Human voltage-gated potassium (Kv) channels are expressed by a 40-member family of genes essential for normal electrical activity and with numerous associations and linkages to excitability disorders. Function-altering sequence variants in the KCNB1 gene, which encodes the neuronally expressed Kv2.1 channel, are associated with neurodevelopmental disorders including developmental delay with or without epileptic activity. Here, we describe a 40-month-old fraternal twin who presented with severe neurodevelopmental delay. Electroencephalogram recordings at 19 months of age revealed poor sleep architecture and the presence of multifocal epileptiform discharges. The individual’s fraternal twin was neurotypical and there was no family history of neurodevelopmental delay or seizures. Whole genome sequencing at 33 months of age for the proband revealed a de novo variant in KCNB1 [c.1154C>T/p.Pro385Leu], encoding a proline to leucine substitution at residue 385, in the extracellular region immediately preceding Kv2.1 transmembrane segment 6 (S6). Cellular electrophysiological analysis of the effects of the gene variant in heterologously expressed Kv2.1 demonstrated that homozygous Kv2.1-P385L channels were completely nonfunctional. Channels generated by 50/50 expression of wild-type Kv2.1 and Kv2.1-P385L to mimic the heterozygous status of the proband revealed a partially dominant-negative, 81% reduction in current magnitude. The dramatic loss of function in Kv2.1 is the most likely cause of the severe developmental delay and seizure activity in the proband, further enriching our phenotypic understanding of KCNB1 developmental encephalopathies.

Preparation of channel subunit cRNA preparation and Xenopus laevis oocyte injection 

cDNA encoding human KCNB1 was sub-cloned into a Xenopus expression vector (pMAX) incorporating Xenopus laevis β-globin 5’ and 3’ UTRs flanking the coding region to enhance translation and cRNA stability by Genscript (Piscataway, NJ, USA). Mutant KCNB1 constructs were generated by Genscript and subcloned into pMAX as above. cRNA transcripts were generated by in vitro transcription using the T7 mMessage mMachine kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions, after vector linearization with PacI. Stage V and VI defolliculated Xenopus laevis oocytes (Xenoocyte, Dexter, MI, USA) were injected with the channel cRNAs (0.2-2 ng) and incubated at 16 ^oC in Barth’s solution containing penicillin and streptomycin, with daily washing, prior to two-electrode voltage-clamp (TEVC) recording.

Two-electrode voltage clamp (TEVC)

TEVC was conducted at room temperature with an OC-725C amplifier (Warner Instruments, Hamden, CT, USA) and pClamp10 software (Molecular Devices, Sunnyvale, CA, USA) 24 hours after cRNA injection. Oocytes, in a small-volume oocyte bath (Warner), were viewed with a dissection microscope for cellular electrophysiology. Extracellular bath solution (in mM): 96 NaCl, 4 KCl, 1 MgCl₂, 0.3 CaCl₂, and 10 HEPES, adjusted to pH 7.6 with TRIS BASE. Solutions were introduced into the oocyte recording bath by gravity perfusion at a constant flow of 1 ml per minute.  Pipettes (1-2 MΩ resistance) were filled with 3 M KCl.  Current-voltage graphs were measured in response to voltage pulses between -80 mV and +40 mV at 10 mV intervals from a holding potential of -80 mV. 

Statistics and Reproducibility

All values are expressed as mean ± SEM.  At least 2 batches of oocytes were used per experiment. Multiple comparison statistics were conducted using a One-way ANOVA. Comparison of two groups was conducted using a t-test; all p values were two-sided.