Abstract 498: Functional Annotation of TNNT2 Variants of Uncertain Significance With Induced Pluripotent Stem Cell-derived Cardiomyocytes

Abstract

The introduction of clinical sequencing is dramatically increasing the discovery of variants of uncertain significance (VUSs) in genes linked to inherited cardiomyopathies. We have established a platform for rapid and efficient insertion of TNNT2 gene variants into an induced pluripotent stem cell (iPSC) line to generate an allelic series of isogenic clones for differentiation into cardiomyocytes (iPSC-CMs) for functional annotation of the variants. We first used CRISPR-Cas9 to introduce known pathogenic variants into iPSCs from a healthy person or to correct pathogenic variants in iPSCs from patients with severe cardiomyopathy. Whereas normal/corrected iPSC-CMs responded to isoproterenol treatment with a 50%-70% increase in spontaneous beating rate as assessed by patch-clamp studies, iPSC-CMs with pathogenic variants had minimal responses (close to 0%). Due to the inefficiency of CRISPR-Cas9 in introducing/correcting variants in iPSCs, we next used dual integrase cassette exchange (DICE) to allow for the introduction of a large number of variants in parallel into a pool of cells. In a single pilot use of the DICE platform, we isolated heterozygous clones with 14 unique variants, >10% of all TNNT2 coding variants cataloged in ClinVar. We found that iPSC-CMs with any of 7 VUSs or likely pathogenic variants were impaired in their response to isoproterenol, in contrast to control DICE-treated iPSC-CMs. Finally, we sought to apply the DICE platform to a patient case in real time. A 65-year-old woman with severe hypertrophic cardiomyopathy underwent gene panel testing that identified a single VUS, TNNT2 E251D. Between the first and second clinic visits (~10 weeks), we were able to use DICE to rapidly and efficiently generate iPSC-CMs with the E251D variant and determine they had normal responses to isoproterenol, suggesting that the variant is not pathogenic. Guided by this finding, we recommended that the patient’s children and grandchildren not undergo cascade genetic screening for the E251D variant. In conclusion, this work establishes the feasibility of rapid functional annotation of cardiomyopathy gene variants, which after further validation could be incorporated into clinical practice as a line of evidence to support variant classification.