In humans, coagulation factor V (FV) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Zebrafish possess many distinct advantages including high fecundity, optical clarity, external development, and homology with the mammalian hemostatic system, features that make it ideal for genetic studies. Our aim was to study the role of FV in zebrafish through targeted mutagenesis and apply the model to the study of human F5 variants. CRISPR-mediated genome editing of the zebrafish f5 locus was performed, generating mutants homozygous for a 49 base pair deletion in exon 4. Thrombus formation secondary to vascular endothelial injury was absent in f52/2 mutant embryos and larvae. Despite this severe hemostatic defect, homozygous mutants survived before succumbing to severe hemorrhage in adulthood. Human F5 variants of uncertain significance from patients with FV deficiency were evaluated, and the causative mutations identified and stratified by their ability to restore thrombus formation in larvae. Analysis of these novel mutations demonstrates variable residual FV function, with minimal activity being required to restore hemostasis in response to laser-induced endothelial injury. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology, although limitations exist. Furthermore, homozygous mutant embryos tolerate what is a severe and lethal defect in mammals, suggesting the possibility of species-specific factors enabling survival, and allowing further study not possible in the mouse. Identification of these factors or other genetic modifiers could lead to novel therapeutic modalities.

Analysis of factor V in zebrafish demonstrates minimal levels needed for early hemostasis / A.C. Weyand, S.J. Grzegorski, M.S. Rost, K.I. Lavik, A.C. Ferguson, M. Menegatti, C.E. Richter, R. Asselta, S. Duga, F. Payvandi, J.A. Shavit. - In: BLOOD ADVANCES. - ISSN 2473-9529. - 3:11(2019 Jun 11), pp. 1670-1680. [10.1182/bloodadvances.2018029066]

Analysis of factor V in zebrafish demonstrates minimal levels needed for early hemostasis

M. Menegatti;R. Asselta;S. Duga;F. Payvandi
Penultimo
;
2019

Abstract

In humans, coagulation factor V (FV) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Zebrafish possess many distinct advantages including high fecundity, optical clarity, external development, and homology with the mammalian hemostatic system, features that make it ideal for genetic studies. Our aim was to study the role of FV in zebrafish through targeted mutagenesis and apply the model to the study of human F5 variants. CRISPR-mediated genome editing of the zebrafish f5 locus was performed, generating mutants homozygous for a 49 base pair deletion in exon 4. Thrombus formation secondary to vascular endothelial injury was absent in f52/2 mutant embryos and larvae. Despite this severe hemostatic defect, homozygous mutants survived before succumbing to severe hemorrhage in adulthood. Human F5 variants of uncertain significance from patients with FV deficiency were evaluated, and the causative mutations identified and stratified by their ability to restore thrombus formation in larvae. Analysis of these novel mutations demonstrates variable residual FV function, with minimal activity being required to restore hemostasis in response to laser-induced endothelial injury. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology, although limitations exist. Furthermore, homozygous mutant embryos tolerate what is a severe and lethal defect in mammals, suggesting the possibility of species-specific factors enabling survival, and allowing further study not possible in the mouse. Identification of these factors or other genetic modifiers could lead to novel therapeutic modalities.
Settore MED/09 - Medicina Interna
11-giu-2019
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/659500
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