Abstract
Absalom NL, Liao VW, Johannesen KM, Gardella E, Jacobs J, Lesca G, Gokce-Samar Z, Arzimanoglou A, Zeidler S, Striano P, Meyer P, Benkel-Herrenbrueck I, Mero I-L, Rummel J, Chebib M, Møller RS, Ahring PK. Nat Commun. 2022;13(1):1822. doi:10.1038/s41467-022-29280-x Many patients with developmental and epileptic encephalopathies present with variants in genes coding for GABAA receptors. These variants are presumed to cause loss-of-function receptors leading to reduced neuronal GABAergic activity. Yet, patients with GABAA receptor variants have diverse clinical phenotypes and many are refractory to treatment despite the availability of drugs that enhance GABAergic activity. Here we show that 44 pathogenic GABRB3 missense variants segregate into gain-of-function and loss-of-function groups and respective patients display distinct clinical phenotypes. The gain-of-function cohort (n = 27 patients) presented with a younger age of seizure onset, higher risk of severe intellectual disability, focal seizures at onset, hypotonia, and lower likelihood of seizure freedom in response to treatment. Febrile seizures at onset are exclusive to the loss-of-function cohort (n = 47 patients). Overall, patients with GABRB3 variants that increase GABAergic activity have more severe developmental and epileptic encephalopathies. This paradoxical finding challenges our current understanding of the GABAergic system in epilepsy and how patients should be treated.
Commentary
GABAA receptors (GABAARs) are the predominant inhibitory ligand-gated ion channels in the brain and thus it is unsurprising that numerous GABAAR variants have been associated with epilepsy syndromes. 1 GABAARs are pentamers and the 5 subunits can arise from 19 polypeptides (α1–6, β1–3, γ1–3, δ, ε, θ, π, ρ1-3). The most common postsynaptic GABAAR are composed of two α subunits, two β subunits, and one γ subunit arranged counterclockwise as γ-β-α-β-α when viewed extracellularly. Numerous variants of the gene that encodes the β3 subunit (GABRB3) have been found in epilepsy patients. Mice with heterozygous Gabrb3 deletion exhibit absence seizures 2 and thus it was expected that patients with GABRB3 variants would present with relatively mild idiopathic generalized epilepsy (IGE) syndromes. However, the GABRB3 variant epilepsy phenotypes are diverse 3 -5 and include both IGE syndromes as well as developmental and/or epileptic encephalopathies, pediatric conditions in which patients exhibit frequent epileptiform activity as well as developmental slowing or regression. 6 Why would GABRB3 variants produce severe epilepsy syndromes if heterozygous deletion produced only absence seizures? Previously, single-channel electrophysiology studies of recombinantly expressed GABAARs containing β3 subunits arising from two different genetic epileptic encephalopathy patients found that the mutations increased GABAAR channel opening probabilities as well as open channel times and burst duration times, that is, the variants increased GABAAR function and thus produced “gain of function” (GOF) effects. 7 Possibly, GOF β3 subunit mutations could produce more severe epilepsy phenotypes than loss-of-function (LOF).
In this study, Absalom et al performed in vitro functional studies in Xenopus oocytes comparing recombinant wild type α1β3γ2 GABAARs with those in which one of the β3 subunits harbored one of 54 GABRB3 epilepsy variants. 8 Rather than expressing 3 separate cRNAs encoding α1, β3, and γ2 subunits, they used a single cRNA that encoded all 5 subunits concatenated together with short (15-27 amino acids) peptide linkers between the subunits. Because most GABAARs have two α subunits and two β subunits, the use of concatenated subunits for characterizing heterozygous α or β subunit GABAAR epilepsy mutations allows expression of GABAARs of a defined stoichiometry with the mutation targeted to a specific α or β subunit in the pentamer, while leaving the other α or β subunit wild type. 9 Here, the β3 mutation was always targeted to the β3 subunit between the γ2 and α1 subunit, while the β3 between the two α1 subunits was wild type.
The authors performed patch clamp electrophysiology recordings and measured the GABA concentrations that evoked half maximal GABA-evoked currents (EC50). Twenty-four variants produced increased EC50 or reduced maximal currents and thus were classified as LOF and 20 variants produced reduced EC50 or increased maximal currents and thus were classified as GOF.
