Β-globin Gene Cluster Research Articles

A Novel εγδβ-Thalassemia Deletion Associated with Severe Anemia at Birth and a β-Thalassemia Intermedia Phenotype Later in Life in Three Generations of a Greek Family

We describe a novel deletion causing heterozygous εγδβ-thalassemia (εγδβ-thal) across three generations of a Greek family. The Greek deletion is about 72 kb in length, spanning from the hypersensitive site 4 (HS4) in the locus control region (LCR) to the 3' end of the β-globin gene, thus encompassing the entire β-globin gene cluster. The deletion caused severe but transient neonatal anemia and a non transfusion-dependent chronic hemolytic anemia state later in life, resembling mild β-thalassemia intermedia (β-TI) rather than β-thalassemia (β-thal) trait, as had been previously reported. Apart from the presentation of clinical and laboratory characteristics, the challenges involving clinical management are also discussed.

Fetal Hemoglobin Modulators May Be Associated With Symptomology of Football Players with Sickle Cell Trait.

This study investigates whether genetic modifiers previously shown to influence adult fetal hemoglobin (HbF) levels and glucose-6-phosphate dehydrogenase deficiency were associated with variable symptomology in a small sample of collegiate football players with sickle cell trait. Survey data on self-assessed symptoms and genotype data from five single nucleotide polymorphisms (SNPs) related to HbF production and two SNPs that cause glucose-6-phosphate dehydrogenase deficiency were collected from current and former college football players. In this sample, SNPs found within the β-globin gene cluster were found to be associated with a previous diagnosis of exertional sickling and experience of extreme heat during and after training. rs10189857 in the BCL11A gene was associated with body mass index and weight and with experiencing extreme thirst during and after training. No significant correlations were found between the other SNPs and symptoms within this sample. These findings show that genetic variation known to affect sickle cell disease symptomology may partly explain why some football players with sickle cell trait experience adverse clinical outcomes during periods of extreme physical exertion and others do not.

Investigation of RFLP Haplotypes β- Globin Gene Cluster in Beta-Thalassemia Patients in Central Iran

Introduction: Beta-thalassemia is one of the most prevalent inherited blood diseases among Iranians. The aim of this study was to elucidate the chromosomal background of beta-thalassemia mutations in Esfahan province, Iran. Materials and Methods: In this study, we investigated three frequent mutations (c.315+1G>A, c.93-21G>A and c.92+5G>C in β-globin gene, the frequency of RFLP haplotypes, and LD between markers at β-globin gene cluster) in 150 beta-thalassemia patients and 50 healthy individuals. The molecular and population genetic investigations were performed on RFLP markers HindIII in the c.315+1G>A of Gγ (HindIIIG) and Aγ (HindIIIA) genes, AvaII in the c.315+1G>A of β-globin gene and BamHI 3' to the β-globin gene. All statistical analyses were performed using Power Marker software and SISA server. Results: Fifty percent of beta-thalasemia patients were associated with these mutations. Haplotype I was the most prevalent haplotype among beta-thalassemia patients (39.33%) and normal individuals (46%). The commonest c.315+1G>A mutation in our population was tightly linked with haplotype III (43.75%) and haplotype I (31.25%). The second prevalent mutation, c.92+5G>C, was 90%, 6.66%, and 3.33% in linkage disequilibrium with haplotypes I, VII, and III, respectively. The c.93-21G>A mutation indicated a strong association with haplotype I (80%). Conclusion: Our study participants like beta-thalassemia patients from Kermanshah province was found to possess a similar haplotype background for common mutations. The emergence of most prevalent mutations on chromosomes with different haplotypes can be explained by gene conversion and recombination. High linkage of a mutation with specific haplotype is consistent with the hypothesis that chromosomes carrying beta-thalassemia mutations experienced positive selection pressure, probably because of the protection against malaria experienced by beta-thalassemia carriers.

