DOI: 10.5061/dryad.jq2bvq8k2
title: A novel loss-of-function KCNB1 gene variant in a twin with global developmental delay and seizures

description: 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&gt;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.
description: <strong><em>Preparation of channel subunit cRNA
 preparation and Xenopus laevis oocyte
 injection </em></strong> cDNA encoding
 human <em>KCNB1 </em>was sub-cloned into a
 <em>Xenopus</em> expression vector (pMAX) incorporating
 <em>Xenopus laevis</em> β-globin 5’ and 3’ UTRs flanking the
 coding region to enhance translation and cRNA stability by Genscript
 (Piscataway, NJ, USA). Mutant <em>KCNB1 </em>constructs were
 generated by Genscript and subcloned into pMAX as above. cRNA transcripts
 were generated by <em>in vitro</em> 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 <em>Xenopus laevis</em>
 oocytes (Xenoocyte, Dexter, MI, USA) were injected with the channel cRNAs
 (0.2-2 ng) and incubated at 16 <sup>o</sup>C in Barth’s
 solution containing penicillin and streptomycin, with daily washing, prior
 to two-electrode voltage-clamp (TEVC) recording.
 <strong><em>Two-electrode voltage clamp (TEVC)
 </em></strong> 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
 MgCl<sub>2</sub>, 0.3 CaCl<sub>2</sub>, 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.  <strong><em>Statistics and
 Reproducibility</em></strong> 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
 <em>p</em> values were two-sided.
description: ## Source Data for manuscript: A novel loss-of-function KCNB1 gene variant
 in a twin with global developmental delay and seizures The datasets
 included are the original Excel files used to generate each panel for
 figures 2-4 in this manuscript. The title of each Excel file is labeled to
 directly correspond to the figure in the manuscript: Figure Number &
 panel &gt; Channel Investigated &gt; Condition &gt; Parameter Measured ---
 The data contained within this repository are those obtained from cellular
 electrophysiology recordings. We used two-electrode voltage clamp (TEVC)
 electrophysiology and the Xenopus laevis oocyte expression system to
 record the electrical activity of wild-type Kv2.1 channels and mutant
 Kv2.1 channels (P385L and P385T) identified by genetic screening of a
 patient exhibiting global developmental delay and seizures. Xenopus laevis
 oocytes were injected with cRNA encoding for Kv2.1, Kv2.1-P385L, and
 Kv2.1-P385T and were incubated at 16 degrees for 24 hrs prior to recording
 using TEVC. These measurements allow us to characterize the biophysical
 properties of the mutants P385L and P385T in order to better understand
 how these mutations may alter the physiology of the patient and provide an
 underlying rationale for their condition. The parameters that we measured
 to characterize the biophysical properties of the Kv2.1 mutants P385L and
 P385T are as follows: &gt; Current-voltage (IV) curve &gt; This is a graph
 representing the relationship between the electrical current (flow of
 ions) and voltage applied across a device (the cell membrane). In
 electrophysiology, I-V curves are used to study the activity of biological
 cells, in this case Xenopus oocytes expressing wild-type and mutant Kv2.1
 channels. The data contained in the I-V curve excel files were measured
 from the peak of the prepulse current generated by a voltage protocol that
 starts at -80 mV and increases in +10 mV increments until +40 mV. All raw
 values are in microamps (uA). &gt; Normalized current-voltage (IV) curve
 &gt; This graph also represents the relationship between the electrical
 current and the voltage applied across a device. However, rather than
 plotting the raw data values for an I-V curve, we normalized the values to
 the peak current value obtained at +40 mV. Raw data values are in
 microamps (uA). &gt; Resting membrane potential &gt; The resting membrane
 potential (RMP) is the electrical potential difference across a
 cell's membrane at rest. The RMP is determined by the concentration
 of ions across the membrane and the membrane permeability to each type of
 ion. Here, we measured the RMP (EM) of unclamped Xenopus laevis oocytes
 expressing Kv2.1, P385L, or P385T channels and reported the values in
 millivolts (mV). &gt; Current density comparisons &gt; These graphs were
 generated by taking the peak current values obtained at +40 mV from the
 I-V curves generated for Kv2.1, Kv2.1-P385L, or Kv2.1-P385T channels.
 These values were then compared using a dot-plot graph in order to compare
 what effect the mutations Kv2.1-P385L and Kv2.1-P385T had on the ability
 of the channel to generate currents compared to wild-type Kv2.1. All
 values are in microamps (uA). &gt; Statistics &gt; All statistical
 analysis were conducted as either paired t-test or one-way ANOVA with
 Dunnett's correction for multiple comparison. &gt; Additional
 Information &gt; Excel files with cells with 'n.a' means not
 applicable. No data was obtained for this cell.