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    Characterisation RyR1 variants linked to malignant hyperthermia : a thesis presented to Massey University in partial fulfilment of the requirements for a Masters of Science in Biochemistry
    (Massey University, 2016) Stephens, Jeremy
    Malignant hyperthermia is a potentially fatal disorder of skeletal muscle manifesting as a rise in body temperature in response inhalational anaesthetics and muscle relaxants. Further clinical signs include muscle rigidity and increased oxygen consumption. The increased metabolism is induced by alterations to Ca2+ homeostasis resulting from the dysregulation of the sarcoplasmic reticulum protein the ryanodine receptor type 1 (RyR1). A large proportion of known malignant hyperthermia linked genetic variants reside within the gene encoding the type 1 ryanodine receptor, RYR1. Malignant hyperthermia can be diagnosed by in vitro contracture testing of biopsied muscle tissue. The use of DNA diagnostic testing is advantageous, however it is limited to only 35 of the proposed 400 RYR1 linked variants known to be associated with malignant hyperthermia. The research described in this thesis reports the functional characterisation of two RYR1 variants linked to malignant hyperthermia, c.641C>T and c.7042_7044delCAG resulting in the amino acid changes p.T214M and p.ΔE2348. The ability of each variant to release Ca2+ in response to a stimulus was examined in a heterologous system. The variant p.ΔE2348 was shown to be hyperactive in response to agonists indicating the variant is the cause of malignant hyperthermia, while the p.T214M variant does not appear to have an effect ryanodine receptor function. To understand the relationship between RyR1 function and any structural alterations induced by the p.T214M and p.ΔE2348 variants, the domain housing each variant was cloned for bacterial expression. Subsequent purification and structural characterisation could be used to explain the role each variant plays with respect to the onset of MH. The RyR1 N-­terminal domain, amino acids 1-­558, and helical domain, amino acids 2091-­2525, were expressed in E. coli and partially purified. The domains were shown to be soluble and stable following expression.
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    Functional and structural characterisation of the malignant hyperthermia associated RYR1 mutation R245W : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 2013) Roesl, Cornelia
    Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle triggered by volatile halogenated anaesthetics. Susceptible individuals can exhibit symptoms including tachycardia, high temperature, hypoxia, hypermetabolism and skeletal muscle rigidity. There are usually no symptoms of MH in normal day to day life although “awake” episodes have been reported as a result of extreme exercise in MH-susceptible subjects. Genetic variants have been associated with the skeletal muscle ryanodine receptor gene (RYR1) in 50-70 % of susceptible patients. At the molecular level susceptible patients are thought to have an increased sensitivity to RYR1 agonists in skeletal muscle compared to non-affected patients that results in disregulated skeletal muscle Ca2+ homeostasis. In 2000 a novel RYR1-mutation, c.G7354T (p.R2452W), associated with MH, was identified in a New Zealand (NZ) family. Subsequently the same mutation was identified in a separate NZ family and has also been reported in the United Kingdom. To date this mutation had not been shown to be causative of MH. Therefore, the aim of this study was to carry out functional analyses to test whether the R2452W mutant receptor alters Ca2+ release compared to wildtype. For this study Ca2+ release assays were carried out in three different cell types: B-lymphoblastoid cells, myotubes and HEK293 cells transfected with full-length human RYR1 cDNA. Cells were exposed to the RYR1-specific agonist 4-chloro-m-cresol, which stimulates Ca2+ release through the receptor while the increase in cytosolic Ca2+ was detected using a membrane-permeable fluorophore. Cells expressing R2452W mutant RYR1 showed altered Ca2+ release from the sarco(endo)plasmic reticulum suggesting a hypersensitive channel. In order to study structure/function relationships of the R2452W mutation within the RYR1 protein, as well as the 3D structure of a central RYR1 domain (amino acid 2144-2489), a region encompassing this substitution was cloned for bacterial expression and subsequently purified. Wildtype and mutant proteins were compared to determine any effects the mutation may have upon the stability of the protein. Wildtype and R2452W RYR1 protein showed no obvious differences in stability but both proteins appeared to oligomerise suggesting this region might be involved in RYR1-RYR1 domain interactions.
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    A functional analysis of RYR1 mutations causing malignant hyperthermia : a thesis presented to Massey University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 2009) Sato, Keisaku
    Malignant hyperthermia (MH) is a rare pharmacogenetic disorder in humans induced by volatile anaesthetics and depolarising muscle relaxants. An MH reaction shows abnormal calcium homeostasis in skeletal muscle leading to a hypermetabolic state and increased muscle contracture. A mutation within the skeletal muscle calcium release channel ryanodine receptor gene (RYR1) is associated with MH and is thought to cause functional defects in the RYR1 channel leading to abnormal calcium release to the sarcoplasm and consequent MH reactions. Mutations within RYR1 are also associated with a rare congenital myopathy, central core disease (CCD). CCD is characterised by muscle weakness and is thought to be caused by insufficient calcium release from the RYR1 channel during excitation-contraction (EC) coupling. To investigate functional effects of RYR1 mutations, the entire coding region of human RYR1 was assembled and cloned into an expression vector. Mutant clones containing RYR1 mutations linked to MH or CCD were also constructed. Wild-type (WT) and mutant RYR1 clones were used for transient transfection of HEK-293 cells. Western blotting was performed after harvesting and expressed WT and mutant RYR1 proteins were successfully detected. Immunofluorescence showed co-localisation of RYR1 proteins and the endoplasmic reticulum in HEK-293 cells. [3H]ryanodine binding assays showed that RYR1 mutants linked to MH were more sensitive to the agonist 4-chloro-m-cresol (4-CmC) and less sensitive to the antagonist Mg2+ compared with WT. Two C-terminal RYR1 mutants T4826I and H4833Y were very significantly hypersensitive to 4-CmC and they may also result in a leaky channel. This hypersensitivity of mutants linked to MH may result in abnormal calcium release through the RYR1 channel induced by triggering agents leading to MH reactions. RYR1 mutants linked to CCD showed no response to 4-CmC showing their hyposensitive characteristics to agonists. This study showed that the human RYR1 proteins could be expressed in HEK-293 cells. Moreover, using the recombinant human RYR1 clone, a single mutation within RYR1 resulted in a functional defect in expressed RYR1 proteins and functions of mutant RYR1 proteins varied from hypersensitive to hyposensitive depending on the mutation and whether it was linked to MH or CCD.