RYR2 Variants in Catecholaminergic Polymorphic Ventricular Tachycardia Patients: Insights From Protein Structure and Clinical Data

Authors, Journal, Affiliations, Type, DOI

• Alexander Chang, Halil Beqaj, Leah Sittenfeld, Marco C. Miotto, Haikel Dridi, Gloria Willson, Carolyn Martinez Jorge, Jaan Altosaar Li, Steven Reiken, Yang Liu, Zonglin Dai, Carl Tchagou, Sana Elsayed, Steven O. Marx, Andrew R. Marks
• Circulation: Arrhythmia and Electrophysiology (AHA), 2025;18:e013757
• Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY
• Systematic review and meta-analysis (PRISMA-guided) of published RYR2 variant data
• DOI: 10.1161/CIRCEP.124.013757

Overview

Largest-ever systematic review and database compilation of CPVT-associated RYR2 variants: 221 publications yielded 1342 patients with RYR2 variants, of whom 964 had a clinical CPVT diagnosis. The database catalogs 263 unique RYR2 protein-coding variants with detailed clinical phenotypes and treatment outcomes. Variants were mapped onto the high-resolution cryo-EM structure of human RYR2 (PDB: 7UA5) to correlate variant location with phenotype severity. The median age of CPVT onset was 11 years (IQR 7–14). Age of onset differed significantly by exon, domain, and subdomain — variants in the core solenoid (CSol) domain and channel pore subdomain had the earliest onset (median 8 years). Variant-specific treatment responses were demonstrated: even at the same amino acid (R420Q vs R420W), age of onset and flecainide/ICD requirements differed significantly. A publicly accessible web app (markslab-cpvtdb.org) was developed to query the database.

Keywords

exons, humans, incidence, phenotype, treatment outcome

Key Takeaways

Background

• CPVT is a malignant inherited arrhythmia causing stress/exercise-induced syncope and SCD in the absence of structural heart disease; mortality 30–50% by age 40.
• Pathogenic RYR2 variants account for ~60% of clinically well-defined CPVT (autosomal dominant).
• RYR2 encodes the homotetrameric intracellular Ca²⁺ release channel mediating excitation-contraction coupling; during β-adrenergic stimulation, PKA phosphorylation at Ser2808 depletes calstabin2, increasing channel open probability.
• β-blockers are cornerstone therapy but near-fatal events remain unacceptably high; flecainide is recommended for refractory cases; Rycals (calstabin2 stabilizers) are in development.
• ~3% of ostensibly healthy individuals carry a rare RYR2 protein-altering variant, creating a VUS "genetic purgatory" that complicates clinical interpretation.

Methods

• Systematic review (PRISMA guidelines) of PubMed, Embase, Scopus through October 2020.
• From 4510 articles → 2570 after deduplication → 882 full-text reviewed → 221 publications included for data extraction.
• Two independent investigators extracted data; disagreements resolved by consensus.
• Variants validated against NM_001035.3 (transcript) and NP_001026.2 (protein) reference sequences.
• Structural mapping onto human RYR2 cryo-EM model (PDB: 7UA5, Miotto et al 2022) using ChimeraX.
• Statistical analysis: Mann-Whitney U test for age-of-onset comparisons (≥5 patients required per group); Fisher exact tests with Benjamini-Hochberg FDR correction for treatment comparisons.
• Web app developed: markslab-cpvtdb.org (Mol* Viewer for 3D visualization).

Results — Database and Clinical Characteristics

• 1342 patients extracted (644 F, 527 M, 171 unreported sex); 964 with CPVT diagnosis, 132 asymptomatic carriers, 246 non-CPVT diagnoses (e.g. LQTS).
• Of CPVT patients: 463 F, 351 M; 186 inherited, 87 de novo variants; 447/491 structurally normal hearts on echo.
• 315/321 patients with documented stress testing had polymorphic VT; 261/270 with syncope had it during exercise/stress; 97/113 with sudden cardiac arrest had it during exercise/stress.
• Median age of onset: 11 years (IQR 7–14), range 0–66 years, right-skewed distribution.
• Age of onset significantly lower in patients with polymorphic VT (median 10y vs 13.5y, P<0.001) and patients with syncope (median 10y vs 14.5y, P<0.001).

Results — Variant Distribution

• 263 unique RYR2 protein-coding variants in CPVT patients; 914/937 missense, 18 deletions, 4 insertions, 1 frameshift.
• Top 3 most frequent variants: p.G357S (99 patients), p.R420Q (52), p.R420W (27).
• 76.43% of variants were reported in ≤3 patients — most are rare.
• 77.13% of substitution variants fall within the 4 historical hotspots (I: exons 3–15, II: 44–50, III: 83–90, IV: 93–105).
• Most commonly affected exons: 90 (43 variants), 47 (12), 8 (10).
• Authors propose exons 37 and 42 should be considered part of hotspot II based on additional unique variants found.

Results — Age of Onset by Exon

• Lowest median age of onset: exon 102 (4y, n=6, P=0.003), exon 45 (4.5y, n=6, P=0.001), exon 88 (6y, n=15, P=0.013).
• Highest median age of onset: exon 93 (15.5y, n=8, P=0.005).
• Caveat: modifications at the same amino acid still differ — p.R420Q (median 10y, n=26) vs p.R420W (median 18.5y, n=8, P=0.012).

