Functional consequences of RyR₁ variants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand

Abstract

Malignant hyperthermia (MH) is an uncommon pharmacogenetic disorder that is asymptomatic until triggered by volatile anaesthetics or depolarising muscle relaxants. Exposure to such a trigger can result in a potentially fatal hypermetabolic crisis in an MH-susceptible individual. With prior diagnosis, MH episodes can be avoided by using alternative anaesthesia. Diagnostic testing requires a morbidly invasive muscle biopsy for those considered at risk based on family history. Linkage of MH-susceptibility to variants in the skeletal muscle calcium release channel ryanodine receptor 1 (RyR₁) has provided an opportunity for DNA testing as an alternative to the muscle biopsy. DNA-based diagnosis is severely limited by the number of diagnostic mutations identified—only 50 mutations have been established as MH-causative from over 300 genetic variants associated with the disorder. Moreover, DNA testing may only diagnose an individual as MH-susceptible; a negative DNA test is insufficient under current guidelines for a negative MH diagnosis. The purpose of this study was to develop molecular tools to investigate the hypothesis that RyR₁ variants associated with MH-susceptibility cause dysregulation of calcium release from intracellular stores. Two experimental approaches were followed with the objective of expanding the capabilities of DNA-based diagnosis for MH. The first technique was the generation of mammalian cell lines stably expressing recombinant RyR₁ variants by use of the Flp-In™ T-REx™ system from Invitrogen, followed by functional analysis. Four of five genetic variants associated with MH or myopathy had altered sensitivities to an RyR₁ agonist and therefore meet the criteria for use as diagnostic variants for MH-susceptibility. The second molecular technique explored was gene editing, with the aim of showing that a single nucleotide change was both necessary and sufficient to cause MH-susceptibility. This was developed by introducing a well-characterised MH-causative variant into the genome of a human skeletal muscle cell line. Preliminary results indicated that gene editing was successful.