A comprehensive framework for the interpretation of TTN missense variants

Missense variants in TTN pose a major challenge in genetic diagnostics due to their high frequency in the general population, the large size of the gene, and the complex multidomain architecture of the titin protein. While the contribution of truncating variants (TTNtv) to titinopathies is well established, the role of rare TTN missense variants remains poorly defined. Advances in computational prediction and functional testing offer new tools to assess their potential pathogenicity, which however are currently not fully utilized for clinical application. We analyzed an international cohort of unsolved myopathy cases selected based on the presence of a rare missense variant in trans with a TTNtv. Clinical data were collected from neuromuscular centers worldwide. In silico predictions were generated using AlphaMissense and complemented by minor allele frequency (MAF) and exon usage information. Additional inclusion criteria were based on a MAF < 0.010 and an AlphaMissense score ≥ 0.792 for the missense variants, in accordance with the latest ClinGen guidelines. Selected missense variants were characterized in vitro through protein expression and cell imaging assays to assess their effects on domain solubility and aggregation. Thirty patients with TTNtv/missense combinations were identified, presenting with heterogeneous myopathic phenotypes, ranging from congenital to adult onset. An in-depth analysis on AlphaMissense predictions highlighted those changes most frequently predicted as possibly pathogenic. Functional assays showed that three selected variants with changes to proline, located in β-sheets of Ig domains, led to impaired folding, cytoplasmic aggregation and co-localisation with proteostasis markers. In our cohort, all non-proline mutations occurred at buried sites, while some proline substitutions affected exposed residues. Notably, the variant p.(Gln7023Pro) was identified in 5 unrelated families sharing a conserved haplotype, indicating a common ancestor. This variant and the previously reported p.(Arg25480Pro) now meet ACMG criteria for classification as likely pathogenic. By integrating clinical, computational, and functional evidence, we propose a framework for interpreting TTN missense variants. Combining multiple lines of evidence is essential for variants’ classification and interpretation, especially given TTN complexity. Advancing diagnostic accuracy will require tailored interpretation guidelines and a global effort in data sharing and functional validation.

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Journal
Genome Medicine
Published
2026-02-26
DOI
https://doi.org/10.1186/s13073-026-01605-1
Primary Topic
Cardiomyopathy and Myosin Studies
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article
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article

A comprehensive framework for the interpretation of TTN missense variants

Siiri Sarv, Mark Holt, C. Genetti, Sheffali Gulati et al.
Genome Medicine
Cardiomyopathy and Myosin Studies
article

A comprehensive framework for the interpretation of TTN missense variants

Siiri Sarv, Mark Holt, C. Genetti, Sheffali Gulati, C. Fiorillo, Pamela Barraza‐Flores, Crystal Nguyen, E. Malfatti, Anna Kuchina, Serena Baratto, Ay Lin Kho, Athanasios Evangeliou, A. Beggs, Federica Ricci, Olof Danielsson, Aysylu F. Murtazina, Ioannis Zaganas, Martin Rees, Victoria Lillback, Minas Drakos, Monica Traverso, Stellan Mörner, Martha Spilioti, Polina Chausova, Enrico Bertini, Tereza Kramářová, Tara Reeves, Maria Francesca Di Feo, Daria Diodato, Gianina Ravenscroft, Aishwarya Dhall, Michela Catteruccia, Bandana Jassal, Jana Zídková, Anna Sarkozy, Balogh István, Michele Iacomino, Carmelo Rodolico, Sonia Messina, Berta Estévez Arias, Alessandro Mussa, Angelina Meybatova, O. A. Shchagina, Federico Zara, Mathias Gautel, Heinz Jungbluth, Katalin Szakszon, Mridul Johari, Johana Kopčilová, Mehar Chand Sharma, Sushan Luo, Katrin Õunap, Mohammed Faruq, Francesca Faravelli, Venugopalan Y. Vishnu, Giorgio Tasca, Sergei A. Kurbatov, Luciano Merlini, Bjarne Udd, Francesco Muntoni, G. M. Luana Mandarà, T. T. N. study group, Adele D’Amico, Giovanni Baranello, Huahua Zhong, Claudio Bruno, Franziska Schnabel, Marina Pedemonte, Daniel Natera-de Benito
article en

