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

Abstract

Purpose

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.

Methods

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 (EC50) 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.

Results

In functional analysis using HEK-293 cells, we found significant reductions in the EC50 of p.Ser2345Thr and p.Ser2345Arg in comparison with wild-type RYR1 (P < 0.001), while the EC50 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 EC50 values for all variants (P < 0.001 for all comparisons).

Conclusions

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.

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Acknowledgements

We thank prof. David H. MacLennan (University of Toronto) for the kind gift of rabbit-RyR1/pcDNA. Part of this work was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.

Funding

This study was supported in part by a Grant-inAid (Grant number 17K16733, 18K08856) for Scientific Research from the Japan Society for the Promotion of Science, Japan.

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Correspondence to Yuko Noda.

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Noda, Y., Yasuda, T., Kanzaki, R. et al. Functional analysis of newly identified RYR1 variants in patients susceptible to malignant hyperthermia. J Anesth (2020). https://doi.org/10.1007/s00540-020-02803-w

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Keywords

  • Malignant hyperthermia
  • Ryanodine receptor type 1
  • Functional analysis