PB0319 - Overexpression of ANKRD26 results in thrombocytopenia that may exacerbate clinical phenotypes of thrombotic thrombocytopenic purpura

Background: Mutations in the 5’-untranslated region (5’-UTR) of ANKRD26 (Ankyrin repeat domain-containing protein-26) gene cause ANKRD26 overexpression during megakaryocyte differentiation, leading to impaired proplatelet formation and thrombocytopenia. Patients with such mutations are at risk of developing hematological malignancies. Little is known about the underlying mechanism of ANKRD26-related thrombocytopenia. Moreover, an association was found between ANKRD family gene mutations and immune thrombotic thrombocytopenic purpura (iTTP).

Aims: To explore the function of ANKRD26 and its pathological role in thrombocytopenia and iTTP.

Methods: We generated the first zebrafish model by introducing mutations in the 5’-UTR of ankrd26 using CRISPR/Cas9. Flow cytometry was used to assess the thrombocyte counts and maturation status in the zebrafish. Western blot and ELISA were employed to determine the levels of ANKRD26 in megakaryoblastic cells (MEG01) and iTTP patients.

Results: Zebrafish carrying a homozygous 2-nt deletion in the 5’-UTR of ankrd26 resulted in an overexpression of Ankrd26 mRNA and protein, and significantly reduced total and mature thrombocyte counts (Fig. 1A-C), recapitulating the clinical presentation of hereditary thrombocytopenia in patients. Additionally, ankrd26-overexpressing zebrafish mutants in the background of Adamts13 deficiency present a further reduction of thrombocyte counts than zebrafish with Adamts13 deficiency or Ankrd26 overexpression alone (Fig. 1D-F). Moreover, in iTTP patients, platelets and plasma ANKRD26 levels were significantly increased compared with those in non-iTTP controls (Fig. 1G-H). Finally, overexpression of PRMT1, an arginine methyltransferase for RUNX1 which is a repressor for ANKRD26 expression, increased ANKRD26 levels in the MEG01 cells.

Conclusion(s): These results indicate that mutations in the 5’-UTR of ANKRD26, resulting in an overexpression of ANKRD26 in megakaryocytes and platelets, may lead to hereditary thrombocytopenia that could exacerbate the clinical phenotype of iTTP resulting from severe ADAMTS13 deficiency. This is the first disease-relevant animal model for ANKRD26-related hereditary thrombocytopenia, which may be useful for further understanding the disease mechanism and testing novel therapeutics.