Objective The Epi4K consortium recently identified four de novo mutations in the γ-aminobutyric acid type A (GABAA) receptor β3 subunit gene GABRB3 and one in the β1 subunit gene GABRB1 in children with epileptic encephalopathies (EEs) Lennox-Gastaut syndrome (LGS) or infantile spasms (IS). Since the etiology of EEs is often unknown, we determined the impact of GABRB mutations on GABAA receptor function and biogenesis. Methods GABAA receptor α1 and γ2L subunits were co-expressed with wild-type and/or mutant β3 or β1 subunits in HEK 293T cells. Currents were measured using whole cell and single channel patch clamp techniques. Surface and total expression levels were measured using flow cytometry. Potential structural perturbations in mutant GABAA receptors were explored using structural modeling. Results LGS-associated GABRB3(D120N, E180G, Y302C) mutations located at β+ subunit interfaces reduced whole cell currents by decreasing single channel open probability without loss of surface receptors. In contrast, IS-associated GABRB3(N110D) and GABRB1(F246S) mutations at β-subunit interfaces produced minor changes in whole cell current peak amplitude but altered current deactivation by decreasing or increasing single channel burst duration, respectively. GABRB3(E180G) and GABRB1(F246S) mutations also produced spontaneous channel openings. Interpretation All five de novo GABRB mutations impaired GABAA receptor function by rearranging conserved structural domains, supporting their role in EEs. The primary effect of LGS-associated mutations was reduced GABA-evoked peak current amplitudes while the major impact of IS-associated mutations was on current kinetic properties. Despite lack of association with epilepsy syndromes, our results suggest GABRB1 as a candidate human epilepsy gene.

Summary Objective The mutant γ-aminobutyric acid type A (GABAA) receptor γ2(Q390X) subunit, (Q351X in the mature peptide) has been associated with the epileptic encephalopathy, Dravet syndrome, and the epilepsy syndrome genetic epilepsy with febrile seizures plus (GEFS+). The mutation generates a premature stop codon that results in translation of a stable truncated and misfolded γ2 subunit that accumulates in neurons, forms intracellular aggregates, disrupts incorporation of γ2 subunits into GABAA receptors and affects trafficking of partnering α and β subunits. Heterozygous Gabrg2+/Q390X knock-in (KI) mice had reduced cortical inhibition, spike wave discharges on EEG, a lower seizure threshold to the convulsant drug pentylenetetrazol (PTZ) and spontaneous generalized tonic-clonic seizures. In this proof of principal study, we attempted to rescue these deficits in KI mice using a γ2 subunit gene (GABRG2) replacement therapy. Methods We introduced the GABRG2 allele by crossing Gabrg2+/Q390X KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin) tagged human γ2HA subunits and compared GABAA receptor subunit expression by western blot and immunohistochemical staining, seizure threshold by monitoring mouse behavior after PTZ-injection, and thalamocortical inhibition and network oscillation by slice recording. Results Compared to KI mice, adult mice carrying both mutant allele and transgene had increased wild-type γ2 and partnering α1 and β2/3 subunits, increased miniature inhibitory postsynaptic current (mIPSC) amplitudes recorded from layer VI cortical neurons, reduced thalamocortical network oscillations, and higher PTZ seizure threshold. Significance Based on these results we suggest that seizures in a genetic epilepsy syndrome caused by epilepsy mutant γ2(Q390X) subunits with dominant negative effects could be rescued potentially by overexpression of wild-type γ2 subunits.

Reduced ocular pigmentation is common in Angelman syndrome (AS) and Prader-Willi syndrome (PWS) and long thought to be caused by OCA2 deletion. GABRB3 is located in the 15q11-13 region flanked by UBE3A, GABRA5, GABRG3 and OCA2. Mutations in GABRB3 have been frequently associated with epilepsy and autism, consistent with its role in neurodevelopment. We report here a robust phenotype in the mouse in which deletion of Gabrb3 alone causes nearly complete loss of retinal pigmentation due to atrophied melanosomes, as evidenced by electron microscopy. Using exome and RNA sequencing, we confirmed that only the Gabrb3 gene was disrupted while the Oca2 gene was intact. However, mRNA abundance of Oca2 and other genes adjacent to Gabrb3 is substantially reduced in Gabrb3−/− mice, suggesting complex transcriptional regulation in this region. These results suggest that impairment in GABRB3 downregulates OCA2 and indirectly causes ocular hypopigmentation and visual defects in AS and PWS.

We thank Lien and colleagues for their letter in response to our recent publication 1 describing a patient with epileptic encephalopathy harboring a novel de novo GABRB1 mutation, T287I. The phenotypes of this patient are similar to those of the patient with the epileptic encephalopathy infantile spasms, and the GABRB1 (F246S) mutation, that was reported by the Epi4K consortium in 2013. 2 In this article, the GABRB1 mutation was found in only the 1 patient, and GABRB1 had not been previously associated with epilepsy. Thus, it was not considered to be a confirmed cause of epilepsy. Our recent publication, 1 however, reported that the GABRB1 (F246S) mutation disrupted GABA A receptor function, suggesting a likely contribution to epilepsy pathogenesis. The current report by Lien et al extends our findings and further strengthens the involvement of GABRB1 in epilepsy syndromes. The GABRB1 (T287I) mutation alters a highly conserved amino acid residue located in the channel pore forming the transmembrane 2 domain of the b1 subunit, and thus, it is likely to disrupt GABA A receptor function. Moreover, the polyphen-2 score of 0.999 for the GABRB1 (T287I) mutation also suggests that this mutation is likely to be damaging. Mutations in GABRB3 and their contribution to epilepsy syndromes are well documented. 3As b1 and b3 subunits are highly homologous and are ubiquitously expressed in the regions involved with generation of seizures such as cortex, hippocampus, and thalamus, it can be anticipated that future studies will reveal additional mutations in GABRB1 in patients with epilepsy.