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Genome editing of factor X in zebrafish reveals unexpected tolerance of severe defects in the common pathway

Zhilian Hu, Yang Liu, Michael C. Huarng, Marzia Menegatti, Deepak Reyon, Megan S. Rost, Zachary G. Norris, Catherine E. Richter, Alexandra N. Stapleton, Neil C. Chi, Flora Peyvandi, J. Keith Joung and Jordan A. Shavit

Key Points

  • Deficiency of coagulation factor X in zebrafish results in a severe hemostatic defect that is surprisingly well-tolerated until adulthood.

  • In vivo analysis of human mutations in zebrafish identifies variants underlying symptomatic factor X deficiency.

Publisher's Note: There is an Inside Blood Commentary on this article in this issue.

Abstract

Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish f10 by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use f10−/− zebrafish to confirm 5 novel human F10 variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of F10 variants. These findings as well as the prolonged survival of f10−/− mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders.

  • Submitted February 7, 2017.
  • Accepted May 23, 2017.
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