Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare, genetically heterogeneous arrhythmogenic disorder characterised by ventricular tachycardia and syncope triggered by physical or emotional stress. Heterozygous missense variants in RYR2 are the most common cause. Here, I undertook analysis to classify the pathogenicity and mechanism of action of RYR2 variants. RYR2 variants were collated with phenotypic data where available. Each variant was assessed based on minor allele frequencies, in silico prediction tools and appraisal of functional studies and classified according to the 2015 American College of Medical Genomics guidelines. Of the 326 identified RYR2 missense variants, 55 (16.9%), previously disease-associated variants were re-classified as benign. Application of the gnomAD database allowed reclassification of 11 variants more than the smaller ExAC database, indicating the utility of larger control datasets in variant classification. In contrast to the gain of function RYR2 variants that cause CPVT, rare RYR2 loss of function variants can also result in ventricular arrhythmias. We used splice prediction tools and an ex vivo splicing assay to investigate whether RYR2 missense variants result in altered splicing. Ten RYR2 variants were consistently predicted to disrupt splicing, however none altered splicing in the splicing assay. The RyR2 Phe4905Leu variant was identified in an 18-year-old male who died during sleep. We performed cascade screening on the probandâ€™s family and functionally analysed full length RyR2 channels mutagenized with the Phe4905Leu variant in HEK 293 cells. Calcium release events in HEK 293 cells expressing eGFP tagged RyR2 Phe4905Leu had a significantly greater duration (P < 0.005), rise rate (P < 0.005), and fall rate (P < 0.005), but a lower amplitude (P < 0.05) compared to cells expressing WT RyR2. Furthermore, Phe4905Leu RyR2 was found to be less sensitive to caffeine compared to WT RyR2. In summary, the reclassification of RYR2 variants as benign is important as family members previously tested to carry these variants may not be at increased risk and those without these variants may have been falsely reassured and remain at risk of arrhythmia or sudden cardiac death. RYR2 missense variants are unlikely to alter splicing. Lastly, functional characterisation of a novel RYR2 variant reported in a family with an unusual phenotype, has generated data to support classification of this variant as pathogenic and facilitate diagnostic cascade screening within the family.
|Date of Award||31 Dec 2020|
- The University of Manchester
|Supervisor||William Newman (Supervisor) & Luigi Venetucci (Supervisor)|