Functional characterisation of genetic variants associated with malignant hyperthermia

Mutations in the skeletal muscle sarcoplasmic reticulum calcium release channel (RYR1) have been identified in
association with malignant hyperthermia(MH)and exertional
heat stroke(EHS). MH is a pharmacogenetic disorder in which
hypermetabolism is triggered upon exposure to volatile anaesthetics. MH has long been linked with EHS, a life threatening increase in body temperature caused by strenuous exercise in warm climates with both conditions being caused by a deregulation of skeletal muscle calcium homeostasis. The availability of genetic testing in MH is limited to variants that have been proven to alter calcium handling in vitro. In this thesis, data on the functional consequences of seven RYR1 variants found in association with MH are presented, one of which has also been identified in an EHS patient. Site-directed mutagenesis was used to introduce the variants into a human RYR1 cDNA clone before expressing wild type and mutant constructs in HEK293 cells. Six of the variants, including the EHS mutation, were found to have an increased sensitivity to caffeine as evidenced through a significant decrease in EC50 as well as exaggerated calcium release at low doses of caffeine as compared to wild type controls. For one variant, p.D3986E, an increase in RYR1 expression was required before the phenotype resembled the other RYR1 variants, hinting at a more complicated role in MH for this variant. The functional data presented in this thesis is supportive of the addition of six RYR1 variants onto the genetic diagnostic panel for MH, increasing the availability of initial genetic testing by 19%. Furthermore, the work presented further supports the link between MH and EHS through the presence of a common RYR1 variant proven to alter calcium handling. Finally, data obtained for the p.D3986E variant lays the foundation for future studies aiming to identify genetic modifiers of the MH phenotype.