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case_images: Interictal EEG findings. Slow background activity with generalized multifocal spikes (blue box), multi-spikes (red arrow), and epileptoid waves.
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case_images: Video electroencephalography (EEG) demonstrated a slow background activity with generalized multifocal spikes, multi-spikes, and fast waves, which was accentuated during sleep (Figure 1).
case_images: Molecular findings in the epilepsy family. (A) Sanger sequencing confirmed a heterozygous variation in the GABRA2 gene (NM_000807: c.923C>T, p.Ala308Val) in the proband, while both parents exhibit the wild-type allele. (B) The GABRA2 variant Ala308Val is positioned in the extracellular region between M2 and M3 (indicated by the red arrow), whereas previously reported variants are primarily located in the transmembrane region (indicated by green circles).
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case_images: This variant was confirmed to be de novo through Sanger sequencing, as it was absent in both healthy parents (Figure 2A). Additionally, this variant is located in proximity to previously reported GABRA2 gene variants (Figure 2B). This variant has not been previously reported, thus categorizing it as a novel variant (Figure 2B).
case_images: Analysis of GABAAR pentameric structure. (A) A pentameric model featuring 2 alpha2 subunits (GABRA2), 2 beta3 subunits (GABRB3), and 1 gamma2 subunit (GABRG2). (B) The structure is rotated 90 degrees for a top-down view. In the wild-type GABRA2, Ala308 forms hydrogen bonds with Pro209 of GABRB3 and with another GABRA2 protein at position 238 of GABRG2. (C) In the mutated GABRA2 (p.Ala308Val), the longer valine side chain disrupts these hydrogen bonds, potentially altering structural stability.
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case_images: Given that inter-subunit interactions are critical for maintaining the integrity of the pentameric receptor complex, the p.Ala308Val variant may compromise the proper assembly and structural stability of the GABAAR (Figure 3).
case_images: Subcellular localization of the GABRA2 Variant in 293T Cells. GABRA2 with FLAG-tag (green), calnexin antibody (red, ER marker), and DAPI (blue, nuclear counterstaining). Confocal microscopy indicated that both wild-type and variant proteins localized to ER vesicles, but the variant showed reduced expression and weaker fluorescence. Normalized relative fluorescence intensity analysis revealed significant differences between GABRA2-WT and GABRA2-MUT groups, ***P < 0.001.
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case_images: Nevertheless, a notable reduction in expression levels was observed, with the fluorescence intensity of GABRA2-MUT being approximately 44.4% lower than that of GABRA2-WT (Figure 4).
case_images: In vitro functional assay. (A) Western blotting analysis indicated that the GABRA2-MUT group exhibited protein expression, but it was significantly lower than the GABRA2-WT group, with a reduction of approximately 70.62% (***P < 0.001). (B) CO-IP results showed that wild-type GABRA2 interacted with GABRB3/GABRG2, while the mutant may disrupt this interaction. The right panel presents normalized CO-IP analysis, ***P < 0.001.
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case_images: T cells that were transfected with either wild-type (GABRA2-WT) or mutant (GABRA2-MUT) expression constructs. The immunoblotting results confirmed protein expression for both variants; however, the GABRA2-MUT group exhibited a significant reduction in expression, approximately 70.62% lower than that of the GABRA2-WT group (Figure 5A). In contrast, the GABRA2-MUT markedly impaired this interaction (Figure 5B).