HbF Levels Research Articles

Domain-Focused CRISPR-Cas9 Screen Identifies Hri Kinase As a Regulator of Fetal Hemoglobin in Adult Human Erythroid Cells

Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit in patients with sickle cell disease (SCD) and some forms of β-thalassemia. While developing genome editing and gene replacement approaches promise long term HbF elevation in SCD patients, there remains a strong need for pharmacologic treatment of these diseases. Two transcription factors, BCL11A and LRF (ZBTB7A) and their co-regulators mediate most of fetal globin transcriptional silencing in adult erythroid cells. However, transcription factors are inherently challenging to inhibit with small molecules.To identify potentially druggable HbF regulators, we carried out a CRISPR-Cas9 based genetic screen in a human erythroid cell line targeting protein kinases as they are inherently amenable to inhibition by small molecules. Most CRISPR-screens published to date have made use of sgRNAs targeting early exons to induce frameshift mutations. It has recently been shown that targeting sgRNAs to functional protein domains significantly improves screening efficiencies, as both in-frame and frameshift mutations contribute to generating hypomorphic alleles. We thus designed a sgRNA library targeting almost all annotated kinase-domains using a newly optimized sgRNA-scaffold that improves on-target editing activity. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1) as an HbF repressor. HRI is an erythroid specific kinase known to phosphorylate the translation initiation factor eIF2α. eIF2α phosphorylation generally tempers protein synthesis, but select transcripts with upstream Open Reading Frames (uORFs) in their 5'UTR are known to escape this inhibition.HRI depletion increased the fraction of HbF+ cells and elevated γ-globin (the fetal β-type globin chain) mRNA and protein levels. These effects were surprisingly specific as evidenced by whole proteome mass spectrometry and RNA-seq. Importantly, HRI depletion did not adversely affect cell growth or maturation. Similar results were obtained in primary CD34+ derived erythroid cells that were HRI depleted. HRI deficiency in erythroid cultures from patients with SCD reduced cell sickling, suggesting that biologically relevant HbF levels might be achieved by targeting HRI.Mechanistically, HRI loss strongly reduces BCL11A expression post-transcriptionally without impacting LRF production. BCL11A appears to be a major effector of HRI function since forced expression of BCL11A in HRI-depleted cells restores HbF repression to a significant degree.Finally, we explored whether pharmacologic HbF inducers that act via distinct mechanisms might further amplify HbF production. Pomalidomide has recently been shown to lower BCL11A mRNA levels and raise HbF production. Our proof-of-concept experiments demonstrate strong cooperativity of HRI depletion and pomalidomide treatment in HbF induction. HRI emerges as an erythroid specific target for HbF induction alone or in combination with mechanistically distinct HbF inducers. DisclosuresNo relevant conflicts of interest to declare.

