Functional Analysis of an^Aγ-Globin Gene Promoter Variant (HBG1: g.-225_-222delAGCA) Underlines Its Role in Increasing Fetal Hemoglobin Levels Under Erythropoietic Stress

Abstract

Hereditary persistence of fetal hemoglobin (HPFH) is a condition characterized by persistent γ-globin gene expression and synthesis of high levels of fetal hemoglobin (Hb F; α2γ2) during adult life. It is usually caused by promoter variants or large deletions affecting the human fetal globin (HBG1 and HBG2) genes. Some of these HPFH-causing variants, such as HBG2: g.-158 C > T, exert their effect only under conditions of erythropoietic stress, typical for β-thalassemia (β-thal) patients. Namely, the presence of HBG2: g.-158 C > T favors a higher Hb F response, while it has little effect in healthy individuals. We analyzed a previously reported deletion residing in the promoter region of the HBG1 gene (HBG1: g.-225_-222delAGCA), both in normal conditions and under conditions of erythropoietic stress. Our results indicate that this deletion is responsible for decreased HBG1 gene expression. Specifically, this deletion was shown to result in drastically reduced reporter gene expression in K562 cells, compared to the wild-type sequence but only under conditions of erythropoietic stress, mimicked by introduction of erythropoietin (EPO) into the cell culture. Also, electrophoretic mobility shift analysis showed that the HBG1: g.-225_-222delAGCA deletion creates additional transcriptional factors' binding sites, which, we propose, bind a transcriptional repressor, thus decreasing the HBG1 gene promoter activity. These results are consistent with in silico analysis, which indicated that this deletion creates a binding site for GATA1, known to be a repressor of the γ-globin gene expression. These data confirm the regulatory role of the HBG1: g.-225_-222 region that exerts its effect under conditions of erythropoietic stress characteristic for β-thal patients.

Differently expressed long noncoding RNAs and mRNAs in TK6 cells exposed to low dose hydroquinone

“…The HBG1 and HBG2 are normally expressed in the fetal liver, spleen and bone marrow. Compared with wild type promoter, the activity of HBG1 gene promoter variant decreased significantly in K562 cells under conditions of erythropoietic stress [ 34 ]. This finding is helpful to analyze the pathogenesis of β-Thalassemia and provide a reference for its treatment.…”

Molecular basis of a high Hb A2/Hb Fβ-thalassemia trait: a retrospective analysis, genotype-phenotype interaction, diagnostic implication, and identification of a novel interaction withα-globin gene triplication

“…These included the -AGCA deletion at −225 to −222 of A γ -globin gene promoter, rs5006884 (C>T) on OR51B6 gene, −158 G γ - Xmn I, BCL11A binding motifs between A γ - and δ -globin genes as well as the whole γ -globin gene sequencing. It has been shown that the four bp deletion (-AGCA) at −225 to −222 of the A γ -globin gene was associated with reduced A γ -globin chain and elevation of G γ chain with slightly increased Hb F level in β -thalassemia subjects ( Manca et al, 1991 ; Ugrin et al, 2016 ). The rs5006884 (C>T) on OR51B6 gene located upstream of β -globin gene cluster might intervene in the tetramer formation of α -globin chain with β - and δ -globin chains and regulated Hb A 2 level in β -thalassemia carriers ( Cyrus et al, 2019 ).…”