Abstract
Hereditary Persistence of Fetal Hemoglobin (HPFH) and δβ-thalassemia are genetic disorders characterized by elevated levels of fetal hemoglobin (HbF) in adulthood. Deletions of variable sizes, and at different positions, that involve the β-globin gene complex on chromosome 11 are associated with these disorders. The distinction between these conditions is made based on clinical and hematological data. Deletional HPFH includes a wide range of conditions, but typically it is characterized in heterozygotes by levels of HbF of 15% to 30% with normal red blood cell indices, while heterozygotes for δβ-thalassemia tend to have elevated levels of HbF that are lower (5% to 20%) and accompanied by mild anemia with hypochromic, microcytic red blood cell indices. MicroRNAs (miRNAs) have been described to have a possible role in globin switching and can modulate transcriptional erythroid-specific regulators. To the best of our knowledge miRNAs have not been analyzed in HPFH and δβ-thalassemia. The aim of this study was to investigate the miRNAs expression profile and possible post-transcriptional role of these molecules in relation to the lack of normal suppression of γ genes in these genetic disorders.
CD34(+)-derived erythroid cells from two HPFH-2 individuals and two δβ-thalassaemia Sicilian type patients (DB) and healthy controls (CTRL) were cultured for 13 days to examine the expression profile of miRNAs. The miRNAs were hybridized using an Agilent miRNA microarray platform and the profiles were obtained through bioinformatics data analysis using GeneSpring software. qPCR analysis was used to validate the miRNA expression (TaqMan® miRNAs assays) and to quantify gene expression of 19 transcription factors. Different databases, such as miRBase, TargetScan, microRNA.org and BioGPS were used to determine the predicted targets of miRNAs data found.
Six miRNAs were up-regulated in HPFH and in DB compared to CTRL: miR-146b-5p, miR-181a, miR-342-3p, miR-362-3p, miR-362-5p and miR-365. Five miRNAs were up-regulated in HPFH, compared to DB: miR-223, miR-630, miR-638, miR-1246 and miR-1290. Nine miRNAs were up-regulated in DB compared to HPFH: miR-10a, miR-21*, miR-33b*, miR-96, miR-128, miR-194, miR-210, miR-424 and miR-1275. The ALK4 and the GATA2 mRNAs were significantly up-regulated in HPFH, compared to DB. The BCL11A and the SH3BGRL2 mRNAs were significantly down-regulated in HPFH compared to DB. In silico analysis and the literature show that several miRNAs have targets related to HbF production or erythropoiesis. These include miR-10a, miR-33b*, miR-96, miR-128, miR-210, miR-223, miR-342-3p, miR-362-3p, miR-424 and miR-630.
The comparison of miRNA and transcription factor profiles suggests differences in the expressions of several miRNAs that may influence γ-globin gene expression. These data may contribute to understanding the phenotypic differences found between deletional HPFH and δβ-thalassemia. Financial support by FAPESP and CNPq/INCTS.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