Reduced Hemolysis and Related Complications in Females with Sickle Cell Disease

Sickle cell disease (SCD) is an inherited erythrocyte disorder characterized by chronic hemolysis and vasculopathy. Females have reduced levels of hemolysis compared to males in both the general population (Jordan A et al, Vox Sang 2016) as well as in SCD (Kanias T et al, Transfusion 2016). This phenomenon may be due to the effects of gender on hemoglobin (Hb) F levels (Dover GJ et al, Blood 1992), red blood cell membrane stability (Jordan A et al, Vox Sang 2016), and nitric oxide (NO) bioavailability (Gladwin MT et al, Circulation 2003). We wished to confirm reduced hemolysis in SCD females and whether this would be associated with reduced hemolytic complications, some of which are associated with mortality. Finally, we wished to see if there were suggestions of improved survival in females compared to males with SCD.We investigated differences in laboratory and clinical features by gender in 395 (155 male, 240 female) SCD patients treated at the University of Illinois at Chicago (UIC) and in 668 (304 male, 364 female) SCD patients from the multicenter Walk-PHaSST cohort. Comparisons of linear and categorical variables were performed using ANOVA and logistic regression analysis, respectively adjusting for age, SCD genotype, and Hb F levels. Median values and interquartile range (IQR) are provided.In SCD patients either not on hydroxyurea or with Hb F levels available prior to initiating hydroxyurea, we observed higher Hb F values in females vs. males from both the UIC (n=329, P=0.004) and Walk-PhaSST (n=368, P=0.003) cohorts (Table 1). Hemolytic markers were lower in females vs. males and these relationships persisted after adjusting for HbF levels, including LDH (UIC: P=0.0004, Walk-PHaSST: P=0.01); total bilirubin (UIC: P=0.008; Walk-PHaSST: P=0.002), AST (UIC: P=0.08; Walk-PHaSST: P=0.0002), and absolute reticulocyte count (UIC: P=0.04, Walk-PHaSST: P=0.01) (Table 1).Less hemolysis would be expected to decrease the occurrence or severity of a hemolytic vasculopathy and its manifestations. Pulse pressures were lower in females than males in both cohorts (UIC: P <0.0001; Walk-PHaSST: P=0.0003). Additional manifestations less frequently observed in females than males included leg ulcer history (UIC: P<0.0001; Walk-PHaSST: P=0.09) and tricuspid regurgitant velocity >3.0m/s (UIC: P=0.2; Walk-PHaSST: P=0.01)(Table 1). The degree of hemolysis is an independent predictor for early mortality in SCD (Kato GJ et al, Blood 2006, Nouriae M et al, Haematologica 2013). There were progressively higher ratios of females:males with increasing age in SCD patients (Cochran's linear trend; UIC: P=0.03, Walk-PHaSST: P=0.005) (Figure 1).We compared gene expression in peripheral blood mononuclear cells by gender in 180 SCD patients from UIC using the Affymetrix Human Exon 2.0 ST array. Restricting analyses to autosomes with an FDR < 0.05, no statistically significant differences in gene expression were observed. We also determined the X-linked G6PD A- status by PCR in 259 SCD patients from the UIC cohort. G6PD A- deficiency was observed in 4% (6/155) of females and 10% (10/104) of males and was not associated with hemolytic markers.In summary, females had a lower degree of hemolysis, decreased vasculopathy, and represented a progressively higher proportion of SCD patients with increasing age in both the UIC and Walk-PHaSST cohorts. Future investigation of loci on the sex chromosomes for effects on Hb F levels and the degree of hemolysis are warranted. Understanding the mechanisms for reduced hemolysis in women with SCD, such as the potential effects of testosterone and estrogen on erythrocyte membrane stabilization, may also help guide future therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High-Resolution CRISPR Functional Genomics Reveals Critical Vulnerabilities of the NuRD Complex for Fetal Hemoglobin Control

