Functional analysis of epilepsy‐associated GABAA receptor mutations using Caenorhabditis elegans

Abstract

AbstractObjectiveGABAA receptor subunit mutations pose a significant risk for genetic generalized epilepsy; however, there are over 150 identified variants, many with unknown or unvalidated pathogenicity. We aimed to develop in vivo models for testing GABAA receptor variants using the model organism, Caenorhabditis elegans.MethodsCRISPR‐Cas9 gene editing was used to create a complete deletion of unc‐49, a C. elegans GABAA receptor, and to create homozygous epilepsy‐associated mutations in the endogenous unc‐49 gene. The unc‐49 deletion strain was rescued with transgenes for either the C. elegans unc‐49B subunit or the α1, β3, and γ2 subunits for the human GABAA receptor. All newly created strains were analyzed for phenotype and compared against existing unc‐49 mutations.ResultsNematodes with a full genetic deletion of the entire unc‐49 locus were compared with existing unc‐49 mutations in three separate phenotypic assays—coordinated locomotion, shrinker frequency and seizure‐like convulsions. The full unc‐49 deletion exhibited reduced locomotion and increased shrinker frequency and PTZ‐induced convulsions, but were not found to be phenotypically stronger than existing unc‐49 mutations. Rescue with the unc‐49B subunit or creation of humanized worms for the GABAA receptor both showed partial phenotypic rescue for all three phenotypes investigated. Finally, two epilepsy‐associated variants were analyzed and deemed to be loss of function, thus validating their pathogenicity.SignificanceThese findings establish C. elegans as a genetic model to investigate GABAA receptor mutations and delineate a platform for validating associated variants in any epilepsy‐associated gene.Plain Language SummaryEpilepsy is a complex human disease that can be caused by mutations in specific genes. Many possible mutations have been identified, but it is unknown for most of them whether they cause the disease. We tested the role of mutations in one specific gene using a small microscopic worm as an animal model. Our results establish this worm as a model for epilepsy and confirm that the two unknown mutations are likely to cause the disease.