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High throughput Characterization of KCNB1 variants Associated with Developmental and Epileptic Encephalopathy

Seok Kyu Kang, Carlos G. Vanoye, Sunita N. Misra, Dennis M. Echevarria, Jeffrey D. Calhoun, John B. O’Connor, Katarina L. Fabre, Dianalee McKnight, Laurie Demmer, Paula Goldenberg, Lauren E. Grote, Isabelle Thiffault, Carol Saunders, Kevin A. Strauss, Ali Torkamani, Jasper van der Smagt, Koen van Gassen, Robert P. Carson, Jullianne Diaz, Eyby Leon, Joseph E. Jacher, Mark C. Hannibal, Jessica Litwin, Neil R. Friedman, Allison Schreiber, Bryan Lynch, Annapurna Poduri, Eric D. Marsh, Ethan M. Goldberg, John J. Millichap, Alfred L. George Jr., Jennifer A. Kearney
doi: https://doi.org/10.1101/637041
Seok Kyu Kang
1Departments of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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Carlos G. Vanoye
1Departments of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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Sunita N. Misra
1Departments of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
2Departments of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
3Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL 60611, USA
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Dennis M. Echevarria
1Departments of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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Jeffrey D. Calhoun
1Departments of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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John B. O’Connor
3Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL 60611, USA
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Katarina L. Fabre
1Departments of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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Dianalee McKnight
4GeneDX, Gaithersburg, MD 20877, USA
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Laurie Demmer
5Department of Pediatrics, Atrium Health’s Levine Children’s Hospital, Charlotte, North Carolina 28232, USA
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Paula Goldenberg
6Medical Genetics, Massachusetts General Hospital for Children, Harvard Medical School, Boston, MA 02114, USA
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Lauren E. Grote
7Division of Clinical Genetics, Children’s Mercy Hospital, Kansas City, MO 64108, USA
8University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108, USA
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Isabelle Thiffault
8University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108, USA
9Center for Pediatric Genomic Medicine, Children’s Mercy Hospital, Kansas City, MO 64108, USA
10Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO 64108, USA
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Carol Saunders
8University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108, USA
9Center for Pediatric Genomic Medicine, Children’s Mercy Hospital, Kansas City, MO 64108, USA
10Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO 64108, USA
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Kevin A. Strauss
11Clinic for Special Children, Strasburg, PA 17579, USA
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Ali Torkamani
12Scripps Translational Science Institute and Scripps Research Institute, La Jolla, CA 92037, USA
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Jasper van der Smagt
13Department of Genetics, University Medical Center Utrecht, Utrecht, The Netherlands
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Koen van Gassen
13Department of Genetics, University Medical Center Utrecht, Utrecht, The Netherlands
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Robert P. Carson
14Monroe Carell Jr. Children’s Hospital at Vanderbilt, Nashville, TN 37232, USA
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Jullianne Diaz
15Rare Disease Institute, Children’s National Medical Center, Washington, DC 20010, USA
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Eyby Leon
15Rare Disease Institute, Children’s National Medical Center, Washington, DC 20010, USA
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Joseph E. Jacher
16Division of Pediatric Genetics, Metabolism & Genomic Medicine, University of Michigan, Ann Arbor, MI 48109, USA
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Mark C. Hannibal
16Division of Pediatric Genetics, Metabolism & Genomic Medicine, University of Michigan, Ann Arbor, MI 48109, USA
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Jessica Litwin
17UCSF Benioff Children’s Hospital, San Francisco, CA 94143, USA
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Neil R. Friedman
18Cleveland Clinic Children’s, Cleveland, OH 44195, USA
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Allison Schreiber
18Cleveland Clinic Children’s, Cleveland, OH 44195, USA
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Bryan Lynch
19Department of Paediatric Neurology and Clinical Neurophysiology, Children’s University Hospital, Dublin, Ireland
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Annapurna Poduri
20Epilepsy Genetics Program, Department of Neurology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
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Eric D. Marsh
21Division of Child Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
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Ethan M. Goldberg
21Division of Child Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
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John J. Millichap
2Departments of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
3Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL 60611, USA
22Departments of Neurology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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Alfred L. George Jr.
1Departments of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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Jennifer A. Kearney
1Departments of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
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  • For correspondence: jennifer.kearney@northwestern.edu
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ABSTRACT

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.

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Posted May 14, 2019.
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High throughput Characterization of KCNB1 variants Associated with Developmental and Epileptic Encephalopathy
Seok Kyu Kang, Carlos G. Vanoye, Sunita N. Misra, Dennis M. Echevarria, Jeffrey D. Calhoun, John B. O’Connor, Katarina L. Fabre, Dianalee McKnight, Laurie Demmer, Paula Goldenberg, Lauren E. Grote, Isabelle Thiffault, Carol Saunders, Kevin A. Strauss, Ali Torkamani, Jasper van der Smagt, Koen van Gassen, Robert P. Carson, Jullianne Diaz, Eyby Leon, Joseph E. Jacher, Mark C. Hannibal, Jessica Litwin, Neil R. Friedman, Allison Schreiber, Bryan Lynch, Annapurna Poduri, Eric D. Marsh, Ethan M. Goldberg, John J. Millichap, Alfred L. George Jr., Jennifer A. Kearney
bioRxiv 637041; doi: https://doi.org/10.1101/637041
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High throughput Characterization of KCNB1 variants Associated with Developmental and Epileptic Encephalopathy
Seok Kyu Kang, Carlos G. Vanoye, Sunita N. Misra, Dennis M. Echevarria, Jeffrey D. Calhoun, John B. O’Connor, Katarina L. Fabre, Dianalee McKnight, Laurie Demmer, Paula Goldenberg, Lauren E. Grote, Isabelle Thiffault, Carol Saunders, Kevin A. Strauss, Ali Torkamani, Jasper van der Smagt, Koen van Gassen, Robert P. Carson, Jullianne Diaz, Eyby Leon, Joseph E. Jacher, Mark C. Hannibal, Jessica Litwin, Neil R. Friedman, Allison Schreiber, Bryan Lynch, Annapurna Poduri, Eric D. Marsh, Ethan M. Goldberg, John J. Millichap, Alfred L. George Jr., Jennifer A. Kearney
bioRxiv 637041; doi: https://doi.org/10.1101/637041

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