Transcriptomic gene profiles in an ex vivo model of erythropoiesis to unravel molecular pathomechanisms in sickle cell disease
Sickle cell disease (SCD) is an autosomal recessive, single gene disorder caused by a point mutation in the beta globin (HBB) gene. The single amino acid substitution of the haemoglobin molecule leads to aberrant ß-chain formation, polymerizing sickled haemoglobin (HbS) molecules together during episodes of deoxygenation and therefore causing intracellular sickling. Hydroxyurea (HU) is still the most indicated drug inducing fetal hemoglobin (HbF) production for SCD treatment, especially for the SCD patients with a severe clinical profile. In this study we could identify transcriptional profiles in erythroid cells of SCD patients, which have been treated with HU in comparison to untreated SCD patients and healthy controls. Our present transcriptomic profile analysis contained a new subset of genes identified for the first time as associated with SCD. In SCD non-treated-derived erythroid cells compared to controls, 398 DEGs were identified, of which 100 genes were up-regulated and 298 genes were down-regulated in the SCD patient's cells. 65 DEGs were identified in SCD HU-treated patient-derived erythroid cells compared to controls, of which 37 genes were upregulated and 28 genes were downregulated. 212 DEGs were identified in SCD HU-treated patient-derived compared to SCD non-treated derived erythroid cells, of which 113 genes were upregulated and 99 genes were downregulated. 141 DEGs were identified in SCD derived erythroid cells compared to controls, of which 58 genes were up-regulated and 83 genes were down-regulated. We found biological processes such as oxidative phosphorylation pathway, Fanconi anaemia pathway, adaptive immune response or inflammatory response to be part of our significantly enriched Gene Ontology (GO) terms. Further genes encoding significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for proteasome, autophagy, immune response, platelet activation, as well as for natural killer cell (NK) cytotoxicity, oxidative phosphorylation, Fanconi anaemia pathway, ribosome biogenesis were upregulated in both SCD non treated and SCD HU-treated patient-derived erythroid cells in comparison to healthy controls. Our findings collectively suggest different as well as common molecular signatures between SCD non-treated, SCD HU-treated compared to HC. We could validate 12 of our top significant genes by qRT-PCR (calneuron 1 (CALN1), nucleoporin 85 (NUP85), tumor protein 63 (TP63), ras and rab interactor 2 (RIN2), solute carrier family 44 member 5 (SLC44A5), phenylethanolamine N-methyltransferase (PNMT), stabilin 1/ clever-1 (STAB1), SLX1 Homolog A, Structure-Specific Endonuclease Subunit (SLX1A), ATPase phospholipid transporting 9A (ATP9A), MSH5-SAPCD1, G protein subunit gamma 4 (GNG4) and glutathione S-transferase theta 2B (GSTT2B)) and compare their mRNA levels in bone marrow and spleen tissue from the Berkely SCD mouse model. In short, our study could identify new genes and pathways that may be involved in the SCD disease pathogenesis and that may be influenced by the HU treatment. The findings encourage future characterization of their roles in SCD to establish these findings as basis for new therapeutic targets.