Hemoglobinopathies resulting from mutations of the adult beta-globin gene affect millions worldwide. Persistently elevated levels of fetal hemoglobin (HbF) are without consequence in healthy individuals but convey a major clinical benefit in patients with sickle cell disease and beta-thalassemia. Fetal globin silencing relies on interactions between DNA binding factors and chromatin readers, writers, and erasers acting within multiprotein nuclear complexes. The nucleosome remodeling and deacetylase (NuRD) complex is an epigenetic chromatin modifier, including ATP-dependent nucleosome remodeling and histone deacetylase activities, that has been implicated in HbF control. BCL11A and ZBTB7A, two key transcription factors required for HbF repression in adult erythroid cells, physically interact with NuRD complex members. In order to identify potential target sites within the NuRD complex with maximal HbF de-repression and minimal cellular toxicity potential we set out to dissect the function of the NuRD complex in adult erythroid precursors by dense mutagenesis. We performed CRISPR-Cas9 mediated saturating mutagenesis of all coding regions of human NuRD complex members in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) adult erythroid cells with HbF expression as the primary readout and cell fitness as a counter-screen. This approach distinguished four classes of NuRD complex members: those required for HbF repression but dispensable for cellular fitness (such as MTA2, MBD2, HDAC2, and GATAD2A), those required for both HbF repression and cell fitness (such as CHD4), those only required for cell fitness (such as RBBP4), and many not essential for either HbF repression or cell fitness. The NuRD paralogs essential for HbF repression correlated with the abundance of peptides retrieved by MTA2 immunoprecipitation followed by mass spectrometry, suggesting a NuRD sub-complex that silences fetal globin. We demonstrated that this tiling CRISPR screen approach identifies residues at which short in-frame deletions result in loss of function, including at catalytic residues of CHD4 and HDAC2, additional NuRD domains and conserved regions of unknown significance. By mapping functional data onto existing structures we detected sequences essential for HbF repression within the ELM2 and BAH domains of MTA2 that mediate key protein-protein interactions with HDAC1/2 and with chromatin. At CHD4, we found numerous critical positions where in-frame alleles resulted in robust HbF elevation to a greater magnitude than disruption of any other NuRD member. However nearly all of these HbF-inducing perturbations at CHD4 were inversely correlated with cell fitness. In contrast, we observed that in-frame deletions within the CHDCT2 domain of CHD4 resulted in profound elevation of HbF level but spared the negative impact on cell growth. Overall, these results demonstrate a sub-complex of NuRD essential for HbF silencing and identify key interfaces including at the potent HbF repressor CHD4 which could serve as selective small molecule development targets for therapeutic HbF re-induction. DisclosuresOrkin:Epizyme Inc.: Consultancy; Bioverativ: Consultancy.

Integrated Molecular Profiling of Juvenile Myelomonocytic Leukemia

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that occurs in infancy and early childhood, characterized by excessive myelomonocytic cell proliferation and granulocyte-macrophage colony-stimulating factor hypersensitivity. Over 80% of JMML patients have been reported to harbor mutually exclusive somatic and/or germline mutations in canonical RAS pathway genes (e.g., PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several poor prognostic factors, including the presence of two or more mutations, acute myeloid leukemia-like (AML-like) gene expression profiles, and selected gene promoter methylation profiles. However, the molecular pathogenesis in 10%-20% of patients and the relationships among biomarkers have not been well defined. This study aimed to assess the molecular pathogenesis in patients and the relationships among several biomarkers and outcomes.Patients and Methods: We performed comprehensive genetic analyses in 150 JMML patients, including 15 with Noonan syndrome-associated myeloproliferative disorder (NS/MPD), using whole-exome sequencing (n = 69, 46%), targeted deep sequencing (n = 92, 61%), RNA sequencing (n = 129, 86%), and genome-wide methylation analysis (n = 106, 71%).Results: We identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in three of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro . Therefore, we administered crizotinib to a patient with the RANBP2-ALK fusion gene. This patient achieved complete molecular remission after the administration of crizotinib for 51 days and successfully bridged to allogeneic stem cell transplantation. This patient survived without disease recurrence for 15 months after transplantation.We performed a genome-wide methylation analysis in 106 patients and combined the data with repository data from normal CD34+ samples (n = 5) and AML CD34+CD38− samples (n = 14) for an unsupervised consensus clustering of CpG methylation profiles, which yielded two distinct subgroups, hypermethylation profile (n = 45, 42%) and hypomethylation profile (n = 61, 58%). All AML samples had a hypermethylation profile, whereas all normal CD34+ and NS/MPD samples had a hypomethylation profile. A gene set enrichment analysis revealed marked enrichment of AML-associated genes in the hypermethylation cluster. The 5-year OS rate was 46.0% [95% confidence interval (CI), 31.0%-59.8%] among patients with a hypermethylation profile and 73.4% (95% CI, 57.6%-84.1%) among those with a hypomethylation profile (p = 0.0028). The 5-year TFS was significantly poorer in the hypermethylation group than in the hypomethylation group [2.2% (95% CI, 0.2%-10.1%) vs. 41.2% (95% CI, 27.1%-54.8%), p = 3.1 × 10−10]. Differential gene expression analysis for the hypermethylation and hypomethylation profiles revealed overexpression of LIN28B and its downstream target HMGA2 in the hypermethylation profile.Lastly, we integrated the molecular analyses of JMML, including genome-wide methylation profiling, RNA sequencing, and resequencing data and found that the hypermethylation profile was highly correlated with the most established risk factors for JMML, including older age, higher HbF level, lower platelet count, PTPN11/NF1 mutations, presence of two or more mutations, LIN28B overexpression, and AML-like expression profiles, as well as poor survival.Discussion: We identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations. Crizotinib represents a promising candidate drug for treating JMML in patients with ALK/ROS1 fusions. In addition, we established a risk profiling system based on genome-wide methylation analysis and showed that the hypermethylation profile is highly associated with poor survival and most established poor prognostic biomarkers for JMML. These finding will contribute to the development of precision treatments for JMML patients. DisclosuresNo relevant conflicts of interest to declare.

