Mirella Memmi

Mutations in the Cardiac Ryanodine Receptor Gene (hRyR2) Underlie Catecholaminergic Polymorphic Ventricular Tachycardia

BackgroundCatecholaminergic polymorphic ventricular tachycardia is a genetic arrhythmogenic disorder characterized by stress-induced, bidirectional ventricular tachycardia that may degenerate into cardiac arrest and cause sudden death. The electrocardiographic pattern of this ventricular tachycardia closely resembles the arrhythmias associated with calcium overload and the delayed afterdepolarizations observed during digitalis toxicity. We speculated that a genetically determined abnormality of intracellular calcium handling might be the substrate of the disease; therefore, we considered the human cardiac ryanodine receptor gene (hRyR2) a likely candidate for this genetically transmitted arrhythmic disorder. Methods and ResultsTwelve patients presenting with typical catecholaminergic polymorphic ventricular tachycardia in the absence of structural heart abnormalities were identified. DNA was extracted from peripheral blood lymphocytes, and single-strand conformation polymorphism analysis was performed on polymerase chain reaction–amplified exons of the hRyR2 gene. Four single nucleotide substitutions leading to missense mutations were identified in 4 probands affected by the disease. Genetic analysis of the asymptomatic parents revealed that 3 probands carried de novo mutations. In 1 case, the identical twin of the proband died suddenly after having suffered syncopal episodes. The fourth mutation was identified in the proband, in 4 clinically affected family members, and in none of 3 nonaffected family members in a kindred with 2 sudden deaths that occurred at 16 and 14 years, respectively, in the sisters of the proband. ConclusionsWe demonstrated that, in agreement with our hypothesis, hRyR2 is a gene responsible for catecholaminergic polymorphic ventricular tachycardia.

Clinical and Molecular Characterization of Patients With Catecholaminergic Polymorphic Ventricular Tachycardia

Background—Mutations in the cardiac ryanodine receptor gene (RyR2) underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmogenic disease occurring in the structurally intact heart. The proportion of patients with CPVT carrying RyR2 mutations is unknown, and the clinical features of RyR2-CPVT as compared with nongenotyped CPVT are undefined. Methods and Results—Patients with documented polymorphic ventricular arrhythmias occurring during physical or emotional stress with a normal heart entered the study. The clinical phenotype of the 30 probands and of 118 family members was evaluated, and mutation screening on the RyR2 gene was performed. Arrhythmias documented in probands were: 14 of 30 bidirectional ventricular tachycardia, 12 of 30 polymorphic ventricular tachycardia, and 4 of 30 catecholaminergic idiopathic ventricular fibrillation;RyR2 mutations were identified in 14 of 30 probands (36% bidirectional ventricular tachycardia, 58% polymorphic ventricular tachycardia, 50% catecholaminergic idiopathic ventricular fibrillation) and in 9 family members (4 silent gene carriers). Genotype-phenotype analysis showed that patients with RyR2 CPVT have events at a younger age than do patients with nongenotyped CPVT and that male sex is a risk factor for syncope in RyR2-CPVT (relative risk=4.2). Conclusions—CPVT is a clinically and genetically heterogeneous disease manifesting beyond pediatric age with a spectrum of polymorphic arrhythmias. &bgr;-Blockers reduce arrhythmias, but in 30% of patients an implantable defibrillator may be required. Genetic analysis identifies two groups of patients: Patients with nongenotyped CPVT are predominantly women and become symptomatic later in life; patients with RyR2 CPVT become symptomatic earlier, and men are at higher risk of cardiac events. These data provide a rationale for prompt evaluation and treatment of young men with RyR2 mutations.

Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by life threatening arrhythmias and mutations in the gene encoding the ryanodine receptor (RyR2). Disagreement exists on whether (1) RyR2 mutations induce abnormal calcium transients in the absence of adrenergic stimulation; (2) decreased affinity of mutant RyR2 for FKBP12.6 causes CPVT; (3) K201 prevent arrhythmias by normalizing the FKBP12.6-RyR2 binding. We studied ventricular myocytes isolated from wild-type (WT) and knock-in mice harboring the R4496C mutation (RyR2R4496C+/−). Pacing protocols did not elicit delayed afterdepolarizations (DADs) (n=20) in WT but induced DADs in 21 of 33 (63%) RyR2R4496C+/− myocytes (P=0.001). Superfusion with isoproterenol (30 nmol/L) induced small DADs (45%) and no triggered activity in WT myocytes, whereas it elicited DADs in 87% and triggered activity in 60% of RyR2R4496C+/− myocytes (P=0.001). DADs and triggered activity were abolished by ryanodine (10 &mgr;mol/L) but not by K201 (1 &mgr;mol/L or 10 &mgr;mol/L). In vivo administration of K201 failed to prevent induction of polymorphic ventricular tachycardia (VT) in RyR2R4496C+/− mice. Measurement of the FKBP12.6/RyR2 ratio in the heavy sarcoplasmic reticulum membrane showed normal RyR2–FKBP12.6 interaction both in WT and RyR2R4496C+/− either before and after treatment with caffeine and epinephrine. We suggest that (1) triggered activity is the likely arrhythmogenic mechanism of CPVT; (2) K201 fails to prevent DADs in RyR2R4496C+/− myocytes and ventricular arrhythmias in RyR2R4496C+/− mice; and (3) RyR2–FKBP12.6 interaction in RyR2R4496C+/− is identical to that of WT both before and after epinephrine and caffeine, thus suggesting that it is unlikely that the R4496C mutation interferes with the RyR2/FKBP12.6 complex.

