Titin Missense Variants as a Cause of Familial Dilated Cardiomyopathy
2023; Lippincott Williams & Wilkins; Volume: 147; Issue: 22 Linguagem: Inglês
10.1161/circulationaha.122.062833
ISSN1524-4539
AutoresFernándo Domínguez, Laura Lalaguna, Inés Martínez-Martín, Jesús Piqueras‐Flores, Torsten B. Rasmussen, Esther Zorio, Giovanna Giovinazzo, Belén Prados, Juan Pablo Ochoa, Belén Bornstein, Esther González-López, Diana Velázquez-Carreras, María Rosaria Pricolo, Francisco Gutiérrez‐Agüera, Juan A. Bernal, Elías Herrero‐Galán, Jorge Alegre‐Cebollada, Enrique Lara‐Pezzi, Pablo García‐Pavía,
Tópico(s)RNA and protein synthesis mechanisms
ResumoHomeCirculationVol. 147, No. 22Titin Missense Variants as a Cause of Familial Dilated Cardiomyopathy Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBTitin Missense Variants as a Cause of Familial Dilated Cardiomyopathy Fernando Domínguez, Laura Lalaguna, Inés Martínez-Martín, Jesús Piqueras-Flores, Torsten Bloch Rasmussen, Esther Zorio, Giovanna Giovinazzo, Belen Prados, Juan Pablo Ochoa, Belen Bornstein, Esther González-López, Diana Velázquez-Carreras, Maria Rosaria Pricolo, Francisco Gutiérrez-Agüera, Juan Antonio Bernal, Elías Herrero-Galán, Jorge Alegre-Cebollada, Enrique Lara-Pezzi and Pablo García-Pavía Fernando DomínguezFernando Domínguez Correspondence to: Fernando Domínguez, MD, PhD, Department of Cardiology, Hospital Universitario Puerta de Hierro, Manuel de Falla, 2, Majadahonda, Madrid, 28222, Spain, Email E-mail Address: [email protected] https://orcid.org/0000-0001-8408-363X Heart Failure and Inherited Cardiac Diseases Unit, Department of Cardiology, Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain (F.D., B.B., E.G.-L., P.G.-P.). Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). CIBER Cardiovascular Diseases (CIBERCV), Madrid, Spain (F.D., E.Z., E.G.-L., E.L.-P., P.G.-P.). , Laura LalagunaLaura Lalaguna https://orcid.org/0000-0002-4737-358X Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Inés Martínez-MartínInés Martínez-Martín https://orcid.org/0000-0001-5818-9545 Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Jesús Piqueras-FloresJesús Piqueras-Flores https://orcid.org/0000-0002-2215-740X Unidad de Cardiopatías Familiares. Servicio de Cardiología. Hospital Universitario de Ciudad Real, Spain (J.P.-F.). Facultad de Medicina de Ciudad Real. Universidad de Castilla La Mancha, Spain (J.P.-F.). , Torsten Bloch RasmussenTorsten Bloch Rasmussen Department of Cardiology, Aarhus University Hospital, Denmark (T.B.R.). , Esther ZorioEsther Zorio https://orcid.org/0000-0003-3608-4210 CIBER Cardiovascular Diseases (CIBERCV), Madrid, Spain (F.D., E.Z., E.G.-L., E.L.-P., P.G.-P.). Unidad de Cardiopatías Familiares, Muerte Súbita y Mecanismos de Enfermedad (CaFaMuSMe), Instituto de Investigación Sanitaria La Fe, Servicio de Cardiología, Hospital Universitario y Politécnico La Fe, Valencia, Spain (E.Z.). , Giovanna GiovinazzoGiovanna Giovinazzo https://orcid.org/0000-0002-5391-0441 Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Belen PradosBelen Prados Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Juan Pablo OchoaJuan Pablo Ochoa Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Belen BornsteinBelen Bornstein https://orcid.org/0000-0003-1791-1567 Heart Failure and Inherited Cardiac Diseases Unit, Department of Cardiology, Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain (F.D., B.B., E.G.-L., P.G.-P.). , Esther González-LópezEsther González-López Heart Failure and Inherited Cardiac Diseases Unit, Department of Cardiology, Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain (F.D., B.B., E.G.-L., P.G.-P.). CIBER Cardiovascular Diseases (CIBERCV), Madrid, Spain (F.D., E.Z., E.G.-L., E.L.-P., P.G.