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

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.

导致氨基酸改变的突变，可同时影响前信使RNA（pre-mRNA）剪接与蛋白质功能。明晰这类突变的联合效应，对于实现精准诊断，以及在分子层面制定最适宜的治疗策略至关重要。本研究鉴定出一系列位于凝血因子IX（FIX）第5外显子的致病性剪接突变，这类突变可通过修饰后的U1小核核糖核蛋白颗粒（U1snRNP particle），经由SRSF2依赖的增强机制实现完全修复。我们发现，伴随部分FIX分泌缺陷的同义突变与错义替换，均可作为该疗法的靶点——经剪接矫正后的蛋白质可维持正常的FIX凝血比活性。由此可见，剪接与蛋白质改变共同明确了凝血FIX第5外显子中多种外显子突变的致病分子机制。此外，本研究结果对剪接转换疗法的开发具有重要意义，尤其针对那些同时影响剪接与蛋白质功能的突变：提升正确剪接蛋白质的产量，即可弥补其基础功能缺陷。