Functional Characterization of Patient-Derived Myotubes Carrying ANO5 and ORAI3 Variants in RYR1-Negative Malignant Hyperthermia Susceptibility

Background/Objectives: Malignant hyperthermia (MH) is a life-threatening pharmacogenetic disorder of skeletal muscle primarily associated with pathogenic variants in RYR1; however, a substantial proportion of MH-susceptible individuals lack identifiable variants in known genes. This study aimed to identify novel genetic contributors in Ca2+-induced Ca2+ release (CICR)-positive patients without RYR1 variants and to evaluate their functional relevance. Methods: Among 29 CICR-positive individuals without pathogenic variants identified by gene panel testing, five patients underwent whole-exome sequencing (WES). In one family, heterozygous variants in ANO5 (p.Arg547Gln) and ORAI3 (p.Arg287Cys) were identified. To evaluate their functional significance, intracellular Ca2+ dynamics were analyzed in primary myotubes derived from Case 165, an affected individual carrying both variants, and compared with those from CICR-negative controls and CICR-positive patients harboring RYR1 variants. Results: Myotubes derived from Case 165 demonstrated enhanced sensitivity to caffeine and 4-chloro-m-cresol, elevated resting intracellular Ca2+ levels, and greater Ca2+ reduction under Ca2+-free conditions compared with CICR-negative controls, resembling the phenotype observed in the RYR1 variant group. In contrast, dantrolene-induced Ca2+ reduction was significantly greater only in the RYR1 variant group. Conclusions: These findings suggest that ANO5 and ORAI3 variants may contribute to abnormal Ca2+ regulation in MH-susceptible individuals without RYR1 mutations and highlight the importance of combining genomic analysis with functional validation to identify novel genetic contributors to MH susceptibility.