Using human induced pluripotent stem cells to characterise a novel nonsense variant in RYR2

Student thesis: Phd


Introduction: Heterozygous pathogenic variants within RYR2 cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Patients with CPVT have structurally normal hearts but develop adrenergically mediated arrhythmias. The majority of pathogenic variants within RYR2 are missense variants which result in a gain of function of RYR2, causing ryanodine receptors to become increasingly sensitive to activation by calcium and have an increased open probability leading to an increased propensity to develop spontaneous calcium waves. More recently several missense variants have been shown to lead to arrhythmias by causing a loss of function. We identified a novel nonsense variant, p.(Arg4790Ter), in RYR2 in a young woman who suffered a cardiac arrest. In order to further characterise this variant and understand the underlying mechanism by which RYR2 variants can cause arrhythmias human induced pluripotent stem cells (hiPSCs) were generated from this patient. Methods: The hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs). Fluorescent calcium imaging was performed on the hiPSC-CMs to assess calcium handling abnormalities at baseline, following adrenergic stimulation and after incubation with different beta-blockers. RT-PCR and western blotting was performed to determine whether the nonsense variant results in the expression of the mutant allele and production of a truncated protein. Allele-specific shRNAs were designed in order to silence the mutant allele. Results: Significantly more hiPSC-CMs derived from the two different clones of the patient line (RYR2 hiPSC-CMs) displayed calcium handling abnormalities at baseline compared to control hiPSC-CMs (70.9% and 64.9% vs 28.9%, p
Date of Award31 Dec 2019
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorSusan Kimber (Supervisor), William Newman (Supervisor) & Luigi Venetucci (Supervisor)


  • RYR2
  • CPVT
  • hiPSC

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