Neuropsychopharmacology (2016) 41, 886–895 © 2016 American College of Neuropsychopharmacology. All rights reserved 0893-133X/16 www.neuropsychopharmacology.org An Integrative Genomic Study Implicates the Postsynaptic Density in the Pathogenesis of Bipolar Disorder ,1 1,3 2 1 Nirmala Akula* , Jens R Wendland , Kwang H Choi and Francis J McMahon 1 Human Genetics Branch, National Institute of Mental Health Intramural Research Program (NIMH-IRP), National Institutes of Health, US 2 Department of Health and Human Services, Bethesda, MD, USA; Department of Psychiatry, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Genome-wide association studies (GWAS) have identified several common variants associated with bipolar disorder (BD), but the — biological meaning of these findings remains unclear. Integrative genomics the integration of GWAS signals with gene expression — data may illuminate genes and gene networks that have key roles in the pathogenesis of BD. We applied weighted gene co-expression network analysis (WGCNA), which exploits patterns of co-expression among genes, to brain transcriptome data obtained by sequencing of poly-A RNA derived from postmortem dorsolateral prefrontal cortex from people with BD, along with age- and sex-matched controls. WGCNA identified 33 gene modules. Many of the modules corresponded closely to those previously reported in human cortex. Three modules were associated with BD, enriched for genes differentially expressed in BD, and also enriched for signals in prior GWAS of BD. Functional analysis of genes within these modules revealed significant enrichment of several functionally related sets of genes, especially those involved in the postsynaptic density (PSD). These results provide convergent support for the hypothesis that dysregulation of genes involved in the PSD is a key factor in the pathogenesis of BD. If replicated in larger samples, these findings could point toward new therapeutic targets for BD. Neuropsychopharmacology (2016) 41, 886–895; doi:10.1038/npp.2015.218; published online 11 November 2015 INTRODUCTION Many of the common genetic variants identified by GWAS lie in the regulatory regions, where they can affect the Bipolar disorder (BD) is a debilitating and highly heritable expression of nearby genes (Maurano et al, 2012). Much of psychiatric disorder whose genetic etiology is largely the impact of genetic variation on gene expression is tissue- unknown. Candidate gene, genome-wide association dependent (Andersson et al, 2014). Non-protein-coding (GWAS), and gene expression studies have all implicated a genes that produce regulatory RNAs also appear to have an variety of genes, but a coherent theory of pathogenesis has important role in fine-tuning of gene expression over not yet emerged. Multiple variants in many genes often come development and in response to environmental stressors together into several gene networks and fewer biological (Barry, 2014). It has also become clear that most genes, pathways. This points to the powerful strategy, known as especially those expressed in brain, produce a number of ‘ ’ integrative genomics (Schadt, 2006), to address the extreme distinct messenger RNA molecules, or transcripts, owing genetic heterogeneity seen in many common illnesses. This to alternative splicing, differential exon usage, and other strategy has proven to be of value in interpreting the posttranscriptional modifications (Barry, 2014). multigenic signals that have been observed in GWAS, copy Thus, the integration of GWAS signals with gene number variation, and de novo mutation studies of many expression data requires a sensitive, tissue-specific approach common neuropsychiatric diseases (Walsh et al, 2008; Choi that can assess differences in transcript abundance, non- et al, 2011; Fromer et al, 2014) but has so far been little coding RNAs, and posttranscriptional modification. Next- studied in BD. generation sequencing of RNA, known as RNA sequencing (RNA-seq), addresses many of these needs better than *Correspondence: Dr N Akula, Human Genetics Branch, National traditional gene expression microarrays, providing a more Institute of Mental Health Intramural Research Program (NIMH-IRP), complete picture of the ‘transcriptome’ (McGettigan, 2013). National Institutes of Health, US Department of Health and Human As a first step toward an integrative genomics strategy in Services, Building 35, Room 1A-100, 35 Convent Drive, Bethesda, BD, we sequenced RNA derived from postmortem brain MD 20892, USA, Tel: +1 301 451 4258, Fax: +1 301 402 7094, E-mail: [email protected] obtained from individuals with BD and matched controls. 