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Clinical/Translational Research

An In Vivo Cardiac Assay to Determine the Functional Consequences of Putative Long QT Syndrome MutationsNovelty and Significance

Chuanchau J. Jou, Spencer M. Barnett, Jian-Tao Bian, H. Cindy Weng, Xiaoming Sheng, Martin Tristani-Firouzi
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https://doi.org/10.1161/CIRCRESAHA.112.300664
Circulation Research. 2013;112:826-830
Originally published February 28, 2013
Chuanchau J. Jou
From the Division of Pediatric Cardiology (C.J.J., S.M.B., M.T.-F.), Nora Eccles Harrison Cardiovascular Research and Training Institute (C.J.J., J.-T.B., M.T.-F.), Department of Pediatrics (H.C.W., X.S.), University of Utah, Salt Lake City, UT.
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Spencer M. Barnett
From the Division of Pediatric Cardiology (C.J.J., S.M.B., M.T.-F.), Nora Eccles Harrison Cardiovascular Research and Training Institute (C.J.J., J.-T.B., M.T.-F.), Department of Pediatrics (H.C.W., X.S.), University of Utah, Salt Lake City, UT.
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Jian-Tao Bian
From the Division of Pediatric Cardiology (C.J.J., S.M.B., M.T.-F.), Nora Eccles Harrison Cardiovascular Research and Training Institute (C.J.J., J.-T.B., M.T.-F.), Department of Pediatrics (H.C.W., X.S.), University of Utah, Salt Lake City, UT.
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H. Cindy Weng
From the Division of Pediatric Cardiology (C.J.J., S.M.B., M.T.-F.), Nora Eccles Harrison Cardiovascular Research and Training Institute (C.J.J., J.-T.B., M.T.-F.), Department of Pediatrics (H.C.W., X.S.), University of Utah, Salt Lake City, UT.
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Xiaoming Sheng
From the Division of Pediatric Cardiology (C.J.J., S.M.B., M.T.-F.), Nora Eccles Harrison Cardiovascular Research and Training Institute (C.J.J., J.-T.B., M.T.-F.), Department of Pediatrics (H.C.W., X.S.), University of Utah, Salt Lake City, UT.
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Martin Tristani-Firouzi
From the Division of Pediatric Cardiology (C.J.J., S.M.B., M.T.-F.), Nora Eccles Harrison Cardiovascular Research and Training Institute (C.J.J., J.-T.B., M.T.-F.), Department of Pediatrics (H.C.W., X.S.), University of Utah, Salt Lake City, UT.
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Abstract

Rationale: Genetic testing for Long QT Syndrome is now a standard and integral component of clinical cardiology. A major obstacle to the interpretation of genetic findings is the lack of robust functional assays to determine the pathogenicity of identified gene variants in a high-throughput manner.

Objective: The goal of this study was to design and test a high-throughput in vivo cardiac assay to distinguish between disease-causing and benign KCNH2 (hERG1) variants, using the zebrafish as a model organism.

Methods and Results: We tested the ability of previously characterized Long QT Syndrome hERG1 mutations and polymorphisms to restore normal repolarization in the kcnh2-knockdown embryonic zebrafish. The cardiac assay correctly identified a benign variant in 9 of 10 cases (negative predictive value 90%), whereas correctly identifying a disease-causing variant in 39/39 cases (positive predictive value 100%).

Conclusions: The in vivo zebrafish cardiac assay approaches the accuracy of the current benchmark in vitro assay for the detection of disease-causing mutations, and is far superior in terms of throughput rate. Together with emerging algorithms for interpreting a positive long QT syndrome genetic test, the zebrafish cardiac assay provides an additional tool for the final determination of pathogenicity of gene variants identified in long QT syndrome genetic screening.

