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High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KCNH2: Deep mutational scan of KCNH2 trafficking

Krystian A. Kozek, Andrew M. Glazer, Chai-Ann Ng, Daniel Blackwell, Christian L. Egly, Loren R. Vanags, Marcia Blair, Devyn Mitchell, Kenneth A. Matreyek, Douglas M. Fowler, Bjorn C. Knollmann, View ORCID ProfileJamie Vandenberg, Dan M. Roden, Brett M. Kroncke
doi: https://doi.org/10.1101/2020.02.17.952606
Krystian A. Kozek
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Andrew M. Glazer
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Chai-Ann Ng
2Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia
3St Vincent’s Clinical School, UNSW Sydney, Darlinghurst, New South Wales, Australia
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Daniel Blackwell
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Christian L. Egly
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Loren R. Vanags
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Marcia Blair
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Devyn Mitchell
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Kenneth A. Matreyek
4Department of Genome Sciences, University of Washington, Seattle, WA, USA
6Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA
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Douglas M. Fowler
4Department of Genome Sciences, University of Washington, Seattle, WA, USA
5Department of Bioengineering, University of Washington, Seattle, WA, USA
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Bjorn C. Knollmann
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Jamie Vandenberg
2Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia
3St Vincent’s Clinical School, UNSW Sydney, Darlinghurst, New South Wales, Australia
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  • ORCID record for Jamie Vandenberg
Dan M. Roden
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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Brett M. Kroncke
1Vanderbilt Center for Arrhythmia Research and Therapeutics, Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
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  • For correspondence: brett.m.kroncke.1@vumc.org
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ABSTRACT

Background KCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease IKr can cause Type 2 Long QT syndrome, usually due to mistrafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would help clinicians identify and treat individuals at risk of a major cardiac event. The volume of reported non-synonymous KCNH2 variants preclude the use of conventional electrophysiologic methods for functional study.

Objective To report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in KCNH2.

Methods We develop a method to quantitate KCNH2 variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region.

Results For 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We additionally compare our results with planar patch electrophysiology and find that loss-of-trafficking variants do not produce IKr, but that some variants which traffic normally may still be functionally compromised.

Conclusions Here, we describe a new method for detecting trafficking-deficient variants in KCNH2 in a multiplexed assay. This new method accurately generates trafficking data for variants in KCNH2 and can be readily extended to all residues in Kv11.1 and to other cell surface proteins.

CLINICAL IMPLICATIONS Hundreds of KCNH2 variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of KV11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning of KCNH2 trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number of KCNH2 variants being found in the population.

Footnotes

  • The authors have no conflicts to disclose

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KCNH2: Deep mutational scan of KCNH2 trafficking
Krystian A. Kozek, Andrew M. Glazer, Chai-Ann Ng, Daniel Blackwell, Christian L. Egly, Loren R. Vanags, Marcia Blair, Devyn Mitchell, Kenneth A. Matreyek, Douglas M. Fowler, Bjorn C. Knollmann, Jamie Vandenberg, Dan M. Roden, Brett M. Kroncke
bioRxiv 2020.02.17.952606; doi: https://doi.org/10.1101/2020.02.17.952606
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High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KCNH2: Deep mutational scan of KCNH2 trafficking
Krystian A. Kozek, Andrew M. Glazer, Chai-Ann Ng, Daniel Blackwell, Christian L. Egly, Loren R. Vanags, Marcia Blair, Devyn Mitchell, Kenneth A. Matreyek, Douglas M. Fowler, Bjorn C. Knollmann, Jamie Vandenberg, Dan M. Roden, Brett M. Kroncke
bioRxiv 2020.02.17.952606; doi: https://doi.org/10.1101/2020.02.17.952606

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