Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level.

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during ß-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during ß-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.

CPVT-associated calmodulin variants N53I and A102V dysregulate Ca2+ signalling via different mechanisms.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that interaction of both Ca2+/CaM-N53I and Ca2+/CaM-A102V with the RyR2 binding domain is decreased. Ca2+/CaM-RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild type (WT). We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events; CaM-A102V exhibited a lower frequency of Ca2+ oscillations. In addition, we show that CaMKIIδ (also known as CAMK2D) phosphorylation activity is increased for A102V, compared to CaM-WT. This paper provides novel insight into the molecular mechanisms of CPVT-associated CaM variants and will facilitate the development of strategies for future therapies.

Molecular genetics in 1991 arrhythmia probands and 2782 relatives in Norway: Results from 17 years of genetic testing in a national laboratory.

The aim of this study was to explore the prevalence of likely pathogenic or pathogenic variants and assess the diagnostic yield from genetic testing for cardiac arrhythmias in Norway since 2003. Data from 1991 probands and 2782 relatives were retrospectively collected from the laboratory information management system at Unit for Cardiac and Cardiovascular Genetics, Oslo University hospital. Of 1991 probands, 57.4% were females, age at genetic testing was 33.1 (±22.7) years, and 32.5% were under the age of 18. A likely pathogenic or pathogenic variant (including 14 novel) was detected in 15.4% in total. Of the 2782 relatives, 53.7% were females, age at genetic testing was 35.6 (±22.5) years, 27.3% were under the age of 18, and 45.3% carried the family variant. Probands and relatives combined, 1/3356 persons in the Norwegian population were heterozygous for an arrhythmia-causing variant. The founder variant p.Q530X (NM_000218.2: c.1588C>T) in KCNQ1 accounted for 34% of all variants in Norway. In conclusion, genetic testing provided a genetic basis of the arrhythmia in 15.4% of the probands. Familial cascade screening identified four times as many variant-positive relatives, allowing early detection and prompt stratification of arrhythmic risk of those variant carriers.

Human Genetics of Cardiac Arrhythmias.

Inherited forms of cardiac arrhythmias mostly are rare diseases (prevalence <1:2000) and considered to be either "primary electrical heart disorders" due to the absence of structural heart abnormalities or "cardiac ion channel disorders" due to the myocellular structures involved. Precise knowledge of the electrocardiographic features of these diseases and their genetic classification will enable early disease recognition and prevention of cardiac events including sudden cardiac death.The genetic background of these diseases is complex and heterogeneous. In addition to the predominant "private character" of a mutation in each family, locus heterogeneity involving many ion channel genes for the same familial arrhythmia syndrome is typical. Founder pathogenic variants or mutational hot spots are uncommon. Moreover, phenotypes may vary and overlap even within the same family and mutation carriers. For the majority of arrhythmias, the clinical phenotype of an ion channel mutation is restricted to cardiac tissue, and therefore, the disease is nonsyndromic.Recent and innovative methods of parallel DNA analysis (so-called next-generation sequencing, NGS) will enhance further mutation and other variant detection as well as arrhythmia gene identification.

Molecular Pathways and Animal Models of Arrhythmias.

Arrhythmias account for over 300,000 annual deaths in the United States, and approximately half of all deaths are associated with heart disease. Mechanisms underlying arrhythmia risk are complex; however, work in humans and animal models over the past 25 years has identified a host of molecular pathways linked with both arrhythmia substrates and triggers. This chapter will focus on select arrhythmia pathways solved by linking human clinical and genetic data with animal models.

A novel RyR2 mutation associated with co-morbid catecholaminergic polymorphic ventricular tachycardia (CPVT) and benign epilepsy with centrotemporal spikes (BECTS).

In this case report, we describe a 14-year-old patient with a novel RyR2 gene mutation (c.6577G > T/p.Val2193Leu), identified through a comprehensive review of medical history, examination findings, and follow-up data. The pathogenic potential of this mutation, which results in the loss of some interatomic forces and compromises the closure of the RyR2 protein pore leading to calcium leakage, was analyzed using the I-TASSER Suite to predict the structural changes in the protein. This mutation manifested clinically as co-morbid catecholaminergic polymorphic ventricular tachycardia (CPVT) and benign epilepsy with centrotemporal spikes (BECTS), a combination not previously documented in the same patient. While seizures were successfully managed with levetiracetam, the patient's exercise-induced syncope episodes could not be controlled with metoprolol, highlighting the complexity and challenge in managing CPVT associated with this novel RyR2 variation.

