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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 Molecular genetic analysis for 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, 2004) Sato, KeisakuMalignant hyperthermia (MH) is a rare pharmacogenetic disorder in humans caused by inhalational general 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 calcium release channel ryanodine receptor of skeletal muscle (RYR1) is one of the causes of MH leading to the abnormally high release of calcium ions into the cytosol during MH reactions. The MH reaction can also be triggered by excess exercise, heat and stress. A New Zealand male, identified as M818, showed a fulminant MH reaction which resulted in death. The reaction was caused by exercise, and he did not have a family history of MH. As this individual did not have any of the mutations within RYR1 found to date in New Zealand families, the entire RYR1 cDNA was screened for a novel mutation that may result in susceptibility to exercise-induced MH. This patient may have had a novel RYRl mutation because exercise-induced MH is quite rare. Screening of this gene, however did not identify any mutations within RYR1 suggesting that the M818 patient may have a mutation in another gene because MH is a heterogeneous disorder with 40-50% of families showing linkage to alternative loci. Heterogeneity of MH can result in discordance between genotype and phenotype. Some MH susceptible patients do not have a RYR1 mutation that is found in other individuals with the same kindred. One or more other genes could be associated with MH for these individuals although alternative loci have not been studied in New Zealand families. A genome-wide scan was performed to search for other candidate loci using a large MH kindred known as the CH family within which discordance has been observed. Non-parametric linkage analysis across all chromosomes identified five weak linkages from one branch, and two strong linkages from another branch of the CH family. Secondary linkage analysis was performed on one candidate locus identified in the genome-wide scan, and a weak linkage and recombination was observed within the shorter region. No candidate genes with obvious relevance to calcium homeostasis or signalling were identified within this region. The existence of alternative causative loci in this family cannot be ruled out however, because the loci identified from the genome-wide scan are very large and contain many genes of unknown function.