Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

BACKGROUND: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. METHODS: An in vitro SCN5A-Brugada syndrome automated patch clamp assay was generated and independently studied at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. RESULTS: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values yielded 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. CONCLUSIONS: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

Multi-site validation of a functional assay to adjudicate SCN5A Brugada Syndrome-associated variants.

Brugada Syndrome (BrS) is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in the cardiac sodium channel gene, SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging and ~79% of SCN5A missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). An in vitro SCN5A-BrS automated patch clamp assay was generated for high-throughput functional studies of NaV1.5. The assay was independently studied at two separate research sites - Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute - revealing strong correlations, including peak INa density (R2=0.86). The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. The assay accurately distinguished benign controls (24/25) from pathogenic controls (23/24). Odds of Pathogenicity values derived from the experimental results yielded 0.042 for normal function (BS3 criterion) and 24.0 for abnormal function (PS3 criterion), resulting in up to strong evidence for both ACMG criteria. The calibrated assay was then used to study SCN5A VUS observed in four families with BrS and other arrhythmia phenotypes associated with SCN5A loss-of-function. The assay revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. This validated APC assay provides clinical-grade functional evidence for the reclassification of current VUS and will aid future SCN5A-BrS variant classification.

Epsilon wave uncovered by exercise test in a patient with desmoplakin-positive arrhythmogenic right ventricular cardiomyopathy.

Epsilon waves are a major criterion for the diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) but are an insensitive sign. Recently, exercise testing has been shown to uncover epsilon waves in asymptomatic patients carrying mutations in the PKP2 gene. We describe a case of an asymptomatic carrier of a mutation in the DSP gene who had a normal baseline electrocardiogram and an exercise-induced epsilon wave. This finding suggests that exercise testing may be valuable for the diagnosis of ARVC and that exercise-induced epsilon waves may be found in various genetic subtypes of this disease.

Evaluation of genes encoding for the transient outward current (Ito) identifies the KCND2 gene as a cause of J-wave syndrome associated with sudden cardiac death.

BACKGROUND: J-wave ECG patterns are associated with an increased risk of sudden arrhythmic death, and experimental evidence supports a transient outward current (I(to))-mediated mechanism of J-wave formation. This study aimed to determine the frequency of genetic mutations in genes encoding the I(to) in patients with J waves on ECG. METHODS AND RESULTS: Comprehensive mutational analysis was performed on I(to)-encoding KCNA4, KCND2, and KCND3 genes, as well as the previously described J-wave-associated KCNJ8 gene, in 51 unrelated patients with ECG evidence defining a J-wave syndrome. Only patients with a resuscitated cardiac arrest or type 1 Brugada ECG pattern were included for analysis. A rare genetic mutation of the KCND2 gene, p.D612N, was identified in a single patient. Co-expression of mutant and wild-type KCND2 with KChIP2 in HEK293 cells demonstrated a gain-of-function phenotype, including an increase in peak I(to) density of 48% (P<0.05) in the heterozygous state. Using computer modeling, this increase in Ito resulted in loss of the epicardial action potential dome, predicting an increased ventricular transmural Ito gradient. The previously described KCNJ8-S422L mutation was not identified in this cohort of patients with ECG evidence of J-wave syndrome. CONCLUSIONS: These findings are the first to implicate the KCND2 gene as a novel cause of J-wave syndrome associated with sudden cardiac arrest. However, genetic defects in I(to)-encoding genes seem to be an uncommon cause of sudden cardiac arrest in patients with apparent J-wave syndromes.

Kinetics of drug interaction with the Kv11.1 potassium channel.

The Kv11.1 potassium channel is the molecular target for the majority of drugs implicated in acquired long QT syndrome, the most common cause of drug-induced sudden cardiac death, and a common reason for drug restriction or withdrawal from the market. While the IC50 for block of Kv11.1 is commonly used to estimate the risk of acquired long QT syndrome, this approach is crude, and it is widely accepted that the kinetics of drug interactions with the channel are a critical component in understanding their mechanism of action and risk profiles. In this study we report the first directly measured kinetics of block and unblock of Kv11.1 by a QT prolonging drug: the antipsychotic clozapine. Our data show that clozapine binding to Kv11.1 is complex. There are at least two kinetically distinct components to both block and unblock, while the kinetics of unblock are dependent on the dose or duration of drug application. Based on these observations, we have proposed a model incorporating kinetically distinct binding to the open and inactivated states of Kv11.1 that can describe the observed kinetic features of clozapine block and correctly predict the overall affinity and apparent nonstate-dependent interaction of clozapine with Kv11.1. Mechanistic insights into drug block of Kv11.1 gained though detailed kinetic analyses such as this have a potential role in development of drugs targeted to specific channel states to reduce unwanted side effects, as well as in the design of better high-throughput preclinical tests for assessing the proarrhythmic effects of QT prolonging drugs.

