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.

ObjectiveTo reveal the prevalence and molecular characterization of (δβ)0‐thalassemia [(δβ)0‐thal] and hereditary persistence of fetal hemoglobin (HPFH) in the Chinese Zhuang population.MethodsA total of 105 subjects with fetal hemoglobin (Hb F) level ≥5% from 14 204 unrelated ones were selected for the study. Multiplex ligation dependent probe amplification was firstly used to analyze dosage changes of the β‐globin gene cluster for associated with (δβ)0‐thal and HPFH mutations. The gap polymerase chain reaction was then performed to identify the deletions using the respective flanking primers. Hematologic data were recorded and correlated with the molecular findings.ResultsTwenty‐one (0.15%) subjects were diagnosed with Chinese Gγ(Aγδβ)0‐thal. Nine (0.06%) were diagnosed with Southeast Asia HPFH (SEA‐HPFH) deletion. Seventy‐five (0.53%) cases remained uncharacterized. Three genotypes for Chinese Gγ(Aγδβ)0‐thal and SEA‐HPFH deletion were identified, respectively. The genotype‐phenotype relationships were discussed.ConclusionOur study for the first time demonstrated that (δβ)0 and HPFH were not rare events, and molecular characterized Gγ(Aγδβ)0‐thal and HFPH mutations in the Chinese Zhuang population. The findings in our study will be useful in genetic counseling and prenatal diagnostic service of β‐thalassemia in this populations.

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of b-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA þ erythroid cells from b-thalassemia-affected individuals with widely varying levels of HbF groups (HbF R 95th percentile or HbF % 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of b-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in b-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of g-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in b-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34 þ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses g-globin expression by directly binding to two consensus motifs regulating g-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for b-hemoglobinopathies.

Background: Thalassemia is one of the most common genetic diseases in southern China. Howerver, population in different regions or different population has their own spectrums of thalassemia. To investigate the prevalence and spectrum features of thalassemia among children in Guangxi. Hematology and genetic analysis were performed on 71,459 children aged 1–10 years in various regions of Guangxi.Results: A total of 11,821 children were diagnoses with thalassemia including 7,615 (10.66%) subjects of α-thalassemia, 3,507 (4.90%) subjects of β-thalassemia, and 699 (0.98%) cases with both α- and β-thalassemia. Nine α-thalassemia mutations and 30 genotypes were identified among the α-thalassemia children. The - -SEA and - -SEA/αα were the most frequent mutation and genotype, respectively. One α-thalassemia fusion gene and a rare 2.4 kb deletion both causing α+-thalassemia were identified, respectively. Thirteen β-thalassemia mutations and 31 genotypes were characterized among the β-thalassemia children, with the most common mutation CD41-42 (-CTTT) accounting for 46.05% of the β-mutations. Two rare mutations IVS-II-5 (G>C), and IVS-I-2 (T>C) were firstly identified. Furthermore, 92 genotypes were identified among 699 children with both α- and β-thalassemia.Conclusions: Our findings highlight the great heterogeneity and the extensive spectrum of thalassemia among children in Guangxi, which provide an available reference for prevention of thalassemia in this area.

Hb Bart's hydrops fetalis is the most severe and generally fatal clinical phenotype of α-thalassemia (α-thal), which is due to the deletion of all four functional α-globin genes of hemoglobin (Hb), resulting in no α-globin chain production (- -/- -). Homozygosity for the - - (Southeast Asian) α-globin gene deletion is the main cause of the Hb Bart's hydrops fetalis in Asia, especially South China. Occasionally, other α-thal deletions can also be found. In this study, we report a case with an atypical form of Hb Bart's hydrops fetalis that was caused by - - and a large novel α-thal deletion (- -) (Guangxi). The fetus with Hb Bart's in our study presented fetal hydrops features in early gestation which was different from that of traditional Hb Bart's hydrops fetalis with a homozygous - - deletion. The early onset of fetal hydrops is attributed to the decreased formation of embryonic Hb Portland (ζ2γ2), which is proposed as a candidate for reactivation in cases of severe α-thal. Our findings indicated that it was important to characterize new or rare mutations, and highlighted the significance of using ultrasonography to identify signs of Hb Bart's hydrops fetalis.

Introduction Although mutations in the human beta‐globin gene cluster are essentially point mutations, several large deletions have been described in recent years. Methods We have identified a novel 223 kb deletion in a Chinese patient by multiplex ligation‐dependent probe amplification and characterized it by next‐generation sequencing, Gap‐PCR, and DNA sequence analysis. Results The deletion extends from the 3′UTR of the δ globin gene (HBD) to 215 kb downstream of the HBB. Compound heterozygous with the typical β‐thalassemia—CD41‐42(‐CTTT) mutation, the proband presented with microcytosis and hypochromic red cells, and required regulate transfusion. The patient was clinically diagnosed with thalassemia major. Conclusion Our study widens the mutation spectrum of β‐thalassemia. In addition, this case may spark future studies of the regulatory regions of the beta‐globin gene cluster.

Background Reactivation 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. Results Here, 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. Conclusions Our 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.

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