The introduction of clinical sequencing is dramatically increasing the discovery of variants of uncertain significance in genes linked to inherited cardiomyopathies. Computational and population-based methods to predict pathogenicity have demonstrated shortcomings that can result in genetic misdiagnoses.1 To help address this situation, we have established a platform for rapid insertion of TNNT2 gene variants into induced pluripotent stem cell-differentiated cardiomyocytes (iPSC-CMs) for functional annotation of the variants.

We first used CRISPR-Cas9-mediated homology-directed repair to introduce known pathogenic TNNT2 variants (R173W2 and K210del3) into iPSCs from a healthy person (DiPS 1016 SevA, termed 1016, obtained from the Harvard Stem Cell Institute iPS Core Facility), correct the R173W variant in iPSCs from a patient with severe dilated cardiomyopathy2 (termed ST, obtained from the Stanford Cardiovascular Institute Biobank), and introduce the K210del variant into the corrected iPSCs. Whereas normal/corrected iPSC-CMs responded to isoproterenol treatment with a 50% to 70% increase in spontaneous beating rate as assessed by patch-clamp studies of ventricular-like cells, iPSC-CMs from patients with pathogenic TNNT2 variants (R173W alone, termed ST; K210del along with an additional DSP C2497X variant, generated from a patient at the University of Pennsylvania Center for Inherited Cardiovascular Disease with severe dilated cardiomyopathy, termed PE; the study was approved by an institutional review committee, and subjects gave informed consent) or with introduced pathogenic TNNT2 variants but not with the solo DSP variant had minimal responses (≈0%) (Figure, A and B), consistent with and extending previous observations.2

Figure. Functional annotation of TNNT2 variants in iPSC-CMs.A, Representative action potentials from patch-clamp studies of iPSC-CMs. B, Response of spontaneous beating rate to 1 µmol/L isoproterenol normalized to pretreatment rate. Error bars indicate SEM; P values calculated by Mann–Whitney U test and adjusted for multiple testing. C, Schematic of DICE procedure. DICE indicates dual-integrase cassette exchange; and iPSC-CMs, induced pluripotent stem cell-differentiated cardiomyocytes.

Because of the inefficiency of CRISPR-Cas9 in introducing/correcting variants in iPSCs by homology-directed repair (often <1% efficiency), we adapted dual-integrase cassette exchange (DICE)4 to allow for multiplex insertion of variants into a pool of cells (Figure, C). With CRISPR-Cas9, we generated an acceptor 1016 line heterozygous for TNNT2 exon 6 replaced with attP sites for the phiC31 and Bxb1 integrases. We generated a library of donor plasmids with phiC31 and Bxb1 attB sites flanking a partial cDNA spanning TNNT2 exons 6 to 17, each with 1 of the 120 TNNT2 coding variants cataloged in ClinVar as of 2015. In a single pilot use of the DICE platform—coelectroporation of the pooled donor plasmid library along with phiC31- and Bxb1-expressing plasmids into the acceptor cell line, fostering stochastic, monoallelic insertion of a single-variant cDNA into the genome of each cell—we isolated heterozygous clones with 14 unique variants, >10% of all ClinVar TNNT2 coding variants (≈5% recombination efficiency). Differentiated iPSC-CMs with R173W or any of 7 other variants, mostly variants of uncertain significance, were impaired in response to isoproterenol, in contrast to control DICE iPSC-CMs with the wild-type cDNA (Figure, B). This finding supports reclassification of the variants of uncertain significance as likely pathogenic.

Last, we applied 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 variant, TNNT2 E251D, which has conflicting interpretations in ClinVar (1 likely benign, 4 variants of uncertain significance, and 1 likely pathogenic); whereas computational algorithms generally predict pathogenicity, the population frequency in the Exome Aggregation Consortium database (0.03%) is higher than what is commonly believed to be consistent with pathogenicity. Between the first and second clinic visits (≈10 weeks), we used DICE to rapidly and efficiently generate iPSCs with the E251D variant (8/61 clones screened) and determine that the iPSC-CMs had normal responses to isoproterenol (Figure, B), suggesting that the variant might not be pathogenic.

To assess whether the E251D variant might elicit a phenotype in a genetic background known to be vulnerable to cardiomyopathy, we subsequently used the version of the ST line in which we had corrected the pathogenic R173W variant (ie, a wild-type iPSC line originating from a patient with severe disease) and introduced E251D with CRISPR-Cas9; differentiated iPSC-CMs had normal responses to isoproterenol (Figure, B). Additional investigation of these E251D iPSC-CMs showed no significant increases in cell size or expression of genes previously reported to be increased in hypertrophic cardiomyopathy iPSC-CMs (NPPA, TNNT2, MYL2, MYL4, and MYH7; data not shown).5 Guided by these findings, we have recommended that the patient’s 2 children and 6 grandchildren not undergo cascade genetic screening for the E251D variant.

Although the gold standard for functional annotation of patient variants might be to generate iPSCs for each patient and correct the variant with CRISPR-Cas9, permitting the phenotypic comparison of matched patient-derived iPSC-CMs with and without the variant, this approach would be unrealistic for the management of a large number of patients because it would be time-consuming and expensive; hence, the advantage of the DICE approach presented here.

In conclusion, this work establishes the feasibility of rapid, real-time functional annotation of cardiomyopathy gene variants, with the prospect of eventual incorporation into clinical practice as an additional line of evidence to support variant classification.

Sources of Funding This work was supported by National Institute of Health grants R01-HL118744 and R01-GM104464 (K.M.) and the Winkelman Family Fund in Cardiovascular Innovation (A.T.O., K.M.).

Disclosures None.

Footnotes

https://www.ahajournals.org/journal/circ Data sharing: The data that support the findings of this study are available from the corresponding authors upon reasonable request. Kiran Musunuru, MD, PhD, MPH, Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, 3400 Civic Center Blvd, Building 421, 11–104 Smilow Center for Translational Research, Philadelphia, PA 19104. Email [email protected] com Chris McDermott-Roe, PhD, Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, 3400 Civic Center Blvd, Building 421, 11–189 Smilow Center for Translational Research, Philadelphia, PA 19104. Email [email protected] upenn. edu