High-throughput phenotyping of heteromeric human ether-à-go-go-related gene potassium channel variants can discriminate pathogenic from rare benign variants

Ng, Chai Ann; Perry, Matthew D.; Liang, Whitney; Smith, Nicola J.; Foo, Brian; Shrier, Alvin; Lukacs, Gergely L.; Hill, Adam P.; Vandenberg, Jamie I.

doi:10.1016/j.hrthm.2019.09.020

Cited by 58 publications

(74 citation statements)

References 31 publications

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“…Identifying ion current kinetics and separating out experimental artefacts is crucial for many cardiac electrophysiology studies. For example, ion channel mutation studies often conclude with a statement such as "there is a 5-10 mV shift in the half-activation potential" Clerx et al (2018); Ng et al (2019). However, given the variability that we observe in patch-clamp data, often the cell to cell variability in half-activation potential can be in the range of 10-15 mV.…”

Section: Discussionmentioning

confidence: 84%

Accounting for variability in ion current recordings using a mathematical model of artefacts in voltage-clamp experiments

Lei

Clerx

Whittaker

et al. 2019

Preprint

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Mathematical models of ion channels, which constitute indispensable components of action potential models, are commonly constructed by fitting to whole-cell patch-clamp data. In a previous study we fitted cell-specific models to hERG1a (Kv11.1) recordings simultaneously measured using an automated high-throughput system, and studied cell-cell variability by inspecting the resulting model parameters. However, the origin of the observed variability was not identified. Here we study the source of variability by constructing a model that describes not just ion current dynamics, but the entire voltage-clamp experiment. The experimental artefact components of the model include: series resistance, membrane and pipette capacitance, voltage offsets, imperfect compensations made by the amplifier for these phenomena, and leak current. In this model, variability in the observations can be explained by either cell properties, measurement artefacts, or both. Remarkably, by assuming that variability arises exclusively from measurement artefacts, it is possible to explain a larger amount of the observed variability than when assuming cell-specific ion current kinetics. This assumption also leads to a smaller number of model parameters. This result suggests that most of the observed variability in patch-clamp data measured under the same conditions is caused by experimental artefacts, and hence can be compensated for in post-processing by using our model for the patch-clamp experiment. This study has implications for the question of the extent to which cell-cell variability in ion channel kinetics exists, and opens up routes for better correction of artefacts in patch-clamp data.

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Section: Discussionmentioning

confidence: 84%

Accounting for variability in ion current recordings using a mathematical model of artefacts in voltage-clamp experiments

Lei

Clerx

Whittaker

et al. 2019

Preprint

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“…Previous studies have implemented a similar automated patch clamping approach on dozens of variants in two other arrhythmia-associated genes, KCNQ1 and KCNH2. 16,17 Therefore, targeted high-throughput patch clamping of ion channel variants is a promising method for reclassifying additional variants in SCN5A and other Mendelian arrhythmia genes. This approach may be extended to study additional SCN5A variants, including gain of function Long QT-associated variants or additional variants observed in population sequencing efforts.…”

Section: High-throughput Approaches To Variant Classificationmentioning

confidence: 99%

“…This approach may be extended to study additional SCN5A variants, including gain of function Long QT-associated variants or additional variants observed in population sequencing efforts. Accurate classification of the large number of variants in arrhythmiaassociated genes will require integrating data from multiple different model systems, such as patch clamping, 16,17 induced pluripotent stem cell-derived cardiomyocytes, [49][50][51] structural and computational models, 12,52,53 and ultra-high-throughput multiplexed assays. [54][55][56] Limitations: This study assays variants in a heterologous expression system.…”

Section: High-throughput Approaches To Variant Classificationmentioning

confidence: 99%

“…Recently, automated high-throughput patch clamp devices have allowed the rapid evaluation of dozens of variants in ion channel genes, including the arrhythmia-associated genes KCNQ1 and KCNH2. [15][16][17] In this study, we use automated patch clamping to study 83 SCN5A variants, identify 45 new partial or total loss of function variants, and reclassify 50/60 variants of uncertain significance.…”

Section: Introductionmentioning

confidence: 99%

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High-throughput reclassification ofSCN5Avariants

Glazer

Wada

Bian

et al. 2019

Preprint

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Rationale: Partial or complete loss of function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada Syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care. Objective:We selected 83 variants for study: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada Syndrome-associated variants, selected on the basis of their frequency in the general population and in patients with Brugada Syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data.Methods and Results: Ten previously studied variants had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wildtype-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 23 variants had partial loss of function (10-50% normalized peak current). The 73 previously unstudied variants were initially classified as likely benign (n=2), likely pathogenic (n=11), or VUS (n=60). After the patch clamp studies, 16 variants were benign/likely benign, 47 were pathogenic/likely pathogenic, and only 10 were still VUS. 8/22 loss of function variants were partially rescuable by incubation at lower temperature or pretreatment with a sodium channel blocker. Structural modeling identified likely mechanisms for loss of function including altered thermostability, and disruptions to alpha helices, disulfide bonds, or the permeation pore. Conclusions:High-throughput automated patch clamping enabled the reclassification of the majority of tested VUS's in SCN5A.

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“…This approach offers a unique opportunity to generate high-throughput experimental data for hundreds or thousands of single codon substitutions in a multiplexed experiment. This provides complementary information to in silico predictions 29,30 and other functional characterization techniques, such as automated patch clamp electrophysiology 31 and CRISPR-generated, or naturally-occurring, variants in induced pluripotent stem cell-derived cardiomyocytes. 32 Here we present a DMS of a transmembrane helix of KCNH2.…”

Section: Introductionmentioning

confidence: 99%

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channelKCNH2: Deep mutational scan ofKCNH2trafficking

Ka¹,

Am²,

Ng³

et al. 2020

Preprint

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Background: KCHN2 encodes the K V 11.1 potassium channel responsible for I Kr , a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease I Kr 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 I Kr , 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.

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High-throughput phenotyping of heteromeric human ether-à-go-go-related gene potassium channel variants can discriminate pathogenic from rare benign variants

Cited by 58 publications

References 31 publications

Accounting for variability in ion current recordings using a mathematical model of artefacts in voltage-clamp experiments

Accounting for variability in ion current recordings using a mathematical model of artefacts in voltage-clamp experiments

High-throughput reclassification ofSCN5Avariants

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channelKCNH2: Deep mutational scan ofKCNH2trafficking

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