Parker, Remai2020-07-012020-07-012019http://hdl.handle.net/10179/15444Malignant 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.enThe AuthorMalignant hyperthermiaGenetic aspectsDiagnosisRyanodineReceptorsCalcium in the bodyThesis320506 Medical biochemistry - proteins and peptides (incl. medical proteomics)Q112949832https://www.wikidata.org/wiki/Q112949832