Interactions of unstable hemoglobin Rush with thalassemia and hemoglobin E result in thalassemia intermedia

ABSTRACTBackground: The clinical consequences and significance of many unstable hemoglobins interacting with other hemoglobinopathies remain unrecognized. Here we first explore molecular and hematological characterizations of previously undescribed compound heterozygosity states for unstable hemoglobin Rush (Hb Rush, Beta 101 Glu > Gln, HBB:c.304G > C) with Hb E and different forms of thalassemia.Methods: Hematological assays, globin gene mutation assays and β-globin gene cluster haplotype were conducted in 11 patients from 8 unrelated Chinese ethnic families with unexplained hemoglobin separation fraction in hemoglobin gel electrophoresis.Results: Hb Rush in various combinations with Hb E, β0-thalassemias and α+-thalassemia were identified. Hb Rush simple heterozygote was generally associated with mild hemolytic anemia, and the compound heterozygotes of Hb Rush and the other β-globin variants led to thalassemia intermedia phenotypes with moderate anemia. Hemoglobin electrophoreses showed that the co-presence of Hb Rush with either Hb E or β0-thalassemias increased proportion of Hb Rush due to relative decrease of other globin chain synthesis. Beta-globin gene cluster haplotype analysis suggested a common origin of the Hb Rush variant in the Chinese families of different ethnic ancestry.Conclusions: Unstable Hb Rush interacting with β-thalassemia result in thalassemia intermedia phenotypes, which demonstrated the clinical significance of Hb Rush and new insights into complex mechanism of clinical heterogeneity of thalassemia.

Nietypowy obraz hemoglobinopatii współistniejącej z talasemią β

Introduction. Inherited hemoglobin disorders, including thalassemia and hemoglobinopathies, are frequently occurring diseases in the world. Thalassemias are autosomal recessive disorders of hemoglobin synthesis. Underlying molecular defects in the α-globin or β-globin gene clusters form the basis of defective hemoglobin synthesis and the various inherited forms of α- halasemias or β-thalasemias. Thalassemia is diagnosted where hemoglobin levels, volume of red blood cells (MCV) and mean cellular hemoglobin (MCH) are significantly reduced. Aim. We want to introduce atypical clinical falieure of hemoglobinopathy. Material and methods. We presented two children with anemia. In first case it was a boy with normal MCV and reduced MCH, next case it was a girl after splenectomy with increased MCV and MCH. In both cases the red cells were hypochromic and also target cells were present in peripheral blood smear. Hemoglobin electrophoresis, measurement of HbA2 and HbF levels were performed. Results. Test results were characteristic for thalasemia β. Extending the diagnostic panel with genetic tests helped to identify in both of children thalassemia α silience carrier (-α/αα). In first case unstabile hemoglobin Hb Köln (Codon 98(295G > A)) was also detected and in second unstabile hemoglobin Hb Bruxelles (deletions del 42/β+). Conclusion. Only the genetic tests made it possible to explain atypical blood smear for thalassemia.

Genetic Modifiers of Fetal Haemoglobin in Sickle Cell Disease.

Fetal haemoglobin (HbF) levels have a clinically beneficial effect on sickle cell disease (SCD). Patients with SCD demonstrate extreme variability in HbF levels (1-30%), a large part of which is likely genetically determined. The main genetic modifier loci for HbF persistence, HBS1L-MYB, BCL11A and the β-globin gene cluster in adults also act in SCD patients. Their effects are, however, modified significantly by a disease pathology that includes a drastically shortened erythrocyte lifespan with an enhanced survival of those red blood cells that carry HbF (F cells). We propose a model of how HbF modifier genes and disease pathology interact to shape HbF levels measured in patients. We review current knowledge on the action of these loci in SCD, their genetic architecture, and their putative functional components. At each locus, one strong candidate for a causative, functional DNA change has been proposed: Xmn1-HBG2 at the β-globin cluster, rs1427407 at BCL11A and the 3bp deletion rs66650371 at HBS1L-MYB. These, however, explain only part of the impact of these loci and additional variants are yet to be identified. Further progress in understanding the genetic control of HbF levels requires that confounding factors inherent in SCD, such as ethnic complexity, the role of F cells and the influence of drugs, are suitably addressed. This will depend on international collaboration and on large, well-characterised patient cohorts with genome-wide single-nucleotide polymorphism or sequence data.

