No abstract

A potential link between GABRD encoding the δ subunit of extrasynaptic GABAA receptors and neurodevelopmental disorders has largely been disregarded due to conflicting conclusions from early studies. However, we identified seven heterozygous missense GABRD variants in 10 patients with neurodevelopmental disorders and generalized epilepsy. One variant occurred in two sibs of healthy parents with presumed somatic mosaicism, another segregated with the disease in three affected family members, and the remaining five occurred de novo in sporadic patients. Electrophysiological measurements were used to determine the functional consequence of the seven missense δ subunit variants in receptor combinations of α1β3δ and α4β2δ GABAA receptors. This was accompanied by analysis of electro-clinical phenotypes of the affected individuals. We determined that five of the seven variants caused altered function of the resulting α1β3δ and α4β2δ GABAA receptors. Surprisingly, four of the five variants led to gain-of-function effects whereas one led to a loss-of-function effect. The stark differences between the gain-of-function and loss-of function effects were mirrored by the clinical phenotypes. Six patients with gain-of-function variants shared common phenotypes: neurodevelopmental disorders with generalized epilepsy, behavioral issues, and various degrees of intellectual disability. Six patients with gain-of-function variants shared common phenotypes: neurodevelopmental disorders with behavioral issues, various degrees of intellectual disability, generalized epilepsy with atypical absences and generalized myoclonic and/or bilateral tonic-clonic seizures. The EEG showed qualitative analogies among the different gain-of-function variant carriers consisting of focal slowing in the occipital regions often preceding irregular generalized epileptiform discharges, with frontal predominance. In contrast, the one patient carrying a loss-of-function variant had normal intelligence, no seizure history but has a diagnosis of autism spectrum disorder and suffering from elevated internalizing psychiatric symptoms. We hypothesize that increase in tonic GABA-evoked current levels mediated by δ-containing extrasynaptic GABAA receptors lead to abnormal neurotransmission, which represent a novel mechanism for severe neurodevelopmental disorders. In support of this, the electro-clinical findings for the gain-of-function GABRD variants resemble the phenotypic spectrum reported in patients with missense SLC6A1 (GABA uptake transporter) variants. This also indicates that the phenomenon of extrasynaptic receptor over-activity is observed in a broader range of patients with neurodevelopmental disorders, since SLC6A1 loss-of-function variants also lead to overactive extrasynaptic δ-containing GABAA receptors. These findings have implications when selecting potential treatment options, since a substantial portion of available anti-seizure medication act by enhancing GABAergic function either directly or indirectly, which could exacerbate symptoms in patients with gain-of-function GABRD variants.

Nicotine acetylcholine receptors can form countless heteromeric stoichiometries from a common set of subunits. Ahring et al. present the limitations of subunit concatenation and establish a refinement that achieves substantiated expression of uniform receptor pools from complex stoichiometric origins.

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.

Subunit concatenation is a powerful technique used to control the assembly of structurally diverse heteromeric receptors such as GABAARs. Liao et al. find that existing GABAAR concatemers do not assemble as expected and describe refinements that allow expression of uniform receptor populations.

Based on our data, we conclude that galantamine is not a positive allosteric modulator of α7 or α4β2 receptors, which represent the majority of nACh receptors in mammalian brain.

Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A (GABAA) receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby GABA has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits GABA transaminase to increase tonic GABA currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different GABAA receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p. Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p. Glu77Lys and p. Thr287Ile variants into a concatenated synaptic and extrasynaptic GABAA receptor construct, to resemble the GABAA receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p. Glu77Lys and p. Thr287Ile variants characterized by increased potency of GABAA without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of GABA that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

Edited by Mike Shipston A number of epilepsy-causing mutations have recently been identified in the genes of the ␣1, ␤3, and ␥2 subunits comprising the ␥-aminobutyric acid type A (GABA A) receptor. These mutations are typically dominant, and in certain cases, such as the ␣1 and ␤3 subunits, they may lead to a mix of receptors at the cell surface that contain no mutant subunits, a single mutated subunit, or two mutated subunits. To determine the effects of mutations in a single subunit or in two subunits on receptor activation, we created a concatenated protein assembly that links all five subunits of the ␣1␤3␥2 receptor and expresses them in the correct orientation. We created nine separate receptor variants with a single-mutant subunit and four receptors containing two subunits of the ␥2 R323Q , ␤3 D120N , ␤3 T157M , ␤3 Y302C , and ␤3 S254F epilepsy-causing mutations. We found that the singly mutated ␥2 R323Q subunit impairs GABA activation of the receptor by reducing GABA potency. A single ␤3 D120N , ␤3 T157M , or ␤3 Y302C mutation also substantially impaired receptor activation, and two copies of these mutants within a receptor were catastrophic. Of note, an effect of the ␤3 S254F mutation on GABA potency depended on the location of this mutant subunit within the receptor, possibly because of the membrane environment surrounding the transmembrane region of the receptor. Our results highlight that precise functional genomic analyses of GABA A receptor mutations using concatenated constructs can identify receptors with an intermediate phenotype that contribute to epileptic phenotypes and that are potential drug targets for precision medicine approaches.