Weyand, A. C. | Search bioRxiv

bioRxiv2019

Analysis of factor V in zebrafish demonstrates minimal levels needed for hemostasis and risk stratifies human variants

In humans, coagulation factor V (F5) deficiency is a rare, clinically heterogeneous bleeding disorder, suggesting that genetic modifiers may contribute to disease expressivity. Complete loss of mouse F5 results in early lethality. 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 F5 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 f5-/- 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 F5 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 F5 function, with minimal activity being required to restore hemostasis. This in vivo evaluation may be beneficial for patients whose factor activity levels lack correlation with bleeding symptomatology. 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.\n\nKey PointsO_LIF5 mutant fish embryos tolerate symptoms lethal in mammals but succumb to bleeding in adulthood\nC_LIO_LIAnalysis of human variants demonstrate that all have some residual function and that minimal F5 activity is required to restore hemostasis\nC_LI

genetics↗

bioRxiv2017

The Function of the COPII Gene Paralogs SEC23A and SEC23B Are Interchangeable In Vivo

SEC23 is a core component of the coat protein-complex II (COPII)-coated vesicle, which mediates transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi1-3. Mammals express 2 paralogs for SEC23 (SEC23A and SEC23B). Though the SEC23 gene duplication dates back >500 million years, both SEC23s are ~85% identical at the amino acid sequence level. In humans, deficiency for SEC23A or SEC23B results in cranio-lenticulo-sutural dysplasia4 or congenital dyserythropoietic anemia type II (CDAII), respectively5. The disparate human syndromes and reports of secretory cargos with apparent paralog-specific dependence6,7, suggest unique functions for the two SEC23 paralogs. Here we show indistinguishable intracellular interactomes for human SEC23A and SEC23B, complementation of yeast SEC23 by both human and murine SEC23A/B paralogs, and the rescue of lethality resulting from Sec23b disruption in zebrafish by a Sec23a-expressing transgene. Finally, we demonstrate that the Sec23a coding sequence inserted into the endogenous murine Sec23b locus fully rescues the mortality and severe pancreatic phenotype previously reported with SEC23B-deficiency in the mouse8-10. Taken together, these data indicate that the disparate phenotypes of SEC23A and SEC23B deficiency likely result from evolutionary shifts in gene expression program rather than differences in protein function, a paradigm likely applicable to other sets of paralogous genes. These findings also suggest the potential for increased expression of SEC23A as a novel therapeutic approach to the treatment of CDAII, with potential relevance to other disorders due to mutations in paralogous genes.

evolutionary biology↗