Functional analysis of newly identified RYR1 variants in patients susceptible to malignant hyperthermia

• Published in: Journal of anesthesia Vol. 34; no. 5; pp. 658 - 665
• Main Authors: Noda, Yuko, Yasuda, Toshimichi, Kanzaki, Rieko, Miyoshi, Hirotsugu, Mukaida, Keiko, Kamiya, Satoshi, Morio, Atsushi, Hamada, Hiroshi, Kawamoto, Masashi, Tsutsumi, Yasuo M
• Format: Journal Article
• Language: English
• Published: Japan 01.10.2020
• Subjects: Malignant hyperthermia; Functional analysis; Ryanodine receptor type 1
• ISSN: 1438-8359
• DOI: 10.1007/s00540-020-02803-w

This study aimed to evaluate whether the three ryanodine receptor type 1 (RYR1) variants (p.Ser2345Thr, p.Ser2345Arg, and p.Lys3367Arg) which we identified in Japanese malignant hyperthermia (MH) patients with a clinical grading scale rank of 6 were causative for MH. We prepared human embryonic kidney (HEK)-293 cells transfected with wild-type RYR1 or one of the RYR1 variants, along with myotubes cultured from muscle pieces. Calcium kinetics were examined by calculating the 340/380-nm ratio under various caffeine and 4-chloro-m-cresol (4CmC) concentrations with the ratiometric dye Fura-2 AM. Half-maximal effective concentration (EC ) values were calculated from dose-response curves. Statistical analysis was based on one-way analysis of variance with a Dunnett's multiple comparison test, using a P value < 0.05 as evidence of statistical significance. In functional analysis using HEK-293 cells, we found significant reductions in the EC of p.Ser2345Thr and p.Ser2345Arg in comparison with wild-type RYR1 (P < 0.001), while the EC of p.Lys3367Arg was not significantly different (P = 0.062 for caffeine and P > 0.999 for 4CmC). On the other hand, functional analysis using myotubes showed significant differences in the EC values for all variants (P < 0.001 for all comparisons). p.Ser2345Thr and p.Ser2345Arg appear capable of causing a calcium metabolism disorder that leads to the onset of MH, and p.Ser2345Arg can be considered as a diagnostic mutation, because it meets the European Malignant Hyperthermia Group criteria. However, patients with p.Lys3367Arg might have mutations in genes other than RYR1 that are capable of causing MH.