Unique Polymorphisms at BCL11A, HBS1L-MYB and HBB Loci Associated with HbF in Kuwaiti Patients with Sickle Cell Disease.

Patients with sickle cell disease (SCD) in Kuwait have elevated HbF levels ranging from ~10–44%; however, the modulating factors are unclear. We investigated the association of single nucleotide polymorphisms (SNPs) at BCL11A, HBS1L-MYB and HBB with HbF levels in 237 Kuwaiti SCD patients, divided into 3 subgroups according to their HbF levels. Illumina Ampliseq custom DNA panel was used for genotyping and confirmed by arrayed primer extension or Sanger sequencing. In the BCL11A locus, the CC genotype of rs7606173 [χ2 = 16.5] and (GG) of rs10195871 [χ2 = 15.0] were associated with Hb-F1 and HbF-2 subgroups, unlike rs1427404-T [χ2 = 17.3], which showed the highest association across the three subgroups. HBS1L-MYB locus revealed 2 previously-described SNPs (rs66650371 [χ2 = 9.5] and rs35795442 [χ2 = 9.2]) and 2 previously-unreported SNPs, (rs13220662 [χ2 = 6.2] and rs1406811 [χ2 = 6.7]) that were associated with the HbF-3 subgroup, making this the key locus elevating HbF to the highest levels. HBB cluster variants were associated with lower levels of HbF (β = −1.1). We report four previously-unpublished variants showing significant association with HbF. Each of the three quantitative trait loci affects HbF levels differently; unique SNPs, especially in HBS1L-MYB, elevate HbF to the highest levels.

HbA2 induction: the merit of pancellularity in sickle cell disease.

HbA2 induction: the merit of pancellularity in sickle cell disease.

BCL11A Polymorphism in Egyptian Children with β-Thalassemia: Relation to Phenotypic Heterogeneity.

Fetal hemoglobin (HbF) is a potent genetic modifier of β-thalassemia phenotype. B-cell lymphoma 11A ( BCL11A ) gene results in significant silencing of HbF. The aim of this study was to assess the prevalence of different BCL11A genotypes among a cohort of Egyptian children with β-thalassemia and to correlate them to HbF and clinical severity score. Eighty-two children with β-thalassemia (aged 12.95 ± 3.63 years) were recruited from the Pediatric Hematology Clinic, Ain Shams University. They were divided based on the clinical severity of β-thalassemia into three subgroups: 20 mild (24.4%), 24 moderate (29.3%), and 38 severe (46.3%). Age, gender, age of diagnosis, initial HbF level, transfusion history, and history of splenectomy were assessed. Anthropometric measures, signs of anemia and hemosiderosis, and the severity score were determined. Laboratory investigations such as complete blood picture, ferritin, and single gene polymorphism genotyping of the rs11886868 were also performed. Our findings showed that 16 children had CC genotype (19.5%), 38 had TC genotype (46.3%), and 28 had TT genotype (34.1%) of the rs#. β-thalassemia children with TT genotype had significantly higher severity scoring than the other two groups ( p < 0.001). Moreover, mean initial HbF was found to be lower in children with TT genotype followed by TC and CC genotypes ( p < 0.001). Increased γ-globin expression associated with BCL11A gene polymorphism is associated with better clinical severity of β-thalassemia.