Brugada syndrome and sudden cardiac death in children

In five children from the same family who died after unexplained cardiac arrest, Brugada syndrome syndrome was suspected based on the transient manifestation of the typical electrocardiogram pattern in one of them. A mutation in the cardiac sodium-channel confirmed the diagnosis of Brugada syndrome, which suggests that this disease may cause sudden death in children.

Long QT syndrome, Brugada syndrome, and conduction system disease are linked to a single sodium channel mutation

The function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na(+) channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (DeltaK1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and DeltaK1500 mutant channel. The mutation shifted V(1/2)h( infinity ) to more negative membrane potentials and increased k(h) consistent with a reduction of inactivation valence of 1. The shift in h( infinity ) was the result of an increase in closed-state inactivation rate (11-fold at -100 mV). V(1/2)m was shifted to more positive potentials, and k(m) was doubled in the DeltaK1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (change in charge, -1 and -2, respectively). For both mutations, V(1/2)h was shifted back toward control; however, V(1/2)m shifted progressively to more positive potentials. The late component of Na(+) current was increased in the DeltaK1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation.

Involvement of the cardiac ryanodine receptor/calcium release channel in catecholaminergic polymorphic ventricular tachycardia.

The cardiac ryanodine receptor (RyR2), the major calcium release channel on the sarcoplasmic reticulum (SR) in cardiomyocytes, has recently been shown to be involved in at least two forms of sudden cardiac death (SCD): (1) Catecholaminergic polymorphic ventricular tachycardia (CPVT) or familial polymorphic VT (FPVT); and (2) Arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Eleven RyR2 missense mutations have been linked to these diseases. All eleven RyR2 mutations cluster into 3 regions of RyR2 that are homologous to the three malignant hyperthermia (MH)/central core disease (CCD) mutation regions of the skeletal muscle ryanodine receptor/calcium release channel RyR1. MH/CCD RyR1 mutations have been shown to alter calcium‐induced calcium release. Sympathetic nervous system stimulation leads to phosphorylation of RyR2 by protein kinase A (PKA). PKA phosphorylation of RyR2 activates the channel. In conditions associated with high rates of SCD such as heart failure RyR2 is PKA hyperphosphorylated resulting in “leaky” channels. SR calcium leak during diastole can generate “delayed after depolarizations” that can trigger fatal cardiac arrhythmias (e.g., VT). We propose that RyR2 mutations linked to genetic forms of catecholaminergic‐induced SCD may alter the regulation of the channel resulting in increased SR calcium leak during sympathetic stimulation. J. Cell. Physiol. 190: 1–6, 2002.

Clinical Phenotype and Functional Characterization of CASQ2 Mutations Associated With Catecholaminergic Polymorphic Ventricular Tachycardia

Background— Four distinct mutations in the human cardiac calsequestrin gene (CASQ2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). The mechanisms leading to the clinical phenotype are still poorly understood because only 1 CASQ2 mutation has been characterized in vitro. Methods and Results— We identified a homozygous 16-bp deletion at position 339 to 354 leading to a frame shift and a stop codon after 5aa (CASQ2G112+5X) in a child with stress-induced ventricular tachycardia and cardiac arrest. The same deletion was also identified in association with a novel point mutation (CASQ2L167H) in a highly symptomatic CPVT child who is the first CPVT patient carrier of compound heterozygous CASQ2 mutations. We characterized in vitro the properties of CASQ2 mutants: CASQ2G112+5X did not bind Ca2+, whereas CASQ2L167H had normal calcium-binding properties. When expressed in rat myocytes, both mutants decreased the sarcoplasmic reticulum Ca2+-storing capacity and reduced the amplitude of ICa-induced Ca2+ transients and of spontaneous Ca2+ sparks in permeabilized myocytes. Exposure of myocytes to isoproterenol caused the development of delayed afterdepolarizations in CASQ2G112+5X. Conclusions— CASQ2L167H and CASQ2G112+5X alter CASQ2 function in cardiac myocytes, which leads to reduction of active sarcoplasmic reticulum Ca2+ release and calcium content. In addition, CASQ2G112+5X displays altered calcium-binding properties and leads to delayed afterdepolarizations. We conclude that the 2 CASQ2 mutations identified in CPVT create distinct abnormalities that lead to abnormal intracellular calcium regulation, thus facilitating the development of tachyarrhythmias.

