Clinical and Functional Characterization of Ryanodine Receptor 2 Variants Implicated in Calcium-Release Deficiency Syndrome

Abstract

Importance: Calcium-release deficiency syndrome (CRDS), which is caused by loss-of-function variants in cardiac ryanodine receptor 2 (RyR2), is an emerging cause of ventricular fibrillation. However, the lack of complex polymorphic/bidirectional ventricular tachyarrhythmias during exercise stress testing (EST) may distinguish it from catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, in the first clinical series describing the condition, mouse and human studies showed that the long-burst, long-pause, short-coupled ventricular extra stimulus (LBLPS) electrophysiology protocol reliably induced CRDS ventricular arrhythmias. Data from larger populations with CRDS and its associated spectrum of disease are lacking.

Objective: To further insight into CRDS through international collaboration.

Design, setting, and participants: In this multicenter observational cohort study, probands with unexplained life-threatening arrhythmic events and an ultrarare RyR2 variant were identified. Variants were expressed in HEK293 cells and subjected to caffeine stimulation to determine their functional impact. Data were collected from September 1, 2012, to March 6, 2021, and analyzed from August 9, 2015, to March 6, 2021.

Main outcomes and measures: The functional association of RyR2 variants found in putative cases of CRDS and the associated clinical phenotype(s).

Results: Of 10 RyR2 variants found in 10 probands, 6 were loss-of-function, consistent with CRDS (p.E4451del, p.F4499C, p.V4606E, p.R4608Q, p.R4608W, and p.Q2275H) (in 4 [67%] male and 2 [33%] female probands; median age at presentation, 22 [IQR, 8-34] years). In 5 probands with a documented trigger, 3 were catecholamine driven. During EST, 3 probands with CRDS had no arrhythmias, 1 had a monomorphic couplet, and 2 could not undergo EST (deceased). Relatives of the decedents carrying the RyR2 variant did not have EST results consistent with CPVT. After screening 3 families, 13 relatives were diagnosed with CRDS, including 3 with previous arrhythmic events (23%). None had complex ventricular tachyarrhythmias during EST. Among the 19 confirmed cases with CRDS, 10 had at least 1 life-threatening event at presentation and/or during a median follow-up of 7 (IQR, 6-18) years. Two of the 3 device-detected ventricular fibrillation episodes were induced by a spontaneous LBLPS-like sequence. β-Blockers were used in 16 of 17 surviving patients (94%). Three of 16 individuals who were reportedly adherent to β-blocker therapy (19%) had breakthrough events.

Conclusions and relevance: The results of this study suggest that calcium-release deficiency syndrome due to RyR2 loss-of-function variants mechanistically and phenotypically differs from CPVT. Ventricular fibrillation may be precipitated by a spontaneous LBLPS-like sequence of ectopy; however, CRDS remains difficult to recognize clinically. These data highlight the need for better diagnostic tools and treatments for this emerging condition.

Conflict of interest statement

Conflict of Interest Disclosures: Ms Orland reported receiving personal fees from My Gene Counsel outside the submitted work. Dr Wilde reported serving as a member of the scientific advisory board from LQT Therapeutics outside the submitted work. Dr Jensen reported receiving grants from the Novo Nordisk Foundation, Denmark, and personal fees from Biosense Webster, Inc, and Abbott Denmark outside the submitted work. Dr Sanatani reported receiving grants from Heart and Stroke Foundation during the conduct of the study and serving as medical advisor for emtelligent outside the submitted work. No other disclosures were reported.

Figures

Figure 1.. Summary of Ryanodine Receptor 2 (RyR2)–Calcium-Release Deficiency Syndrome (CRDS) Case Ascertainment and Outcomes in the Multicenter Cohort

CPVT indicates catecholaminergic polymorphic ventricular tachycardia; GOF, gain of function; LOF, loss of function; PVC, premature ventricular complex; and WT, wild-type.

Figure 2.. Location of Ryanodine Receptor 2 (RyR2) Sequence Variants in the 3-Dimensional (3-D) Structure of RyR2

A, Schematic diagram of the linear sequence of RyR2 showing major structural domains of RyR2 (blue boxes). Orange boxes indicate 4 disease-associated variant clusters (variant hotspots). RyR2 variants identified and characterized in this study are indicated beneath their corresponding domains. B, Locations of RyR2 variants in the 3-D structure of RyR2 (Protein Data Bank identification code 6JI0). Locations of the cytosolic calcium ion (Ca2+) binding sites, U-motif, C-terminal domain (CTD), S2 to S3 loop, and S1 to S6 helixes are also depicted. The 3-D locations of RyR2 residues, Q2275 and E4451, have not been resolved; therefore, the p.Q2275H and p.E4451del variants are not shown in the 3-D structure.

Figure 3.. Responses of Wild-Type (WT) Ryanodine Receptor (RyR2) and Mutants to Caffeine Activation

The association between caffeine-induced calcium ion (Ca2+) release and the cumulative caffeine concentration in HEK293 cells transfected with WT RyR2 and mutants. The amplitude of caffeine-induced Ca2+ release at each caffeine concentration was normalized to that of the maximum caffeine peak for each experiment. Data are shown as the mean (SEM) for 5-12 patients (P < .05 vs WT [1-way analysis of variance with a Bonferroni post hoc test]). The caffeine response curves of loss-of-function RyR2 variants (p.Q2275H, p.E4451del, p.F4499C, p.V4606E, p.R4608Q, and p.R4608W) are depicted in blue; gain-of-function RyR2 variants (p.G3988D, p.G4772S, and p.D4956E), in orange; and WT-like variant (p.I4867T), in gray.

Figure 4.. Device Recordings of Captured Calcium-Release Deficiency Syndrome (CRDS) Arrhythmic Events

A, Electrogram leading up to ventricular fibrillation (VF) in the genetically affected first-degree relative of proband 3 showing sinus tachycardia with stable cycle length, 2 premature ventricular complexes (PVCs), a long pause, a sinus beat, and a shorter coupled PVC, precipitating polymorphic ventricular tachycardia/VF. B, Dot plot of appropriate implantable cardioverter-defibrillator high-rate detection in second VF episode in proband 1 with RyR2-p.Q2275H variant. A indicates atrial; V, ventricular.