Prognosis and clinical management of asymptomatic family members with RYR2-mediated catecholaminergic polymorphic ventricular tachycardia: a review.

Despite its low prevalence, the potential diagnosis of catecholaminergic polymorphic ventricular tachycardia (CPVT) should be at the forefront of a paediatric cardiologists mind in children with syncope during exercise or emotions. Over the years, the number of children with a genetic diagnosis of CPVT due to a (likely) pathogenic RYR2 variant early in life and prior to the onset of symptoms has increased due to cascade screening programmes. Limited guidance for this group of patients is currently available. Therefore, we aimed to summarise currently available literature for asymptomatic patients with a (likely) pathogenic RYR2 variant, particularly the history of CPVT and its genetic architecture, the currently available diagnostic tests and their limitations, and the development of a CPVT phenotype - both electrocardiographically and symptomatic - of affected family members. Their risk of arrhythmic events is presumably low and a phenotype seems to develop in the first two decades of life. Future research should focus on this group in particular, to better understand the development of a phenotype over time, and therefore, to be able to better guide clinical management - including the frequency of diagnostic tests, the timing of the initiation of drug therapy, and lifestyle recommendations.

Genetic and clinical characteristics of catecholaminergic polymorphic ventricular tachycardia in a Taiwanese nationwide cohort.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare and lethal arrhythmia. Ryanodine receptor 2 (RYR2) mutation accounts for ∼60% of CPVT patients which is inherited in an autosomal dominant pattern. OBJECTIVE: This study aimed to identify CPVT-related mutations and clinical characteristics among Taiwanese CPVT patients and compare to other cohorts worldwide. METHODS: Clinical and genetic data were obtained from the Sudden Arrhythmia Death Syndrome Registry in Taiwan (SADS-TW). Forty clinically diagnosed Taiwanese CPVT patients were included. RESULTS: This is the first nationwide CPVT cohort in Taiwan. Among the 29 Taiwanese patients with CPVT-related gene mutations, 55% had RYR2 mutations, a rate similar to other ethnicities. Three out of 12 RYR2 variants were unreported. Exercise-induced symptoms including syncope and cardiac arrest were more frequent in East Asian cohorts (Taiwanese 79%, Japanese 91%), compared to Caucasian cohorts (59%) (p = 0.002). CONCLUSION: The discovery of diverse RYR2 mutations in the Taiwanese CVPT population demonstrates the importance of genetic testing in different ethnicities.

Primary Electrical Heart Disease-Principles of Pathophysiology and Genetics.

Primary electrical heart diseases, often considered channelopathies, are inherited genetic abnormalities of cardiomyocyte electrical behavior carrying the risk of malignant arrhythmias leading to sudden cardiac death (SCD). Approximately 54% of sudden, unexpected deaths in individuals under the age of 35 do not exhibit signs of structural heart disease during autopsy, suggesting the potential significance of channelopathies in this group of age. Channelopathies constitute a highly heterogenous group comprising various diseases such as long QT syndrome (LQTS), short QT syndrome (SQTS), idiopathic ventricular fibrillation (IVF), Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and early repolarization syndromes (ERS). Although new advances in the diagnostic process of channelopathies have been made, the link between a disease and sudden cardiac death remains not fully explained. Evolving data in electrophysiology and genetic testing suggest previously described diseases as complex with multiple underlying genes and a high variety of factors associated with SCD in channelopathies. This review summarizes available, well-established information about channelopathy pathogenesis, genetic basics, and molecular aspects relative to principles of the pathophysiology of arrhythmia. In addition, general information about diagnostic approaches and management is presented. Analyzing principles of channelopathies and their underlying causes improves the understanding of genetic and molecular basics that may assist general research and improve SCD prevention.

Trans-2,3-enoyl-CoA reductase-like-related catecholaminergic polymorphic ventricular tachycardia with regular ventricular tachycardia and response to flecainide.

INTRODUCTION: We describe a unique case of TECRL-CPVT presented with cardiac arrest. METHODS: Post resuscitation, the patient developed regular ventricular tachycardia featuring a left purkinje system morphology. RESULTS: There was clear suppression of arrhythmia with the addition of flecainide and isolated ventricular ectopy causing secondary T-wave changes. CONCLUSION: A high index of suspicion was required to eventually make the diagnosis through whole exome sequencing.

Genome Editing and Inherited Cardiac Arrhythmias.

