Abstract Number: OC 56.1
Meeting: ISTH 2021 Congress
Background: A major aim of Hemophilia A (HA) studies is the definition of F8 variants’ causative role and the association with bleeding phenotypes. The pathogenic significance of missense variants is generally attributed to quantitative or qualitative changes in protein expression. Nevertheless, these changes may exert pleiotropic effects and also impair mRNA splicing due to the overlapping of the amino acid and splicing codes.
Aims: To combine and compare in silico and in vitro analyses to systematically characterize the pleiotropic effects of F8 exon 19 variants on both protein biology and mRNA splicing.
Methods: Analysis of nucleotide variants through bioinformatic tools, combining results from multiple algorithms, and transient expression of minigenes and recombinant FVIII variants in cellular models, to evaluate their impact on splicing and protein features.
Results: Data on thirty variants provided evidence for higher prediction ability of in vitro assays. Whereas bioinformatics provided qualitative indications, recombinant expression provided better quantitative prediction (60% vs 90%), essential for relationships with the degree of bleeding severity. Importantly, the knowledge of the specific pathogenic molecular mechanisms led to the development of tailored correction approaches. In particular, a single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants.
Conclusions: We extensively characterized and provided molecular insights for a large panel of HA-causing variants by combining in silico and in vitro analysis, demonstrating the pleiotropic effects of several exonic changes. Our data suggest caution during variants classification based on nucleotide location or bioinformatic prediction and highlight the importance of experimental characterization to dissect the molecular mechanisms underlying HA, which might pave the way for the development of new individualized therapeutic strategies, also translatable to other genetic diseases.
To cite this abstract in AMA style:Lombardi S, Peretto L, Merlin S, Follenzi A, McVey JH, Maestri I, Bernardi F, Pinotti M, Balestra D. Deep Molecular Mechanisms of F8 exon 19 Variants and Translational Approaches in Hemophilia A [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/deep-molecular-mechanisms-of-f8-exon-19-variants-and-translational-approaches-in-hemophilia-a/. Accessed September 23, 2021.
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