Abstract 539: Molecular and Functional Characterization of Novel Hypertrophic Cardiomyopathy Susceptibility Mutations in TNNC1-Encoded Troponin C
Background Hypertrophic cardiomyopathy (HCM) is one of the most common causes of sudden death in young athletes. The most common genetic subtype of HCM, sarcomeric-HCM, involves 8 genes that encode essential cardiac myofilament proteins. A critical component of the thin filament, TNNC1-encoded human cardiac troponin C (HcTnC) plays a vital role in cardiomyocyte contraction through binding of Ca2+, yet TNNC1 has not been established as an HCM-susceptibility gene. Herein, we sought to determine whether genetic perturbations in HcTnC may play a role in the pathogenesis of HCM.
Methods Utilizing polymerase chain reaction, denaturing high performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice site mutation analysis of TNNC1 was performed on 1025 unrelated patients with HCM (39% female, 49 ± 18 years at diagnosis, maximal left ventricular wall thickness 22.3 ± 7 mm) enrolled consecutively at the Mayo Clinic from 1998 to 2007. To assess the implications of the identified mutations on cardiac muscle contraction, TnC-depleted porcine cardiac skinned fibers were reconstituted with the recombinant HcTnC mutants or wild-type to assess Ca2+ sensitivity and maximal steady state force development.
Results Four sarcomeric-HCM negative patients hosted novel missense mutations in TNNC1: A8V, C84Y, E134D, and D145E. Each mutation involved residues conserved completely across species and was absent in 1000 reference alleles (400 white, 100 black). In situ studies demonstrated that each of the HcTnC mutants increased the Ca2+ sensitivity of force development (A8V > C84Y > D145E >>> E134D~WT). In addition, differing abilities of the mutants to recover force upon reconstitution (A8V = D145E >>> C84Y = E134D = WT) was observed.
Conclusion We present the first large cohort analysis of TNNC1 in HCM and detail the discovery of four novel HCM-associated mutations. In this cohort, TNNC1-HCM is similar in frequency to 3 of the 8 genes that currently comprise the commercially available genetic test for sarcomeric-HCM. Our findings, that mutations in HcTnC alter Ca2+ sensitivity of contraction to varying degrees within the same domains of HcTnC, suggest that the role of calcium mishandling in the pathogenesis of HCM warrants further scrutiny.