Help or hinderance, are CryoEM structures sufficient evidence for precision medicine in epilepsy?

Abstract

Epilepsy comprises of a heterogenous group of neurological disorders underpinned by a broad array of genetic, functional and pharmacological mechanisms. Among these, mutations in GABAA receptor subunits have emerged as important contributors to early-onset epilepsies. Although hundreds of pathogenic variants have now been identified across subunits such as GABRA1-4, GABRB2, GABRB3, GABRG2 and GABRD, interpretation remains limited by the lack of functional annotation. This project aimed to evaluate whether two clinically used antiepileptic drugs, lamotrigine (LTG) and levetiracetam (LEV), could serve as potential precision therapies for patients with GABAA receptor variants, particularly those resulting in receptor loss-of-function (LOF).

To address this, the research combined electrophysiological assays in Xenopus laevis oocytes with a retrospective clinical outcome analysis of patients carrying known GABAA receptor variants. Modulation and competition assays were performed using clinically relevant including supraphysiological doses of LTG and LEV, alongside diazepam and flumazenil as positive and negative controls respectively. In parallel, patient-reported data were collected via clinical surveys and analysed to identify patterns in therapeutic response across individuals stratified by variant effect – LOF or GOF (gain-of-function).

Results from clinical analysis indicated that benzodiazepines, as expected, were effective in LOF cases but often ineffective or harmful in GOF cases. LEV showed a similar pattern, suggesting some alignment with GABAergic mechanism, while LTG did not display any significant association with functional class. Electrophysiological testing revealed that neither LTG nor LEV produced potentiation of GABA currents in a manner consistent with direct positive allosteric modulation. Moreover, LEV required supraphysiological concentrations to elicit measurable effects, and LTG showed no enhancement at all.

These findings suggest that, despite CryoEM structural data implying direct binding at GABAA sites, LTG and LEV do not function as positive modulators under physiological conditions. As such, their utility as precision therapies for GABAA related epilepsies may be limited. The study underscores the importance of aligning structural and functional evidence before clinical translation. More broadly, this research advocates for classifying variants by functional effect rather than gene alone and highlights the potential of predictive tools like GENTLY to accelerate this transition. As precision medicine becomes increasingly viable, such approaches will be essential to guide treatment decisions in genetically stratified epilepsies.

Date of Thesis submission	2026
Original language	English
Awarding Institution	Western Sydney University
Sponsors	Australian Government Research Training Program Scholarship
Supervisor	Nathan Absalom (Supervisor)