  • arrhythmia
  • channelopathy
  • gene mutation
  • genetic testing
  • genetics polymorphism
  • ion channels
  • long QT syndrome
  • model organism
  • sudden death

Introduction

Mutations in KCNH2 (human ether-a-go-go related gene [hERG1]), the gene encoding the rapidly activating, delayed rectifier K+ current (IKr), account for 30% to 45% of mutation-positive Long QT Syndrome (LQTS).1,2 Commercial genetic testing for the known LQTS disease-causing genes is now the standard of care in the work-up of a new patient with clinical LQTS. Policy experts agree that a major obstacle to the interpretation of genetic findings is the lack of robust functional assays to determine the pathogenicity of identified gene variants.3,4 The current gold-standard assays for functional characterization of LQTS mutants (noncardiac mammalian cell expression systems, such as HEK293, CHO cells) are laborious and unable to meet the demands of LQTS genetic testing. Thus, there is an urgent need to develop the tools to characterize the functional consequences of LQTS gene variants in a high-throughput fashion to improve counseling and clinical care.

The zebrafish is a powerful vertebrate genetic model to explore the molecular basis of cardiac development, disease, and arrhythmia. Although the zebrafish heart is 2-chambered, fundamental electric properties are similar to higher mammals, including embryonic/adult heart rates,5,6 action potential parameters,7 QT interval,6,7 and the relationship between QT interval and heart rate.7 We previously validated a zebrafish model of human LQT-2 and showed that the zebrafish orthologue of hERG1, kcnh2 is essential for cardiac repolarization in embryonic zebrafish ventricle. Homozygous kcnh2 mutations cause ventricular asystole as a consequence of membrane depolarization. Submaximal IKr blockade prolongs ventricular action potential duration and causes 2:1 atrioventricular block (AVB), whereas maximal IKr blockade causes membrane depolarization leading to cessation of action potential generation.7 We sought to take advantage of the observation that kcnh2 channels provide the principal repolarizing current in embryonic ventricle to design an in vivo system to discriminate deleterious mutations in hERG1 variants.

Methods

Kcnh2 antisense morpholino (MO) was injected alone or coinjected with wild-type (WT) or mutant hERG1 cRNA in WT zebrafish embryos at 1- to 2-cell stage. Cardiac repolarization phenotypes (normal 1:1 atrioventricular [AV] conduction, 2:1 AVB, or ventricular asystole) were screened under light microscopy at 48-hours postfertilization. Statistical analyses were performed as described in the online-only Data Supplement.

Results

Morpholino kcnh2 Knockdown Disrupts Cardiac Repolarization

Kcnh2 is expressed very early in cardiac development in the bilateral cardiac primordia before the onset of cardiac contraction and specifically localized to the cardiac chambers at 48-hours postfertilization (Figure 1A). MO-knockdown of kcnh2 caused a spectrum of repolarization defects, similar to those observed in the zebrafish knch2 mutants, breakdance, and silent ventricle.7,8 Ninety-one percent of MO-injected embryos (n=210) displayed a repolarization-deficient phenotype (2:1 AVB or ventricular asystole), confirming the robust nature of the MO-induced kcnh2-knockdown. WT hERG1 RNA injection restored normal repolarization in 55% of MO-injected embryos, and shifted the severity of the repolarization defect from ventricular asystole to 2:1 AVB (Figure 1B). To define the cellular correlates of our observed repolarization phenotypes, we directly measured Vm in explanted embryonic ventricle. Spontaneous ventricular action potentials recorded from embryos manifesting 2:1 AVB were significantly prolonged compared with embryos that displayed normal repolarization (1:1 AV conduction; Figure 1C and the online-only Data Supplement Table I). The resting ventricular Vm recorded from embyros displaying a ventricular asystole phenotype was markedly depolarized and spontaneous action potentials were not observed (Figure 1C), similar to that recorded in the complete loss-of-function knch2 mutant, silent ventricle.7 MO-injected embyros displaying a normal repolarization phenotype captured the pacing stimulus in a 1:1 fashion, whereas those displaying a 2:1 AVB phenotype were unable to pace in a 1:1 manner as a consequence of action potential duration prolongation (Figure 1D). Taken together, these data demonstrate that kcnh2 MO-knockdown produces repolarization-deficient phenotypes that can be rescued by injection of WT hERG1 RNA.

Figure 1.
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Figure 1.

