Pathogenicity assessment of seven RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia in the Netherlands.

Background: Malignant hyperthermia (MH) susceptibility is associated with variants in RYR1, the gene encoding the skeletal muscle ryanodine receptor-1 (RyR1), in 70-75% of patients. Functional characterisation demonstrating an increased sensitivity to RyR1 agonists is necessary among other criteria for inclusion in the European Malignant Hyperthermia Group list of MH susceptibility diagnostic variants.

Methods: Seven variants in the RYR1 gene, p.Glu342Lys, p.Leu2288Ser, p.Phe2340Leu, p.Arg2676Trp, p.Val3324Ala, p.Phe4076Leu, and p.Trp5020Cys, identified in MH-susceptible individuals were introduced into the cDNA for the human RYR1 gene. These variants were tested in cultured human embryonic kidney HEK293 cells for their effect on calcium release in response to the RyR1 agonist 4-chloro-m-cresol. Calcium release of each variant was compared with wild-type and benign and pathogenic controls. Each variant was subjected to curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 Variant Curation Expert Panel guidelines.

Results: Six of seven RYR1 variants (p.Glu342Lys, p.Leu2288Ser, p.Phe2340Leu, p.Arg2676Trp, p.Val3324Ala, p.Phe4076Leu) showed hypersensitivity to 4-chloro-m-cresol compared with wild-type. The p.Trp5020Cys variant did not release calcium in response to 4-chloro-m-cresol. All variants had minor allele frequencies <0.1%. Rare exome variant ensemble learner scores of p.Glu342Lys, p.Leu2288Ser, p.Phe4076Leu, and p.Trp5020Cys were >0.85, supporting pathogenicity.

Conclusions: The variants p.Glu342Lys, p.Leu2288Ser p.Phe2340Leu, and p.Arg2676Trp are pathogenic or likely pathogenic for MH and can be used for presymptomatic testing for MH susceptibility. As current knowledge on the p.Val3324Ala, p.Phe4076Leu, and p.Trp5020Cys variants remains insufficient, they are still classified as variants of uncertain significance.