Pleiotropic Effects of Different Exonic Nucleotide Changes at the Same Position Contributing to Hemophilia B Phenotypic Variation

Background The disease-causing effects of genetic variations often depend on their location within a gene. Exonic changes generally lead to alterations in protein production, secretion, activity, or clearance. However, due to the overlap between proteins and splicing codes, missense variants can also affect mRNA splicing, thus adding a layer of complexity and influencing disease phenotypes. Objectives To extensively characterize a panel of 13 exonic variants in the F9 gene occurring at six different FIX positions and associated with varying severity of Hemophilia B (HB). Methods Computational predictions, splicing analysis, and recombinant FIX assays were exploited to characterize F9 variants. Results We demonstrated that 5/13 (38%) of selected F9 exonic variants have pleiotropic effects. Although bioinformatic approaches accurately classified effects, extensive experimental assays were required to elucidate and deepen the molecular mechanisms underlying the pleiotropic effects. Importantly, their characterization was instrumental in developing tailored RNA therapeutics based on engineered U7snRNA to mask cryptic splice sites and compensatory U1snRNA to enhance exon definition. Conclusion Overall, albeit a multi-tools bioinformatics approach suggested the molecular effects of multiple HB variants, the deep investigation of molecular mechanisms revealed insights into the HB phenotype-genotype relationship, enabling accurate classification of HB variants. Importantly, knowledge of molecular mechanisms allowed the development of tailored RNA therapeutics, which can also be translated to other genetic diseases.