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Title: Defining molecular mechanisms of calcium dysregulation in malignant hyperthermia susceptibility
Author: Dodds, Rachel Emma
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Malignant hyperthermia (MH) presents in susceptible individuals following exposure to volatile anaesthetic agents, manifesting as Ca2+ dysregulation via aberrant activity of the ryanodine receptor 1 (RYR1) – the major Ca2+ release unit of skeletal muscle sarcoplasmic reticulum (SR). Progress in the characterisation of RYR1 variants identified in MH susceptible individuals has been hindered by characterisation processes that rely on the cloning and heterologous expression of the large, ~15 kb RYR1 gene. A novel CRISPRCas9 based method for variant characterisation was developed to circumvent existing limitations of variant characterisation. The p.D3986E and p.S1728F variants were introduced into C2C12 myoblasts. In addition to the desired integration of the repair template, this resulted in a range of erroneous integrations and undesired editing events. Nonetheless, this work provides a substantial foundation for future development of CRISPR-Cas9 genome editing in the MH field and highlights key areas for future optimisation. It has been speculated that variants in genes other than the RYR1 and CACNA1S could contribute to MH susceptibility in the proportion of MH cases for which no genetic cause has been ascertained. Next generation sequencing of fifty genes associated with Ca2+ handling, performed by the Leeds MH Unit, identified two calsequestrin-1 (CASQ1) variants, p.I138T and p.F186Y, in an MH individual and an exertional heat illness (EHI) individual, respectively. An additional variant, CASQ1 p.E364K was identified in an MHShc individual by colleagues in the Australian population. EHI has significant clinical overlap with MH and MH-associated RYR1 variants have been detected in EHI patients. CASQ1 is thought to be the major Ca2+ binding protein of skeletal muscle SR and a regulator of RYR1 activity. CASQ1 null mice have been shown to exhibit an MH and EHI-like phenotype in response to halothane and heat, respectively. In this project, CASQ1 variants p.F186Y and p.I138T were shown to have reduced Ca2+ binding and polymerisation capacities compared CASQ1 WT and CASQ1 p.E364K. These data suggest that the highly conserved CASQ1 p.F186Y and p.I138T variants could act to influence the MH/EHI phenotype.
Supervisor: Hopkins, Philip ; Shaw, Marie-Anne ; Bilmen, Jonathan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available