FUNctional characterisation of type 1 ryanodine receptor variants : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Manawatū, New Zealand
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2021
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Massey University
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Abstract
Malignant hyperthermia (MH) is a pharmacogenetic disorder that presents as a hypermetabolic response to volatile anaesthetics such as isoflurane, desflurane and sevoflurane, as well as the depolarising muscle relaxant succinylcholine. MH reactions are characterised by a wide variety of clinical symptoms, including but not limited to muscle rigidity, tachycardia, hyperthermia, and hyperkalaemia. If left untreated, an MH reaction can result in cardiac arrest; therefore, those with a family history of MH are encouraged to be tested for susceptibility. The current diagnostic procedure is the in vitro contracture test (IVCT), wherein the contractile response of a patient muscle biopsy after exposure to caffeine and halothane is measured. While considered the “gold-standard” for MH susceptibility testing, the reliance of the test on an excised muscle specimen makes it traumatic for the patient, and a financial burden on the healthcare system. As a result, a cheaper and less traumatic genetic test has been under development to one day replace the IVCT. Genetic testing for MH susceptibility focuses on the allelic heterogeneity of a Ca²⁺ release channel known as the type 1 ryanodine receptor (RYR1). RyR1 functions to regulate Ca²⁺ release for the purposes of muscle contraction. Currently, between 50 and 86% of MH reactions can be attributed to variants in the RYR1 gene. In order for MH susceptibility to be accurately diagnosed using a genetic test, known variants of RyR1 must meet a set of requirements to determine their pathogenicity, including being functionally characterised and shown to be hypersensitive to agonists. Over the course of this project, six RyR1 variants, from families known to be susceptible to MH, were functionally characterised. Of these, one was located in the N-terminal hot spot domain (p.Phe539Leu), three were located in the central hot spot domain (p.Gly2183Glu, p.Cys2237Tyr, p.Arg2458Leu), while two flanked the central domain (p.Arg1707Cys, p.Pro2793Leu). The results obtained indicate that the substitutions p.Cys2237Tyr and p.Pro2793Leu produce hypersensitive channels, and while p.Pro2793Leu remains a variant of unknown significance, p.Cys2237Tyr has met the criteria to be classified as likely pathogenic for MH and should therefore be added to the list of MH diagnostic variants.