OC 45.2 - Mutations in the 5’-untranslated region of ANKRD26 result in thrombocytopenia and hematological malignancy in zebrafish

Background: Mutations in 5’-untranslated region (5’-UTR) of ANKRD26 (ankyrin repeat domain-containing protein-26) are associated with inherited thrombocytopenia-2 (THC2), which predisposes to myeloid malignancies. However, no animal model thus far has been established to demonstrate the direct link between ANKRD26 mutations and THC2; and the mechanism underlying ANKRD26 mutations associated THC2 is not fully understood.

Aims: To create a zebrafish model to demonstrate the direct causative effect of mutations in 5'-UTR of ANKRD26 in thrombocytopenia and possible modification of thrombotic thrombocytopenic purpura.

Methods: Using CRISPR/cas9 to create mutations in 5'-UTR of anrkd26 in zebrafish.

Results: Our results demonstrate that a deletion of 2-basepairs (bp) (Δ2) or 3-bp plus 26-bp insertion (Δ3+26) in the 5’-UTR of ankrd26 resulted in an increased expression of ankrd26 mRNA and protein (Figure 1). Zebrafish carrying a homozygous mutation in 5’-UTR of ankrd26 exhibited spontaneous thrombocytopenia and enhanced thrombus formation under flow (Figure 2). Ankrd26Δ2/Δ2 zebrafish also showed a reduced survival rate, likely resulting from hematological malignancy (not shown). Proteomic study on young thrombocytes from ankrd26Δ2/Δ2 zebrafish revealed a dramatic upregulation of ninjurin1, a proinflammatory and cell rupture-related protein (not shown). Moreover, zebrafish with double mutations (e.g., ankrd26+/Δ2/adamts13-/- or ankrd26 Δ2/Δ2/adamts13-/-) exhibited more severe thrombocytopenia than those with either genotype alone (not shown). Finally, we found significantly elevated ANKRD26 protein in the peripheral blood buffy coat (e.g., leukocytes and platelets) and plasma from patients with immune thrombotic thrombocytopenic purpura (TTP) compared with the non-TTP controls (not shown).

Conclusion(s): We conclude that we have generated the first zebrafish model of THC2; and the overexpression of ankrd26 protein resulting from genetic or acquired etiology may modify TTP phenotypes by causing more severe thrombocytopenia. Our findings may help develop a targeted therapy for THC2 and perhaps for other inflammatory and thrombotic disorder such as immune TTP.