Functional analysis and classification of six RYR1 variants in Japanese patients with malignant hyperthermia

Malignant hyperthermia (MH) is a life-threatening pharmacogenetic disorder triggered by volatile anaesthetics or depolarising neuromuscular blockers, characterised by dysregulated calcium homeostasis in skeletal muscle. Dysfunction of ryanodine receptor type 1 (RYR1) because of genetic variants plays a central role in MH. Functional evaluation of newly identified RYR1 variants is essential for accurate presymptomatic diagnosis of MH susceptibility. This study aimed to assess the function of novel RYR1 variants detected in Japanese patients.

Six previously uncharacterised RYR1 variants were introduced into full-length rabbit RYR1 cDNA and expressed in human embryonic kidney (HEK-293) cells. Calcium release in response to caffeine and 4-chloro-m-cresol (4-CmC) was measured using Fura-2 AM. Expression of variants and wild-type (WT) RYR1 was confirmed by immunoblotting. Concentration-response curves (EC₅₀ values) were analysed using Prism 9.5. Statistical significance was determined by an extra sum-of-squares F test (global fit) (P < 0.01 indicates statistical significance). Data are presented as 95% confidence intervals for EC₅₀ values (n ≥ 14).

All six variants (p.Ile2358Thr, p.Asp2431His, p.Asp2431Glu, p.Pro2366Arg, p.Arg2454Gly, p.Glu2545Asp) showed hypersensitivity to caffeine and 4-CmC. Best-fit EC₅₀ values for caffeine (1.82-2.50 mM) and 4-CmC (74.6-103.6 μM) were significantly lower than WT (4.28 mM and 178 μM, respectively; P<0.01).

The RYR1 variant p.Pro2366Arg was classified as pathogenic or likely pathogenic, whereas p.Ile2358Thr, p.Asp2431His and p.Arg2454Gly remained as variants of uncertain significance but as likely pathogenic, and p.Asp2431Glu and p.Glu2545Asp remained variants of uncertain significance.