Embryonic knch2 expression and the effects of kcnh2-knockdown on cardiac repolarization. A, Left, Whole-mount in situ hybridization reveals kcnh2 expression in the bilateral cardiac primordia (12 somite stage) before the onset of cardiac contraction and specific expression in the heart at 48-hours postfertilization. Right, B,The percentage of embryos displaying normal (1:1 atrioventricular [AV] conduction) or abnormal repolarization (2:1 atrioventricular block [AVB]), or ventricular asystole) 48 hours after injection of anti-kcnh2 morpholino (MO) alone or together with wild-type (WT) human ether-a-go-go related gene [hERG] RNA. C, Examples of Vm recordings from explanted embryonic ventricle of MO-injected embryos that displayed normal 1:1 AV conduction, 2:1 AVB, or ventricular asystole phenotypes. D, External pacing (cycle length 500 ms) of explanted ventricle from MO-injected embryos displaying 1:1 or 2:1 AV conduction. AVB indicates atrioventricular block; and MO, morpholino.

An In Vivo Cardiac Assay to Determine the Functional Consequences of hERG1 Variants

Next, we tested the ability of previously described LQTS hERG1 mutations (n=40) and hERG1 polymorphisms (n=10) to restore normal repolarization in the kcnh2-knockdown embryonic zebrafish (Figure 2). A spectrum of normal repolarization rescue was observed for the 40 putative LQTS hERG1 mutants, but the degree of rescue was statistically lower than WT hERG1 rescue (the online-only Data Supplemental Table II). All 10 hERG1 single nucleotide polymorphisms rescued the kcnh2 MO-knockdown to a similar degree as WT hERG1 (P>0.05, generalized linear mixed model). To confirm the electrophysiological basis for the repolarization phenotypes observed, we measured ventricular Vm in kcnh2-MO knockdown embyros injected with WT, N470D, A614V, and A1116V hERG1 (the online-only Data Supplemental Figure I). These data confirm that 2:1 AVB is the result of marked action potential duration prolongation, whereas ventricular asystole is because of marked depolarization.

Figure 2.
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Figure 2.

Functional effects of putative long QT (LQT) 2 mutations and polymorphisms, as determined by zebrafish cardiac assay. Percentage of embryos manifesting normal repolarization (1:1 atrioventricular [AV] conduction) plotted for anti-kcnh2 morpholino (MO)-injected (MO alone), wild-type (WT) human ether-a-go-go related gene [hERG]+MO, and mutant hERG+MO. The location of the variant on the channel is listed below for the putative LQT-2 variants. Note that error bars represent +95% confidence intervals. N=152 to 488 in each group. C-term indicates C-terminus; N-term, N-terminus; polymorph, poly-morphism; and TM, transmembrane domain.

Forty-nine of 50 hERG1 variants have been functionally characterized using the current gold-standard in vitro heterologous expression system. When compared with the benchmark assay, the cardiac assay correctly identified a nondisease-causing variant in 9 of 10 cases (negative predictive value 90%), whereas correctly identifying a disease-causing variant in 39/39 cases (positive predictive value 100%). The only variant incorrectly assigned by the in vivo assay was T436M, which was predicted to be a pathogenic variant.

As an alternative comparison between the 2 assays, we normalized the values obtained using the in vivo zebrafish cardiac assay to the gold-standard in vitro assay as follows. For each hERG1 variant, the fraction of embryos with normal repolarization was normalized to WT value and plotted against the fraction of WT current measured in vitro, using published values (Figure 3). We used hierarchal cluster analysis to group the values into 2 or more unique subsets that represent concordance between the 2 assays. The data were best fit by 2 distinct clusters that distinguish pathogenic from nonpathogenic variants (the online-only Data Supplemental Figure II). For example, variants with current magnitude <0.5 of WT clustered tightly with variants, whose repolarization rescue was <0.5 of WT. Variants with current magnitude >0.7 of WT clustered with a broader range of rescued repolarization values, but were generally concordant with rescue values >0.67 of WT. One variant fell outside of the 95% confidence interval ellipse of either cluster (T474I). Although T474I generates functional channels in vitro, it was considered a pathogenic mutation based on altered kinetic properties9 and was correctly identified as disease-causing by the in vivo cardiac assay.

