β-hemoglobinopathies (β-thalassemias and sickle cell disease) are the most common genetic disorders in the human population. Sustained expression of γ-globin, the β-like globin expressed during the fetal stages of development, is a major mitigating factor for disease severity. Worldwide, over 300,000 affected children are born every year. Symptoms arise soon after birth when adult hemoglobin replaces fetal hemoglobin (HbF). In patients with hereditary persistence of HbF, symptoms are milder, and life expectancy is prolonged. To date, hydroxyurea is the only drug available with proven efficacy as an HbF-inducer, but only a subset of β-hemoglobinopathy patients responds to this treatment. The global burden of β-hemoglobinopathies calls for the development of novel, accessible, and affordable treatment options.
The promoters of the HBG1 and HBG2 genes, encoding γ-globin, are normally repressed in adult erythroid cells. This repression mechanism involves a network of transcription factors and epigenetic modifications. The latter are targeted by pharmacological compounds aimed at alleviating γ-globin suppression such as RN-1 which inhibits the histone demethylase LSD1, and butyrates which inhibit histone deacetylases. In the 1980s a small number of end-stage β-thalassemia patients were treated with the DNA methylation inhibitor 5-azacytidine. This resulted in a remarkable increase in γ-globin expression. Due to concerns about toxicity of 5-azacytidine this experimental treatment was discontinued. Since 5-azacytidine is a general demethylating agent, it remained unclear whether the increase in γ-globin expression was due to demethylation of the HBG1/2 promoters, or whether indirect effects were involved. Despite decades of research, this remained a controversy.
With the research conducted under LSBR 1627, we aimed to resolve this controversy. In agreement with the literature, we found that treatment of adult erythroid cells with Decitabine, a 5-azacytidine analog, resulted in demethylation of the HBG1/2 promoters and induction of HbF expression. Upon withdrawal of Decitabine, methylation of the HBG1/2 promoters was quickly restored to pre-treatment levels and HbF expression was silenced. We then studied a patient in which one of the two copies of the BCL11A had been inactivated. BCL11A is a repressor protein that binds to the HBG1/2 promoters in adult erythroid cells. We observed that in the patient expression of BCL11A was reduced by ~50%; this was accompanied by sustained expression of HbF at 18% of total Hb. Despite this high level of HbF expression, the HBG1/2 promoters were fully methylated. Thus, our data show that promoter methylation has no direct role in repression of the HBG1/2 genes. Consequently, HbF induction upon Decitabine treatment is not a direct result of demethylation of the HBG1/2 promoters. Finally, we created an HbF reporter cell line representing adult erythroid cells. With this cell line, HbF expression can be precisely assessed using a simple assay that is suitable for automation. This facilitates development of novel pharmacological compounds and genetic therapies for β-hemoglobinopathy patients.