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Item Establishing 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.Item 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(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.Item Preliminary investigation of the C-terminal mutations that cause malignant hyperthermia : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry(Massey University, 2000) Jones, Angela MarieMalignant hyperthermia (MH) is a genetic disorder characterised by abnormal muscle contractures, hypermetabolism and hyperthermia. It is referred to as 'malignant' as it can lead to death when under anaesthetic if not recognised and treated immediately. The molecular basis of MH is an abnormality in the calcium release mechanism of the sarcoplasmic reticulum. Abnormal calcium release causes the physiological symptoms of an MH crisis. Genetic linkage studies have led to the identification of the ryanodine receptor/Ca2+ -release channel as a causative factor in MH. The ryanodine receptor is a large protein which is regulated by a number of ligands including Ca2+ , Mg2+ , ATP, ryanodine and calmodulin. Most mutations in the ryanodine receptor gene that cause MH are located near two main regulatory regions on the receptor. Three mutations have recently been identified that are located in the regulatory region of the C-terminal domain. The biochemical properties of one of these mutations have been studied. The current research project began to investigate the biochemical characteristics of the other two mutations in the C-terminal domain in relation to their ryanodine binding and calcium release properties. Sarcoplasmic reticulum vesicles were isolated from skeletal muscle samples, and an attempt to identify ryanodine receptors by 3 H-ryanodine binding was made. RT-CR using RNA extracted from a skeletal muscle sample was used to construct the cDNA for the C-terminal transmembrane domain of the ryanodine receptor. This cDNA was cloned into a mammalian expression vector and introduced into COS cells. RT-PCR was also used to produce the cDNA encoding a small polypeptide to an antigenic region in the C-terminal domain of the ryanodine receptor for the preparation of antibodies. Although it appeared that there may have been ryanodine receptors in the SR vesicle preparation as determined by immunoblotting, 3 H-Ry binding to the ryanodine receptors was unable to confirm the presence of the receptors in the SR vesicles. Initial expression studies of the C-terminal domain in COS cells were inconclusive. Partial cleavage of a small antigenic polypeptide was obtained which could be used to produce antibodies to the Cterminal domain of the ryanodine receptor.Item A pilot study for the development of a diagnostic test for malignant hyperthermia : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1986) Kerr, Robin GillianThe only definitive diagnostic test for Malignant Hyperthermia, a genetic disease that effects skeletal muscle, is the caffeine-contracture test. Alternative tests are being sought since this test is not totally satisfactory. It requires muscle biopsy, an invasive procedure and often produces results difficult to interpret. A test that could be used for wide spread screening of all patients about to undergo an operation would reduce the incidence of unexpected Malignant Hyperthermic episodes induced by anaesthetics, the most common cause of an episode. In this project the effect of mild stress induction on skeletal muscle, ischaemia produced by a tourniquet is studied. The tourniquet effect on a sample of five pre-diagnosed Malignant Hyperthermia susceptible subjects is compared to the effect on a sample of twelve normal subjects. The effect was determined by the measurment of serum metabolites before and after tourniquet application. The variables measured were creatine kinase, lactate dehydrogenase, AMP deaminase, total solids, total protein, potassium, osmolality, inorganic pyrophosphatase, creatine and erythrocyte pyrophosphatase. Between the two groups AMP-deaminase, creatine and osmolality showed no difference in response to tourniquet application. Inorganic pyrophosphate rose in the Malignant Hyperthermia group after tourniquet application but remained unaltered in the normal groups. All other Malignant Hyperthermia variables moved in a negative direction with respect to the normal levels. That is if the normal metabolites level rose the Malignant hyperthermia metabolites stayed the same, or if the normal levels stayed the same the MH levels dropped. A measurement of resting metabolite levels showed Creatine kinase was higher in the MH subjects compared to the normal subjects levels but creatine and pyrophosphatase were lower in the MH subjects. These differences may form the basis of a diagnostic test.Item Adenine nucleotide metabolism in human blood platelets: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Clinical Biochemistry at Massey University(Massey University, 1985) Farndale, Bruce MathewAt present, a skeletal muscle biopsy provides the most specific test for susceptibility to Malignant Hyperthermia (MH). This procedure is unsuitable for large scale screening of individuals and a simpler, less invasive test to distinguish MR-susceptible (MHS) people from those not possessing the genetic defect, is highly desirable. Platelets contain a calcium-activated contractile system, a calcium-storing and releasing system, and an active ATP-generating system. It is thus logical to assume that the same processes occur in platelets as those in muscle during MR-induced accelerated metabolism. In this research, [8-14 c]adenine incorporation into blood platelet adenine nucleotides was investigated with a view to using differences between platelets from normal and MHS individuals as the basis for a clinical test. It was assumed that under resting and/or halothane-stimulated conditions, nucleotide turnover in MHS platelets is significantly abnormal, and that the turnover abnormality is reflected in differences in adenine incorporation to platelet nucleotides via the salvage pathway. MHS platelets