Sinus Bradycardia and Long QT Syndrome: Double Heterozygosity for Variants in KCNH2 and HCN4

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): the Dutch Research Council: NWO Talent Scheme Background A large multigenerational family harboring a pathogenic KCNH2 variant (L69P) was identified. This gene encodes the hERG channel, responsible for the cardiac rapid delayed rectifier K+ current (IKr). Pathogenic variants in KCNH2 can cause Long QT syndrome type 2 (LQTS2). Interestingly, in addition to LQTS2 we observed a high incidence of bradycardia in this family. Bradycardia is a known feature of some types of LQTS. However, evidence of bradycardia in LQTS2 is limited to a few sporadic cases. Purpose This study aims to identify the genetic variants and biophysiological changes in ion channel function, explanatory of the phenotypes LQTS2 and bradycardia. Methods An overview of the phenotype and genetic information of the index and family members was generated , including symptoms and electrocardiogram (ECG) parameters. QTc was calculated using Bazetts’ correction. Segregation of the identified genetic variants with LQTS and bradycardia were determined by LOD score. Biophysiological properties of the encoded ion channels was measured by manual whole-cell patch-clamp experiments. Results On the basis of linkage analysis, the pathogenic variant KCNH2-p.L69P was found to be unrelated to the bradycardia. Therefore, Sanger sequencing of HCN4, encoding the channel responsible for the hyperpolarization-activated current (If), an important current for sinoatrial node automaticity, was performed. We identified the HCN4-p.R666W variant in multiple family members, which co-segregated with bradycardia (LOD-score 3.2). Patients carrying both variants had more severe phenotypes than carriers of a single variant. Patch-clamp experiments in HEK293A-cells expressing wild type, or KCNH2-p.L69P show a reduced current density, and an altered time component of the fast deactivation, explaining the observed LQTS2. Functional assays of HCN4-p.R666W will elucidate the biophysiological changes possibly underlying the bradycardia. Conclusion we present a large multigenerational family that harbors a likely pathogenic variant in HCN4 in conjunction with a pathogenic variant of KCNH2. Double carriers were more affected than single carriers, arguing for continued alertness and deep phenotyping even in families with known pathogenic variants. Furthermore, we identified functional changes in the kinetics of the hERG channel encoded by KCNH2-p.L69P, elucidating the molecular mechanism underlying LQTS2 in this family.

Long QT syndrome (LQTS) type 3 although less common than the first two forms, differs in that arrhythmic events are less likely triggered by adrenergic stimuli and are more often lethal. Effective pharmacological treatment is challenged by interindividual differences, mutation dependence, and adverse effects, translating into an increased use of invasive measures (implantable cardioverter-defibrillator, sympathetic denervation) in patients with LQTS type 3. Previous studies have demonstrated the therapeutic potential of polyclonal KCNQ1 antibody for LQTS type 2. Here, we sought to identify a monoclonal KCNQ1 antibody that preserves the electrophysiological properties of the polyclonal form. Using hybridoma technology, murine monoclonal antibodies were generated, and patch clamp studies were performed for functional characterization. We identified a monoclonal KCNQ1 antibody able to normalize cardiac action potential duration and to suppress arrhythmias in a pharmacological model of LQTS type 3 using human-induced pluripotent stem cell-derived cardiomyocytes.NEW & NOTEWORTHY Long QT syndrome is a leading cause of sudden cardiac death in the young. Recent research has highlighted KCNQ1 antibody therapy as a new treatment modality for long QT syndrome type 2. Here, we developed a monoclonal KCNQ1 antibody that similarly restores cardiac repolarization. Moreover, the identified monoclonal KCNQ1 antibody suppresses arrhythmias in a cellular model of long QT syndrome type 3, holding promise as a first-in-class antiarrhythmic immunotherapy.

Injectable contraceptive Depo-Provera induces erratic beating patterns in patient-specific induced pluripotent stem cell–derived cardiomyocytes with long QT syndrome type 2

Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

Long QT syndrome (LQTS) type 3 is characterized by prolonged ventricular repolarization due to persistent sodium inward current secondary to a mutation in SCN5a, the gene encoding for the α-subunit of the sodium channel. We speculated that by disrupting calcium homeostasis the persistent inward sodium current in patients with LQTS type 3 might cause derangement of diastolic function. We aimed to identify functional myocardial alterations in a family with a sodium channelopathy with a phenotype of both LQTS type 3 and Brugada syndrome. The study group comprised 12 SCN5a mutation carriers (SCN5a-1795insD), 9 females and 3 males, mean age 35.7 ± 7.3 years, and 12 healthy controls. In addition to conventional echocardiographic measurements, two-dimensional speckle tracking was performed to assess tissue properties. Mean e' was lower in the patients compared with the controls (5.6 ± 0.75 vs. 6.7 ± 0.98 cm/s, P = 0.006). Onset QRS to maximum s' was longer in the patients than in the controls (0.20 ± 0.04 vs. 0.15 ± 0.05 s, P = 0.007), and the number of segments with post-systolic shortening was higher (6.58 ± 2.54 vs. 1.83 ± 1.64, P < 0.001). Patients in this family with LQTS type 3 showed post-systolic shortening, as well as both left and right ventricular diastolic dysfunction. The underlying mechanism remains to be elucidated but the persistent sodium inward current leading to calcium overload might play a role, in particular regarding diastolic dysfunction.

