Abstract / Summary
Although synonymous variants are generally regarded as functionally neutral and are frequently classified as benign or variants of uncertain significance (VUS) in clinical genetic testing, accumulating evidence indicates that they can contribute to disease through multiple post-transcriptional mechanisms. However, their functional significance in PROS1 remains poorly understood. Here, we systematically investigated synonymous PROS1 variants to define their molecular mechanisms, clinical relevance, and therapeutic potential. Fourteen synonymous PROS1 variants were evaluated using an integrated framework combining computational prediction with experimental validation. Analyses included in silico assessment of splicing, mRNA stability, and codon usage, together with minigene splicing assays, mRNA stability measurements, protein expression analyses, anticoagulant activity assays, clinical genotype–phenotype correlation, and mechanism-guided RNA rescue experiments. Synonymous PROS1 variants disrupted gene expression through multiple post-transcriptional mechanisms, including aberrant pre-mRNA splicing (6/14 variants, 42.9%), reduced mRNA stability (2/14 variants, 14.3%), and impaired translational efficiency (2/14 variants, 14.3%), resulting in decreased Protein S expression and anticoagulant activity. Clinical relevance was further supported by three additional patients carrying spliceogenic PROS1 variants, who exhibited reduced Protein S levels and thrombotic manifestations consistent with the experimentally identified functional defects. Integration of multi-layer functional evidence together with clinical genotype–phenotype data enabled ACMG/AMP-based reclassification of several variants from VUS to likely pathogenic or benign. Furthermore, mechanism-guided RNA correction using engineered U1 snRNA and antisense U7 snRNA partially restored normal splicing in selected spliceogenic variants, supporting the feasibility of variant-specific RNA therapeutics. This study demonstrates that synonymous PROS1 variants are not silent sequence changes but can exert pathogenic effects through aberrant splicing, altered mRNA stability, and impaired translational efficiency. Integration of multi-layer functional and clinical evidence improved ACMG/AMP-based variant interpretation, enhanced the molecular diagnosis and clinical interpretation of Protein S deficiency, and supported the reclassification of multiple synonymous variants. Moreover, successful correction of selected spliceogenic variants using engineered U1 and U7 snRNAs highlights the therapeutic potential of mechanism-guided RNA-based splice correction for inherited thrombophilia. Not applicable.