Epigenetic Insights and Potential Modifiers as Therapeutic Targets in β-Thalassemia.

Thalassemia, an inherited quantitative globin disorder, consists of two types, α– and β–thalassemia. β–thalassemia is a heterogeneous disease that can be asymptomatic, mild, or even severe. Considerable research has focused on investigating its underlying etiology. These studies found that DNA hypomethylation in the β–globin gene cluster is significantly related to fetal hemoglobin (HbF) elevation. Histone modification reactivates γ-globin gene expression in adults and increases β–globin expression. Down-regulation of γ–globin suppressor genes, i.e., BCL11A, KLF1, HBG-XMN1, HBS1L-MYB, and SOX6, elevates the HbF level. β–thalassemia severity is predictable through FLT1, ARG2, NOS2A, and MAP3K5 gene expression. NOS2A and MAP3K5 may predict the β–thalassemia patient’s response to hydroxyurea, a HbF-inducing drug. The transcription factors NRF2 and BACH1 work with antioxidant enzymes, i.e., PRDX1, PRDX2, TRX1, and SOD1, to protect erythrocytes from oxidative damage, thus increasing their lifespan. A single β–thalassemia-causing mutation can result in different phenotypes, and these are predictable by IGSF4 and LARP2 methylation as well as long non-coding RNA expression levels. Finally, the coinheritance of β–thalassemia with α–thalassemia ameliorates the β–thalassemia clinical presentation. In conclusion, the management of β–thalassemia is currently limited to genetic and epigenetic approaches, and numerous factors should be further explored in the future.

Acyclovir induces fetal hemoglobin via downregulation of γ-globin repressors, BCL11A and SOX6 trans-acting factors

Pharmacological reactivation of developmentally silenced fetal hemoglobin (HbF) is an attractive approach to ameliorate the clinical manifestations of β-thalassemia and sickle cell anemia. Hydroxyurea, the only HbF inducer, has obtained regulatory approval. However, hydroxyurea non-responders and associated myelosuppression making its widespread use undesirable. A high level of HbF with safe and effective agents remains an elusive therapeutic goal for this global health burden. This study demonstrated the effect of acyclovir on γ-globin expression and erythropoiesis, associated with increased HbF production. In vitro, human erythroleukemia cells and human CD34+ erythroid progenitors, and in vivo β-YAC transgenic mice were used as experimental models. We found that acyclovir significantly induces expression of the γ-globin gene and HbF synthesis in CD34+ erythroid progenitors, without affecting terminal erythroid differentiation and erythroid cell proliferation. In contrast to other HbF inducers, no associated cytotoxicity with acyclovir was observed. Further, we reported the effect of acyclovir on γ-globin gene transcriptional regulators including BCL11A, FOP1, KLF1 SOX6, and GATA-1. Significant downregulation of the γ-globin repressors BCL11A and SOX6 was observed at both mRNA and protein levels. Whereas, GATA-1, a master erythroid transcription factor, was upregulated in acyclovir treated human CD34+ erythroid culture. Similarly, the HbF inducing effect of acyclovir in β-YAC transgenic mice revealed a good in vitro correlation, with a substantial increase in fetal globin mRNA, and F cells population. These findings collectively suggest acyclovir as an effective HbF inducer and pave the way to evaluate its clinical efficacy in treating β-globin disorders.

Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.