It was remarkable that this study found that such a high percentage (81%) of variants produced significant changes EC50. and could thus be dichotomized as LOF or GOF. This high positive rate with the functional assay allowed the authors to determine whether the GABRB3 variants’ GOF/LOF classification correlated with the patients’ clinical symptoms. They found that GOF/LOF designation was highly predictive of several clinical features with the GOF variants conferring more severe phenotypes. Patients with GOF variants exhibited their first seizure much earlier (2.5 months, confidence interval [CI] = 1.6-3.0) compared with LOF variants (10.5 months, CI = 9-15) months. Moreover, severe intellectual disability occurred in 94% of patients with GOF mutations and only 18% of patients with LOF variants (Odds ratio = 80; CI = 10-856). Patients with GOF variants were also significantly more likely to experience focal seizures, hypotonia, microcephaly, and intractable seizures than those with LOF variants. It would also have been of interest if the authors determined if a quantifiable measurement of GOF (i.e., EC50) correlated with any of the continuous outcome measurements (e.g., age of onset) to reveal if there is a “dose–response” relationship between GOF and outcome.
Although two GABRB3 GOF variants had previously been linked to severe epileptic encephalopathy syndromes, 7 this paper is very important in that it screened such a large number of variants, successfully dichotomized the majority (81%) of them as LOF/GOF, and established a striking linkage between LOF/GOF classification with clinical outcome with the GOF group exhibiting worse clinical features. Future studies of course would be useful to provide more in vitro characterization of the variants. Would the same LOF/GOF characterization persist if the variants were expressed in the β3 subunit between the two α1 subunits, instead of between the γ2 and α1 subunits? It would also be informative to determine the effects of the variants on GABAAR physiology and protein expression using subunits expressed as separate polypeptides rather than in a concatemer. Although most missense variants do not alter GABAAR subunit expression, 10 variants that introduce charged residues within a transmembrane domain can prevent proper protein folding 11 and this effect may be masked when they are incorporated in a large concatenated GABAAR. 9 Moreover, it is important to learn the effects of the variant β3 subunits coexpressed with α4 and δ subunits as is typical for extrasynaptic receptors as well as with the α3 subunit given its prominence early in development. Finally, future studies should evaluate the time course of GABAAR current kinetics using a methodology with fast solution exchange to model synaptic-like conditions.
Aside from molecular characterization, it is critical to determine the mechanisms by which the GOF variants worsen clinical outcome and whether any therapeutic strategies may mitigate it. It has been established that enhanced tonic inhibition in thalamic relay nuclei worsens generalized seizures. 12,13 If GABRB3 variants produced seizures by increasing tonic inhibition in the relay nuclei, therapeutic thalamic stimulation protocols that reduce inhibition may improve seizures. However, increased thalamic inhibition may not be the most likely mechanism by which GOF worsen outcome, at least after the neonatal/infancy periods. While β3 subunit is expressed in prenatal thalamus, it has very low expression in thalamus later in development.
The strong expression of β3 subunit early in ontogeny could suggest that the GOF variants produce changes in brain structure and connectivity in prenatal/early postnatal life that then persist as the patient ages. In the developing brain, GABAAR activation modulates neuron progenitor cell proliferation, migration, and morphological maturation, 14 and thus all these processes could be altered by GOF GABRB3 variants. Because structural/connectivity changes due to GABRB3 GOF variants may occur early in development when β3 subunit expression is high and then persist even when β3 subunit expression is downregulated, therapeutic strategies that decrease GABAergic inhibition after these structural changes occur (early postnatal life) may not be successful; early identification of neonates with possible GOF variants would be crucial.
In summary, Absalom et al report a very important study due to its functional screening of many GABRB3 epilepsy-associated variants, the successful classification of them as LOF and GOF, and the elucidation of the striking relationship between GOF classification and poor clinical outcome. Future studies will undoubtedly further reveal the biophysical and cell trafficking characteristics of these variants and, importantly, elucidate their neuropathophysiological mechanisms to help us reduce the pain of these severe epileptic encephalopathies.