took up less adenine and assimilated it into nucleotides at a slower rate than normal platelets. However, after two hours, 20% more labelled ATP was extracted from MHS platelets than normal, with a concomittant decrease in ADP levels. Halothane had little effect on normal platelets but caused a 10% decrease in incorporation into ATP in MHS platelets. AMP labelling was lower than normal in MHS platelets, indicating increased deamination of this nucleotide. Specific radioactivities of nucleotides were not measured since [ 14 c]adenine distributes evenly among metabolic ATP, ADP, and AMP; therefore, the total radioactivities were used as a measure for the levels of adenine nucleotides within the metabolic pool. From the limited number of individuals screened, results suggest that MHS platelets have a higher basal ATP turnover rate than normal. When challenged with halothane the adenylate energy charge decreased, causing an increased nucleotide turnover rate which in turn led to a decreased ATP level due to the increased deamination of AMP. The appearance of more hypoxanthine and inosine than normal in the extraplatelet medium is consistent with the above sequence of events. The platelet-halothane bioassay displays a limited ability to distinguish between normal and MHS individuals and may have the potential to become a less invasive equivalent to the "ATP-depletion test" in muscle.Item 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, 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.Item 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, CorneliaMalignant 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.Item A genetic test for malignant hyperthermia : a dissertation presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry(Massey University, 2000) Brown, Rosemary LMalignant Hyperthermia (MH) is an inherited disorder of skeletal muscle in which an abnormality in the regulation of calcium release from internal stores can result in a fatal hypermetabolic reaction on exposure to general anesthetics. Mutations in the gene encoding the skeletal muscle ryanodine receptor/ calcium release channel (RYR1) have been linked to MHS in 50% of overseas families examined, and at least five additional MH susceptibility loci have since been proposed. Current diagnosis of MH in New Zealand relies on the in vitro contracture testing (IVCT) of excised muscle bundles with caffeine and halothane. The genetic basis of MH in NZ families was investigated, with the goal of developing genetic tests to replace the muscle biopsy test. A search for previously published RYR1 mutations in susceptible members of 33 NZ MH families revealed three RYR1 mutations; Arg163Cys, Gly341Arg, and Gly2434Arg, which co-segregated completely with susceptibility to MH (MHS). None of the 17 published RYR1 mutations were detected in a local MHS Maori family in which several anaesthetic deaths have occurred. This is the largest characterised MH family in the world. An examination of the segregation of a panel of chromosome 19q markers with MHS in over 200 members of this family revealed that MHS was linked to the RYR1-flanking markers. This implicated the involvement of a novel RYR1 defect. The entire 15.3 kb RYR1 coding region was combed for mutations by RT-PCR and automatic sequence analysis. A novel point mutation was detected that changed threonine 4826 to isoleucine in the C-terminal region of the RyR1 protein. This mutation was not found in 220 chromosomes from the normal population, or in 94 members of the family who had been diagnosed MHN (normal). A screen for the mutation in 210 key family members revealed a direct correlation between inheritance of the mutation and highly abnormal muscle contracture results in 36 individuals. 22 MHS individuals lacked the mutation; consequently the false positive rate of the IVCT and the possible segregation of at least one additional MHS gene complicated genetic linkage analysis. These problems were addressed by investigating increasingly stringent models for MH diagnosis. Four additional novel RYR1 mutations were detected in other MHS families investigated by sequence analysis of cDNA and genomic DNA, Arg401Cys, Arg2452Trp, Arg2454His and His4833Tyr. The detection of the Thr4826Ile and His 4833Tyr mutations established the channel domain of the ryanodine receptor as a new MHS domain. Genetic testing for MHS can now be applied with caution to predict MH susceptibility in approximately 40 % of at-risk individual in NZ, thus reducing the number of patients requiring an expensive and invasive surgical procedure.Item Loss of heterozygosity of the H4833Y mutation on RYR1 gene causing malignant hyperthermia : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North(Massey University, 2010) Balasubramanain, DianaMalignant hyperthermia is a potentially fatal pharmacological disorder and is triggered by volatile anaesthetics in predisposed individuals. Mutations in the RYR1 gene, encoding the skeletal muscle calcium receptor channel have been linked to MH susceptibility. Over 200 point mutations have been have been found to date in the RYR1 gene linked to MHS worldwide. EBV-immortalization is regularly used worldwide as an effective procedure for inducing long-term growth of human B lymphocytes. In the current study, it was observed that immortalized lymphocytes from MHS patients heterozygous for the missense mutation H4833Y when initially cultured expressed both wild type and mutant allele but after a few weeks of culture they seemed to lose the mutant allele. High resolution melting assays and hybridization probe assays showed the loss of heterozygosity and this was confirmed using DNA sequencing. Genotyping and haplotype analysis using three intragenic RFLPs and two (CA)n repeat microsatellite markers tightly linked to the RYR1 gene showed a definite change in the haplotype, suggesting more widespread changes in the genome upon short-term culture of EBV-immortalized B-lymphocytesItem 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, KeisakuMalignant 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.