Pinpointing functional noncoding DNA sequences and defining their contributions to health-related traits is a major challenge for modern genetics. We developed a high-throughput framework to map noncoding DNA functions with single-nucleotide resolution in four loci that control erythroid fetal hemoglobin (HbF) expression, a genetically determined trait that modifies sickle cell disease (SCD) phenotypes. Specifically, we used the adenine base editor ABEmax to introduce 10,156 separate A•T to G•C conversions in 307 predicted regulatory elements and quantified the effects on erythroid HbF expression. We identified numerous regulatory elements, defined their epigenomic structures and linked them to low-frequency variants associated with HbF expression in an SCD cohort. Targeting a newly discovered γ-globin gene repressor element in SCD donor CD34+ hematopoietic progenitors raised HbF levels in the erythroid progeny, inhibiting hypoxia-induced sickling. Our findings reveal previously unappreciated genetic complexities of HbF regulation and provide potentially therapeutic insights into SCD.

Fetal-haemoglobin enhancing genotype at BCL11A reduces HbA2 levels in patients with sickle cell anaemia.

Understanding the interplay of genetic factors with haemoglobin expression and pathological processes in sickle cell disease is important for pharmacological and gene‐therapeutic interventions. In our nascent study cohort of Nigerian patients, we found that three major disease‐modifying factors, HbF levels, α‐thalassaemia deletion and BCL11A genotype, had expected beneficial haematological effects. A key BCL11A variant, while improving HbF levels (5.7%–9.0%), also led to a small, but significant decrease in HbA2. We conclude that in general, interventions boosting HbF are likely to reduce HbA2 in patients’ erythroid cells and that such therapeutic strategies might benefit from a parallel stimulation of HbA2 through independent mechanisms.

Molecular analysis of the erythroid phenotype of a patient with BCL11A haploinsufficiency

AbstractThe BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G>T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the γ-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient's cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of γ-globin expression in β-hemoglobinopathy patients. Our data strongly support this approach.

Fetal hemoglobin levels in premature newborns. Should we reconsider transfusion of adult donor blood?

PurposeThe aim of this study was to assess the percent decrease in fetal hemoglobin (HbF) after transfusion of adult-derived donor packed red blood cell (pRBC) units in extremely low gestational age newborns (ELGANs). MethodsControl percent fetal hemoglobin (%HbF) levels were measured in newborn cord blood or peripheral blood samples in non-transfused patients prior to elective surgery. ELGANs were followed prospectively and %HbF was measured on residual post-test complete blood count (CBC) specimens. ELGAN %HbF values were compared to the control population and transfusions were recorded. ResultsInitial mean %HbF in ELGANs (n=16) was 92.2±1.3% (range 90.2-95.1%), which is similar to the control group (n=25). Mean levels dropped to 61.1±11.1% (range 34.2-73.2%) after a single pRBC transfusion (n=9) and to a mean of 35.6±6.3% after an additional transfusion (n=5). %HbF levels trended upwards if no additional transfusions were given, but levels still remained lower than expected for gestational age through discharge (n=85 samples). ConclusionsPercent fetal hemoglobin concentrations in ELGANs decrease precipitously after transfusion with adult donor pRBCs. Further studies are needed to evaluate the benefit of maintaining higher fetal hemoglobin concentrations in these patients and whether administration of HbF rather than adult donor pRBCs would improve patient outcomes.

Impairment of Cerebrovascular Hemodynamics in Patients With Severe and Milder Forms of Sickle Cell Disease.

