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Publication
Pathophysiological characterization of megakaryopoiesis and erythropoiesis: whole genome sequencing and functional studies
Book - Dissertation
My PhD project aimed to gain more insight into megakaryopoiesis and erythropoiesis by studying patients with inherited thrombocytopenia and inherited anemia, respectively.Firstly, in 2016, my host lab identified a germline heterozygous missense variant E527K in SRC in a large pedigree with dominant thrombocytopenia using whole genome sequencing. Patients that carry the E527K SRC variant present with thrombocytopenia accompanied with bleeding symptoms, a paucity of alpha granules, and a variety of other variable symptoms including myelofibrosis, osteoporosis and facial dysmorphism. The E527K SRC variant was found to result in increased kinase activity and defects in platelet formation, but downstream pathways because of increased kinase activity in megakaryocytes (MK) still remained unknown. At the beginning of my PhD project, I was able to describe the clinical and laboratory findings in a second thrombocytopenia patient with the same gain-of-function (GOF) variant E527K in SRC. Although many functional assays were already performed to prove causality of this GOF variant in SRC for the clinical phenotype of the patients of this large pedigree, the confirmation in this patient from an independent family significantly strengthened these findings and allowed improved delineation of this novel platelet disorder. Next, we wanted to obtain insights in the MK defect introduced by the SRC GOF variant using transcriptomic and proteomic approaches. For these experiments, hematopoietic stem cells (HSC) were isolated from healthy controls before transduction with WT-SRC and E527K-SRC lentiviral vectors and differentiation to MK. The RNAsequencing data showed that many of the most significant downregulated genes in E527K-SRC MK were involved in platelet activation, signaling and alpha granules, which nicely accorded with the platelet function defect described in these patients. The combined pathway analysis using both RNAsequencing and proteomics datasets also identified IFNα/β signaling as most significantly enriched downregulated pathway in E527K-SRC MK. We performed several validation studies in cell models and found that interferon-stimulated genes (ISG) play a role during megakaryopoiesis by acting downstream of SRC signaling. The exact mechanisms of how SRC can change ISG to influence megakaryopoiesis still remain unknown and require further studies.Secondly, whole exome sequencing was performed for a patient with severe anemia. She and her affected sister were born from consanguineous parents and rare homozygous WES variants were selected as candidates. The most plausible candidate was a homozygous missense variant in DAP3 that codes for the mitochondrial Death-Associated Protein 3. Therefore, the second part of my PhD project was to study the role of DAP3 in erythropoiesis, by performing functional studies in HSC-derived erythroblasts, RNAsequencing, and dap3 depletion experiments in Zebrafish. HSC from the patient showed reduced erythroblast colony formation with mainly smaller colony sizes. Flow cytometry analysis revealed a significant erythroblast maturation delay for patient-derived cultures with reduced levels of functional mitochondria. The analysis of RNAsequencing data for A-to-I RNA editing showed a significantly reduced editing frequency for 127 sites near 40 genes in patient cells. Reactome pathway analysis of these genes showed an enrichment of cell cycle and RNA metabolism pathways. Finally, dap3 depleted zebrafish embryos presented with reduced erythrocyte numbers, reduced expression of gata1 and embryonic globin genes, and increased numbers of apoptotic cells. In summary, our data revealed an important role for DAP3 during erythropoiesis via regulation of mitochondrial health and evidence for A-to-I RNA editing in this process.
Publication year:2021
Accessibility:Embargoed