Haplotype Analysis of Three Common β-Thalassemia Mutations in Syrian Patients

β-Globin haplotypes were used to investigate the origin of three common β-globin mutations, IVS-I-110 (G>A); HBB: c.93-21G>A, IVS-I-1 (G>A); HBB: c.92 + 1G>A and codon 39 (C>T); HBB: c.118C > T in Syrian patients. Haplotype analysis was done for 49 unrelated patients with β-thalassemia (β-thal) and 20 unrelated healthy subjects by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) for the β-globin gene cluster of the following polymorphic restriction sites: HincII 5' to ε, HindIII 5' to Gγ, HindIII 5' to Aγ, HincII in ψβ, HincII 3' to ψβ, AvaII in β, and HinfI 3' to β. The IVS-I-110 mutation was associated with three haplotypes: I [+ – – – – + +] (79.4%), V [+ – – – – + –] (5.9%) and VII [+ – – – – – +] (14.7%), while, the two mutations IVS-I-1 and codon 39 were be linked to a single haplotype V (100.0%) and II [– + + – + + +] (100.0%), respectively. The normal chromosomes (βA/βA) were associated with four haplotypes, I (50.0%), II (7.5%), V (32.5%) and VII (10.0%). In the Syrian population, the IVS-I-110 mutation was associated with multi haplotypes, whereas the IVS-I-1 and codon 39 mutations have a single origin. More studies for the other mutations will be very useful for genetic epidemiological studies in Syria.

Time estimations by network of beta globin gene cluster haplotypes linked with Hb D-Los Angeles [β121 (GH4) Glu→Gln GAA→CAA] mutation in the world populations.

Backgroundβ‐Globin gene cluster haplotypes associated with the Hb D‐Los Angeles mutation have been reported in many different locations in different populations including Italy, Iran, Thailand, Belgium, Mexico, Holland, and Turkey. In this study, we have identified genetic relationships and formation periods between the haplotypes reported in the world regarding the Hb D‐Los Angeles.MethodsWe comparatively analyzed the RFLP (restriction fragment length polymorphism) data in Denizli region and world populations using Arlequin 3.5 statistical software program. The data obtained from the Arlequin software were then entered into the Phylogenetic Network software to calculate the age estimates and to discover possible links between the haplotypes.ResultsWe observed the frequencies of the β‐globin gene haplotypes for the seven polymorphic restriction sites around the world and calculated the estimated time of haplotypes using Network software on the basis of ancestral haplotypes. We performed the phylogenetic network analysis of the haplotypes linked with Hb D‐Los Angeles mutation by processing the data of frequency and age estimations with Network software.ConclusionOur period of time results suggests that HAP1 was formed before modern human migration to Asia and/or independent origin of the Hb D‐Los Angeles mutation from other populations. Considering that the population in Denizli region started the Hb D‐Los Angeles mutation past about 40,000 years ago, it can be said that HAP1, HAP15, and HAP21 belong to the gene pool with an external effect. Our period of time results of HAP6 is compatible with published dating results.

Benzene metabolite 1,2,4-benzenetriol changes DNA methylation and histone acetylation of erythroid-specific genes in K562 cells.

1,2,4-Benzenetriol (BT) is one of the phenolic metabolites of benzene, a general occupational hazard and ubiquitous environmental air pollutant with leukemogenic potential in humans. Previous studies have revealed that the benzene metabolites phenol and hydroquinone can inhibit hemin-induced erythroid differentiation in K562 cells. We investigated the roles of DNA methylation and histone acetylation in BT-inhibited erythroid differentiation in K562 cells. When K562 cells were treated with 0, 5, 10, 15 or 20µM BT for 72h, hemin-induced hemoglobin synthesis decreased in a concentration-dependent manner. Both 5-aza-2'-deoxycytidine (5-aza-CdR, DNA methyltransferase inhibitor) and trichostatin A (TSA, histone deacetylases inhibitor) could prevent 20µM BT from inhibiting hemin-induced hemoglobin synthesis and the mRNA expression of erythroid genes. Exposure to BT changed DNA methylation levels at several CpG sites of erythroid-specific genes, as well as the acetylation of histone H3 and H4, chromatin occupancy of GATA-1 and recruitment of RNA polymerase II at α-globin and β-globin gene clusters after hemin induction. These results demonstrated that BT could inhibit hemin-induced erythroid differentiation, where DNA methylation and histone acetylation also played important roles by down-regulating erythroid-specific genes. This partly explained the mechanisms of benzene hematotoxicity.