Figure 3.
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Figure 3.

Comparison of in vivo zebrafish cardiac assay with in vitro mammalian cell assay. For each human ether-a-go-go related gene [hERG] variant, the fraction of embryos with normal repolarization is normalized to wild-type (WT) value and plotted against fraction of WT current assayed in mammalian cells, using published values. Cluster analysis identified 2 distinct subsets that represent concordance between the assays to distinguish pathogenic and benign variants. T474I fell outside the 95% confidence interval (CI) ellipses (dotted gray lines) of the 2 clusters. Although T474I generates functional channels in vitro, it was considered a pathogenic mutation based on altered kinetic properties9 and was correctly identified as disease-causing by the in vivo cardiac assay.

Finally, we tested the ability of the in vivo cardiac assay to corroborate the clinical observation that inheriting a common polymorphism (K897T) and an LQT-2 variant (A490T) in cis orientation reduces the severity of the LQTS phenotype compared with subjects with the A490T mutation alone.10 The A490T single mutant behaved as a pathogenic variant in the in vivo assay, whereas the double mutant A490T-K897T rescued repolarization similar to WT hERG1 (the online-only Data Supplemental Figure III). Taken together, our results suggests that replacing the endogenous kcnh2 gene with the human orthologue has predictive value for testing putative disease-causing LQT-2 variants, and corroborates an interesting clinical observation that a common polymorphism modifies the severity of a specific LQTS phenotype.

Discussion

Although genetic testing for channelopathies is now a standard and integral component of clinical cardiology, a major obstacle to the interpretation of genetic findings is the lack of robust functional assays. Here, we describe the utility of an in vivo cardiac assay to distinguish between disease-causing and benign hERG1 variants, and demonstrate the proof-of-principle concept that replacing an endogenous gene with the human orthologue has predictive value for testing genetic variants. As compared with the current benchmark in vitro assay, the in vivo cardiac assay correctly distinguished a pathogenic from a benign variant with high accuracy (positive predictive value 100%, negative predictive value 90%). T436M represented the only discrepant variant between the 2 assays. T436M was originally reported in a single LQTS individual as a de novo variant11 and has not been subsequently reported in the literature as either a rare polymorphism or an LQTS mutation. Until additional genetic information is available, the pathogenicity of this rare variant and the superiority of the individual assay remain to be determined.

The in vivo cardiac assay provides several practical advantages over the current gold-standard in vitro assays. Although the in vitro assays reveal important biophysical characteristics of the mutant channel, in silico modeling is required to infer the consequences of altered channel gating/conductance on cardiac action potential duration. By contrast, the in vivo assay provides a functional readout in the form of normal/abnormal repolarization. For example, although the abnormal gating properties of the T474I variant were predicted to cause the LQTS phenotype,12 the in vivo assay confirmed the abnormal nature of cardiac repolarization. In addition, the in vivo assay is less laborious and high-throughput than the in vitro assays, and thus, can bridge the knowledge gap between variant identification and functionality. The Inherited Arrhythmia Database (www.fsm.it/cardmoc/) lists 335 nonsynonymous hERG coding variants, the vast majority of whose function is not known. Moreover, the importance of functionally characterizing LQTS variants is further underscored by recent report that previously-associated LQTS genetic variants were identified in 3% of 5400 participants of a whole-exome sequencing project,13 implying a high false-positive designation as disease-causing.

Temperature is known to play an important role in protein processing/trafficking in heterologous expression systems. Lowering the incubation temperature rescues the cell surface expression of several trafficking-deficient hERG1 mutations. Zebrafish larvae were raised and repolarization was assayed at 25°C to 28°C (whereas mammalian cells are incubated at 36°C). We were initially concerned that these temperature-sensitive mutants might traffick and function normally in the zebrafish assay. However, the temperature-sensitive mutants, N470D and R752W, did not restore repolarization in the zebrafish cardiac assay, as expected for disease-causing mutations. These results imply that, in addition to temperature, there may be cell-specific mechanisms that influence trafficking in vivo.

