014-23 – Investigating epilepsy using a novel unc-49 (GABAA receptor) mutant.

Genetic epilepsy accounts for approximately 30-40% of total epilepsy cases; however, often associated genes and pathological mechanisms remain elusive. A landmark paper found that missense mutations in human GABAA receptors are common in genetic epilepsy patients. Here, we aim to create a humanised worm model that can be used to study novel mutations. The worm homologue for the human GABAA receptor is called unc-49 and is required for coordinated locomotion. Via a singular promoter, unc-49 generates three distinct subunits that co-assemble to form a heteromeric receptor. Many unc-49 mutants have previously been studied; however, none are complete true deletions. Therefore, we used CRISPR-Cas9 to produce an unc-49 knockout. Through behavioural assays, we confirmed that our novel null mutant elicits the same phenotypic deficits as known unc-49 mutants. Next, we questioned if we could rescue the null mutant by introducing wildtype unc-49 cDNA or human GABAA receptor cDNA. Functional analysis highlighted a partial rescue effect in both strains, whereby the rescue strains outperformed the null mutant but did not fully resemble wildtype. Combined, despite the partial completeness of our rescue strains, we can produce a viable humanised worm model. Finally, in vivo we investigated two highly conserved novel human epilepsy-linked mutations (C167W and G254D) that were produced in endogenous unc-49. Functional analysis found that both mutants displayed phenotypic defects that are consistent with the unc-49 null mutant, suggesting that they may be pathogenic. In conclusion, our true unc-49 null mutant can be partially rescued using wildtype unc-49 or human GABAA receptor cDNA. Also, our selected human epilepsy-linked point mutations displayed phenotypic deficits consistent with the unc-49 null mutant. Such findings highlight the possibility of using C. elegans as an efficient way of investigating novel mutations to assess pathogenicity.