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Nonsense-mediated mRNA decay caused by a frameshift mutation in a large kindred of type 2 long QT syndrome

Gene-specific paradoxical QT responses during rapid eye movement sleep in women with congenital long QT syndrome

Variants in KCNH2, encoding the human ether a-go-go (hERG) channel that is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to long QT syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making. A genotype-phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual-, and automated calibrated patch clamp. HEK293a cells expressing (i) wild-type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, Hm) or (iii) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz) were used for manual patching. Automated patch clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines. Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2 or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch clamp showed a reduced current density by 69.8 and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared with KCNH2-WT. Assessment of KCNH2-p.S906L-Hz by calibrated automatic patch clamp assay showed a reduction in current density by 35.6%. The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2.

Background/Objectives: Long QT Syndrome type 2 (LQT2) is a cardiac channelopathy linked to pathogenic variants in the KCNH2 gene, which encodes the Kv11.1 potassium channel, essential for cardiac repolarization. Variants affecting splice sites disrupt potassium ion flow, prolong QT interval, and increase the risk of arrhythmias and sudden cardiac death (SCD). Understanding genotype-phenotype correlations is key, given the variability of clinical manifestations even within families sharing the same variant. We aimed to evaluate new pathogenic variants by analyzing genotype-phenotype correlations in informative families. Methods: Genetic and clinical assessments were performed on index cases and family members carrying KCNH2 pathogenic variants, referred for genetic testing between 2010 and June 2023. The next-generation sequencing (NGS) of 210 cardiovascular-related genes was conducted. Clinical data, including demographic details, family history, arrhythmic events, electrocardiographic parameters, and treatments, were collected. Results: Among 390 patients (152 probands) tested for LQTS, only 2 KCNH2 variants had over 5 carriers. The detailed clinical information of 22 carriers of this KCNH2 p.Ser261fs. has already been reported by our research group. Moreover, we identified 12 carriers of the KCNH2 c.77-2del variant, predicted to disrupt a splice site and not previously reported. Segregation analysis showed its high penetrance, supporting its classification as pathogenic. Conclusions: The newly identified KCNH2 c.77-2del variant is a pathogenic, as strongly supported by the segregation analysis. Our findings underscore the importance of further research into splice site variants to enhance clinical management and genetic counseling for affected families.

Mexiletine shortened QTc interval in an age-dependent manner but suppressed ventricular arrhythmias at all age in children with long QT syndrome type 8

See related article, pages 841–847 The study by Malan et al1 in this issue of the Circulation Research presents elegant data about induced pluripotent stem cell (iPSC)-derived myocytes2 from a mouse model of long QT syndrome (LQTS) type 3.3 The study is an important contribution that adds to a highly innovative field that is trying to define the role of iPSC technology in the understanding of inherited arrhythmias. In this accompanying editorial, I provide an overview of the previous studies in the field, comment on the contribution of Malan et al,1 and conclude by discussing some of the challenges in the field. The technique to differentiate cardiac myocytes from pluripotent stem cells is quite recent and it was introduced by the seminal article published in 2006 by Takahashi and Yamanaka,2 who used a combination of four retrovirally transduced transcription factors ( Oct3/4 , Sox2 , Klf4 , and c-Myc ) to generate iPSCs from mouse somatic cells. The importance of this technology was immediately recognized and it was rapidly applied to human cells.4 In the past few years, several investigators have refined protocols to optimize differentiation of iPSCs into cardiac myocytes and to characterize their properties. In analogy with myocardial cells derived from human embryonic cells,5 iPSC-derived myocytes differentiate into three cell types: nodal, atrial, and ventricular myocytes and present physiological adaptation of action potential duration to changes in heart rate and normal response to beta adrenergic stimulation.6 Furthermore, the presence of key ion channels7 and regulators of intracellular calcium physiology,8 as well as the preservation of the sarcomeric structure,9 have been confirmed in iPSC-derived myocytes. This comprehensive set of data provided a solid background to test the hypothesis that the differentiation of iPSC-derived cardiac cells from patients with inherited cardiac …

DETECTION OF CONGENITAL LONG QT SYNDROME WITH ARTIFICIAL INTELLIGENCE

Alternating Cycle Lengths Increases Dispersion of Action Potential Durations (APD) in Transgenic Rabbit Model of Long QT Syndrome Type 2

Generation and characterization of an induced pluripotent stem cell (iPSC) line (NUIGi003-A) from a long QT syndrome type 2 (LQT2) patient harbouring the KCNH2 c.2464G>A pathogenic variant.