Inherited arrhythmic disorders are a group of heterogeneous diseases predisposing to life-threatening arrhythmias and sudden cardiac death. Their diagnosis is not always simple due to incomplete penetrance and genetic heterogeneity. Furthermore, the available treatments are usually invasive and merely preventive. Genome editing and especially CRISPR/Cas9 technologies have the potential to correct the genetic arrhythmogenic substrate, thereby offering a cure for these fatal diseases. To date, genome editing has allowed reproducing cardiac arrhythmias in vitro, providing a robust platform for variant pathogenicity, mechanistic, and drug-testing studies. However, in vivo approaches still need profound research regarding safety, specificity, and efficiency of the methods.

Importance of exercise stress testing in evaluation of unexplained cardiac arrest survivor.

BACKGROUND: In sudden cardiac arrest survivors without an immediately identifiable cause, additional extensive yet individualised testing is required. METHODS: We describe 3 survivors of sudden cardiac arrest in whom exercise stress testing was not performed during the initial hospital admission. RESULTS: All 3 patients were incorrectly diagnosed with long QT syndrome based on temporary sudden cardiac arrest-related heart rate-corrected QT interval prolongation, and exercise stress testing was not performed during the initial work-up. When they were subjected to exercise stress testing during follow-up, a delayed diagnosis of catecholaminergic polymorphic ventricular tachycardia (CPVT) was made. As a result, these patients were initially managed inappropriately, and their family members were initially not screened for CPVT. CONCLUSION: In sudden cardiac arrest survivors without an immediately identifiable cause, omission of exercise stress testing or erroneous interpretation of the results can lead to a delayed or missed diagnosis of CPVT, which may have considerable implications for survivors and their family.

Clinical Characteristics, Genetic Basis and Healthcare Resource Utilisation and Costs in Patients with Catecholaminergic Polymorphic Ventricular Tachycardia: A Retrospective Cohort Study.

Background: This study examined the clinical characteristics, genetic basis, healthcare utilisation and costs of catecholaminergic ventricular tachycardia (CPVT) patients from a Chinese city. Methods: This was a territory-wide retrospective cohort study of consecutive CPVT patients at public hospitals or clinics in Hong Kong. Healthcare resource utilisation for accident and emergency (A&E), inpatient and outpatient attendances were analysed over 19 years (2001-2019) followed by calculations of annualised costs (in USD). Results: Sixteen patients with a median presentation age (interquartile range (IQR) of 11 (9-14) years old) were included. Fifteen patients (93.8%) were initially symptomatic. Ten patients had both premature ventricular complexes (PVCs) and ventricular tachycardia/fibrillation (VT/VF). One patient had PVCs without VT/VF. Genetic tests were performed on 14 patients (87.5%). Eight (57.1%) tested positive for the ryanodine receptor 2 (RyR2) gene. Seven variants have been described elsewhere (c.14848G > A, c.12475C > A, c.7420A > G, c.11836G > A, c.14159T > C, c.10046C > T and c.7202G > A). c.14861C > G is a novel RyR2 variant not been reported outside this cohort. Patients were treated with beta-blockers (n = 16), amiodarone (n = 3) and verapamil (n = 2). Sympathectomy (n = 8) and implantable-cardioverter defibrillator implantation (n = 3) were performed. Over a median follow-up of 13.3 years (IQR: 8.4-18.1) years, six patients exhibited incident VT/VF. At the patient level, the median (IQR) annualised costs for A&E, inpatient and outpatient attendances were $ 66 (40-95), $ 10521 (5240-66887) and $ 791 (546-1105), respectively. Conclusions: All patients presented before the age of 19. The yield of genetic testing was 57%. The most expensive attendance type was inpatient stays, followed by outpatients and A&E attendances.

Unnatural verticilide enantiomer inhibits type 2 ryanodine receptor-mediated calcium leak and is antiarrhythmic.

