Browsing by Author "Stephens, Jeremy"
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- ItemCharacterisation 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, JeremyMalignant 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.
- ItemEstablishing systems to characterise MH pathogenic RyR1 variants : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry(Massey University, 2021) Stephens, JeremyMalignant hyperthermia (MH) is a potentially fatal, autosomal dominant, metabolic disorder triggered in susceptible individuals upon exposure to volatile anaesthetics. Following the onset of an MH episode, a patient will enter a hypermetabolic state, displaying the symptoms of intense muscle contraction, metabolic acidosis, increased oxygen consumption. Prolonged episodes can result in rhabdomyolysis. If left untreated, MH can manifest as an increase in body temperature and death by cardiac arrest. MH is diagnosed by the invasive in vitro contracture test, which requires a muscle tissue biopsy. DNA screening has been implemented and is commonly used to diagnose a genetic predisposition to MH; however, the test is currently limited to fifty variants confirmed to be pathogenic out of approximately 350 variants linked to the disorder. DNA-based tests are limited because of the technical difficulties associated with functional analysis. Thus, additional variants must be functionally characterised. The structural implications of MH-linked variants potentially leading to the onset of MH are not yet well defined. Potential structural changes induced by pathogenic variants have been modelled in silico, where variants were mapped to the rabbit RyR1 structure characterised by cryo electron microscopy. However, this does not confirm the role the variants play in the structural and functional alteration of the channel. To address, this a functionally significant region of RyR1 was cloned for recombinant expression in E. coli. The RyR1 region was shown to be soluble and efforts were made to purify the protein. However, the protein could not be purified to an extent acceptable for either biochemical analysis or crystallisation trials or for subsequent X-ray crystallography. A number of pathogenic variants were instead modelled in silico to provide some insights into their potential pathogenic functional role. The viability of a new cell-based system for the functional characterisation of variants was also tested. Patient derived myoblasts were immortalised using lentivirus transduction with the cDNA for human telomerase and cyclin dependent protein kinase 4. The genome editing tool CRISPR Cas 9 was then used to successfully introduce the pathogenic variant c.14497C>T p.his 4833 tyr into the genome of MH negative myoblasts. Functional characterisation of the introduced variant has yet to be performed.