Functional Annotation of TNNT2 Variants of Uncertain Significance With Genome-Edited Cardiomyocytes
2018; Lippincott Williams & Wilkins; Volume: 138; Issue: 24 Linguagem: Inglês
10.1161/circulationaha.118.035028
ISSN1524-4539
AutoresWenjian Lv, Lyon Qiao, Nataliya Petrenko, Wenjun Li, Anjali Owens, Chris McDermott‐Roe, Kiran Musunuru,
Tópico(s)Congenital heart defects research
ResumoHomeCirculationVol. 138, No. 24Functional Annotation of TNNT2 Variants of Uncertain Significance With Genome-Edited Cardiomyocytes Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBFunctional Annotation of TNNT2 Variants of Uncertain Significance With Genome-Edited Cardiomyocytes Wenjian Lv, PhD, Lyon Qiao, MS, Nataliya Petrenko, MS, Wenjun Li, BS, Anjali T. Owens, MD, Chris McDermott-Roe, PhD and Kiran Musunuru, MD, PhD, MPH Wenjian LvWenjian Lv Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. , Lyon QiaoLyon Qiao Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. , Nataliya PetrenkoNataliya Petrenko Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. , Wenjun LiWenjun Li Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. , Anjali T. OwensAnjali T. Owens Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. , Chris McDermott-RoeChris McDermott-Roe 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 E-mail Address: [email protected] Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. and Kiran MusunuruKiran Musunuru 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 E-mail Address: [email protected] Department of Medicine, Division of Cardiology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. Originally published10 Dec 2018https://doi.org/10.1161/CIRCULATIONAHA.118.035028Circulation. 2018;138:2852–2854The 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.2Download figureDownload PowerPointFigure. 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 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 FundingThis 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.).DisclosuresNone.Footnoteshttps://www.ahajournals.org/journal/circData 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]comChris 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.eduReferences1. Manrai AK, Funke BH, Rehm HL, Olesen MS, Maron BA, Szolovits P, Margulies DM, Loscalzo J, Kohane IS. Genetic misdiagnoses and the potential for health disparities.N Engl J Med. 2016; 375:655–665. doi: 10.1056/NEJMsa1507092CrossrefMedlineGoogle Scholar2. Wu H, Lee J, Vincent LG, Wang Q, Gu M, Lan F, Churko JM, Sallam KI, Matsa E, Sharma A, Gold JD, Engler AJ, Xiang YK, Bers DM, Wu JC. Epigenetic regulation of phosphodiesterases 2A and 3A underlies compromised β-adrenergic signaling in an iPSC model of dilated cardiomyopathy.Cell Stem Cell. 2015; 17:89–100. doi: 10.1016/j.stem.2015.04.020CrossrefMedlineGoogle Scholar3. 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Musunuru K and Kathiresan S (2019) Genetics of Common, Complex Coronary Artery Disease, Cell, 10.1016/j.cell.2019.02.015, 177:1, (132-145), Online publication date: 1-Mar-2019. December 11, 2018Vol 138, Issue 24 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.118.035028PMID: 30565988 Originally publishedDecember 10, 2018 Keywordsgene mutationgeneticscardiomyocytecardiomyopathygenome editingstem cellPDF download Advertisement SubjectsCardiomyopathyGeneticsStem CellsTranslational Studies
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