Functional impact of PROC variants on splicing and protein C activity: evidence for clinical reclassification and RNA...

Inherited protein C deficiency is an established genetic risk factor for venous thromboembolism. The pathogenic relevance and clinical interpretation of exonic and near-splice PROC variants, however, remain poorly understood.

We systematically investigated 15 PROC variants located at exon-intron boundaries (±20 bp) or within coding regions, integrating splicing, protein function, and computational prediction to refine variant classification and explore RNA-based correction.

Variant effects on splicing and protein function were first evaluated using in silico prediction. Experimental validation included minigene splicing assays, rescue with engineered U1 and U7 snRNAs, and analyses of protein expression, secretion, and activity, complemented by structural modeling. Five splicing prediction tools were benchmarked against experimental data, and findings were incorporated into the American College of Medical Genetics and Genomics/Association for Molecular Pathology framework.

Several variants induced complex splicing defects, including exon skipping, activation of cryptic donor sites, and generation of multiple aberrant isoforms. SpliceAI provided the best binary prediction of splice disruption, although it underestimated isoform diversity. Some exon-edge variants exerted combined effects on splicing and protein function. Integration of transcript- and protein-level data enabled reclassification of previously uncertain variants. Both U1 and U7 snRNAs partially restored normal splicing in selected cases, demonstrating the feasibility of splice-directed correction.

Exonic and near-splice PROC variants constitute a heterogeneous group of pathogenic alleles that are often missed by in silico prediction. Functional assays at both RNA and protein levels enhance American College of Medical Genetics and Genomics/Association for Molecular Pathology-based variant classification and support the development of RNA-targeted therapeutic approaches in hereditary thrombophilia.