Abstract 793: Identification and Characterization of a Novel Mutation in JPH2-Encoded Junctophilin-2 in an Italian Hypertrophic Cardiomyopathy Cohort
Background Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden death in young athletes. While genes encoding 8 key myofilament proteins represent the most common genetic subtype of HCM known as sarcomeric HCM, we established mutations in JPH2-encoded junctophilin-2 as a novel pathogenic mechanism for HCM based on its role in stabilizing the cardiac dyad for efficient calcium-induced calcium-release (CICR). Here, we sought to validate JPH2 as a novel HCM-susceptibility gene by analyzing an independent cohort of patients with HCM.
Methods Utilizing polymerase chain reaction, denaturing high performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice site mutation analysis of JPH2 was performed on 203 unrelated Italian patients with HCM (73 female, 43±18 years at diagnosis, maximal left ventricular wall thickness 23±6 mm) enrolled consecutively in Florence, Italy since 2002. To assess the implications of mutations on intracellular calcium, HL-1 cardiac cells were transfected with wild type and mutant JPH2 and loaded with Fluo-4-AM for live cell imaging by confocal microscopy.
Results A novel missense mutation, E169K-JPH2, was discovered in 1 patient and was absent in 1200 reference alleles (500 white, 100 Italian) involving a residue completely conserved across species. The subject, who lacked sarcomeric mutations, was diagnosed at 6 months of age and demonstrated massive left ventricular hypertrophy (32 mm) and outflow obstruction by adolescence. Paroxysmal atrial fibrillation and sinoatrial block necessitated implantation of a pacemaker-ICD. Expression of E169K-JPH2 in HL-1 cells precipitated a >50% reduction in the amplitude of individual calcium oscillations and reduced frequency of spontaneous calcium oscillations during contraction suggesting reduced CICR.
Conclusion This study represents the first independent validation study of JPH2 as a novel HCM-susceptibility gene. Mutant JPH2 may induce ineffective CICR through desensitization of the ryanodine receptor (RyR2) and physical uncoupling of RyR2 to the L-type calcium channel. Importantly, these observations warrant increased scrutiny of calcium mishandling as a pathogenic mechanism underlying sarcomeric-negative HCM.