Results — Structural Mapping and Domain-Level Analysis

• Four historical hotspots cluster in the RYR2 core; the outer cytoplasmic shell has less CPVT influence.
• Core solenoid (CSol) domain: lowest median age of onset among all domains (8y, IQR 6–14, n=74, P=0.001 vs other domains). CSol is a signal transduction domain connecting regulatory cytoplasmic domains with the transmembrane domain.
• N-terminal domain (NTD): higher median age of onset (12y, IQR 9–16, n=97, P=0.006 vs other domains) — furthest hotspot from the pore, less allosteric influence.
• CSol(exEF-hand) subdomain: lowest median age among subdomains (8y, IQR 6–13, n=69, P<0.001 vs other subdomains).
• Channel pore subdomain: significantly lower age of onset (8y, IQR 6–13, P=0.019 vs other subdomains) — intrinsic pore function role.
• EF-hand subdomain: highest median age (28y, n=5, P=0.187 NS) — minor role in channel opening, major role in Ca²⁺ release termination/inactivation.

Results — Structural Mechanisms of Exemplar Variants

• p.G357S: disturbs local β-turn secondary structure (glycine-stabilized), affecting NTD-B domain interaction with CSol.
• p.R420Q/W: disrupts interaction network centered on R420, affecting N-terminal solenoid and NTD-B interaction. Q420 induces larger conformational changes than W420, correlating with significantly earlier onset (10y vs 18.5y, P=0.012).
• p.S2246L: substitutes small polar serine with large hydrophobic leucine, introducing steric impediments affecting intradomain stability and increasing domain flexibility.
• All three sites operate via different local structural mechanisms but share a common global consequence: loss of inter-domain interactions and increased flexibility → leaky channels (confirmed by single-channel Ca²⁺ imaging and knock-in mouse models).

Results — Therapeutics

• 432/601 (71.9%) CPVT patients with treatment records were on β-blockers.
• 129 patients on β-blockers required additional pharmacological therapy (flecainide most common); 129 required invasive therapy (ICD most common, then LCSD, catheter ablation).
• Variant-specific flecainide use (post-2013): p.S2246L 3/4, p.R420W 7/9, p.C2277R 6/8 — significantly more than p.G357S 0/91 and p.G2337V 0/21.
• Variant-specific ICD use: p.S2246L 7/9, p.R420Q 13/24, p.R420W 5/17 — significantly higher than p.G357S 1/94 and p.G2337V 1/21.
• Variant-specific β-blocker effectiveness: p.G2337V 10/11 reported effective vs p.R420W 5/17 (P=0.031) and p.C2277R 2/8 (P=0.047).
• p.R420W required significantly more combination flecainide + BB than p.R420Q at the same residue position (P=0.008).

Discussion

• De novo variant patients had greater proportion of sudden cardiac arrest vs inherited — suggests genetic testing in suspected CPVT even without family history.
• Age of onset as a clinical proxy for relative pathogenicity is supported: p.N4178S, p.A4091T, p.G3946S (early onset) correspond to Kapplinger et al's Near-Definite Pathogenic Variants.
• Inter-family heterogeneity example: p.M3978I — every case symptomatic; p.G155R — nearly all asymptomatic except one 16-year-old sudden death while playing sports.
• Intra-variant heterogeneity greatest for frequently reported variants (e.g. p.R420Q ranges from cardiac arrest to asymptomatic).
• Flecainide mechanism debate: Liu et al showed antiarrhythmic effect without affecting Ca²⁺ homeostasis (Na⁺ channel mechanism); Kryshtal et al demonstrated direct RYR2 inhibition; Bannister et al found no direct RYR2 effect. Consensus: antiarrhythmic benefit likely primarily Na⁺ channel-mediated.
• CaMKII inhibition and Rycal calstabin2 stabilization identified as promising future therapeutic targets.

Limitations

• Only missense variants affecting single exon/domain/subdomain were analyzed for age of onset associations (2.5% of cases were non-missense).
• Treatment comparisons limited by small sample sizes per variant.
• Publication bias: case reports/series tend to report novel variants or unique cases.
• Inconsistencies in patient features reported across articles.
• p.G357S frequency inflated by inclusion of a large multi-generational family study.
• Flecainide analysis restricted to post-2013 (when expert consensus endorsed its use).
• Statistical model required ≥5 patients per group — many rare variants excluded from domain/exon comparisons.

Key Concepts Mentioned

• concepts/Bidirectional-Ventricular-Tachycardia — hallmark CPVT arrhythmia; 315/321 patients with stress testing had polymorphic VT
• concepts/Sudden-Cardiac-Death — 30–50% CPVT mortality by age 40; positive family history in 349/492 patients
• concepts/Variant-Reclassification — VUS "genetic purgatory" in RYR2; database foundation for ML pathogenicity predictor
• concepts/Left-Cardiac-Sympathetic-Denervation — invasive therapy option for β-blocker-refractory CPVT
• concepts/Cascade-Family-Screening — HRS/EHRA consensus recommendation for all CPVT probands
• concepts/Sports-Cardiology-SDM — de novo variant SCA implications for family screening and exercise guidance

Key Entities Mentioned

• entities/RYR2 — 263 variants cataloged; domain-specific structure-function analysis; calstabin2 binding; PKA phosphorylation at Ser2808
• entities/CPVT — 964-patient clinical database; median onset 11y; variant-specific treatment responses
• entities/CASQ2 — second CPVT gene (~5%, AR); calsequestrin-2 in RYR2 calcium release unit
• entities/Flecainide — most common add-on antiarrhythmic; mechanism debate (Na⁺ channel vs direct RYR2 inhibition)

Wiki Pages Updated

• sources/RYR2-CPVT-CircEP-2025 — created
• entities/RYR2 — updated with domain-specific age of onset, 263-variant catalog, structural mechanisms, variant-specific treatment data
• entities/CPVT — updated with 964-patient database statistics, median onset, variant-specific therapeutic data
• wiki/sourceindex.md — updated
• wiki/wikiindex.md — updated