Pathogenicity and pathophysiology assessments of sarcomere gene variants identified in the clinic

PROJECT SUMMARY/ABSTRACT A variant of unknown/uncertain significance (VUS) is a change in an individual’s genetic sequence that has an unknown effect on the individual’s health. It is estimated that several hundred thousand individuals in the US carry VUSs in the gene encoding cardiac troponin T, TNNT2, an essential component of the thin filament of the sarcomere—the basic contractile unit of the cardiomyocyte. Because pathogenic TNNT2 variants cause at least two forms of heart failure with distinct prognoses and treatment responses, this VUS pathogenicity knowledge gap has tremendous consequences in the clinic. Previous functional studies of a small number of pathogenic TNNT2 variants have supported a myofilament tension-based pathogenesis model whereby TNNT2 variants that increase myofilament tension through increasing thin filament calcium (Ca2+) sensitivity cause hypertrophic cardiomyopathy (HCM), a disorder with impaired cardiac relaxation that is the most common cause of sudden cardiac death in young athletes. In contrast, TNNT2 variants that decrease myofilament tension through decreasing Ca2+ sensitivity cause dilated cardiomyopathy (DCM), a disorder with impaired cardiac contraction that has a mortality rate of 50% at five years due to progressive heart failure. The central goal of our proposal is to reclassify the pathogenicity of all TNNT2 VUSs and identify their pathophysiology, which would more precisely inform heart failure risk type and treatment responses in the clinic. In Aim 1, we will study the 251 TNNT2 VUSs from ClinVar, an extensive database of genomic variation in humans linked to disease status. We will use a recently developed experimental platform designed to comprehensively interrogate the function of TNNT2 variants within the context of human cardiomyocytes (iPS-CMs) differentiated from induced pluripotent stem (iPS) cells and validated using 3-dimensional cardiac microtissue assays exhibiting advanced cellular maturation, multicellular architecture, and biophysical inputs. In Aim 2, we will perform a deep mutational scan of all possible missense TNNT2 variants using iPS-CMs expressing a transcriptional reporter assay, flow cytometry, and next-generation sequencing that we previously validated to predict the pathogenicity and pathophysiology of gold-standard TNNT2 variants with >91% accuracy. Finally, in Aim 3, we will interrogate the pathogenicity in vivo of two VUSs that function like pathogenic variants in vitro, using C57BL6/J inbred mice and myotropic adeno-associated viruses (MyoAAVs) delivering physiological troponin T levels. Our study will provide essential pathogenicity and pathophysiology determinations for VUSs in an important heart failure gene, establish new insights into troponin T functions, and provide a blueprint to resolve VUSs in other heart failure genes.