Abstract
BackgroundWe aimed to clarify the emerging epigenetic landscape in a group of genes classified as “modifier genes” of the β-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/β-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented.ResultsWe examined the CpG islands’ DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients’ monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU.ConclusionsThis is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5′ CpG sequences of all studied human HBB cluster “modifier genes.” Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the β-type hemoglobinopathy patients.
Highlights
Thalassemias and sickle cell anemia (SCA) are still of the most common genetic disorders among human population even after many decades of worldwide prenatal control attempts
Detection and recording of γ-globin expression in the presence of hydroxyurea Total RNA collected from colony-forming unit assay (CFU) cultures was subjected to a quantitative PCR assay to detect γ-globin mRNA expression profile
Variation in γ-globin levels were observed in biological replicate experiments with CFU cultures from the same donor, which prompted us to refine the HU concentrations used in culture conditions (50–100 μM)
Summary
Thalassemias and sickle cell anemia (SCA) are still of the most common genetic disorders among human population even after many decades of worldwide prenatal control attempts. Clinical practice has shown that, despite identical disease-causing mutations, the pathological phenotype of disease varies enormously between individual patients This is partly related to genetic variants of the genetic loci coding for the hemoglobin subunits alpha and beta (HBA and HBB) including their remote regulatory regions (LCRs—locus control regions) [1, 2] and the co-interaction with other parameters such as epigenetic regulatory mechanisms [3]. These genetic disorders have an early onset, within a few months after birth, which is accompanied by high financial burdens to healthcare systems due to the expensive therapeutic protocols applied to patients. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented
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