Dissertação de Mestrado Investigação Biomédica 2022 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Primary immunodeficiencies (PID) are a group of chronic diseases caused by defects in the development and/or function of immune system components that can lead to recurrent infections, autoimmunity, aberrant inflammation, and increased risk of hematologic malignancies. Such disorders are mainly caused by de novo or inherited genetic mutations that affect the development of hematopoietic cells. However, many of the mutations responsible for primary hematopoietic disorders remain to be identified. A novel candidate gene for these disorders is DIPK2B (Divergent Protein Kinase Domain 2B; also known as DIA1R, Deleted in Autism 1 Related, Cxorf36), a gene of unknown function that was recently shown to be mutated in patients with PID. We hypothesize that mutations in dipk2b may cause an aberrant expansion of hematopoietic progenitors and an imbalance in blood cell populations that can lead to hematologic defects. To address this hypothesis, we investigated the hematopoietic defects of dipk2b morphant and mutant zebrafish. The effect of dipk2b knockdown by morpholino injection was assessed by imaging and flow cytometry analysis of transgenic zebrafish embryos expressing lineage-specific reporters in different hematopoietic cell populations, such as erythromyeloid progenitors, hematopoietic stem/progenitor cells, endothelial cells, neutrophils and macrophages. Moreover, we analyzed the hematopoietic cell populations in the kidney marrow, spleen, and peripheral blood of adult dipk2b mutant fish by flow cytometry. Our observations in morphant and mutant transgenic embryos suggest that dipk2b regulates the development of erythromyeloid progenitor cells as well as the formation of blood vessels. In adult zebrafish, the hematopoietic cell populations in the kidney (the main hematopoietic organ in adult fish) and spleen did not show a clear phenotype in 6 months and one-year old dipk2b mutant fish. However, dipk2b deficiency appears to increase the percentage of lymphocytes in peripheral blood, which may be associated with phenotypes frequently observed in PID patients, such as increased susceptibility to infections and/or to lymphodysplasia and leukemia. Altogether, our results indicate that dipk2b has a significant role during zebrafish hematopoietic development but not during adult hematopoiesis. This study provides the basis for future investigation into the role of DIPK2B in the pathogenesis of hematologic and immune disorders such as PID.