Abstracts

Rationale: Neuronal KCNQ/Kv7 channels composed of KCNQ2/Kv7.2 and KCNQ3/Kv7.3 subunits are voltage-gated potassium channels that potently inhibit neuronal excitability. Close to 200 mutations in Kcnq2 and Kcnq3 genes are associated with neonatal epilepsy in humans including self-limiting Benign Familial Neonatal Epilepsy (BFNE) and Epileptic Encephalopathy (EE) characterized by refractory seizures and developmental delay with severe comorbidities psychomotor retardation (rikee.org). However, it remains unknown whether these mutations cluster at specific protein domains within the coding region of Kv7.2, and whether such specific distribution correlates with clinical severity and disruption of the functional domains. Methods: In this study, we used nonrandom mutation clustering (NMC) and resampling algorithms on missense and single amino acid epilepsy mutations in Kv7.2 to statistically identify mutation hotspots. To better visualize the location of pathogenic mutations, structural modeling was performed based on cryo-EM structure of Kv7.1. Results: Statistical analyses and structural modeling revealed that pathogenic EE variants of K_v7.2 are significantly clustered at the voltage-sensing domain S4 and the central pore conduction domains including the pore loop and S6.  In the intracellular C-terminal tail of K_v7.2, these mutations are also enriched at helix B that mediates calmodulin (CaM) binding, and helix B-C linker. In contrast, pathogenic variants of K_v7.3 mostly cause BFNE and cluster in the pore. We characterized the effects of 4 missense EE mutations located in mutation hotspots. Despite their localization in distinct protein domains within K_v7.2, our functional and expression characterization revealed that reductions in PIP₂ sensitivity and axonal surface expression of heteromeric K_v7.2/ K_v7.3 channels serve as the major pathogenetic mechanisms underlying EE. Conclusions: Our study identified five critical functional domains as mutation hotspots and discovered major pathogenic mechanisms underlying previously uncharacterized EE mutations in K_v7.2. Current efforts include statistical and functional analyses of K_v7.2 mutations that cause neurodevelopmental delay (NDD). Our studies are significant because such genotype-phenotype correlations will help develop mutation-specific therapeutic strategies for EE and NDD. Funding: This research is funded by NIH RO1 Grant (NS083402, PI: Chung) and Epilepsy Foundation Grant (C4107, PI: Chung).