Center of HOPE, Mason, OH, USA. 195Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Edinburgh, UK. 196Human Genetics Branch, Intramural Research Program, National Institute of Mental Health, Bethesda, MD, USA. 197Division of Mental Health and Addiction, University of Oslo, Institute of Clinical Medicine, Oslo, Norway. 198Psychiatry, St Olavs University Hospital, Trondheim, Norway. 199Psychosis Research Unit, Aarhus University Hospital - Psychiatry, Risskov, Denmark. 200Munich Cluster for Systems Neurology (SyNergy), Munich, Germany. 201University of Liverpool, Liverpool, UK. 202Research/Psychiatry, Veterans Affairs San Diego Healthcare System, San Diego, Author Manuscript CA, USA. 203Mental Health Services in the Capital Region of Denmark, Mental Health Center Copenhagen, University of Copenhagen, Copenhagen, Denmark. 204Division of Psychiatry, Haukeland Universitetssjukehus, Bergen, Norway. 205Faculty of Medicine and Dentistry, University of Bergen, Bergen, Norway. 206Department of Clinical Neuroscience and Center for Molecular Medicine, Karolinska Institutet at Karolinska University Hospital, Solna, Sweden. 207Human Genetics and Computational Biomedicine, Pfizer Global Research and Development, Groton, CT, USA. 208University of Melbourne, Melbourne, VIC, Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 26 Author Manuscript Australia. 209United Arab Emirates University, College of Medicine and Health Sciences, Department of Pathology, Al-Ain, United Arab Emirates. 210United Arab Emirates University, Zayed Center of Health Sciences, Al-Ain, United Arab Emirates. 211Psychiatry, Harvard Medical School, Boston, MA, USA. 212Division of Clinical Research, Massachusetts General Hospital, Boston, MA, USA. 213Department of Complex Trait Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands. 214Department of Clinical Genetics, Amsterdam Neuroscience, Vrije Universiteit Medical Center, Amsterdam, The Netherlands. 215Department of Neurology and Neurosurgery, McGill University, Faculty of Medicine, Montreal, QC, Canada. 216Department of Psychiatry and Behavioral Sciences, SUNY Upstate Medical University, Syracuse, NY, USA. 217Department of Biomedical and Author Manuscript NeuroMotor Sciences, University of Bologna, Bologna, Italy. 218Department of Neuroscience, SUNY Upstate Medical University, Syracuse, NY, USA. 219Psychiatric and Neurodevelopmental Genetics Unit (PNGU), Massachusetts General Hospital, Boston, MA, USA. 220Faculty of Medicine, University of Iceland, Reykjavik, Iceland. 221Department of Psychiatry, Hospital Namsos, Namsos, Norway. 222Department of Neuroscience, Norges Teknisk Naturvitenskapelige Universitet Fakultet for naturvitenskap og teknologi, Trondheim, Norway. 223Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 224Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 225Department of Psychiatry, McGill University, Montreal, QC, Canada. 226Department of Psychiatry, Sankt Olavs Hospital Universitetssykehuset i Trondheim, Trondheim, Norway. 227Clinical Institute of Neuroscience, Hospital Clinic, Author Manuscript University of Barcelona, IDIBAPS, CIBERSAM, Barcelona, Spain. 228Department of Psychology, Emory University, Atlanta, GA, USA. 229Institute of Biological Psychiatry, Mental Health Services, Copenhagen University Hospital, Copenhagen, Denmark. 230Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark. 231Center for GeoGenetics, GLOBE Institute, University of Copenhagen, Copenhagen, Denmark. 232Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia. 233Psychiatry, Indiana University School of Medicine, Indianapolis, IN, USA. 234Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA. 235Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA. 236These authors contributed equally to this work: Niamh Mullins, Andreas J. Forstner. 237These authors jointly supervised this work: Eli A. Author Manuscript Stahl, Andrew McQuillin, Arianna Di Florio, Roel A. Ophoff, Ole A. Andreassen. Acknowledgements We thank the participants who donated their time, life experiences and DNA to this research and the clinical and scientific teams that worked with them. We are deeply indebted to the investigators who comprise the PGC. The PGC has received major funding from the US National Institute of Mental Health (PGC3: U01 MH109528; PGC2: U01 MH094421; PGC1: U01 MH085520). Statistical analyses were carried out on the NL Genetic Cluster Computer (http://www.geneticcluster.org) hosted by SURFsara and the Mount Sinai high performance computing cluster (http://hpc.mssm.edu), which is supported by the Office of Research Infrastructure of the National Institutes Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 27 of Health under award numbers S10OD018522 and S10OD026880. 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Manhattan plot of genome-wide association meta-analysis of 41,917 bipolar disorder cases and 371,549 controls. The x-axis shows genomic position (chromosomes 1–22 and X), and the y-axis shows statistical significance as −log10(P value). P values are two-sided and based on an inverse variance weighted fixed effects meta-analysis. The red line shows the genome-wide significance threshold (P < 5 × 10−8). SNPs in genome-wide significant loci are colored green for loci previously associated with bipolar disorder (BD) and yellow for novel Author Manuscript associations from this study. The genes labeled are those prioritized by integrative eQTL analyses or notable genes in novel loci (MHC, CACNB2, KCNB1). Author Manuscript Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 34 Author Manuscript Author Manuscript Figure 2 |. Phenotypic variance in bipolar disorder explained by polygenic risk scores. Variance explained is presented on the liability scale, assuming a 2% population prevalence of bipolar disorder. For European ancestries, the results shown are the weighted mean R2 values across all 57 cohorts in the PGC3 meta-analysis, weighted by the effective n per cohort. The numbers of cases and controls are shown from left to right under the barplot for each study. GWAS PT, the color of the bars represents the P value threshold used to select SNPs from the discovery GWAS; GAIN-AA, Genetic Association Information Network African American cohort; AA-GPC, African American Genomic Psychiatry Cohort. Author Manuscript Author Manuscript Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 35 Author Manuscript Author Manuscript Figure 3 |. Relationships between bipolar disorder and modifiable risk factors based on genetic correlations, generalized summary statistics-based Mendelian randomization, and bivariate Author Manuscript gaussian mixture modeling. Venn diagrams depict MiXeR results of the estimated number of influencing variants shared between bipolar disorder (BD) and each trait of interest (grey), unique to BD (blue) and unique to the trait of interest (orange). The number of influencing variants and standard error are shown in thousands. The size of the circles reflects the polygenicity of each trait, with larger circles corresponding to greater polygenicity. The estimated genetic correlation (rg) between BD and each trait of interest and standard error from LD Score