Fast Temperature-Gradient COLD PCR for the enrichment of the paternally inherited SNPs in cell free fetal DNA; an application to non-invasive prenatal diagnosis of β-thalassaemia

ObjectiveTo develop a sensitive, specific, simple, cost-effective and reproducible platform for the non-invasive prenatal detection of paternally inherited alleles for β-thalassaemia. The development of such an assay is of major significance in order to replace currently-applied invasive methods containing inherent fetal loss risks.MethodsWe present a fast Temperature-Gradient Co-amplification at Lower Denaturation Temperature Polymerase Chain Reaction (fast TG COLD PCR) methodology for the detection of the paternally-inherited fetal alleles in maternal plasma. Two single-nucleotide polymorphisms (SNPs), rs7480526 (G/T) and rs968857 (G/A) that are located on the β-globin gene cluster and exhibit a high degree of heterozygosity in the Cypriot population were selected for evaluation. Seventeen maternal plasma samples from pregnancies at risk for β-thalassemia were analysed for the selected SNPs using the novel fast TG COLD PCR assay.ResultsUsing fast TG COLD PCR, the paternally inherited allele in cell free fetal DNA was correctly determined for all the 17 maternal plasma samples tested, showing full agreement with the Chorionic Villus Sampling (CVS) analysis.ConclusionsOur findings are encouraging and demonstrate the efficiency and sensitivity of fast TG COLD PCR in detecting the minor paternally-inherited fetal alleles in maternal plasma for the development of a NIPD assay for β-thalassaemia.

First Identification of the 3.5 kb Deletion (NC_000011.10: g.5224302-5227791del3490bp) on the β-Globin Gene Cluster in a Chinese Family

β-Thalassemia (β-thal) is one of the most common autosomal recessive disorders worldwide. It is caused mainly by point mutations or, more rarely, deletions on the β-globin gene, leading to reduced (β+) or absent (β0) synthesis of the β chains of hemoglobin (Hb). Molecular characterization of β-thal is essential for the prevention of this disease in the population. In China, more than 46 different mutations have been found, while approximately five large deletional types of β-thal have been reported. Here we described a large deletional mutation of the β-globin gene cluster previously unreported in the Chinese population, the 3.5 kb deletion (NC_000011.10: g.5224302-5227791del3490bp) removing the β-globin gene promoter and the whole β-globin gene leading to a β0-thal phenotype.

A survey of genetic fetal-haemoglobin modifiers in Nigerian patients with sickle cell anaemia.

Genetic variants at three quantitative trait loci (QTL) for fetal haemoglobin (HbF), BCL11A, HBS1L-MYB and the β-globin gene cluster, have attracted interest as potential targets of therapeutic strategies for HbF reactivation in sickle cell anaemia (SCA). We carried out the first systematic evaluation of critical single nucleotide polymorphisms at these disease modifier loci in Nigerian patients with SCA. Common variants for BCL11A and HBS1L-MYB were strongly associated with HbF levels. At both loci, secondary association signals were detected, illustrating the mapping resolution attainable in this population. For BCL11A, the two independent sites of association were represented by rs1427407 (primary site, p = 7.0 x 10−10) and rs6545816 (secondary site, conditioned on rs1427407: p = 0.02) and for HBS1L-MYB by rs9402686 (HMIP-2B, p = 1.23 x 10−4) and rs66650371 (HMIP-2A, p = 0.002). Haplotype analysis revealed similarities in the genetic architecture of BCL11A and HBS1L-MYB in Nigerian patients. Variants at both loci also alleviated anaemia. The variant allele for the γ globin gene promoter polymorphism XmnI-HBG2 was too infrequent in our patients to be evaluated in this relatively small study. Studying the large and diverse SCA patient populations in African countries such as Nigeria will be key for a clearer understanding of how these loci work and for the discovery of new disease modifier genes.

