The Fontan EV Score: A Circulating Extracellular Vesicle-Based Risk Stratification Tool for Fontan-Associated Liver Disease

Abstract

BackgroundFontan-associated liver disease (FALD) is a universal complication of the Fontan palliation characterized by chronic congestion and progressive hepatic fibrosis. Current diagnostics rely on invasive biopsies or non-specific biochemical and imaging biomarkers that fail to capture early fibrogenesis, creating a critical need for non-invasive biomarkers to stratify disease severity. MethodsWe utilized a translational ovine Fontan model (n = 19) to investigate circulating serum extracellular vesicles (sEVs) as reporters of hepatic pathology. Longitudinal serum samples paired with liver elastography were collected, and sEVs were subjected to multi-omic profiling including small RNA sequencing and proteomics. Regularized regression was used to identify transcriptomic predictors, which were integrated with time post-surgery into an ordinal logistic regression framework to construct the Fontan EV Score (FES). Model performance was evaluated on a held-out test cohort and benchmarked against established serological fibrosis indices. To validate the biological relevance of the FES panel, TGF-{beta}-treated human liver organoids were generated and scored miRNA expression was assessed. ResultsThe sEV proteome exhibited robust separation by surgical physiology, while the small RNA cargo was primarily stratified by fibrotic status. Bioinformatic analysis confirmed a high hepatic origin for these transcripts and identified enrichment of inflammatory pathways including Toll-like receptor and Interleukin-17 cascades in fibrotic subjects. The FES, incorporating time post-surgery and eleven small RNA biomarkers, demonstrated high predictive accuracy in the independent testing cohort with an AUC of 0.876 for moderate and 0.963 for severe fibrosis, substantially outperforming APRI (AUC = 0.618) and FIB-4 (AUC = 0.731). In TGF-{beta}-treated human liver organoids, several scoring miRNAs, including miR-125a-5p and miR-193b-5p, were directionally responsive to profibrotic stimulation. ConclusionsCirculating sEVs carry a liver-associated molecular cargo that can be leveraged for the non-invasive prediction of FALD severity. The FES provides a biologically validated scoring system that substantially outperforms existing serological indices and offers a new avenue for early detection and risk stratification of FALD Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LIFontan-associated liver disease (FALD) is a nearly universal consequence of the Fontan circulation, driven by chronic venous hypertension and reduced cardiac output. C_LIO_LICurrent surveillance tools, including transaminases, composite serological indices (APRI, FIB-4), and elastography, have limited sensitivity and specificity for detecting and staging hepatic fibrosis in the Fontan population. C_LIO_LICirculating small extracellular vesicles (sEVs) carry tissue-derived molecular cargo and have shown diagnostic potential in other liver diseases, but their utility in FALD has not been explored. C_LI What New Information Does This Article Contribute?O_LIMulti-omic profiling of circulating sEVs in a translational ovine Fontan model reveals that the small RNA cargo is stratified by fibrotic status and enriched for inflammatory pathways associated with hepatic stellate cell activation. C_LIO_LIThe Fontan EV Score (FES), integrating time post-surgery with eleven circulating small RNA biomarkers, predicts FALD severity with substantially greater accuracy than APRI and FIB-4. C_LIO_LITGF-{beta}-treated human liver organoids confirm that several FES-associated miRNAs are directly responsive to profibrotic stimulation, providing biological validation independent of Fontan hemodynamics. C_LI This study demonstrates that circulating sEVs function as non-invasive reporters of hepatic fibrogenesis in the Fontan circulation and introduces the first EV-based scoring system for FALD risk stratification. The FES achieved an AUC of 0.876 for moderate and 0.963 for severe fibrosis in an independent test cohort, outperforming established serological indices that were originally developed for viral hepatitis but which perform poorly in congestive hepatopathy. By combining molecular biomarker discovery with in vitro functional validation, this work establishes a foundation for developing targeted, non-invasive diagnostics to guide surveillance and clinical decision-making in the growing Fontan patient population.