regression is shown below the corresponding Venn diagram, with an accompanying scale (-1 to +1). The arrows above and below the Venn diagrams indicate the results of generalized summary statistics- based Mendelian randomization (GSMR) of BD on the trait of interest, and the trait of interest on BD, respectively. The GSMR effect size and standard error is shown inside the corresponding arrow. Solid arrows indicate a significant relationship between the exposure and the outcome, after correction for multiple comparisons (P < 1.47 × 10−3), and dashed Author Manuscript arrows indicate a non-significant relationship. Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 36 Table 1 | Genome-wide significant loci for bipolar disorder from meta-analysis of 41,917 cases and 371,549 controls Author Manuscript Previous Previous A1 freq report^ Name for report^ for A1/ in for BD novel locus psychiatric Locus CHR BP SNP P OR s.e. A2 controls (citation) + disorders A/ 1 1 61105668 rs2126180 1.6E-09 1.058 0.009 G 0.457 LINC01748 2 1 163745389 rs10737496 7.2E-09 1.056 0.009 C/T 0.444 NUF2 CDG G/ LMAN2L 3* 2 97416153 rs4619651 4.8E-11 1.068 0.010 A 0.670 (PGC2) CDG G/ SCN2A 4 2 166152389 rs17183814 2.7E-08 1.108 0.019 A 0.924 (PGC2) C/ 5 2 169481837 rs13417268 2.1E-08 1.064 0.011 G 0.758 CERS6 A/ Author Manuscript 6 2 193738336 rs2011302 4.3E-08 1.055 0.010 T 0.377 PCGEM1 CDG A/ intergenic 7 2 194437889 rs2719164 4.9E-08 1.053 0.010 G 0.564 (PGC2) CDG TRANK1 8* 3 36856030 rs9834970 6.6E-19 1.087 0.009 C/T 0.481 (PGC2) SCZ, CDG ITIH1 9* 3 52626443 rs2336147 3.6E-13 1.070 0.009 T/C 0.498 (PGC2) SCZ, CDG T/ 10 3 70488788 rs115694474 2.4E-08 1.068 0.012 A 0.799 MDFIC2 C/ CD47 11 3 107757060 rs696366 4.5E-08 1.053 0.009 A 0.550 (PGC2) G/ 12* 4 123076007 rs112481526 1.9E-09 1.065 0.011 A 0.256 KIAA1109 MD A/ ADCY2 13* 5 7542911 rs28565152 2.0E-09 1.070 0.011 G 0.238 (PGC2) Author Manuscript T/ 14* 5 78849505 rs6865469 1.7E-08 1.060 0.010 G 0.274 HOMER1 G/ SSBP2 15 5 80961069 rs6887473 8.8E-09 1.062 0.011 A 0.739 (PGC2) 16* 5 137712121 rs10043984 3.7E-08 1.062 0.011 T/C 0.236 KDM3B CDG 17 5 169289206 rs10866641 2.8E-11 1.065 0.009 T/C 0.575 DOCK2 MD, SCZ, G/ CDG, 18* 6 26463575 rs13195402 5.8E-15 1.146 0.018 T 0.919 MHC MOOD POU3F2 19* 6 98565211 rs1487445 1.5E-15 1.078 0.009 T/C 0.487 (PGC2) CDG SYNE1 A/ (Green 20 6 152793572 rs4331993 2.0E-08 1.056 0.010 T 0.382 2013) Author Manuscript G/ RPS6KA2 21* 6 166995260 rs10455979 4.2E-09 1.057 0.010 C 0.500 (PGC2) MAD1L1 (Hou 2016, G/ Ikeda MD, SCZ, 22* 7 2020995 rs12668848 1.9E-09 1.059 0.010 A 0.575 2017) CDG Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 37 Previous Previous A1 freq report^ Name for report^ for A1/ in for BD novel locus psychiatric Author Manuscript Locus CHR BP SNP P OR s.e. A2 controls (citation) + disorders A/ THSD7A 23* 7 11871787 rs113779084 1.4E-13 1.079 0.010 G 0.299 (PGC2) G/ 24* 7 21492589 rs6954854 5.9E-10 1.060 0.009 A 0.425 SP4 G/ SCZ, CDG, 25 7 24647222 rs12672003 2.7E-09 1.096 0.016 A 0.113 MPP6 MOOD A/ SRPK2 SCZ, ASD, 26 7 105043229 rs11764361 3.5E-09 1.063 0.010 G 0.668 (PGC2) CDG C/ 27 7 131870597 rs6946056 3.7E-08 1.055 0.010 A 0.623 PLXNA4 A/ MRPS33 28 7 140676153 rs10255167 1.6E-08 1.068 0.012 G 0.778 (PGC2) CDG G/ SCZ, ALC, 29* 8 9763581 rs62489493 2.6E-11 1.094 0.014 C 0.128 miR124–1 ASD Author Manuscript SCZ, ALC, 30* 8 10226355 rs3088186 2.1E-08 1.058 0.010 T/C 0.287 MSRA ASD RP1– SCZ, A/ 84O15.2 ADHD, 31 8 34152492 rs2953928 6.3E-09 1.124 0.020 G 0.067 (lincRNA) CDG G/ 32* 8 144993377 rs6992333 1.6E-09 1.062 0.010 A 0.410 PLEC MD, CDG, 33 9 37090538 rs10973201 2.5E-08 1.101 