Session: 101. Red Cells and Erythropoiesis, Excluding Iron: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Methods: We obtained bone marrow mononuclear cells from five patients diagnosed with PNH and five healthy controls. CD59+ and CD59- cells from PNH patients were isolated using flow cytometry. Subsequently, deep single-cell RNA sequencing was conducted on all hematopoietic cell lines. A thorough research was conducted to explore the increased erythroid differentiation in the bone marrow PNH clone.
Results: In this study, we used single-cell sequencing to identify seven red blood cell clusters based on maturation marker expression levels such as GYPA. The red blood cell clusters were characterized as MEP, BEU-E, CFU-E, ProE, BasoE, PolyE, and OrthoE. For further analysis, we classified CD59+ cells in PNH patients as positive (P), CD59- cells as negative (N), and cells from healthy controls as Control (C). Initially, we analyzed the proportions of the three cell groups. Our results showed that there were no significant differences between the groups at the erythroid progenitor cell stage. In the PolyE group, the N content was higher compared to C but here was no significant difference when compared to the P group. In the OrthoE group, both N and P groups decreased as compared to the C group, with no significant difference between the N and P groups. Subsequently, we conducted a detailed analysis of the differential genes in the PolyE and OrthoE groups. In poly E, compared to control group, the upregulated pathways in N group are mainly enriched in processes such as oxygen binding, drug metabolism, and cofactor metabolism. Conversely, the downregulated pathways in group N are mainly enriched in pathways related to cell cycle and immune activation. In comparison with group C, the downregulated pathways in group P are mainly enriched in cell cycle, IL-17 signaling pathway, neutrophil extracellular trap formation, drug metabolism, and DNA fragmentation induced by apoptosis.
In orthoE, a comparison between the C group and the N group reveals distinct enrichment patterns. The N group exhibits upregulated pathways related to DNA damage, DNA methylation, PRC2 methylated histones, DNA processes, and cellular senescence. Conversely, downregulated pathways in the N group are associated with eukaryotic translation elongation, rRNA processing, and cytoplasmic solute functions. In a similar vein, contrasting the N group with the P group uncovers differing pathway enrichments. The N group displays upregulated pathways linked to ribosomes, IL-17 signaling, and NF-κB activation in B cells, while downregulated pathways are associated with GPI-anchored proteins, cytokines, and immune regulatory interactions. Furthermore, comparing the P group with the C group reveals distinct pathway enrichments. The P group shows upregulated pathways related to mitochondrial autophagy, megakaryocyte development, platelet formation, and red blood cell functions. Conversely, downregulated pathways in the P group are associated with oxidative phosphorylation, ribosomal functions, and amino acid metabolism.
Subsequently, we discovered genes with differential expression at various stages of erythroid development in the C, N, and P groups. Following enrichment cross-analysis, we hypothesize that NT5C3A and TANK may play influential roles in the process of erythroid cell differentiation in PNH patients. Further research is needed to understand the particular mechanisms involved.
Conclusion: We observed abnormalities in the erythroid differentiation of PNH patients. Furthermore, NT5C3A and TANK are likely to play important roles during the erythroid cell differentiation process in PNH patients.
Keywords: PNH, erythroid differentiation, single-cell RNA sequencing
Disclosures: No relevant conflicts of interest to declare.
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