Importance: Genetic risk variants associated with bipolar disorder (BD) are likely to result in transcriptomic changes. RNA sequencing (RNAseq) studies in multiplex BD families are limited. Objective: To identify candidate genes and pathways involved in BD. Design, settings, and participants: This cohort study includes Australian families with three or more members diagnosed with BD. RNAseq was performed on total mRNA from lymphoblastoid cell lines of eight multiplex BD families. Patients were enrolled between May 1999 and October 2004. Data were analysed from June 2022 to March 2024. Exposures: Differential gene expression changes and co-expressed gene networks in affected and unaffected relatives. Main outcomes and measures: Quantified read counts were examined for differential gene expression and Weighted Gene Co-expression Network Analysis (WGCNA). Differentially Expressed Genes (DEGs) were validated through: i) gene-based association using BD Genome-Wide Association Study (GWAS) summary statistics; ii) Polygenic Priority Score (PoPS); and iii) replication using RNAseq data from brain tissues of 71 BD and 252 controls. DEGs and co-expressed modules were examined for enriched categories via Gene-Set Enrichment Analysis (GSEA) and Over-Representation Analysis (ORA), and were further validated through gene-set analysis using GWAS data. Results: Sixty significant DEGs were found after comparing 16 BD (37.5% female) and 15 unaffected relatives (46.7% female), with the long non-coding RNA (lncRNA) LINC01237 being the most significant. Gene expression patterns of DEGs were correlated between lymphoblastoid cell lines and brain tissues (r=0.83). After validation, five DEGs were prioritised, including ENSG00000279277, a lncRNA mapping in a GWAS locus for BD. Enrichment analyses of DEGs pointed to nervous system and gated channel activities. WGCNA identified five expression modules associated with BD. The most significant module showed enriched categories associated with ion transmembrane transport and hypoplasia on corpus callosum, together with a protein-protein interaction network related to solute carriers at plasma membrane. Conclusions and relevance: This study implicates a role for lncRNAs in the pathophysiology of BD. Alterations in ion homeostasis, driven by the dysregulation of gated channels, appear to be a plausible impaired process in BD. In addition, the corpus callosum emerges as a key brain structure with a potential involvement in BD.

Janice M. Fullerton received honoraria from Illumina for contribution to a Speakers Bureau in 2023 and had travel expenses paid by Novo Nordisk Fonden in 2023 (not related to this work). No other disclosures were reported.

This study was supported by the NAB Foundation Stronger Minds grant (Cooper and Fullerton), The Australian National Health and Medical Research Council (NHMRC) Program Grant 1037196 (to Mitchell and Schofield), NHMRC Project Grant 1063960 (Fullerton and Schofield), NHMRC Investigator Grants 1176716 (Schofield) and 1177991 (Mitchell), NHMRC & Medical Research Futures Fund Grant 1200428 (to Fullerton) and Spanish Ministry of science and Innovation (grants: RyC2018-024106-I, PID2020-114996RB-I00, CNS2022-135318, PID2023-149154OB-I00 to Toma and PID2021-123141OB-I00 to Lucas). I Garcia-Ortiz was supported by the Fundacion Tatiana Perez Guzman el Bueno Fellowship. Dr Fullerton was supported by philanthropic donations from Betty C. Lynch OAM (dec) and Janette Mary O Neil, and was a recipient of the Janette Mary O Neil Research Fellowship.

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All experiments were carried out following regulations and guidelines approved by the University of New South Wales Human Research Ethics Committee (initial approval HREC04144; extensions HREC10078, HC15503, HC16347). The study does not include any clinical trial.

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