Abstract
BackgroundReactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for β-thalassemia and sickle cell disease. Although many HbF regulators have been identified, the methylation patterns in β-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined.ResultsHere, we evaluated DNA methylation patterns of the β-globin cluster from peripheral bloods of 105 β0/β0 thalassemia patients and 44 normal controls. We also recruited 15 bone marrows and 4 cord blood samples for further evaluation. We identified that the CpG sites in the locus control region (LCR) DNase I hypersensitive site 4 and 3 (HS4-3) regions, and γ- and β-globin promoters displayed hypomethylation in β0/β0-thalassemia patients, especially for the patients with high HbF level, as compared with normal controls. Furthermore, hypomethylations in most of CpG sites of the HS4-3 core regions were also observed in bone marrows (BM) of β0/β0-patients compared with normal controls; and methylation level of γ-globin promoter -50 and + 17 CpG sites showed lower methylation level in patients with high HbF level compared with those with low HbF level and a negative correlation with HbF level among β0-thalassemia patients. Finally, γ-globin promoter + 17 and + 50 CpG sites also displayed significant hypomethylation in cord blood (CB) tissues compared with BM tissues from normal controls.ConclusionsOur findings revealed methylation patterns in β-globin cluster associated with β0 thalassemia disease and γ-globin expression, contributed to understand the epigenetic modification in β0 thalassemia patients and provided candidate targets for the therapies of β-hemoglobinopathies.
Highlights
Reactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for β-thalassemia and sickle cell disease
Since DNA methylation can be associated with aging progression and significant differences in age between β-thalassemia patients and normal controls, we included age as a covariate in analysis of covariance (ANCOVA) of methylation difference between β-thalassemia patients and normal controls and observed that hypomethylation in β-thalassemia patients remained significance in most of CpG sites around the locus control region (LCR) HS4 region, and γ- and β-globin promoters (Fig. 1b, shown with stars above indicated sites)
Given that DNA in peripheral blood lymphocyte (PB) mainly derived from the lymphocyte cells, which will have some limitations for DNA methylation research with high tissue heterogeneity, we examined DNA methylation in bone marrows (BM) GYPA (Glycophorin A) positive cells and observed that significant hypomethylation at most of CpG sites in core regions of HS4-HS3 and the 5′ flanking sequences, especially in the HS4 region, in β0/β0-patients compared with the normal controls (Fig. 2a)
Summary
Reactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for β-thalassemia and sickle cell disease. Many HbF regulators have been identified, the methylation patterns in β-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined. Mei Hsu et al have analyzed the methylation status of regions in the murine β-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types [17] They found a 20-kb domain, extended from the region just past the LCR to before β-major and encompassed the embryonic genes εy, βh, and βh0, is hypomethylated only in primitive erythroid cells. Epigenetic-based underlying mechanism driving the fetal-to-adult hemoglobin switch have been studied and reported in mouse and human, the systematic scanning of methylation patterns in β-globin cluster remains to be determined, especially in β-thalassemia patients. We uncovered the methylation patterns of β-globin cluster, and explored its roles in regulating HbF gene expression in PB, BM, or CB samples from human β-thalassemia patients or normal controls
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