Exercise testing in asymptomatic gene carriers exposes a latent electrical substrate of arrhythmogenic right ventricular cardiomyopathy.

OBJECTIVES: The aim of this study was to determine if exercise testing could expose a latent electrical substrate of arrhythmogenic right ventricular cardiomyopathy (ARVC) in asymptomatic gene carriers. BACKGROUND: Management of asymptomatic ARVC gene carriers is challenging because of variable penetrance of disease and the recognition that sudden cardiac death may be the first clinical manifestation. METHODS: Exercise-induced abnormalities during exercise treadmill testing (ETT) were initially compared in 60 subjects: 30 asymptomatic ARVC gene carriers and 30 healthy controls. In phase 2 of the study, ETT results of 25 patients with ARVC with histories of sustained ventricular arrhythmia or cardiac arrest were evaluated to determine if ETT abnormalities in asymptomatic gene carriers were common to patients with a malignant electrical form of the disease. RESULTS: Depolarization abnormalities during ETT were found to develop more frequently in asymptomatic gene carriers compared with healthy controls: epsilon waves appeared in 4 of 28 (14%) compared with 0 of 30 (0%) (p = 0.048), premature ventricular contractions in 17 of 30 (57%) compared with 3 of 30 (10%) (p = 0.0003), and new QRS terminal activation duration ≥ 55 ms in 7 of 22 (32%) compared with 2 of 29 (7%) (p = 0.03). Superior axis premature ventricular contractions occurred only in gene carriers. In the second phase of the study, the frequency of these abnormalities was found to be high in patients with symptomatic ARVC: new epsilon waves appeared in 3 of 18 (17%), superior axis premature ventricular contractions in 21 of 25 (84%), and new terminal activation duration ≥ 55 ms in 8 of 12 (67%). CONCLUSIONS: Exercise testing exposes a latent electrical substrate in asymptomatic ARVC gene carriers that is shared by patients with ARVC with histories of ventricular arrhythmia. ETT may be useful in guiding treatment decisions, exercise prescription, and prioritizing medical surveillance in asymptomatic ARVC gene carriers.

Genetics of cardiac electrical disease.

Few tragedies compare to the sudden death of a family member. Sadly, this may represent the first sign of a familial vulnerability to such events. One common cause is an inherited cardiac arrhythmia syndrome. Sufferers are prone to premature sudden cardiac death due to altered ion channel function in the heart. Typical causes include Brugada syndrome, long QT syndrome, short QT syndrome, catecholaminergic polymorphic ventricular tachycardia, and the newly recognized early repolarization syndrome. Our knowledge of the genetic underpinnings of each of these disorders has increased markedly in recent years. Genetic screening is now a routine part of clinical care and promises more accurate diagnosis and efficient family screening. This review summarizes the diagnosis and management of each of the listed syndromes in the context of currently available genetic testing.

hERG K(+) channels: structure, function, and clinical significance.

The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the rapid component of the delayed rectifier K(+) channel, Kv11.1, which are expressed in the heart, various brain regions, smooth muscle cells, endocrine cells, and a wide range of tumor cell lines. However, it is the role that Kv11.1 channels play in the heart that has been best characterized, for two main reasons. First, it is the gene product involved in chromosome 7-associated long QT syndrome (LQTS), an inherited disorder associated with a markedly increased risk of ventricular arrhythmias and sudden cardiac death. Second, blockade of Kv11.1, by a wide range of prescription medications, causes drug-induced QT prolongation with an increase in risk of sudden cardiac arrest. In the first part of this review, the properties of Kv11.1 channels, including biogenesis, trafficking, gating, and pharmacology are discussed, while the second part focuses on the pathophysiology of Kv11.1 channels.

Anticoagulation bridging around device surgery: compliance with guidelines.

