Objective Pathogenic variants in KCNB1, encoding the voltage‐gated potassium channel KV2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell‐surface expression. Methods We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high‐throughput functional assays. Specifically, we investigated the biophysical properties and cell‐surface expression of variant KV2.1 channels expressed in heterologous cells using high‐throughput automated electrophysiology and immunocytochemistry–flow cytometry. Results Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild‐type KV2.1. Quantification of protein expression also identified variants with reduced total KV2.1 expression or deficient cell‐surface expression. Interpretation Our study establishes a platform for rapid screening of KV2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899–912

Background -The explosive growth in known human gene variation presents enormous

Background The explosive growth in known human gene variation presents enormous challenges to current approaches for variant classification that have implications for diagnosis and treatment of many genetic diseases. For disorders caused by mutations in cardiac ion channels as in congenital arrhythmia syndromes, in vitro electrophysiological evidence has high value in discriminating pathogenic from benign variants, but these data are often lacking because assays are cost-, time- and labor-intensive. Methods We implemented a strategy for performing high throughput, functional evaluations of ion channel variants that repurposed an automated electrophysiological recording platform developed previously for drug discovery. Results We demonstrated success of this approach by evaluating 78 variants in KCNQ1, a major gene involved in genetic disorders of cardiac arrhythmia susceptibility. We benchmarked our results with traditional electrophysiological approaches and observed a high level of concordance. This strategy also enabled studies of dominant-negative behavior of variants exhibiting severe loss-of-function. Overall, our results provided functional data useful for reclassifying more than 65% of the studied KCNQ1 variants. Conclusions Our results illustrate an efficient and high throughput paradigm linking genotype to function for a human cardiac ion channel that will enable data-driven classification of large numbers of variants and create new opportunities for precision medicine.

Aims The goal of this study was to determine the number of scans needed for novice learners to attain proficiency in B-line quantification compared with expert interpretation. Methods and results This was a prospective, multicentre observational study of novice learners, physicians and non-physicians from three academic institutions. Learners received a 2 h lung ultrasound (LUS) training session on B-line assessment, including lecture, video review to practice counting and hands-on patient scanning. Learners quantified B-lines using an eight-zone scanning protocol in patients with suspected acute heart failure. Ultrasound (US) machine settings were standardized to a depth of 18 cm and clip length of 6 s, and tissue harmonics and multibeam former were deactivated. For quantification, the intercostal space with the greatest number of B-lines within each zone was used for scoring. Each zone was given a score of 0-20 based on the maximum number of B-lines counted during one respiratory cycle. The B-line score was determined by multiplying the percentage of the intercostal space filled with B-lines by 20. We compared learner B-line counts with a blinded expert reviewer (five US fellowship-trained faculty with > 5 years of clinical experience) for each lung zone scanned; proficiency was defined as an intraclass correlation of > 0.7. Learning curves for each learner were constructed using cumulative sum method for statistical analysis. The Wilcoxon rank-sum test was used to compare the number of scans required to reach proficiency between different learner types. Twenty-nine learners (21 research associates, 5 residents and 3 non-US-trained emergency medicine faculty) scanned 2629 lung zones with acute pulmonary oedema. After a mean of 10.8 (standard deviation 14.0) LUS zones scanned, learners reached the predefined proficiency standard. The number of scanned zones required to reach proficiency was not significantly different between physicians and non-physicians (P = 0.26), learners with no prior US experience vs. > 25 prior patient scans (P = 0.64) and no prior vs. some prior LUS experience (P = 0.59). The overall intraclass correlation for agreement between learners and experts was 0.74 and 0.80 between experts. Conclusions Our results show that after a short, structured training, novice learners are able to achieve proficiency for quantifying B-lines on LUS after scanning 11 zones. These findings support the use of LUS for B-line quantification by non-physicians in clinical and research applications.

Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV2.1, are associated with developmental and epileptic encephalopathies (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. We evaluated a series of 17 KCNB1 variants associated with DEE or neurodevelopmental disorder (NDD) to rapidly ascertain channel dysfunction using high-throughput functional assays.Specifically, we investigated the biophysical properties and cell-surface expression of variant KV2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage-dependence of activation and/or inactivation, as homotetramers or when co-expressed with wild-type KV2.1. Quantification of protein expression also identified variants with reduced total KV2.1 expression or deficient cell-surface expression.Our study establishes a platform for rapid screening of functional defects of KCNB1 variants associated with DEE and other NDDs, which will aid in establishing KCNB1 variant pathogenicity and may enable discovery of targeted strategies for therapeutic intervention based on molecular phenotype.