Ca2+ leak via ryanodine receptor type 2 (RyR2) can cause potentially fatal arrhythmias in a variety of heart diseases and has also been implicated in neurodegenerative and seizure disorders, making RyR2 an attractive therapeutic target for drug development. Here we synthesized and investigated the fungal natural product and known insect RyR antagonist (-)-verticilide and several congeners to determine their activity against mammalian RyR2. Although the cyclooligomeric depsipeptide natural product (-)-verticilide had no effect, its nonnatural enantiomer [ent-(+)-verticilide] significantly reduced RyR2-mediated spontaneous Ca2+ leak both in cardiomyocytes from wild-type mouse and from a gene-targeted mouse model of Ca2+ leak-induced arrhythmias (Casq2-/-). ent-(+)-verticilide selectively inhibited RyR2-mediated Ca2+ leak and exhibited higher potency and a distinct mechanism of action compared with the pan-RyR inhibitors dantrolene and tetracaine and the antiarrhythmic drug flecainide. ent-(+)-verticilide prevented arrhythmogenic membrane depolarizations in cardiomyocytes without significant effects on the cardiac action potential and attenuated ventricular arrhythmia in catecholamine-challenged Casq2-/- mice. These findings indicate that ent-(+)-verticilide is a potent and selective inhibitor of RyR2-mediated diastolic Ca2+ leak, making it a molecular tool to investigate the therapeutic potential of targeting RyR2 hyperactivity in heart and brain pathologies. The enantiomer-specific activity and straightforward chemical synthesis of (unnatural) ent-(+)-verticilide provides a compelling argument to prioritize ent-natural product synthesis. Despite their general absence in nature, the enantiomers of natural products may harbor unprecedented activity, thereby leading to new scaffolds for probe and therapeutic development.

Transient ventricular bigeminy during emergence from general anesthesia in a pediatric patient with catecholaminergic polymorphic ventricular tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare genetic disease that is characterized by ventricular arrhythmias and sudden death, induced by exogenous and endogenous catecholamine. We performed general anesthesia for dental treatment of multiple teeth in a 7-year-old boy with CPVT. To avoid sympathetic tone, anesthesia was maintained by total intravenous anesthesia, but ventricular bigeminy was induced by stimulation on emergence form general anesthesia. Although bigeminy in the present case might have been less likely to induce a fatal arrhythmia, we should keep in mind that a small amount of sympathetic tone may induce arrhythmias in a patient with CPVT.

Therapeutic Approaches of Ryanodine Receptor-Associated Heart Diseases.

Cardiac diseases are the leading causes of death, with a growing number of cases worldwide, posing a challenge for both healthcare and research. Therefore, the most relevant aim of cardiac research is to unravel the molecular pathomechanisms and identify new therapeutic targets. Cardiac ryanodine receptor (RyR2), the Ca2+ release channel of the sarcoplasmic reticulum, is believed to be a good therapeutic target in a group of certain heart diseases, collectively called cardiac ryanopathies. Ryanopathies are associated with the impaired function of the RyR, leading to heart diseases such as congestive heart failure (CHF), catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular dysplasia type 2 (ARVD2), and calcium release deficiency syndrome (CRDS). The aim of the current review is to provide a short insight into the pathological mechanisms of ryanopathies and discuss the pharmacological approaches targeting RyR2.

The arrhythmogenic N53I variant subtly changes the structure and dynamics in the calmodulin N-terminal domain, altering its interaction with the cardiac ryanodine receptor.

Mutations in the genes encoding the highly conserved Ca2+-sensing protein calmodulin (CaM) cause severe cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia or long QT syndrome and sudden cardiac death. Most of the identified arrhythmogenic mutations reside in the C-terminal domain of CaM and mostly affect Ca2+-coordinating residues. One exception is the catecholaminergic polymorphic ventricular tachycardia-causing N53I substitution, which resides in the N-terminal domain (N-domain). It does not affect Ca2+ coordination and has only a minor impact on binding affinity toward Ca2+ and on other biophysical properties. Nevertheless, the N53I substitution dramatically affects CaM's ability to reduce the open probability of the cardiac ryanodine receptor (RyR2) while having no effect on the regulation of the plasmalemmal voltage-gated Ca2+ channel, Cav1.2. To gain more insight into the molecular disease mechanism of this mutant, we used NMR to investigate the structures and dynamics of both apo- and Ca2+-bound CaM-N53I in solution. We also solved the crystal structures of WT and N53I CaM in complex with the primary calmodulin-binding domain (CaMBD2) from RyR2 at 1.84-2.13 Å resolutions. We found that all structures of the arrhythmogenic CaM-N53I variant are highly similar to those of WT CaM. However, we noted that the N53I substitution exposes an additional hydrophobic surface and that the intramolecular dynamics of the protein are significantly altered such that they destabilize the CaM N-domain. We conclude that the N53I-induced changes alter the interaction of the CaM N-domain with RyR2 and thereby likely cause the arrhythmogenic phenotype of this mutation.