Novel Insight Into the Natural History of Short QT Syndrome

Objectives This study intends to gain further insights into the natural history, the yield of familial and genetic screening, and the arrhythmogenic mechanisms in the largest cohort of short QT syndrome (SQTS) patients described so far. Background SQTS is a rare genetic disorder associated with life-threatening arrhythmias, and its natural history is incompletely ascertained. Methods Seventy-three SQTS patients (84% male; age, 26 ± 15 years; corrected QT interval, 329 ± 22 ms) were studied, and 62 were followed for 60 ± 41 months (median, 56 months). Results Cardiac arrest (CA) was the most frequent presenting symptom (40% of probands; range, <1 month to 41 years). The rate of CA was 4% in the first year of life and 1.3% per year between 20 and 40 years; the probability of a first occurrence of CA by 40 years of age was 41%. Despite the male predominance, female patients had a risk profile superimposable to that of men (p = 0.49). The yield of genetic screening was low (14%), despite familial disease being present in 44% of kindreds. A history of CA was the only predictor of recurrences at follow-up (p < 0.0000001). Two patterns of onset of ventricular fibrillation were observed and were reproducible in patients with multiple occurrences of CA. Arrhythmias occurred mainly at rest. Conclusions SQTS is highly lethal; CA is often the first manifestation of the disease with a peak incidence in the first year of life. Survivors of CA have a high CA recurrence rate; therefore, implantation of a defibrillator is strongly recommended in this group of patients.

Gene-Specific Therapy With Mexiletine Reduces Arrhythmic Events in Patients With Long QT Syndrome Type 3

Background Long QT syndrome type 3 (LQT3) is a lethal disease caused by gain-of-function mutations in the SCN5A gene, coding for the alpha-subunit of the sodium channel NaV1.5. Mexiletine is used to block late sodium current and to shorten QT interval in LQT3 patients. Objectives The aim of this study was to determine whether mexiletine prevents arrhythmic events (arrhythmic syncope, aborted cardiac arrest, or sudden cardiac death) in LQT3 patients. Methods The endpoint of this retrospective cohort study, which studied consecutive LQT3 patients who were referred to our center and treated with mexiletine, was to evaluate the antiarrhythmic efficacy of mexiletine by comparing the number of arrhythmic events per patient and the annual rate of arrhythmic events during observation periods of equal duration before and after the beginning of therapy with mexiletine. Results The study population comprised 34 LQT3 patients, 19 (56%) of whom were male. The median age at beginning of treatment with mexiletine was 22 years, and median QTc interval before therapy 509 ms. The median duration of oral mexiletine therapy was 36 months, at an average daily dose of 8 ± 0.5 mg/kg. Mexiletine significantly shortened QTc (by 63 ± 6 ms; p < 0.0001) and reduced the percentage of patients with arrhythmic events (from 22% to 3%; p = 0.031), the mean number of arrhythmic events per patient (from 0.43 ± 0.17 to 0.03 ± 0.03; p = 0.027), and the annual rate of arrhythmic events (from 10.3% to 0.7%; p = 0.0097). Conclusions Besides shortening QTc interval, mexiletine caused a major reduction of life-threatening arrhythmic events in LQT3 patients, thus representing an efficacious therapeutic strategy.

Clinical utility gene card for: Catecholaminergic polymorphic ventricular tachycardia (CPVT)

Name of the disease (synonyms): Synonyms: Familial polymorphic ventricular tachycardia (FPVT), catecholamine-induced polymorphic ventricular tachycardia (CPVT). Includes: RYR2-related catecholaminergic ventricular tachycardia, CASQ2-related catecholaminergic ventricular tachycardia OMIM# of the disease: 604772, 611938 Name of the analysed genes or DNA/chromosome segments: RyR2, cardiac ryanodine receptor OMIM# of the gene(s): 180902, 114251 Two genes are clearly associated with CPVT (RyR2 autosomal dominant, and CASQ2 autosomal recessive). Triadin mutations have been also shown in two CPVT families but data need confirmation. Therefore, clinical testing is not indicated. Review of the analytical and clinical validity as well as of the clinical utility of DNA-based testing for mutations in the RyR2 gene in diagnostic, predictive and prenatal settings and for risk assessment in relatives.