-P.). , Diana Velázquez-CarrerasDiana Velázquez-Carreras Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Maria Rosaria PricoloMaria Rosaria Pricolo https://orcid.org/0000-0002-6362-780X Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Francisco Gutiérrez-AgüeraFrancisco Gutiérrez-Agüera Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Juan Antonio BernalJuan Antonio Bernal https://orcid.org/0000-0002-9933-5550 Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Elías Herrero-GalánElías Herrero-Galán https://orcid.org/0000-0003-1916-6185 Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Jorge Alegre-CebolladaJorge Alegre-Cebollada https://orcid.org/0000-0003-0542-0170 Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). , Enrique Lara-PezziEnrique Lara-Pezzi https://orcid.org/0000-0002-2743-1033 Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). CIBER Cardiovascular Diseases (CIBERCV), Madrid, Spain (F.D., E.Z., E.G.-L., E.L.-P., P.G.-P.). and Pablo García-PavíaPablo García-Pavía Pablo Gracía-Pavía, MD, PhD, Department of Cardiology, Hospital Universitario Puerta de Hierro, Manuel de Falla, 2, Majadahonda, Madrid, 28222, Spain, Email E-mail Address: [email protected] https://orcid.org/0000-0002-5470-2257 Heart Failure and Inherited Cardiac Diseases Unit, Department of Cardiology, Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain (F.D., B.B., E.G.-L., P.G.-P.). Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain (F.D., L.L., I.M.-M., G.G., B.P., J.P.O., D.V.-C., M.R.P., F.G.-A., J.A.B., E.H.-G., J.A.-C., E.L.-P., P.G.-P.). CIBER Cardiovascular Diseases (CIBERCV), Madrid, Spain (F.D., E.Z., E.G.-L., E.L.-P., P.G.-P.). Universidad Francisco de Vitoria (UFV), Pozuelo de Alarcón, Spain (P.G.-P.). Originally published30 May 2023https://doi.org/10.1161/CIRCULATIONAHA.122.062833Circulation. 2023;147:1711–1713High-throughput sequencing technologies have revolutionized the identification of genetic variants responsible for genetic diseases such as dilated cardiomyopathy (DCM). However, a causal genetic variant is still not identified in ≈60% of DCM.1 Although truncating variants in TTN (TTNtv) are the main genetic cause of DCM, the role of rare missense variants in TTN as a cause of DCM remains unknown. We describe 2 families with DCM in which clinical and in vitro investigations support that missense variants affecting a conserved cysteine of TTN are causative of DCM. The study was approved by the local ethics committee, and the data that support the findings of this study are available from the corresponding author on reasonable request.A Spanish family with 12 individuals with DCM was studied. Three phenotype-positive distant relatives underwent exome sequencing and shared a variant (Chr2(GRCh38):g.178741559A>T) in TTN predicted to cause the substitution of a cysteine by a serine: p.Cys3892Ser (NM_001267550.2:c.11674T>A). The affected amino acid is highly conserved (PhastCons100way score of 1.000, PhyloP100way score of 9.293). Several in silico predictors suggest that the change is deleterious, including the meta predictor (score, 0.708).2 Exome sequencing did not reveal additional shared rare variants in other cardiomyopathy-associated genes.The proband was a man who underwent cardiac transplantation at 57 years of. Among 36 family members, 14 were carriers and 12 had DCM (86%; Figure A). Median age at DCM diagnosis was 33 years (interquartile range, 18–45). Mean left ventricular (LV) ejection fraction was 43±6% and mean LV end-diastolic diameter was 57±4 mm. The 2 carriers without DCM exhibited LV ejection fraction in the lower limit of normal range. Twenty-two relatives were noncarriers and showed normal phenotype (median age, 51 years; interquartile range, 24–60; LV ejection fraction, 62±5%; LV end-diastolic diameter, 46±2 mm). Family-specific 2-point logarithm of the odds score was 3.96 (dominant model, 80% penetrance).Download figureDownload PowerPointFigure. Evidence supporting