3Current address: PharmaTherapeutics Clinical Research, Worldwide Our initial analysis of these data detected many differentially Research and Development, Pfizer Inc., Cambridge, MA 02139, USA. expressed (DE) genes in BD. These genes have important Received 6 May 2015; revised 25 June 2015; accepted 10 July 2015; roles in neuroplasticity, circadian rhythms, and GTPase accepted article preview online 27 July 2015 binding (Akula et al, 2014). When we integrated these data Integrative genomic study of bipolar disorder N Akula et al 887 with the results from previous GWAS of BD, we found TopHat (TopHat version2.0.4; http://tophat.cbcb.umd. that DE genes in the GTPase pathway were also enriched edu/) (Trapnell et al, 2009) was used to map the reads to for single nucleotide polymorphisms (SNPs) that were the reference human genome (hg19). A total of 2.3 billion associated with BD. This suggested that differential expres- mapped reads from NISC1 and 4.3 billion mapped reads sion of these genes was not just a consequence of BD or its from NISC2 were included in the downstream analyses. treatment, but also reflected inherited genetic variation HTSeq (http://www-huber.embl.de/users/anders/HTSeq/ associated with disease risk. However, that study was limited doc/overview.html) was used to obtain the read counts per by analysis methods that focused on individual genes and gene based on Ensemble gene annotation (http://ftp.ensembl. transcripts, without regard to their correlated patterns of org/pub/release-67/gtf/homo_sapiens/). expression. Here, we performed a complete re-analysis of the same RNA-seq data using methods that exploit the correlated Gene Selection for Co-Expression Analysis patterns of expression among groups of genes. We used A total of 17 296 genes and 16 919 genes were selected after weighted gene correlation network analysis (WGCNA) QC in NISC1 and NISC2, respectively. Of these, 16 571 genes (Langfelder and Horvath, 2008), a widely used method that were common to both these datasets and were thus included finds modules of highly correlated genes, relates these in the downstream co-expression analysis. Read counts were modules to one another, and tests the influence of sample normalized using DESeq (Anders and Huber, 2010), and the phenotypes on gene expression correlations. WGCNA has resulting variance-stabilized transformed data were used been widely used to identify co-expressed gene networks in in the downstream analysis. Weighted gene co-expression various human brain regions (Oldham et al, 2008), animals network analysis (WGCNA) was used to identify the (Fuller et al, 2007; Langfelder et al, 2012), and in human co-expression modules (sub-networks) in BD (http://labs. phenotypes, including schizophrenia (Torkamani et al, genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/ 2010), autism (Voineagu et al, 2011), cancer (Clarke et al, WGCNA/). The co-expression analysis pipeline is shown is 2013), aggressive behavior (Malki et al, 2014), BD (Chen Supplementary Figure S1. et al, 2013a), and psoriasis (Li et al, 2014). However, aside from one study of a few gene networks (Hong et al, 2013), WGCNA has not yet been applied to the complete brain Weighted Gene Co-Expression Network Analysis transcriptome in BD as revealed by RNA-seq. (WGCNA) WGCNA detected a number of robust gene expression modules, several of which were enriched for GWAS signals. In WGCNA networks, genes are represented as nodes and Functional analysis of genes within one of these modules edges represent the correlation in expression (edge-weight) revealed significant enrichment of several functionally between gene pairs. The connection strength (adjacency) related sets of genes, especially those involved in the between two genes is calculated by raising the correlation to a postsynaptic density (PSD). These results provide convergent specific power, β, which must be estimated with each dataset. support for the hypothesis that dysregulation of genes Supplementary Figure S2 shows the relationship between the involved in the PSD is a key factor in the pathogenesis of β and scale free topology fitting index in NISC1 and NISC2. BD. If replicated in larger samples, these findings could point At β = 12, networks created by WGNCA showed 475% scale toward new therapeutic targets for BD. free topology in both datasets, so a value of 12 was used in this study. We used biweight correlation instead of the default Pearson correlation, because it is robust and resistant to outliers (Gaiteri et al, 2014). WGCNA identifies MATERIALS AND METHODS co-expressed genes and puts them into networks or modules. Samples and RNA-Seq Minimum module size was set to 50 genes per module. Here, we used the terms co-expressed networks and co-expressed RNA extracted from the dorsolateral prefrontal cortex of modules interchangeably, but we recognize that these terms postmortem brains of