Browsing by Author "Parker, Remai"
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- ItemCharacterisation of C-terminal RyR1 variants linked to neuromuscular disorders : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Manawatū, New Zealand(Massey University, 2015) Parker, RemaiIntracellular calcium influences a large array of cellular processes in skeletal muscle cells and as a result the movement of free calcium is tightly-regulated by a diverse set of calcium channels and accessory proteins. The main store of calcium in skeletal muscle cells is the sarcoplasmic reticulum, from which the ryanodine receptor one calcium channel (RyR1) controls calcium release. Changes in calcium homeostasis often result in the manifestation of neuromuscular disorders, most notably central core disease (CCD) and malignant hyperthermia (MH). While CCD is usually apparent from the presence of certain physical characteristics, MH is typically asymptomatic unless exposed to a trigger, at which point the disease rapidly manifests as a crisis event which is potentially fatal. Currently, the diagnosis of these disorders requires the testing of a muscle biopsy, which is an expensive and invasive procedure, and thus a genetic test would be an ideal diagnostic alternative. For the most part, CCD and MH cases are linked to the inappropriate release of calcium by defective RyR1 channels – located in the calcium storage organelle membrane – but both are complex disorders with variable penetrance and genetic heterogeneity. A hypoactive RyR1 is thought to cause CCD while a hyperactive RyR1 is thought to cause MH, and yet individuals have been observed to be carriers of both diseases. Most of these instances have been linked to variants in the C-terminal region of RyR1, corresponding to the transmembrane portion of the channel. This research described in this thesis focused on the functional analysis of five C-terminal domain RyR1 variants identified in patients with neuromuscular disorders. The ability of the variant RyR1 channels to release calcium in response to a stimulus in a heterologous system was measured and compared with that of the wild type channel. Moreover, one of these variants was also examined in several B-lymphoblastoid cell lines taken from carriers of the variant. Of the five variants tested in the heterologous system, four different phenotypes were observed, reinforcing the theory that these disorders are caused by a variety of factors that combine to produce a complex phenotype.
- ItemFunctional 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(Massey University, 2019) Parker, RemaiMalignant 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.