In patients with sickle cell disease (SCD), cerebral blood flow (CBF) is elevated to counteract anemia and maintain oxygen supply to the brain. This may exhaust the vasodilating capacity of the vessels, possibly increasing the risk of silent cerebral infarctions (SCI). To further investigate cerebrovascular hemodynamics in SCD patients, we assessed CBF, arterial transit time (ATT), cerebrovascular reactivity of CBF and ATT (CVRCBF and CVRATT) and oxygen delivery in patients with different forms of SCD and matched healthy controls. We analyzed data of 52 patients with severe SCD (HbSS and HbSβ0-thal), 20 patients with mild SCD (HbSC and HbSβ+-thal) and 10 healthy matched controls (HbAA and HbAS). Time-encoded arterial spin labeling (ASL) scans were performed before and after a vasodilatory challenge using acetazolamide (ACZ). To identify predictors of CBF and ATT after vasodilation, regression analyses were performed. Oxygen delivery was calculated and associated with hemoglobin and fetal hemoglobin (HbF) levels. At baseline, severe SCD patients showed significantly higher CBF and lower ATT compared to both the mild SCD patients and healthy controls. As CBFpostACZ was linearly related to CBFpreACZ, CVRCBF decreased with disease severity. CVRATT was also significantly affected in severe SCD patients compared to mild SCD patients and healthy controls. Considering all groups, women showed higher CBFpostACZ than men (p < 0.01) independent of baseline CBF. Subsequently, post ACZ oxygen delivery was also higher in women (p < 0.05). Baseline, but not post ACZ, GM oxygen delivery increased with HbF levels. Our data showed that baseline CBF and ATT and CVRCBF and CVRATT are most affected in severe SCD patients and to a lesser extent in patients with milder forms of SCD compared to healthy controls. Cerebrovascular vasoreactivity was mainly determined by baseline CBF, sex and HbF levels. The higher vascular reactivity observed in women could be related to their lower SCI prevalence, which remains an area of future work. Beneficial effects of HbF on oxygen delivery reflect changes in oxygen dissociation affinity from hemoglobin and were limited to baseline conditions suggesting that high HbF levels do not protect the brain upon a hemodynamic challenge, despite its positive effect on hemolysis.

Epigenetic effects toward new insights as potential therapeutic target in B-thalassemia

BackgroundFetal hemoglobin (HbF) induction has shown promise for the treatment of β-hemoglobinopathies. HbF induction in β-thalassemia could overcome ineffective hematopoiesis and thus terminate transfusion dependency for formerly transfusion dependant patients. Several miRNAs have been found to reactivate γ-globin expression and increase HbF. In this study, we aimed to investigate the expression of 4 miRNAs (miR-15a, miR-16-1, miR-96, and miR-486-3p) in high HbF thalassemia patients and correlate their levels with the patients’ HbF levels then, in order to predict the exact role of the studied miRNAs in hematopoiesis, a bioinformatic analysis was carried out. We went through this bioinformatic analysis to determine the network of genes regulated by miRNAs and further investigate the interaction between all of them through their involvement in hematopoiesis. In this study, the differential expression was measured by qRT-PCR for 40 patients with high HbF and compared to 20 healthy controls. Bioinformatics was conducted involving functional annotation and pathway enrichment analyses. ResultsThe studied microRNAs were significantly deregulated in thalassemia patients in correlation with HbF. Functional annotation and pathway enrichment analyses revealed a major role of miR-486-3p and miR-15a in HbF induction. ConclusionMiR-486-3p and miR-15a are crucial for HbF induction. Further validating studies are needed.

Association of XmnI Polymorphism with Fetal Hemoglobin Level in Sudanese Patients with Sickle Cell Disease

