The GABRG2 F343L allele causes spontaneous seizures in a novel transgenic zebrafish model that can be treated with suberanilohydroxamic acid (SAHA)

Abstract

Background: Mutations in the γ-aminobutyric acid type A (GABA A ) receptor γ2 subunit gene, GABRG2, have been associated frequently with epilepsy syndromes with varying severities. Recently, a de novo GABRG2 mutation, c.T1027C, p.F343L, was identified in a patient with an early onset epileptic encephalopathy (EOEE). In vitro, we demonstrated that GABA A receptors containing the mutant γ2(F343L) subunit have impaired trafficking to the cell surface. Here, we aim to validate an in vivo zebrafish model of EOEE associated with the GABRG2 mutation T1027C.Methods: We generated a novel transgenic zebrafish (AB strain) that overexpressed mutant human γ2(F343L) subunits and provided an initial characterization of the transgenic Tg(hGABRG2 F343L ) zebrafish.Results: Real-time quantitative PCR and in situ hybridization identified a significant up-regulation of c-fos in the mutant transgenic zebrafish, which has a well-established role in epileptogenesis. In the larval stage 5 days postfertilization (dpf), freely swimming Tg(hGABRG2 F343L ) zebrafish displayed spontaneous seizure-like behaviors consisting of whole-body shaking and hyperactivity during automated locomotion video tracking, and seizures can be induced by light stimulation. Using RNA sequencing, we investigated transcriptomic changes due to the presence of mutant γ2L(F343L) subunits and have found 524 genes that are differentially expressed, including up-regulation of 33 genes associated with protein processing. More specifically, protein network analysis indicated histone deacetylases (HDACs) as potential therapeutic targets, and suberanilohydroxamic acid (SAHA), a broad HDACs inhibitor, alleviated seizure-like phenotypes in mutant zebrafish larvae.Conclusions: Overall, our Tg(hGABRG2 F343L ) overexpression zebrafish model provides the first example of a human epilepsy-associated GABRG2 mutation resulting in spontaneous seizures in zebrafish. Moreover, HDAC inhibition may be worth investigating as a therapeutic strategy for genetic epilepsies caused by missense mutations in GABRG2 and possibly in other central nervous system genes that impair surface trafficking.

A novel GABRG2 variant in Sunflower syndrome: A case report and video EEG monitoring

“…Preclinical studies showed that zebrafish overexpressing mutant human GABRG2 (p.(Phe343Leu)) subunits mimicked the human DEE phenotype. 29,30 Moreover, gabrg2 −/− zebrafish mutants are prone to light-induced seizures and respond well to VPA 31 ; mimicking the phenotype and drug response of our patient.…”

“…Most Sunflower syndrome patients have difficult to treat epilepsy, which could also be the case for our patient although longer follow‐up is needed to confirm this constellation. Preclinical studies showed that zebrafish overexpressing mutant human GABRG2 (p.(Phe343Leu)) subunits mimicked the human DEE phenotype 29,30 . Moreover, gabrg2 −/− zebrafish mutants are prone to light‐induced seizures and respond well to VPA 31 ; mimicking the phenotype and drug response of our patient.…”

Differential inflammation responses determine the variable phenotypes of epilepsy induced by GABRG2 mutations

“…To detect the effect of locations of mutations on phenotypes, we compared the locomotive activity of different mutant GABRG2 transgenic zebrafish. In the previous research, we generated F343L and I107T transgenic zebrafish with spontaneous seizures 6,10 . In this research, the distance traveled in 30 min at 5 dpf was compared in the above three transgenic lines and the data showed that all mutant zebrafish traveled more than the WT control group, showing an epileptic phenotype.…”

Identification of microsatellite instability and immune-related prognostic biomarkers in colon adenocarcinoma

“…HOXC9 controls various cellular processes linked to differentiation via activating and repressing the transcription of different gene sets (41). Mutations in GABRG2 have been associated with epilepsy syndromes with varying severities (42). CALB2 is expressed in most poorly differentiated colon carcinomas (43).…”