Analysis of the Genotypes in a Chinese Population with Increased Hb A2 and Low Hematological Indices

Increased Hb A2 is considered the most reliable hematological finding for the identification of β-thalassemia (β-thal) carriers. The aim of this study was to determine the underlying genetic factors associated with a high Hb A2 level in a Chinese population. Subjects were recruited from couples preparing for pregnancy who participated in the thalassemia screening program during a 2-year period. DNA analyses were used for diagnosis of β-thal and other genetic factors. A total of 5985 adults who screened positive for β-thal were recruited. Of these, 5933 (99.1%) were detected to have a β-thal mutation. In the remaining 52 (0.9%) individuals without mutations involving the β-globin gene cluster, 16 were found to have Krüppel-like factor 1 (KLF1) gene variants, and two had an α-globin gene triplication. There were still 34 individuals with unknown genetic factors for their raised Hb A2 values. The results of this study indicate that genetic factors other than β-thal can rarely contribute to the elevation of Hb A2. These subjects usually have borderline microcytic red cell indices and Hb A2 values.

Estimating the age of Hb G-Coushatta [β22(B4)Glu→Ala] mutation by haplotypes of β-globin gene cluster in Denizli, Turkey.

BackgroundHb G‐Coushatta variant was reported from various populations’ parts of the world such as Thai, Korea, Algeria, Thailand, China, Japan and Turkey. In our study, we aimed to discuss the possible historical relationships of the Hb G‐Coushatta mutation with the possible migration routes of the world. For this purpose, associated haplotypes were determined using polymorphic loci in the beta globin gene cluster of hemoglobin G‐Coushatta and normal populations in Denizli, Turkey.MethodsWe performed statistical analysis such as haplotype analysis, Hardy–Weinberg equilibrium, measurement of genetic diversity and population differentiation parameters, analysis of molecular variance using F‐statistics, historical‐demographic analyses, mismatch distribution analysis of both populations and applied the test statistics in Arlequin ver. 3.5 software program.ResultsThe diversity of haplotypes has been shown to indicate different genetic origins for two populations. However, AMOVA results, molecular diversity parameters and population demographic expansion times showed that the Hb G‐Coushatta mutation develops on the normal population gene pool. Our estimated τ values showed the average time since the demographic expansion for normal and Hb G‐Coushatta populations ranged from approximately 42,000 to 38,000 ybp, respectively.ConclusionOur data suggest that Hb G‐Coushatta population originate in normal population in Denizli, Turkey. These results support the hypothesis that the multiple origin of Hb G‐Coushatta and indicate that mutation may have been triggered the formation of new variants on beta globin haplotypes.

Gene Turnover and Diversification of the α- and β-Globin Gene Families in Sauropsid Vertebrates.

The genes that encode the α- and β-chain subunits of vertebrate hemoglobin have served as a model system for elucidating general principles of gene family evolution, but little is known about patterns of evolution in amniotes other than mammals and birds. Here, we report a comparative genomic analysis of the α- and β-globin gene clusters in sauropsids (archosaurs and nonavian reptiles). The objectives were to characterize changes in the size and membership composition of the α- and β-globin gene families within and among the major sauropsid lineages, to reconstruct the evolutionary history of the sauropsid α- and β-globin genes, to resolve orthologous relationships, and to reconstruct evolutionary changes in the developmental regulation of gene expression. Our comparisons revealed contrasting patterns of evolution in the unlinked α- and β-globin gene clusters. In the α-globin gene cluster, which has remained in the ancestral chromosomal location, evolutionary changes in gene content are attributable to the differential retention of paralogous gene copies that were present in the common ancestor of tetrapods. In the β-globin gene cluster, which was translocated to a new chromosomal location, evolutionary changes in gene content are attributable to differential gene gains (via lineage-specific duplication events) and gene losses (via lineage-specific deletions and inactivations). Consequently, all major groups of amniotes possess unique repertoires of embryonic and postnatally expressed β-type globin genes that diversified independently in each lineage. These independently derived β-type globins descend from a pair of tandemly linked paralogs in the most recent common ancestor of sauropsids.

Haplotype Analysis of β-Thalassaemia Major and Carriers with Filipino β°-Deletion in Sabah, Malaysia.

