TRANSCRIPTOME ANALYSIS OF RETICULOCYTES FROM SICKLE CELL ANEMIA PATIENTS WITH HIGH AND LOW FETAL HEMOGLOBIN LEVELS

Abstract

Objectives: Fetal hemoglobin (HbF) levels have long been associated with a milder clinical course in sickle cell anemia (SCA). Given the importance of HbF in SCA pathophysiology, studies have focused on describing how HbF ameliorates the clinical phenotype and identifying previously uncharacterized modulators of gamma globin expression. Here, we performed a transcriptomic analysis of reticulocytes from SCA patients with high and low HbF levels to unravel genetic programs associated with high HbF levels. Methods: Eight individuals with SCA, older than 18 years and regularly followed at a single reference center in northeast Brazil, were recruited. All patients were without clinical presentation of acute symptoms and not undergoing hydroxyurea treatment. RNA was extracted from the patients’ reticulocytes, and the RNA-seq libraries were sequenced in paired-end mode on the NextSeq 500. To compare the degree of contamination with leukocytes, we employed the CIBERSORTx tool, which revealed two samples with high leukocyte contamination that were excluded from further analysis. Three individuals with high baseline HbF (median: 20.9%) and three with low baseline HbF (median: 5.2%) were used gene expression analysis. Functional enrichment analysis was performed with Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Metabolic Flux Balance Analysis (METAFlux). Results and discussion: We identified 293 differentially expressed genes between the conditions (247 upregulated and 46 downregulated in the HbF high group). GSEA associated patients with high HbF levels with the terms “regulation of reactive oxygen species (ROS) process”, “lipid biosynthetic process”, and “cellular oxidant detoxification”, while HbF low samples were associated with “transcription by RNA polymerase”, “methylation”, and “regulation of translational initiation”. These results suggest that high HbF levels drive a molecular program in reticulocytes associated with ROS generation and cellular detoxification pathways. We further identified the leading-edge gene subsets from the significant GSEA analysis. A total of 176 genes were consistently upregulated in several GSEA processes associated with high HbF levels, including SOD2, PRDX2, SYK, and LYN. GO analysis of the core members of high-scoring gene sets showed several pathways involved in the regulation of response to stimulus, response to oxidative stress, and regulation of key metabolic processes that were overexpressed in the high HbF group. Moreover, the processes that were downregulated in the high HbF group were related to RNA metabolic processes, regulation of gene expression, and methylation, suggesting a mechanism of genetic silencing in reticulocytes with low HbF levels in SCA. To further validate these findings, METAFlux analysis, revealed a clear distinction between the metabolic programs, with up-regulation of terms associated with the downregulation of ROS generation and fatty acid detoxification in high HbF group. Conclusions: SCA patients with high HbF levels show improved oxidative stress management and metabolic activity, while those with low HbF have increased gene silencing. These findings offer insights into potential therapeutic targets to ameliorate disease severity through HbF modulation.