Tackling Non-Canonical Splicing in Arrhythmogenic Cardiomyopathy to Reduce the Uncertain Significance Variants Burden

Background . Splice-altering variants (SAVs), particularly those outside canonical splice sites, are an underappreciated contributor to inherited cardiovascular diseases. In arrhythmogenic cardiomyopathy (ACM), these variants frequently remain classified as of uncertain significance (VUS) due to limited predictive power and lack of transcript-level evidence, constraining genetic yield and clinical management. Our study aimed to determine the functional impact of SAVs in ACM genes and refine their classification using ACMG/AMP and ClinGen SVI criteria. Methods . SAVs identified in 200 ACM probands underwent SpliceAI prediction, GTEx cardiac exon-usage annotation, and functional assessment using pSPL3-based minigene assays. Aberrant transcripts were quantified using Percent Splicing Alteration (PSA). Segregation data and ACMG/AMP criteria refined by ClinGen SVI were applied to integrate functional and clinical evidence for classification. Results. Aberrant splicing was confirmed in 9/20 variants (45%), including synonymous, missense, and non-canonical intronic changes. SpliceAI scores correlated strongly with PSA values (R²=0.86). Case–control burden testing revealed significant enrichment of splice-altering variants in DSP , DSG2 , DSC2 and FLNC . Integrating predictive algorithms with experimental validation and segregation analysis markedly enhances reclassification of 16/20 variants (80%). Conclusion. Splicing defects beyond canonical sites significantly shape ACM genetic landscape. Integrating predictive models with experimental validation clarifies uncertain variants bridging the gap between genomic uncertainty and clinical decision-making.