Abstract

Missense variants in TTN pose a major challenge in genetic diagnostics due to their high frequency in the general population, the large size of the gene, and the complex multidomain architecture of the titin protein. While the contribution of truncating variants (TTNtv) to titinopathies is well established, the role of rare TTN missense variants remains poorly defined. Advances in computational prediction and functional testing offer new tools to assess their potential pathogenicity, which however are currently not fully utilized for clinical application. We analyzed an international cohort of unsolved myopathy cases selected based on the presence of a rare missense variant in trans with a TTNtv. Clinical data were collected from neuromuscular centers worldwide. In silico predictions were generated using AlphaMissense and complemented by minor allele frequency (MAF) and exon usage information. Additional inclusion criteria were based on a MAF < 0.010 and an AlphaMissense score ≥ 0.792 for the missense variants, in accordance with the latest ClinGen guidelines. Selected missense variants were characterized in vitro through protein expression and cell imaging assays to assess their effects on domain solubility and aggregation. Thirty patients with TTNtv/missense combinations were identified, presenting with heterogeneous myopathic phenotypes, ranging from congenital to adult onset. An in-depth analysis on AlphaMissense predictions highlighted those changes most frequently predicted as possibly pathogenic. Functional assays showed that three selected variants with changes to proline, located in β-sheets of Ig domains, led to impaired folding, cytoplasmic aggregation and co-localisation with proteostasis markers. In our cohort, all non-proline mutations occurred at buried sites, while some proline substitutions affected exposed residues. Notably, the variant p.(Gln7023Pro) was identified in 5 unrelated families sharing a conserved haplotype, indicating a common ancestor. This variant and the previously reported p.(Arg25480Pro) now meet ACMG criteria for classification as likely pathogenic. By integrating clinical, computational, and functional evidence, we propose a framework for interpreting TTN missense variants. Combining multiple lines of evidence is essential for variants’ classification and interpretation, especially given TTN complexity. Advancing diagnostic accuracy will require tailored interpretation guidelines and a global effort in data sharing and functional validation.

Genome MedicineVol. 18(1)
Linköping University (SE), University of Messina (IT), Boston Children's Hospital (US), University of Debrecen (HU), University of Helsinki (FI), Harvard University (US), University of Crete (GR), Inserm (FR), Tampere University (FI), King's College London (GB), Université Paris-Est Créteil (FR), Guy's and St Thomas' NHS Foundation Trust (GB), Harry Perkins Institute of Medical Research (AU), Great Ormond Street Hospital (GB), Voronezh State Medical Academy named after N.N. Burdenko (RU), Great Ormond Street Hospital for Children NHS Foundation Trust (GB), AHEPA University Hospital (GR), Istituto Giannina Gaslini (IT), Newcastle upon Tyne Hospitals NHS Foundation Trust (GB), Tartu University Hospital (EE), Centre Hospitalier Universitaire Sainte-Justine (CA), Assistance Publique – Hôpitaux de Paris (FR), Azienda Usl 8 Arezzo (IT), St. Luke's Hospital (GR), Vaasa Central Hospital (FI), Bambino Gesù Children's Hospital (IT), Research Centre for Medical Genetics (RU), NIHR Newcastle Biomedical Research Centre (GB), Ospedale Regina Margherita (IT), University Hospital of Heraklion (GR), Evelina London Children's Healthcare (GB), Institut Mondor de Recherche Biomédicale (FR), Huashan Hospital (CN), Crete University Press (GR), Folkhälsans Forskningscentrum (FI), Dana-Farber/Boston Children's Cancer and Blood Disorders Center (US), University College London (GB), University of Turin (IT), University of Genoa (IT), Umeå University (SE), Leipzig University (DE), University of Bologna (IT)
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