IntroductionCatecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an inherited arrhythmogenic cardiac condition which is characterised by episodes of ventricular tachycardia (VT) typically triggered by stress, exercise or emotion. CPVT displays incomplete penetrance and variable expressivity, even within families. The majority of cases of CPVT are due to mutations within the cardiac ryanodine receptor gene (RYR2) and most of these mutations are missense mutations causing a gain of function of RYR2. To our knowledge, no nonsense mutations in RYR2 associated with a cardiac phenotype have been reported. MethodsWe identified a novel heterozygous nonsense mutation of RYR2, p.(Arg4790Ter), in a young woman who suffered a cardiac arrest during sexual intercourse. Other individuals in the family were found to carry the mutation, some of whom were entirely asymptomatic and had normal cardiac evaluation suggesting incomplete penetrance. Human induced pluripotent stem cells (hiPSCs) were generated from the young woman who carries this nonsense mutation. These hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs). Quantitative PCR and allele-specific quantitative PCR were undertaken to determine whether the mutation results in haploinsufficiency. Laser confocal calcium imaging was performed on the hiPSC-CMs to identify calcium handling abnormalities. ResultsQuantitative PCR showed that the total expression of RYR2 within the RYR2 hiPSC-CMs is approximately half of that seen in the control hiPSC-CMs, however allele-specific quantitative PCRs showed that both alleles are expressed suggesting that the p.(Arg4790Ter) mutation does not result in haploinsufficiency. The RYR2 hiPSC-CMs displayed significantly more calcium transient abnormalities at baseline compared to control hiPSC-CMs. More RYR2 hiPSC-CMs developed calcium transient abnormalities in response to isoproterenol and the RYR2 hiPSC-CMs also demonstrated calcium waves at lower external calcium concentrations compared to control cells suggesting a lower threshold for store overload induced calcium release (SOICR). Treatment with carvedilol corrected calcium transient abnormalities in the RYR2 hiPSC-CMs at baseline. DiscussionThe RYR2 hiPSC-CMs display a clearly abnormal calcium handling phenotype. The p.(Arg4790Ter) mutation appears to result in a decreased threshold for SOICR, similar to many previously reported gain of function missense mutations in RYR2. The RYR2 hiPSC-CMs express approximately 50% less RYR2 than control hiPSC-CMs, however both alleles are expressed suggesting that the p.(Arg4790Ter) mutation does not result in haploinsufficiency. Further work is needed to understand the mechanism that results in reduced RYR2 expression in these cells and whether this reduction in RYR2 might explain the reduced penetrance of the mutation within the family, meaning that the combination of the reduced expression with the mutation might be needed for this mutation to result in a clinical phenotype.