BACKGROUND: Current guidelines recommend bridging anticoagulation in patients undergoing cardiac rhythm device surgery with a "moderate to high risk" of thromboembolism. Patients at "low risk" are advised to stop oral anticoagulation without bridging to the procedure. This study examines real world adherence to accepted guidelines and the clinical sequelae of nonadherence. METHODS: We performed a review of all patients undergoing device surgery receiving chronic anticoagulation over a prespecified time period of 14 months. Patients were classified per American College of Chest Physician guidelines as "moderate/high risk" or "low risk" of thromboembolism. We then compared perioperative management of anticoagulation to guideline recommendations and assessed the rate of perioperative bleeding and thromboembolism. RESULTS: One hundred and twenty-nine patients were included in this study. Sixty-two (48%) were classified as "moderate/high risk" and 67 (52%) "low risk." In the "moderate/high risk" group 47/62 (76%) received perioperative anticoagulation but only 25/62 (40%) were bridged both pre- and postprocedure or maintained on uninterrupted warfarin. In the "low risk" group, 22/67 (33%) received bridging therapy. Device pocket hematoma or perioperative bleeding occurred in 10/129 (8%) with 4/10 receiving inappropriate bridging for a calculated low risk of thromboembolism. There were no perioperative thromboembolisms. CONCLUSIONS: Our study identified significant underutilization of bridging, particularly in the postoperative period, in patients at "moderate/high risk" of thromboembolism. Conversely, bridging was overused in "low risk" patients and associated with bleeding complications. Physicians should be urged to follow current expert guidelines in regard to bridging anticoagulation for cardiac rhythm device surgery. (PACE 2012;35:1480-1486).

The genetics of cardiac disease associated with sudden cardiac death: a paper from the 2011 William Beaumont Hospital Symposium on molecular pathology.

Sudden cardiac death due to ventricular arrhythmia most commonly occurs in the setting of coronary artery disease. However, a number of inherited syndromes have now been identified that carry a significant risk of sudden cardiac death and that are disproportionately represented in the young. Arrhythmia in such conditions may result from genetically mediated structural heart disease (eg, hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy) or from altered function of cardiac ion channels in the absence of overt structural disease (eg, Brugada syndrome and long QT syndrome). The past 15 years have seen considerable progress in our understanding of the genetic underpinnings of these disorders. With the advent of clinical genetic testing as a routine part of clinical care, a new knowledge base is required of practicing cardiologists and genetic testing facilities, particularly related to the rational ordering of genetic testing and the interpretation of results. This review addresses the latest findings in regard to the genetic causes of inherited syndromes associated with sudden cardiac death and summarizes recently published guidelines for the genetic testing of affected individuals and their families.

The role of atrial natriuretic peptide in modulating cardiac electrophysiology.

Since the discovery of atrial natriuretic peptide (ANP) in 1981, significant progress has been made in understanding the mechanism of its release and its role in salt and water balance in the body. It has also become clear that ANP plays a key role in cardiac electrophysiology, modulating the autonomic nervous system and regulating the function of cardiac ion channels. The clinical importance of this role was established when mutations in NPPA, the gene encoding ANP, were identified as a cause of familial atrial fibrillation. This review examines our current understanding of the electrophysiological effects of ANP, and their physiological relationship to clinical studies linking ANP and atrial fibrillation.

Human ether-a-go-go related gene (hERG) K+ channels: function and dysfunction.

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in regulating cardiac excitability and maintenance of normal cardiac rhythm. Mutations in hERG cause a third of all cases of congenital long QT syndrome, a disorder of cardiac repolarisation characterised by prolongation of the QT interval on the surface electrocardiogram, abnormal T waves, and a risk of sudden cardiac death due to ventricular arrhythmias. Additionally, the hERG channel protein is the molecular target for almost all drugs that cause the acquired form of long QT syndrome. Advances in understanding the structural basis of hERG gating, its traffic to the cell surface, and the molecular architecture involved in drug-block of hERG, are providing the foundation for rational treatment and prevention of hERG associated long QT syndrome. This review summarises the current knowledge of hERG function and dysfunction, and the areas of ongoing research.

Drug binding to the inactivated state is necessary but not sufficient for high-affinity binding to human ether-à-go-go-related gene channels.

Drug block of the human ether-à-go-go-related gene K(+) channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.