Titin truncations: dissection of genotype and cardiac phenotype. A. Roberts1, 2, 3, 12, J. Ware2, 3, 8, 9, D. Herman8, 9, S. Schafer4, J. Baksi2, 3, R. Buchan2, 3, R. Walsh2, 3, S. John2, 3, S. Wilkinson2, 3, L. Felkin2, 3, A. Bick8, 9, F. Mazzarotto2, 3, M. Radke6, M. Gotthardt6, 7, P. Barton2, 3, N. Hubner4, 5, 7, J. Seidman8, 9, C. Seidman8, 9, 10, S. Cook2, 3, 11, 12, 1) Clinical Sciences Centre, Imperial College London, UK; 2) NIHR Cardiovascular Biomedical Research Unit at Royal Brompton & Harefield NHS Foundation Trust and Imperial College London, UK; 3) National Heart and Lung Institute, Imperial College London, UK; 4) Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine, Berlin, Germany; 5) Charité-Universitätsmedizin, Berlin, Germany; 6) Neuromuscular and Cardiovascular Cell Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany; 7) DZHK (German Centre for Cardiovascular Research), partner site Berlin, Germany; 8) Department of Genetics, Harvard Medical School, Boston, USA; 9) Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; 10) Cardiovascular Division, Brigham and Womens Hospital and Howard Hughes Medical Institute, USA; 11) National Heart Centre Singapore, Singapore; 12) Duke-National University of Singapore, Singapore.

   TTN truncating variants (TTNtv) cause severe dilated cardiomyopathy (DCM), but sometimes occur in healthy individuals, posing significant challenges for the interpretation of these variants in an era of accessible genome sequencing. The mechanism by which TTNtv impact clinical outcomes is poorly understood. Here, we integrated the power of quantitative cardiac MRI and capacity of next generation sequencing to assess the relationship between TTN genotype and cardiac phenotype. We sequenced TTN in 4,440 subjects including 308 healthy volunteers, 3,603 Framingham Heart Study (FHS) and Jackson Heart Study (JHS) participants, 374 prospective, unselected DCM cases and 155 end-stage retrospective DCM cases including 84 for whom left ventricular (LV) tissue was available for RNA and protein studies.
   
   TTNtv were identified in 1.4% of controls (healthy volunteers, FHS and JHS participants), in 13% of unselected and 22% of end-stage DCM cases (OR 16.6, P=4.8x10-45, DCM vs controls). To improve TTN transcript annotations, we determined average cardiac TTN exon usage de novo from RNA-sequencing. TTNtv in DCM cases were enriched in highly utilised exons and isoforms (P=2.5x10-4) compared to controls. We estimate that TTNtv in highly utilised exons have >93% probability of pathogenicity (likelihood ratio 14) in DCM cases. TTNtv-positive DCM patients had more depressed LV ejection fraction (LVEF: P=0.02), thinner LV walls (P<0.02), and a higher incidence of sustained ventricular tachycardia (P=0.001). C-terminus TTNtv were associated with lower LVEF vs N-terminus (=-187%, p=0.006) and were more common in end-stage disease. TTNtv-positive FHS subjects had increased risk for DCM (RR=16, p=0.008). No change was detected in total TTN mRNA or protein levels in TTNtv-positive hearts.
   
    Incorporation of variant position and exon-specific expression improves interpretation of TTNtv. Most individuals with TTNtv do not develop DCM, but TTNtv in highly utilised, particularly C-terminus exons commonly cause DCM, likely through dominant-negative mechanisms. In DCM patients, presence and position of TTNtv may aid prognostication and management.

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