Gain-of-function and loss-of-function GABRB3 variants lead to distinct clinical phenotypes in patients with developmental and epileptic encephalopathies

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.


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Sample size As this is a rare disease, the sample size for clinical parameters were determined solely by the availability of patients with epilepsy and a known variant in the GABRB3 gene. For logEC S0 analysis, a power calculation was performed with the mean and standard deviation of a wild-type dataset, and a logEC S0 shift of 0.2 that was estimated from previous mutant analysis (e.g. Absalom et al, Brain Comm, 2021). A minimum sample size of 9 was determined, and we therefore rounded up to 10 for variants, and a minimum wild-type value of n(wt)=n(var)*sqrt(number of variants), as suggested for the Dunnett's post-hoc test (72). As wild-type experiments were run concurrently with variants, this value was exceeded.
A similar calculation for maximum currents with a change of 0.5 estimated a minimum sample size of 14. However, as a non-parametric test were used this value is not necessarily accurate, hence we increased the minimum value to 20.
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Researchers performing the initial data analysis had access to the identity of each variant, but not the clinical information until experiments were complete.

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'l 1 We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Xenopus laevis females of 1-5 years of age were used solely to extract tissue (i.e. oocytes) -no animal experimentation was J performed.

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This study did not involve wild animals J Field-collected samples This study did not involve field samples. ) For electrophysiology experiments, data were excluded where the holding current were significantly altered during the experiment, the initial three applications of control did not yield consistent current or excessive run up or run down of currents from the same GABA concentration were observed.
A minimum of two batches of oocytes on two separate recording days were used for each experiment and similar numbers of experiments were taken from each run.
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A protocol for Xenopus laevis extraction was approved by the Animal Ethics Committee of The University of Sydney (AEC No. l 2016/970) in accordance with the National Health and Medical Research Council of Australia code for the care and use of animals.
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Data collection

Outcomes
The data was not collected as part of a clinical trial. This is a retrospective descriptive study and not part of a clinical trial. For 74 patients, data were collected from previous publications. For the 11 patients with unpublished data, GABRB3 variants were collected through collaborations with epilepsy and genetic centres in Europe and Canada in some cases facilitated via GeneMatcher.