Clinical and functional characterization of the GABRB3 p.Met80Val variant in early-onset epilepsy with long-term follow-up.

Background: GABRB3 encodes the β3 subunit of the GABA receptor, which is a crucial component in inhibitory neurotransmission within the central nervous system. GABRB3 variants are associated with developmental and epileptic encephalopathy 43. Noteworthy, GABRB3 variants can result in both gain-of-function and loss-of-function effects. However, their precise functional and clinical implications remain unknown.

Methods: Whole-exome sequencing, validation of the identified GABRB3 variants using Sanger sequencing, and structural modeling were done to assess the potential impact of these variants on receptor function. Functional analyses included quantification of GABRB3 protein expression levels, subcellular localization using fluorescence microscopy, and electrophysiological recordings of α1β3γ2 and α1β3(M80V)γ2 receptor complexes to evaluate channel properties.

Results: A heterozygous de novo GABRB3 variant (NM_000814.6: c.238 A > G, p.Met80Val) was identified in a 16-year-old female who developed absence seizures at one year and exhibited persistent EEG abnormalities over the subsequent decade. She exhibited mild intellectual disability, poor academic performance, and limited language skills but maintained school attendance and social engagement. Structural modeling suggested that the p.Met80Val variant compromises the structural integrity of the protein. Functional assays revealed a 2.6-fold increase in GABRB3 protein expression and enhanced fluorescence intensity, with most of the protein localized in the cytoplasm. Electrophysiological recordings demonstrated significantly increased current amplitude, heightened GABA sensitivity, and reduced zinc sensitivity. These findings indicated that the p.Met80Val variant altered the receptor conformation or its zinc-binding site, weakening zinc-mediated inhibition.

Conclusion: This study reports the ninth case of a recurrent GABRB3 p.Met80 variant and highlights its potential as a hotspot missense variant. The findings underscore its pathogenicity after a long follow-up period of more than ten years supported by continuous EEG monitoring. These findings enhance our understanding of the functional changes among GABRB3 variants and their role in the pathogenesis of epilepsy.