the TTN missense variants p.Cys3575Ser and p.Cys3575Arg as a cause of dilated cardiomyopathy. A, Pedigree of a large Spanish kindred with 14 TTN p.Cys3575Ser carriers (12 with DCM) and 22 noncarriers. Blue asterisks show individuals who underwent Exome sequencing. B, Pedigree of the Danish family with the TTN p.Cys3575Arg variant, with 3 carriers (2 with DCM) and 1 noncarrier. C, Amplitude of contraction of WT (green) and homozygous Cys3575Ser (red) induced pluripotent stem cell–induced cardiomyocytes (***P=0.0001, Mann-Whitney). D, Far-ultraviolet circular dichroism spectra at 25 ºC of WT (green) and Cys3575Ser (red) recombinant I21 protein domains. E, Thermal unfolding curves of recombinant I21 WT (green) and Cys3575Ser (red) domains. DCM indicates dilated cardiomyopathy; MRE, molar residue ellipticity; TTN, titin; and WT, wild type.A Danish family with the same mutated residue but with Cys replaced by Arg (NM_001267550.2:c.11674T>C) was identified. In silico evaluation also predicted the variant to be pathogenic, with a REVEL score of 0.688. The proband was a female (LV ejection fraction of 20%, LV end-diastolic diameter 75 mm) who underwent heart transplantation at 17 years of age. Her father (obligate carrier) was diagnosed with DCM at 54 and died at 73 years of age. A 52-year-old half-sister exhibited the variant without DCM (Figure B).To further characterize the p.Cys3892Ser variant, induced pluripotent stem cell (iPSC) lines were generated using CRISPR/Cas9 to introduce a point mutation T>A c:11,674 in the titin (TTN) allele of a wild-type human iPSC line (HDF-iPS-SV10, Spanish National Stem Cell Bank), wild-type human iPSC, hetero- and homozygous human iPSC-TTNCys3892Ser lines were differentiated to cardiac lineage.Video for single-cell contraction amplitude showed deficient contraction in homozygous p.Cys3892Ser cardiomyocytes (Figure C). We did not detect decreased contraction in heterozygous p.Cys3892Ser cells, consistent with other DCM iPSC lines.3Circular dichroism spectroscopy was used to study thermal denaturation of recombinant purified TTN I21 domains with and without the p.Cys3892Ser variant. Circular dichroism signal at 215 nm was monitored as temperature increased from 25 to 85 ºC at a rate of 30 ºC/h. The recombinant TTN I21 domain containing p.Cys3892Ser preserved the global fold of the wild-type protein (Figure D) but was not stable at physiological temperatures (Figure E). We also attempted to study variant p.Cys3892Arg by circular dichroism. However, this was not possible due to the insolubility of the mutant domain, which is probably caused by the higher destabilizing nature of the Cys to Arg substitution (data are not shown).We provide strong evidence supporting that TTN missense variants involving the conserved cysteine p.Cys3892 can cause DCM. Cysteine is one of the least abundant and most conserved amino acids. Cys3892 corresponds to a conserved cysteine of the cardiac specific I21 domain, present in 27 species ranging from zebrafish to humans. None of the 2 variants have been described in the literature or in ClinVar, and they are not reported in gnomAD. Regarding molecular mechanisms of pathogenicity, it is tempting to speculate that TTN domain destabilization could lead to reduced titin levels (haploinsufficiency) or saturation of protein quality control systems, both of which have been linked to pathogenicity in DCM TTN truncating variants. The Spanish family exhibited a logarithm of the odds score of 3.96. Logarithm of the odds scores of 3 support odds 20:1 in favor of linkage, and a variant strongly related to the phenotype.Our study is the first to unequivocally confirm that TTN missense variants can cause DCM. Gerull et al4 reported that a missense TTN mutation could cause DCM in a moderately large family with a logarithm of the odds score of 