Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function

摘要

Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects. Author Summary Clarification of if an exonic variant has an effect on splicing and/or on protein function is an important aspect in clinical genetics and in development of appropriate therapeutic strategies, and most of published evidence consider splicing and protein function separately. In exons, the presence of dense splicing regulatory and amino acidic coding information implies that mutations may have a double pathogenic effect acting on splicing and/or on protein function. To address this issue we focused on coagulation factor IX (FIX) exon 5, where we identified natural mutations that induce different degree of exon skipping. All exon skipping mutations were completely corrected by a novel splicing-switching therapeutic approach based on modified U1 snRNP. To detect the substitutions that might benefit from this correction, we investigated splicing recovered mutations for FIX protein secretion and specific activity. This analysis identified synonymous mutations causing remarkable exon skipping and missense mutations with a partial effects on both splicing and secretion, but compatible with normal FIX coagulant properties, as target variants for the splicing-switching therapy.