0.017 C/T 0.110 ZCCHC7 MOOD 34* 9 141066490 rs62581014 2.8E-08 1.067 0.012 T/C 0.366 TUBBP5 T/ 35* 10 18751103 rs1998820 4.1E-08 1.087 0.015 A 0.886 CACNB2 SCZ, CDG ANK3 36* 10 62322034 rs10994415 1.1E-11 1.125 0.017 C/T 0.082 (PGC2) Author Manuscript 37 10 64525135 rs10761661 4.7E-08 1.053 0.009 T/C 0.472 ADO ADD3 (Charney 2017, 38* 10 111648659 rs2273738 1.6E-11 1.096 0.014 T/C 0.135 PGC2) G/ FADS2 MD, CDG, 39* 11 61618608 rs174592 9.9E-14 1.074 0.010 A 0.395 (PGC2) MOOD A/ 40 11 64009879 rs4672 3.4E-09 1.107 0.017 G 0.083 FKBP2 A/ PACS1 41* 11 65848738 rs475805 2.0E-09 1.070 0.011 G 0.767 (PGC2) PC (PGC1, 42* 11 66324583 rs678397 5.5E-09 1.056 0.009 T/C 0.457 PGC2) A/ SHANK2 43* 11 70517927 rs12575685 1.2E-10 1.067 0.010 G 0.327 (PGC2) MD Author Manuscript ODZ4 44 11 79092527 rs12289486 3.3E-08 1.086 0.015 T/C 0.115 (PGC1) C/ CACNA1C SCZ, CDG, 45* 12 2348844 rs11062170 1.9E-15 1.081 0.010 G 0.333 (PGC2) MOOD G/ 46 13 113869045 rs35306827 3.6E-09 1.068 0.011 A 0.775 CUL4A Nat Genet. Author manuscript; available in PMC 2021 November 17. Mullins et al. Page 38 Previous Previous A1 freq report^ Name for report^ for A1/ in for BD novel locus psychiatric Author Manuscript Locus CHR BP SNP P OR s.e. A2 controls (citation) + disorders G/ 47 14 99719219 rs2693698 2.0E-08 1.055 0.009 A 0.551 BCL11B SCZ, CDG G/ 48* 15 38973793 rs35958438 3.8E-08 1.066 0.012 A 0.772 C15orf53 CDG A/ STARD9 49* 15 42904904 rs4447398 2.6E-09 1.086 0.014 C 0.131 (PGC2) T/ 50 15 83531774 rs62011709 1.4E-08 1.064 0.011 A 0.747 HOMER2 SCZ ZNF592 51* 15 85149575 rs748455 5.0E-11 1.070 0.010 T/C 0.719 (PGC2) SCZ, CDG G/ 52 15 91426560 rs4702 3.5E-09 1.059 0.010 A 0.446 FURIN SCZ, CDG G/ 53 16 9230816 rs28455634 2.6E-10 1.065 0.010 A 0.620 C16orf72 Author Manuscript G/ GRIN2A 54 16 9926348 rs7199910 1.7E-08 1.057 0.010 T 0.312 (PGC2) SCZ, CDG T/ 55 16 89632725 rs12932628 6.7E-09 1.058 0.010 G 0.487 RPL13 56 17 1835482 rs4790841 3.1E-08 1.075 0.013 T/C 0.151 RTN4RL1 T/ ERBB2 57 17 38129841 rs11870683 2.8E-08 1.059 0.010 A 0.650 (Hou 2016) T/ ERBB2 58 17 38220432 rs61554907 1.6E-08 1.091 0.015 G 0.124 (Hou 2016) G/ HDAC5 59* 17 42191893 rs228768 2.8E-10 1.067 0.010 T 0.294 (PGC2) T/ STK4 60* 20 43682551 rs67712855 4.2E-11 1.070 0.010 G 0.687 (PGC2) A/ WFDC12 61* Author Manuscript 20 43944323 rs6032110 1.0E-09 1.059 0.009 G 0.512 (PGC2) 62* 20 48033127 rs237460 4.3E-09 1.057 0.009 T/C 0.412 KCNB1 CDG G/ 63 20 60865815 rs13044225 8.5E-09 1.056 0.010 A 0.440 OSBPL2 MD, SCZ, CDG, 64 22 41153879 rs5758064 2.0E-08 1.054 0.009 T/C 0.523 SLC25A17 MOOD CHR, chromosome; BP, GRCh37 base pair position; SNP, single nucleotide polymorphism; OR, odds ratio; s.e., standard error, A1, tested allele; A2, other allele; freq, frequency; BD, bipolar disorder; CDG, Cross-disorder GWAS of the Psychiatric Genomics Consortium; MD, major depression; SCZ, schizophrenia; MOOD, mood disorders; ASD, autism spectrum disorder; ALC, alcohol use disorder or problematic alcohol use; ADHD, attention deficit/hyperactivity disorder. * Locus overlaps with genome-wide significant locus for bipolar I disorder. ^ Previous report refers to previous association of a SNP in the locus with the psychiatric disorder at genome-wide significance. PGC1 = PMID Author Manuscript 21926972; PGC2 = PMID 31043756; Hou 2016 = PMID 27329760; Ikeda 2017 = PMID 28115744; Green 2013 = PMID 22565781; Charney 2017 = PMID 28072414. + Novel loci are named using the nearest gene to the index SNP. P values are two-sided and based on an inverse variance weighted fixed effects meta-analysis. Nat Genet. Author manuscript; available in PMC 2021 November 17.