A Genetic Variant Ameliorates β-Thalassemia Severity by Epigenetic-Mediated Elevation of Human Fetal Hemoglobin Expression

Abstract

A delayed fetal-to-adult hemoglobin (Hb) switch ameliorates the severity of b-thalassemia and sickle cell disease. The molecular mechanism underlying the epigenetic dysregulation of the switch is unclear. To explore the potential cis-variants responsible for the Hb switching, we systematically analyzed an 80-kb region spanning the b-globin cluster using capture-based next-generation sequencing of 1142 Chinese b-thalassemia persons and identified 31 fetal hemoglobin (HbF)-associated haplotypes of the selected 28 tag regulatory single-nucleotide polymorphisms (rSNPs) in seven linkage disequilibrium (LD) blocks. A Ly1 antibody reactive (LYAR)-binding motif disruptive rSNP rs368698783 (G/A) from LD block 5 in the proximal promoter of hemoglobin subunit gamma 1 (HBG1) was found to be a significant predictor for b-thalassemia clinical severity by epigenetic-mediated variant-dependent HbF elevation. We found this rSNP accounted for 41.6% of b-hemoglobinopathy individuals as an ameliorating factor in a total of 2,738 individuals from southern China and Thailand. We uncovered that the minor allele of the rSNP triggers the attenuation of LYAR and two repressive epigenetic regulators DNA methyltransferase 3 alpha (DNMT3A) and protein arginine methyltransferase 5 (PRMT5) from the HBG promoters, mediating allele-biased g-globin elevation by facilitating demethylation of HBG core promoter CpG sites in erythroid progenitor cells from b-thalassemia persons. The present study demonstrates that this common rSNP in the proximal A g-promoter is a major genetic modifier capable of ameliorating the severity of thalassemia major through the epigenetic-mediated regulation of the delayed fetal-to-adult Hb switch and provides potential targets for the treatment of b-hemoglobinopathy.

DNA methylation patterns of β-globin cluster in β-thalassemia patients

“…However, we found the methylation patterns in γ-globin promoter showed difference between TFH and TFL group of β-thalassemia. The methylation level of most CpG sites in γ-globin promoter were hypomethylated, especially in + 17 and + 50 sites, in the BM tissues of TFH patients, compared with TFL patients, which was consistent with our previous report [12]. The γ-globin promoter methylation, not LCR region DNA methylation, might be the main effect to γ-globin expression, and the methylation alteration in the γ-globin promoter may be a driving factor leading to remote regulation between globin promoter and LCR regions [19].…”

“…The hypomethylated patterns in globin promoters might make it easier to access for some transcription factors mediating the looping with globin promoter. In fact, our previous study showed that a SNP rs368698783 was associated with the methylation level on + 17 CpG site in γ-globin promoter [ 12 ], by recruiting DNMT3A to the promoter and further regulating the γ-globin expression, supporting the important role of γ-globin promoter methylation in regulation of γ-globin expression.…”

“…To evaluate the methylation status of the β-globin clusters in β-thalassemia patients, we first screened the methylation patterns of total 97 CpG sites on the β-globin cluster, including ε-, γ-, δ-, and β-globin promoters, the core sequences of HS4 and HS3 and the 5′ flanking sequences of the LCR region, the CpGs-rich region of the HS2-HS1 intergenic region, the CpG islands (CpGI) in the endogenous retrovirus 9 (ERV-9) located at upstream of HS5, and the predicted binding region of the transcription factor BCL11A on the gene cluster located about 3.5 kb upstream of δ-globin gene [ 9 ] (Fig. 1 a), in PB of 105 β 0 /β 0 thalassemia patients and 44 normal controls using bisulfite sequencing method as previously described [ 12 ]. We observed that most of CpG sites around the LCR HS4-HS3 regions, and γ- and β-globin promoter displayed significant hypomethylation in β 0 /β 0 -thalassemia patients compared with normal controls (Fig.…”

“…Reactivating of HbF was reported to be a therapeutic target for β-thalassemia and SCD. Several genetic modulators [2][3][4][5][6][7] and cis-regulatory elements [6,[8][9][10][11][12] involved in the regulation of human fetal hemoglobin (HbF), and concomitant α-thalassemia have been identified as ameliorators of β-thalassemia [13]. DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-aza) has been applied to hemoglobinopathy patients (mainly in SCD patients) through improvement of HbF expression in the 1980s and 1990s [14,15], which supported that DNA methylation plays an important role in human globin switching.…”

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“…Specifically, HbF blocks HbS from dimerizing, thereby preventing hemoglobin polymer chaining and decreasing the probability of a vaso-occlusion [1,17]. Hydroxyurea, thalidomide, sodium butyrate, and decitabine, a DNMT1 targeting agent, have been previously shown to work through induction of HbF and subsequent protection against VOCs [5,[18][19][20][21][22].…”