There are several limitations inherent in this study. The zebrafish is not a mammalian organism, and it remains unclear that the behavior of the hERG1 mutants in zebrafish heart fully recapitulates that in humans. Moreover, the functional readouts from the zebrafish cardiac assay do not provide a mechanistic understanding of hERG1 channel dysfunction. The zebrafish system does not provide fine details of channel behavior that might suggest a susceptibility to disease, such as a variant that might reduce repolarization reserve in the setting of another diseasing-causing mutation. Despite the caveats listed, the in vivo zebrafish cardiac assay does provide a yes/no screen for the high-throughput evaluation of novel hERG1 gene variants. Together with emerging algorithms for interpreting a positive LQTS genetic test,14,15 the zebrafish cardiac assay may provide an additional tool for the final determination of pathogenicity of gene variants identified in commercial LQTS genetic screening.

Sources of Funding

This work was supported by an American Heart Association Fellow to Faculty award (C.J.J.), the NHLBI Bench to Bassinet Program (M.T.F.) and the generous support of the Nora Eccles Treadwell Foundation.

Disclosures

None.

Footnotes

  • In December 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.300664/-/DC1.

  • APD
    action potential duration
    AV
    atrioventricular
    CI
    confidence interval
    hERG
    human ether-a-go-go related gene
    Hpf
    hours postfertilization
    IKr
    rapidly activating, delayed rectifier K+ current
    LQTS
    Long QT Syndrome
    Vm
    membrane potential

  • Received July 26, 2012.
  • Revision received January 3, 2013.
  • Accepted January 9, 2013.
  • © 2013 American Heart Association, Inc.

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Novelty and Significance

What Is Known?

  • A major obstacle to the interpretation of long QT syndrome (LQTS)–associated genetic variants is the lack of robust functional assays that determine the pathogenicity of the identified variants.

  • Current functional assays using noncardiac heterologous expression systems are costly and labor-intensive.

What New Information Does This Article Contribute?

  • The current study addresses a significant and increasing clinical problem with a straightforward assay, using the zebrafish as a model organism.

  • The zebrafish cardiac assay approaches the accuracy of the current functional assays, and is far superior in terms of throughput rate.

  • The zebrafish model provides practical advantages over noncardiac expression systems, including a functional readout of repolarization that is based on specific electrophysiological correlates.

Next-generation sequencing has revolutionized our ability to study the contribution of genetic variation to human disease. To keep pace with these advances, there is an urgent need for practical, functional assays that distinguish pathogenic from benign variants. The present work describes and validates an in vivo cardiac assay to determine the functional consequences of putative LQTS mutations, using the zebrafish model organism. Our findings support the proof-of-principle concept that replacing an endogenous gene with its human orthologue has predictive value for testing disease-causing gene variants. Together with emerging algorithms for interpreting a positive LQTS genetic test, our work provides a framework to bridge the gap between variant identification and disease risk.

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Circulation Research
March 1, 2013, Volume 112, Issue 5
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    An In Vivo Cardiac Assay to Determine the Functional Consequences of Putative Long QT Syndrome MutationsNovelty and Significance
    Chuanchau J. Jou, Spencer M. Barnett, Jian-Tao Bian, H. Cindy Weng, Xiaoming Sheng and Martin Tristani-Firouzi
    Circulation Research. 2013;112:826-830, originally published February 28, 2013
    https://doi.org/10.1161/CIRCRESAHA.112.300664

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    An In Vivo Cardiac Assay to Determine the Functional Consequences of Putative Long QT Syndrome MutationsNovelty and Significance
    Chuanchau J. Jou, Spencer M. Barnett, Jian-Tao Bian, H. Cindy Weng, Xiaoming Sheng and Martin Tristani-Firouzi
    Circulation Research. 2013;112:826-830, originally published February 28, 2013
    https://doi.org/10.1161/CIRCRESAHA.112.300664
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  • Arrhythmia and Electrophysiology
    • Arrhythmias
  • Basic, Translational, and Clinical Research
    • Animal Models of Human Disease

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