Abstract 916: Molecular and Functional Identification of a New Pathogenic Mechanism in Hypertrophic Cardiomyopathy: Mutations in JPH2-encodeded Jynctophilin-2
Background: JPH2-encoded junctophilin 2 is a cardiac specific junctophilin and a member of a newly characterized family of junctional membrane complex (JMC) proteins. Junctophilin 2 plays an essential role in physically approximating the L-type calcium channel and the sarcoplasmic reticulum ryanodine receptor calcium release channel to allow calcium-induced-calcium-release. JPH2 knockout mice show disrupted calcium transients, altered JMC formation, cardiomyopathy, and embryonic lethality. We considered JPH2 as a novel candidate gene in the pathogenesis of hypertrophic cardiomyopathy (HCM).
Methods: After comprehensive analysis for 15 established HCM-susceptibility genes that encode myofilament and Z-disc proteins of the cardiac sarcomere, 225 unrelated patients with HCM remained genotype negative. Open reading frame/splice site analysis was performed on the 5 translated exons of JPH2 using PCR, DHPLC and direct DNA sequencing for this cohort of genotype negative HCM (N = 225, 125 male, average age at diagnosis 45.2 ± 19 years, mean left ventricular wall thickness (MLVWT) 20.8 ± 6 mm). Novel JPH2 mutations were engineered by site-directed mutagenesis and functionally characterized using immunofluorescent microscopy and Northern blot analysis to determine protein localization and cell hypertrophy. Intracellular calcium release was measured by live confocal microscopy.
Results: Overall, three novel JPH2 missense mutations (S101R, Y141H and S165F) were found in 3 patients (2 male, average age at diagnosis 26.8 ± 3 years, MLVWT 26± 11 mm). All three mutations involved non-conservative amino-acid substitutions of highly conserved residues, localized to functionally important domains and were absent in 1000 ethnically matched reference alleles. Functional analysis of engineered JPH2 mutations resulted in altered intracellular JPH2 localization and vacuolization, hypertrophy of cardiomyocytes and attenuation of spontaneous calcium waves.
Conclusions: This study provides the first evidence implicating perturbations in JPH2 in human disease. We provide molecular and functional evidence implicating JPH2-encoded junctophilin 2 and dysregulation of calcium signaling as a novel pathogenic mechanism for HCM.