Detecting transthyretin protein misfolding before amyloid deposition: a structural proteomics blood test for early identification of transthyretin amyloid (ATTR) cardiomyopathy

Background Transthyretin amyloid cardiomyopathy (ATTR-CM) is an increasingly recognized cause of heart failure driven by transthyretin (TTR) misfolding and amyloid deposition. Effective therapies exist that slow progression, but diagnosis often occurs at advanced stages when substantial myocardial damage is already present 1. Current diagnostics primarily detect established amyloid burden or downstream injury rather than underlying molecular events 3. Here, we describe the development of the Amyfold plasma assay, a minimally invasive Covalent Protein Painting (CPP)-based assay2 that enables direct detection of TTR misfolding in human plasma - the earliest and most actionable pathogenic step in ATTR-CM, which precedes amyloid fibril formation by years. Methods The Amyfold assay quantifies solvent accessibility patterns reflecting circulating TTR conformational states. Performance was evaluated using recombinant TTR, biopsy-derived amyloid fibrils and plasma samples (up to 850 samples from up to 212 individuals). Analytical validation included intra-assay precision and interlaboratory reproducibility testing. Translational evaluation was performed in blinded validation studies including wtATTR-CM, hereditary ATTR (hATTR), and a longitudinal cohort of genetically confirmed asymptomatic ATTR carriers (288 samples, 32 patients). Misfolding signatures were correlated with cardiac functional and structural measures including IVS thickness, myocardial biomarkers (NT-proBNP, hsTn), disease progression, and treatment response. Results Analytical validation demonstrated robust performance (intra-assay CV 2.3%; interlaboratory CV 7.7%). Circulating TTR misfolding signatures differentiated ATTR-CM from controls and correlated strongly with interventricular septal thickness (R² = 0.82), high-sensitivity troponin (R² = 0.80), and left ventricular ejection fraction (R² = 0.72) in a blinded validation study with confirmed wtATTR-CM patients. Discriminatory analysis yielded 95% sensitivity and specificity for ATTR-related cardiac involvement, independent of sex. Observed conformational patterns aligned with cryo-EM structures. Longitudinal analyses revealed distinct molecular trajectories, identifying progressive misfolding dynamics in some asymptomatic carriers, while others exhibited substantial baseline misfolding, suggesting capture beyond the earliest disease phase. Conclusion Plasma-based structural profiling of circulating transthyretin protein identifies upstream disease related-TTR misfolding as a potential early marker for ATTR-CM development associated with structural remodeling, myocardial injury, and functional decline in ATTR-CM. Clinical Implications. Blood-based detection of TTR misfolding may facilitate diagnostic workup of suspected ATTR-CM patients, enable minimally invasive early identification of at risk individuals, and support monitoring of disease progression and treatment response in ATTR-CM.