Abstracts

Rationale: Gamma-aminobutyric acid (GABA) acts as the principal inhibitory neurotransmitter in the central nervous system, mediating its activity mainly via GABA_A receptors composed of different subunits. A wealth of evidence has identified genetic variations in these receptors as responsible for different types of epilepsy, including the GABRB2, GABRB3 and GABRD genes that encode for the β2, β3 and δ subunits. The GABRA6 gene encodes for the α6 subunit that combines with β2 or β3, and δ subunits to form GABA_A receptors. As genes encoding other proteins in the receptor complex have been associated with childhood epilepsies, GABRA6 variants have also reported to be pathogenic.


Methods: In this study, we evaluated whether GABRA6 variants are likely to be associated with epilepsy. We conducted a comparative analysis of benign variant (gnomAD v4) distribution in key functional domains across different GABA_A receptor genes. We then chose several variants based on their allele frequency in gnomad, homology to pathogenic variants on other subunits and previous reports of pathogenic variants. We then injected RNA encoding wild-type or variant α6 with β2 and γ2, or β2 and δ subunits in a 20:1:20 ratio into Xenopus oocytes, and measured concentration-response curves to GABA and estimated the maximum open probabilities of GABA. We then compared the population frequencies to estimated disease prevalence to determine the validity of an association with developmental and epileptic encephalopathies.

Results: Benign variants within the GABRA6 gene are evenly distributed even across key structural regions, unlike other epilepsy-associated GABA_A receptor genes. This suggest a tolerance for genetic variation that does not correlate with severe epileptic phenotypes. We then evaluated the functional outcomes of benign and proposed epilepsy-associated GABRA6 variants with two-electrode voltage clamp electrophysiology. Both loss and gain of function variants were identified, with a maximum 12-fold increase and 3-fold decrease in the logEC₅₀ at α6β2γ2 receptors, and a maximum 10-fold reduction and 6-fold increase in maximum open probabilities. While the allele count of loss-of-function variants exceeded the maximum credible allele count, the allele count of gain-of-function variants did not exceed the maximum count.


Conclusions: These results support the null hypothesis that loss-of-function GABRA6 variants are not associated with childhood epilepsy. However, we cannot rule out a role for gain-of-function GABRA6 variants, consistent with their association with more severe epilepsies in other subunits.

Funding: NHMRC Ideas Grant.