CaMKII inhibition has dual effects on spontaneous Ca2+ release and Ca2+ alternans in ventricular cardiomyocytes from mice with a gain-of-function RyR2 mutation.

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca2+/calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca2+ alternans. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. We studied spontaneous Ca2+ release events and Ca2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca2+ release events in RyR2-R2474S cardiomyocytes exposed to the ß-adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca2+ alternans and increased Ca2+ alternans ratio compared with both an inactive analog of KN-93 and with vehicle-treated controls. This increased propensity for Ca2+ alternans was explained by prolongation of Ca2+ release refractoriness. Importantly, the increased propensity for Ca2+ alternans in KN-93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild type. In conclusion, inhibition of CaMKII efficiently reduces spontaneous Ca2+ release but promotes Ca2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca2+ release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca2+ alternans should include investigation of both phenomena.NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca2+ release. Prolonged refractoriness of Ca2+ release leads to arrhythmogenic Ca2+ alternans. CaMKII inhibition promotes Ca2+ alternans by prolonging Ca2+ release refractoriness.

Novel cases of pediatric sudden cardiac death secondary to TRDN mutations presenting as long QT syndrome at rest and catecholaminergic polymorphic ventricular tachycardia during exercise: The TRDN arrhythmia syndrome.

TRDN mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT) but may present with abnormal electrocardiogram (ECG) findings provoking a diagnosis of long QT syndrome (LQTS). We report two novel cases of sudden cardiac death in children due to mutations of TRDN, providing further insight into this rare and aggressive inherited arrhythmia syndrome. Whole exome sequencing (WES) was performed in two unrelated children who experienced cardiac arrest during exercise and were negative for targeted testing of LQTS. WES identified a novel homozygous splice-site mutation in both patients, denoted c.22+1G>T, absent from gnomAD and suggesting a founder variant in the Iranian population. We now summarize the genetic architecture of all reported TRDN-related patients, including 27 patients from 21 families. The average age-onset was 30 months (range 1-10) for all cases. Adrenergic-mediated cardiac events were common, occurring in 23 of 27 cases (85%). LQTS was diagnosed in 10 cases (37%), CPVT in 10 (37%) cases, and in 7 cases. No phenotypic diagnosis was provided. Five cases (15%) had evidence for associated skeletal myopathy. Four missense TRDN variants (24%) were observed in diseased cases, while the remaining variants reflect putative loss-of-function (LOF) mutations. No disease phenotype was reported in 26 heterozygous carriers. In conclusion, TRDN mutations cause a rare autosomal recessive arrhythmia syndrome presenting with adrenergic-mediated arrhythmic events, but with ECG abnormalities leading to a diagnosis of LQTS in a proportion of cases. Heterozygous carriers are free of disease manifestations.

Novel variants in TECRL cause recessive inherited CPVT type 3 with severe and variable clinical symptoms.

INTRODUCTION: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by adrenergically stimulated ventricular tachycardia. The most common form of CPVT is due to autosomal dominant variants in the cardiac ryanodine-receptor gene (RYR2). However, trans-2,3-enoyl-CoA reductase-like (TECRL) was recently suggested to be a novel candidate gene for life-threatening inherited arrhythmias. Patients previously reported with pathogenic changes in TECRL showed a special mixed phenotype of CPVT and long-QT-syndrome (LQTS) termed CPVT type 3 (CPVT3), an autosomal recessive disorder. METHODS AND RESULTS: We implemented TECRL into our NGS panel diagnostics for CPVT and LQTS in April 2017. By December 2018, 631 index patients with suspected CPVT or LQTS had been referred to our laboratory for genetic testing. Molecular analysis identified four Caucasian families carrying novel variants in TECRL. One patient was homozygous for Gln139* resulting in a premature stop codon and loss-of-function of the TECRL protein. Another patient was homozygous for Pro290His, probably leading to an altered folding of the 3-oxo-5-alpha steroid 4-dehydrogenase domain of the TECRL protein. The LOF-variant Ser309* and the missense-variant Val298Ala have been shown to be compound heterozygous in another individual. NGS-based copy number variation analysis and quantitative PCR revealed a quadruplication of TECRL in the last individual, which is likely to be a homozygous duplication. CONCLUSION: The data from our patient collective indicate that CPVT3 occurs much more frequently than previously expected. Variants in TECRL may be causative in up to 5% of all CPVT cases. According to these findings, the default analysis of this gene is recommended if CPVT is suspected.