ObjectiveThe Filipino β°-deletion has been reported as a unique mutation in East Malaysia with a severe phenotype due to the complete absence of β-globin chain synthesis. In this study, the haplotype patterns of the β-globin gene cluster were used to relate the human genetic variation to this specific β-thalassaemia mutation.MethodsThe 376 study subjects included 219 β-thalassaemia major (β-TM) patients with homozygous Filipino β°-deletion and 157 carriers with heterozygous Filipino β°-deletion from 10 government hospitals in different regions of Sabah. Genomic DNA was isolated from whole blood using silica membrane based DNA purification protocol. Polymerase chain reaction restriction fragment length polymorphism analysis (PCR-RFLP) was conducted on five markers within the β-globin gene cluster to construct the haplotype patterns.ResultsFour haplotypes (Haplotype I–IV) were identified with Haplotype I as the predominant haplotype with the highest frequency of 0.98, followed by Haplotype II, III and Haplotype IV with 0.02. Haplotype I was strongly linked with the Filipino β°-deletion among the indigenous population.ConclusionHaplotype I as the predominant haplotype suggests the patients with the Filipino β°-deletion in Sabah have a similar origin.

Comparison of Fetal and Adult Erythroid Chromosomal Architectures Identifies a Novel Fetal Hemoglobin Regulatory Region

Chromatin structure is tightly intertwined with transcription regulation. The extent to which global chromatin architecture is subjected to alterations at different developmental stages within the same cell lineage has not been examined in great depth. Erythropoiesis offers an ideal model system to study the molecular mechanisms of gene regulation within the same cell lineage during development. Here, we comparatively defined via RNA-seq the transcriptomes, and via Hi-C and Capture-C the chromosome architectures of primary human fetal and adult erythroid cells.Overall, fetal and adult chromosomal conformations displayed a high degree of similarity. This includes the maintenance of A and B compartments representing active and inactive chromatin regions, respectively. Only ~5% of the genome switched compartments from A to B or vice versa, in agreement with the highly similar gene expression profiles. Moreover, topologically associating domains (TADs) were extensively preserved from fetal to adult stages. The developmentally regulated β-globin gene cluster is contained within one topologically associating domain (TAD) but folds into a three sub-TADs structure, the central one of which encompasses the β-globin locus. Notably, although the three sub-TAD structures are flanked by tissue invariant CTCF bound sites, they engage in looped contacts only in erythroid cells, indicating that erythroid specific transcription factors are required for CTCF mediated boundary contacts.At a finer scale, Capture-C detected distinct folding patterns at the developmentally controlled β-globin locus, including the expected stage-specific interactions between the enhancer (LCR) and the fetal γ-globin and adult β-globin genes. Importantly, we identified new developmental stage-specific chromatin contacts involving a region compassing a pseudogene (HBBP1) that resides between the fetal and adult globin genes. Specifically, HBBP1 engages in fetal stage-specific contacts with DNase hypersensitive sites HS5 and 3'HS1 while contacting the embryonic ε-globin gene at the adult stage. Deletion of a 2.3kb fragment encompassing HBBP1 (but not its transcriptional silencing) leads to strong reactivation of γ-globin gene expression in an adult erythroid cell line. This is accompanied by an architecturally restructured locus, including increased LCR-γ-globin chromatin interactions. Notably, the effects of HBBP1 deletion on chromatin architecture and gene expression closely mimic those of deleting the fetal globin repressor BCL11A, implicating BCL11A in the function of the HBBP1 region.In sum, our results identify a new segment, distinct from previously described regions linked to hereditary persistence of fetal hemoglobin, which engages in functionally important chromatin contacts. Since the HBBP1 region resides quite distantly from the structural globin genes, it might be a useful target for therapeutic genome editing without risking damage to the globin genes. Finally, our study highlights the power of high resolution chromosome architectural analysis to identify new regulatory regions. DisclosuresNo relevant conflicts of interest to declare.

Genetic variation of Krüppel-like factor 1 (KLF1) and fetal hemoglobin (HbF) levels in β0-thalassemia/HbE disease.

