Functional characterization and in vitro pharmacological rescue of a novel KCNA2 variant associated with...

Mutations in KCNA2, which encodes Kv1.2, have been reported to be associated with developmental and epileptic encephalopathy (DEE), however, little is known about the underlying mechanisms. Herein, we identified a novel KCNA2 mutation (c.1175C > T, p.S392F) in two unrelated patients with DEE. We further investigated the functional consequences of this mutation by western-blotting, immunocytochemistry, cell death assay, staining as well as patch clamp, and assessed its sensitivity to 4-aminopyridine (4-AP) in the Chinese hamster ovary cells and cortical neurons. Our findings revealed that the p.S392F induced a hyperpolarizing shift both in inactivation and inactivation curves relative to wild-type (WT) channels suggesting it to be a mixed GOF and LOF variant, and was partly rescued by 4- AP. It also induced cell death of which was rescued by 4-AP. Moreover, primary neurons expressing the p.S392F mutation showed a reduction in neuronal firing. The application of 4-AP decreased neuronal firing in S392F transfected neurons. Besides, analysis of the clinical features of eleven patients with mixed GOF and LOF variants from our hospital and literature revealed epilepsy in 100.00 % of the cases, developmental delay/intellectual disability in 100.00 %, speech delay in 100.00 %, and ataxia in 36.36 %, of whom three patients could be partly improved by 4-AP. Thus, this study adds a novel recurrent mixed GOF and LOF variant, new underlying mechanisms and unique genotype-phenotype associations. Moreover, it highlights the therapeutic role of 4-AP.