Abstracts

Rationale: We previously reported de novo mutations of KCNB1, encoding Kv2.1, as a novel cause of epileptic encephalopathy type 26 (EIEE26) in three unrelated patients. To define the range of mutation effects, we studied a series of six new cases with a molecular diagnosis of EIEE26 ascertained by clinical exome sequencing.Methods: Patients were referred for clinical exome sequencing for epileptic encephalopathy or developmental delay of unknown cause. De-identified clinical summaries and genetic results were collected. To determine the effect of these KCNB1 variants on Kv2.1 channel function, we performed functional studies in a heterologous expression system. KCNB1 variants were introduced into a full-length human Kv2.1 cDNA expression construct by site-directed mutagenesis. Expression of wild-type (WT) or mutant Kv2.1 in CHO-K1 cells was achieved by electroporation. Following 72 hour recovery, automated planar array patch clamp recording was performed. Cell surface biotinylation experiments were performed to determine the relative expression of WT and mutant channel protein at the plasma membrane.Results: Six individuals with de novo heterozygous KCNB1 missense mutations were studied. Most patients had overlapping clinical phenotypes that included multiple seizure types, global developmental delay, intellectual disability, hypotonia and movement difficulties. The effect of KCNB1 variants on Kv2.1 channel function were determined by automated high throughput electrophysiology. Functional analysis of homomeric mutant channels demonstrated impaired Kv2.1 function ranging from severe reduction in current amplitude to altered voltage-dependence of activation and inactivation. Biochemical experiments demonstrated a range of defects, including mild to severe reductions of Kv2.1 protein expression, reduced trafficking to the cell surface, and diminished phosphorylation.Conclusions: EIEE26 presents in infancy with multiple seizure types, developmental delay, hypotonia, and movement disorders. All KCNB1 mutations described to date have arisen de novo. Functional studies demonstrate that KCNB1 mutations associated with epileptic encephalopathy result in a spectrum of effects that result mainly in loss of Kv2.1 function.