Investigation of the molecular mechanism of platelet formation in CYCS-associated thrombocytopenia

Mutations in genes which play a role in the processes of megakaryopoiesis and platelet production cause thrombocytopenia. Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. The role of cytochrome c in megakaryopoiesis and platelet formation is unknown, and it is unclear how mutations in cytochrome c cause thrombocytopenia.

The goal of this project is to understand how CYCS mutations cause thrombocytopenia. We are using the human leukemic K562 cell line as a model system to study the impact of CYCS mutations on megakaryocyte differentiation and mitochondrial function. One of the CYCS pathogenic variants (c.145T>C; p.Tyr49His) was introduced into K562 cells using the CRISPR-Cas9 base-editing method. K562 cells have three copies of CYCS-containing chromosome 7 and we isolated CYCS^WT/WT/WT, CYCS^WT/WT/Y49H, CYCS^WT/Y49H/Y49H, CYCS^{Y49H/Y49H/Y49H} clonal lines, confirmed by sequencing and mass spectrometry. K562 cells undergo megakaryocyte differentiation in response to PMA. We monitored the expression of megakaryocyte markers CD61 and CD41a by flow cytometry in response to PMA in all clonal lines. The K562-CYCS^{Y49H/Y49H/Y49H} line showed faster maturation compared to all other clonal lines and the parental K562 cells. We assessed mitochondrial function using the Seahorse XFe96 Analyzer. Our results show that the mutation does not impact mitochondrial function. Finally, we have introduced the Y49H mutation into iPSCs. Investigations of the impact of the Y49H variant on apoptosis showed no significant differences in clonal lines. Our results suggest that the pathogenic c.145T>C; p.Tyr49His CYCS variant enhances megakaryocyte maturation.

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