2.73, which is less than the value of 3 usually considered to prove association.A recent study showing that, in 530 patients with DCM, almost 7% had rare TTN missense variants predicted to be deleterious by bioinformatics filtering. However, they were not over-represented in DCM compared with ExAC, and authors concluded that TTN missense variants should be classified as likely benign.5 Our results confront with this conclusion, because we proved that missense variants involving Cys3892 are causative of DCM, supporting the pathogenic role of certain missense TTN variants.Article InformationSources of FundingThis study has been funded by Mutual Medical (Research Award 2017) and Instituto de Salud Carlos III through the project PI20/0320 (Cofunded by European Regional Development Fund/European Social Fund "A Way to Make Europe"/"Investing in Your Future"). DR Alegre-Cebollada acknowledges funding from the Ministerio de Ciencia e Innovación (MCIN) through grants PID2020-120426GB-I00 and BIO2017-83640-P (Agencia estatal de investigación [National Research Agency]/Fondo europeo de desarollo regional; ERDF [European Regional Development Fund], Unión europea [European Union]). "The project that gave rise to these results received the support of a fellowship from the La Caixa Foundation to Dr Martinez-Martin (ID 100010434). The fellowship code is LCF/BQ/DR20/11790009. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCIN and the Pro CNIC Foundation.Nonstandard Abbreviations and AcronymsDCMdilated cardiomyopathyiPSCinduced pluripotent stem cellLVleft ventricularTTNtitinDisclosures None.FootnotesFor Sources of Funding and Disclosures, see page 1713.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Fernando Domínguez, MD, PhD, Department of Cardiology, Hospital Universitario Puerta de Hierro, Manuel de Falla, 2, Majadahonda, Madrid, 28222, Spain, Email fdominguezrodriguez@gmail.comPablo Gracía-Pavía, MD, PhD, Department of Cardiology, Hospital Universitario Puerta de Hierro, Manuel de Falla, 2, Majadahonda, Madrid, 28222, Spain, Email pablogpavia@yahoo.esReferences1. Escobar-Lopez L, Ochoa JP, Mirelis JG, Espinosa MA, Navarro M, Gallego-Delgado M, Barriales-Villa R, Robles-Mezcua A, Basurte-Elorz MT, Gutiérrez García-Moreno L, et al. Association of genetic variants with outcomes in patients with nonischemic dilated cardiomyopathy.J Am Coll Cardiol. 2021; 78:1682–1699. doi: 10.1016/j.jacc.2021.08.039CrossrefMedlineGoogle Scholar2. Morales A, Kinnamon DD, Jordan E, Platt J, Vatta M, Dorschner MO, Starkey CA, Mead JO, Ai T, Burke W, et al; DCM Precision Medicine study of the DCM Consortium. Variant interpretation for dilated cardiomyopathy: refinement of the American College of Medical Genetics and Genomics/ClinGen Guidelines for the DCM Precision Medicine Study.Circ Genom Precis Med. 2020; 13:e002480. doi: 10.1161/CIRCGEN.119.002480LinkGoogle Scholar3. Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S, Gorham J, Yang L, Schafer S, Sheng CC, et al. HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy.Science. 2015; 349:982–986. doi: 10.1126/science.aaa5458CrossrefMedlineGoogle Scholar4. Gerull B, Gramlich M, Atherton J, McNabb M, Trombitás K, Sasse-Klaassen S, Seidman JG, Seidman C, Granzier H, Labeit S, et al. Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy.Nat Genet. 2002; 30:201–204. doi: 10.1038/ng815CrossrefMedlineGoogle Scholar5. Akinrinade O, Heliö T, Lekanne Deprez RH, Jongbloed JDH, Boven LG, van den Berg MP, Pinto YM, Alastalo TP, Myllykangas S, Spaendonck-Zwarts KV, et al. Relevance of titin missense and non-frameshifting insertions/deletions variants in dilated cardiomyopathy.Sci Rep. 2019; 9:4093. doi: 10.1038/s41598-019-39911-xCrossrefMedlineGoogle Scholar eLetters(0) eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. 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