Beyond Hotspots: Functional Characterization of the Novel p.Asp2730Tyr Mutation in RYR1 Associated With Malignant...

Malignant hyperthermia (MH) is a life-threatening pharmacogenetic disorder triggered by certain anesthetics, characterized by muscle rigidity, elevated body temperature, and hypermetabolic crisis. This condition is primarily associated with genetic mutations in ryanodine receptor 1 ( RYR1 ), which encodes the pivotal calcium release channel in the sarcoplasmic reticulum of skeletal muscle. While numerous hotspot mutations in RYR1 have been identified, the functional impact of nonhotspot mutations on channel activity related to MH remains insufficiently investigated. In this study, we identified a known pathogenic mutation (p.Arg2508His) and a novel variant (p.Asp2730Tyr), both located outside the conventional MH hotspots, in 2 patients with clinical suspicion of MH. Our objective was to investigate the functional implications of the p.Asp2730Tyr mutation in RYR1 on calcium release dynamics related to MH.

We engineered a recombinant wild-type (WT) plasmid (pcDNA3.1-3Myc-His-RYR1-WT) to express the full-length mouse skeletal muscle RYR1 using seamless multi-fragment cloning techniques. Two RYR1 mutations, p.Arg2508His (used as positive control) and p.Asp2730Tyr, were separately introduced into the WT plasmid, generating 2 mutant constructs (pcDNA3.1-3Myc-His-RYR1-p.Arg2508His and pcDNA3.1-3Myc-His-RYR1-p.Asp2730Tyr). We utilized 293T cells expression system to express either the WT or mutant forms of mouse RYR1 . Fluo-4 calcium imaging was conducted to evaluate the alterations in calcium release in response to RYR1 agonists, caffeine or 4-chloro-m-cresol (4CmC), for each mutation compared to WT.

Cells transfected with the p.Arg2508His or p.Asp2730Tyr mutation demonstrated a leftward shift in the caffeine and 4CmC concentration-response curves compared to WT, suggesting an increased channel sensitivity to caffeine and 4CmC ( P < .001). The mean ± standard error of the mean (SEM) of the EC50 values for caffeine-induced calcium release was 2.56 ± 0.04 mM in WT, which significantly decreased to 1.32 ± 0.13 mM for p.Arg2508His ( P < .001) and 1.12 ± 0.09 mM for p.Asp2730Tyr ( P < .001). For 4CmC, the EC50 values were 43.2 ± 1.90 μM in WT, 17.2 ± 0.76 μM for p.Arg2508His ( P < .001), and 21.8 ± 1.04 μM for p.Asp2730Tyr ( P < .001), indicating enhanced calcium release in both mutations.

The p.Asp2730Tyr mutation, situated beyond the established RYR1 hotspot regions, significantly alters calcium release dynamics related to MH. A comprehensive investigation into the structural conformations, functional assays, and in vivo mechanisms associated with this mutation could yield a more profound understanding of the molecular underpinnings of MH pathogenesis.