Distinct metabolic signatures associated with TTN truncating variants in familial dilated cardiomyopathy

Background Dilated cardiomyopathy (DCM) is a leading cause of heart failure and heart transplantation, exhibiting marked genetic and clinical heterogeneity. Truncating variants in TTN represent the most common identifiable genetic cause of DCM; however, the mechanisms linking variant position, molecular consequences, and systemic metabolic remodeling remain incompletely understood. We integrated genetic analysis with targeted plasma metabolomics to investigate genotype-associated metabolic signatures in families with inherited DCM. Methods Two unrelated multigenerational families affected by inherited DCM (11 affected and 19 unaffected relatives) underwent clinical phenotyping, whole-exome sequencing, segregation analysis, and targeted plasma LC–MS/MS metabolomic profiling, the latter performed on 28 individuals (9 affected, 19 unaffected) following exclusion of two participants whose blood was sampled after heart transplantation. Differential metabolite analysis, multivariate modeling, pathway enrichment, and exploratory machine-learning approaches were applied to identify genotype-associated metabolic signatures. Results Whole-exome sequencing identified two ultra-rare heterozygous. TTN frameshift variants (p.(Pro28946Leufs*6) and p.(Glu29179Asnfs*8)) that co-segregated with DCM and were located in constitutively expressed (high-PSI) distal A-band exons. Pooled comparison of affected individuals (n = 9) and unaffected relatives (n = 19), following exclusion of two participants whose blood was sampled after heart transplantation, identified three amino acids — glutamic acid, alanine, and proline — as significantly lower in affected individuals (all FDR < 0.05), with pathway enrichment implicating amino-acid and nitrogen-handling pathways rather than fatty-acid β-oxidation; no acylcarnitine reached FDR-corrected significance in this comparison. Within-family analyses revealed a mixed signature in the family carrying the more proximal variant (four amino acids lower, five acylcarnitines higher relative to controls), whereas the family carrying the more distal variant showed no metabolite reaching significance. Direct comparison between the two families identified seven differentially abundant metabolites, including arginine (higher in the distal-variant family; FDR = 0.0146). Exploratory multivariate and machine-learning analyses supported discrimination both between affected individuals and controls (Random Forest AUC = 0.91, 95% CI: 0.60–1.00) and between the two variant groups (Random Forest AUC up to 1.00, 95% CI: 1.00–1.00). Conclusion This study identifies two TTN truncating variants associated with familial DCM and shows that two closely positioned distal A-band variants, observed in families with similar clinical phenotypes, were associated with distinct plasma metabolic signatures. These findings expand the understanding of genotype-dependent metabolic heterogeneity in TTN-associated DCM and highlight the potential of integrating genomic and metabolomic data for molecular stratification and refined genotype–phenotype characterization in inherited cardiomyopathies. Validation in larger cohorts is required to determine the potential clinical relevance of these metabolic signatures.