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Variable phenotypes, including developmental encephalopathy with (DEE) or without seizures and myoclonic epilepsy and ataxia due to potassium channel mutation, are caused by pathogenetic variants in KCNC1, encoding for Kv3.1 channel subunits. In vitro, channels carrying most KCNC1 pathogenic variants display loss-of-function features. Here, we describe a child affected by DEE with fever-triggered seizures, caused by a novel de novo heterozygous missense KCNC1 variant (c.1273G>A; V425M). Patch-clamp recordings in transiently transfected CHO cells revealed that, compared to wild-type, Kv3.1 V425M currents (1) were larger, with membrane potentials between −40 and +40 mV; (2) displayed a hyperpolarizing shift in activation gating; (3) failed to inactivate; and (4) had slower activation and deactivation kinetics, consistent with a mixed functional pattern with prevalent gain-of-function effects. Exposure to the antidepressant drug fluoxetine inhibited currents expressed by both wild-type and mutant Kv3.1 channels. Treatment of the proband with fluoxetine led to a rapid and prolonged clinical amelioration, with the disappearance of seizures and an improvement in balance, gross motor skills, and oculomotor coordination. These results suggest that drug repurposing based on the specific genetic defect may provide an effective personalized treatment for KCNC1-related DEEs.

Objective Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. Methods Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. Results The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. Interpretation We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype–phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023, Objective: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. Methods: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. Results: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. Interpretation: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype–phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023.

KCNQ2-encoded Kv7.2 subunits play a critical role in balancing neuronal excitability. Mutations in KCNQ2 are responsible for highly-heterogenous epileptic and neurodevelopmental phenotypes ranging from self-limited familial neonatal epilepsy (SeLFNE) to severe developmental and epileptic encephalopathy (DEE). Pathogenic KCNQ2 variants cluster at the voltage sensor domain (VSD), the pore domain, and the C-terminal tail. Although several knock-in mice harboring Kcnq2 pore variants have been developed, no mouse line carrying Kcnq2 voltage-sensor mutations has been described. KCNQ2-R207W is an epilepsy-causing mutation located in the VSD, mainly affecting voltage-dependent channel gating. To study the physiological consequence of Kcnq2 VSD dysfunction, we generated a Kcnq2-R207W mouse line and analyzed the pathological and pharmacological phenotypes of mutant mice. As a result, both homozygous (Kcnq2RW/RW) and heterozygous (Kcnq2RW/+) mice were viable. While Kcnq2RW/RW mice displayed a short lifespan, growth retardation, and spontaneous seizures, Kcnq2RW/+ mice survived and developed normally, although only a fraction (9/64; 14%) of them showed behavioral-and ECoG-confirmed spontaneous seizures. Kcnq2RW/+ mice displayed increased susceptibility to evoked seizures, which was dramatically ameliorated by treatment with the novel KCNQ opener pynegabine (HN37). Our results show that the Kcnq2-R207W mouse line, the first harboring a Kcnq2 voltage-sensor muta-tion, exhibits a unique epileptic phenotype with both spontaneous seizures and increased susceptibility to evoked seizures. In Kcnq2-R207W mice, the potent KCNQ opener HN37, currently in clinical phase I, shows strong anticonvulsant activity, suggesting it may represent a valuable option for the severe phenotypes of KCNQ2-related epilepsy.