Background: Sickle cell disease (SCD) is an inherited blood disorder that affects red blood cells. The studyof various modulating factors, and genetic factors affecting the clinical severity of the SCD is an interestingresearch focus especially in communities with a distinct genetic background. The XmnI polymorphism isa common genetic variation that was reported in previous studies to increase fetal hemoglobin (HbF) level.This was a descriptive cross-sectional study, conducted in El-Obeid city in Northern Kordofan state, westernSudan, during the period from August to November 2016. The Xmn1 polymorphic site was determined bypolymerase chain reaction. Data was analyzed using SPSS software program version 20. P-value of 0.05and below was considered of significance. In present study HbF level among normal individuals AA , shownsignificant difference (p&lt;0.05) between presence of Xmnl +/+ and absence of Xmnl -/- site. in patients withSS , the HbF level was higher in those who had one or two Xmnl sites as compared to those with the siteabsent . In patients with sickle cell trait AS and AA, only the presence of the one and two Xmnl site (+/+)compared to the absence of the site (-/-) was associated with significant increase in the HbF level. There isa close link between the Xmn1 polymorphism site and HbF level. A wide range of HbF level was obtainedboth in the present and absence of this site. Further studies with a large sample size as well as analysis BShaplotypes among the patient with sickle cell anemia population are needed for better understand of possibleassociation .

Epigenetic inactivation of ERF reactivates γ-globin expression in β-thalassemia

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 β-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+ erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-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 β-hemoglobinopathies.

Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

BCL11A, the major regulator of HbF(α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult-type HbA (α2β2). To uncover how BCL11A initiates repression, we used CRISPR/Cas9, dCas9, dCas9-KRAB, and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established upon BCL11A depletion, and precedes γ-globin derepression and LCR-globin loop formation. Our findings reveal that the switch from fetal-to-adult globin gene expression within the >50 kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.

The rare hemoglobin variant Hb Mizuho: report of a Swiss family and literature review

Hb Mizuho is a very rare unstable hemoglobin; here, we describe the clinical history of three Swiss family members with Hb Mizuho together with a systematic review of the previously six published cases. The clinical history of the adult woman we report here is unique since this is the first Hb Mizuho presenting with Moyamoya complications and the first case reported with long-term erythrocyte exchange. The literature review showed that Hb Mizuho was mainly reported as a de novo mutation, with the exception of children descended from known cases. All published patients with this unstable hemoglobin showed severe hemolytic anemia with the exception of one; all were regularly transfused. Patients with higher HbF levels might require fewer transfusions. All patients underwent splenectomy at a median age of 4 years and had variable clinical improvement; some achieved complete resolution of transfusion dependency after splenectomy. Iron overload in Hb Mizuho patients seems to be mainly attributed to transfusions and has less to do with ineffective erythropoiesis. Diagnosis might be challenging; a normal hemoglobin electrophoresis should not rule out the diagnosis of unstable hemoglobin in patients with otherwise unexplained hemolytic anemia. This series shows the enormous utility of using molecular techniques for diagnosis.

Modulatory effect of single nucleotide polymorphism in Xmn1, BCL11A and HBS1L-MYB loci on foetal haemoglobin levels in β-thalassemia major and Intermedia patients.

To evaluate the influence of certain genetic modifiers on foetal haemoglobin levels in thalassemia major and thalassemia intermedia. The cohort study was conducted from November 2018 to August 2019, at Department of Haematology, University of Health Sciences, Lahore and comprised beta thalassemia intermedia and thalassemia major patients who were referred by various healthcare facilities across Punjab, Pakistan. Foetal haemoglobin was quantified by high performance liquid chromatography. Primary mutation analysis and single nucleotide polymorphisms were done by amplification refractory mutation system-based polymerase chain reaction. Data was analysed using SPSS 20. Of the 116 patients, 52(45%) had beta thalassemia intermedia and 64(55%) had thalassemia major. Foetal haemoglobin levels were primarily influenced by alleles of the HBG2 (rs7482144) and BCL11A (rs766432) genes, but single nucleotide polymorphism of HBS1L-MYB (rs9399137) had no significant role (p>0.05). The rs7482144 single nucleotide polymorphism explained 8.3% of the variation in the foetal haemoglobin levels, while 5% of trait variation was explained by rs766432. There was found a clear association between foetal haemoglobin level and single nucleotide polymorphisms in HBG2 (rs7482144) and BCL11A (rs766432) genes. This correlation was additive and was seen both in thalassemia major and thalassemia intermedia cohorts.