OC 76.2 - A zebrafish model of dysfibrinogenemia caused by hotspot mutations in the human fibrinogen gamma chain

Background: Mutations affecting fibrinogen quality lead to unpredictable bleeding and thrombotic events in patients with dysfibrinogenemia. With dominant inheritance, dysfibrinogenemia is caused by heterozygous missense mutations in fibrinogen genes. Mutation of arginine 301 to cysteine or histidine in the fibrinogen gamma chain (γR301C, γR301H), affecting D:D interactions between adjacent fibrin monomers and consequently fibrin polymerization, are frequently detected dysfibrinogenemia-associated mutations.

Aims: To generate a zebrafish model of dysfibrinogenemia caused by γR301 mutations. Once established, the model should allow us to study the effects of additional genetic variation on the dysfibrinogenemia phenotype.

Methods: A morpholino antisense oligonucleotide was used to reduce zebrafish fibrinogen γ chain expression. Fibrinogen γR295C and γR295H cDNA, orthologous to human γR301 mutations, were expressed using transgenesis. In a second approach, a large deletion in the zebrafish fgg gene was generated using CRISPR-Cas9 genome editing. Laser-induced injury was used to assess coagulation and venous thrombosis in zebrafish larvae.

Results: Transient morpholino (MO)-induced knock-down of fibrinogen γ mRNA prevented laser-induced venous thrombosis measured as time to occlusion (TTO) in zebrafish larvae. This was rescued by expression of γ cDNA but not with γR295C or γR295H cDNA, where TTO was prolonged compared to control larvae (mean TTO in controls 29s, MO + γ cDNA 29s, MO + γR295C 56s and MO + γR295H 46s, n=7 to 16). We also produced zebrafish with an 8.1kb deletion in the fgg gene. With laser injury, homozygous mutant larvae showed no blood coagulation or thrombosis, and will now serve as a stable knock-out background to study the effects of γR295C and γR295H expression.

Conclusion(s): Using fibrinogen γ chain mRNA knock-downs we demonstrate dysfunctional clotting in larval zebrafish expressing γR295C and γR295H after laser injury. Our stable γ chain mutant with γR295C or γR295H expression will allow us to study the phenotypic effects of further genetic variation in dysfibrinogenemia.