KCNT2-related disorders: phenotypes, functional and pharmacological properties

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 fourteen novel or previously untested variants. Methods: 25 patients harbouring 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 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 (nine new, ten reported previously): 16 missense, one in-frame deletion of a single amino acid, one nonsense, and one frameshift. Among tested variants, eight showed gain-of-function (GoF), and six 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