Heterogeneity of HbF levels in β0-thalassemia/HbE disease has been reported to be associated with variations in clinical manifestations of the disease, and several genetic-modifying factors beyond the β-globin gene cluster have been identified as HbF regulators. Down-regulation or heterozygous mutations of Krüppel-like factor 1 (KLF1) is associated with elevated HbF levels in non-thalassemia subjects. This study confirms that experimental down-regulation of KLF1 in β0-thalassemia/HbE-derived erythroblasts significantly increases HbF production (up to 52.3±2.4%), albeit with slightly delayed erythroid terminal differentiation. KLF1 exome sequencing of 130 Thai β0-thalassemia/HbE patients without co-inheritance of α-thalassemia found six patients with KLF1 heterozygous mutations including rs2072596 (p.F182L; n=5) and rs745347362 (p.P284L; n=1) missense mutations. However, while these patients had high HbF levels (38.1±7.5%), they were all associated with a severe clinical phenotype. These results suggest that while reduction of KLF1 expression in β0-thalassemia/HbE erythroblasts can increase HbF levels, it is not sufficient to alleviate the clinical phenotype.

SIRT1 activates the expression of fetal hemoglobin genes.

High fetal hemoglobin (HbF, α2 γ2 ) levels ameliorate the clinical manifestations of sickle cell disease and β thalassemia. The mechanisms that repress HbF expression and silence γ-globin genes in adults are incompletely characterized and only a single HbF inducer, hydroxyurea, is approved for treatment, and only in patients with sickle cell disease. We identified SIRT1, a protein deacetylase, as a new inducer of γ-globin. SIRT1 knockdown decreased, while SIRT1 ectopic expression upregulated γ-globin gene (HBG) expression in primary human erythroid cells and in K562 cells. The small molecule SIRT1 activators SRT2104 and SRT1720 enhanced HBG expression in cord blood human erythroblasts and reactivated silenced HBG in adult human erythroblasts. Furthermore, SIRT1 binds in the β-globin gene cluster locus control region (LCR) and HBG promoters, promotes the looping of the LCR to HBG promoter, and increases the binding of RNA polymerase II and H4K16Ac in the HBG promoter. SIRT1 suppressed the expression of the HBG suppressors BCL11A, KLF1, HDAC1 and HDAC2. Lastly, SIRT1 did not change the proliferation of human erythroid progenitor cells or the expression of differentiation marker CD235a. These data suggest that SIRT1 activates HBG expression through facilitating LCR looping to the HBG promoter, inhibiting the expression of transcriptional suppressors of HBG, and indirectly increasing histone acetylation in the HBG promoter. SIRT1 is a potential therapeutic target for γ-globin gene induction, and small molecule SIRT1 activators might serve as a lead compound for the development of new HbF inducers.

Association between beta globin haplotypes, HBBP1 and HMOX1 polymorphisms in relation to HbF among sickle cell anemia patients: a study in Southwest Iran

The haplotypes of β-globin gene cluster in patients with sickle cell anemia are associated with different clinical manifestations and fetal hemoglobin (HbF) levels. In addition, SNP in some genes within the β-globin gene cluster (HBBP1) and outside it (HMOX1) has been recently reported to be related to HbF levels. In this study, rs2071348 and rs2071746 polymorphisms in HBBP1 and heme oxygenase 1 (HMOX1) genes associated with different haplotypes and HbF levels in patients with sickle cell anemia were evaluated in comparison with healthy subjects. In this case-control study, 150 patients with sickle cell anemia (90 homozygous and 60 sickle-cell trait) were evaluated and compared with 50 healthy individuals referred to Shafa tertiary hospital in Ahvaz city of Khuzestan Province, Iran. HbF level was measured by alkali and acid electrophoresis. PCR-RFLP and ARMS-PCR methods were used to determine β-globin gene haplotypes and HBBP1 gene polymorphism, respectively. Arab-Indian was the most common genotype found in both homozygous and sickle-cell trait patients with a respective prevalence of 55.5 and 50% followed by homozygous Benin with 11.1 and 18.3% prevalence, respectively. The highest and lowest level of HbF were observed in Arab-Indian (24.1 ± 6.35) and Benin/Benin haplotypes (7.63 ± 2.29), respectively. A significant relationship was found between HbF level with rs2071746 A>T polymorphism of HMOX1 gene (P < 0.019) as well as with rs2071348 polymorphism of HBBP1 gene (P < 0.034). However, there was no significant relationship between haplotypes with gene polymorphisms. Arab-Indian was the most common haplotype in this region. The HbF level was significantly higher in patients with Arab-Indian haplotype, patients with C genotype in rs2071348 polymorphism locus of HBBP1 gene, and patients with T genotype in rs2071746 polymorphism locus of HMOX gene.