High HbF Research Articles

The relationship between the variations in Gγ and Aγ promotors and the Hereditary Persistence of Fetal Hemoglobin (HPFH).

In the present study, sixty-two samples that have 1.5% and upper level of fetal hemoglobin (HbF), were examined to investigate the relationship between HbF level and non-deletional mutations in both Gγ (G gamma) globin (HBG2) and Aγ (A gamma) globin (HBG1) genes. Four variations were observed in the promotor of Gγ gene, which are -158C/T, -309A/G, -369C/G, and -567T/G. Also, four variations were observed in the 5'-UTR (untranslated regions) and promotor of Aγ gene, which are +25G/A, -369G/C, -499T/A, and -588G/A. One -222/-225 AGCA del homozygous and six variations as heterozygous in A gamma globin gene promotor region were also observed. The results of the current study suggested that there was a significant relationship between high HbF levels and two variations (-309A/T and -369C/G) in Gγ gene promotor. Additionally, a significant relationship between two variations (+25G/A and -499T/A) in Aγ gene promotor was also observed. Furthermore, the persons who carry these variations with high levels of HbF indicated that there might be a haplotype effect between these variations.

Clinical and haematological profile of children with sickle cell anaemia admitted to a rural medical college of Chhattisgarh, India

Background: Sickle cell disease (SCD) is the most common single gene disorder resulting in hemolytic anemia. Aim of the study was to describe the clinico-haematological profile of children with sickle cell anaemia admitted to Paediatric ward/PICU with any acute clinical event and to find out the association between high HbF level, frequency of crises episodes and requirement of blood transfusion in sickle cell anemia.Methods: Hospital based descriptive study. Retrospective data analysis was done from medical records of patients between 0-15 years age group admitted to hospital from March 2014 to August 2017.Results: Total 68 clinical events were recorded in 60 patients during the study period. More than half of the children were in 0-5 years age group. Mean age of diagnosis was 2.79 years. Severe anemia requiring blood transfusion was the most common cause of hospitalization followed by painful crises. Mean Hb level in the children was 6.65(±2.38). More than one third of children had associated nutritional anemia. Children with high HbF level were found to have less number of painful crises episodes.Conclusions: Severe anemia followed by painful crises were the commonest presentations requiring admission in our hospital. Chronic anemia with microcytosis and high HbF level is the hematological profile of the present study group. Children with high HbF level suffered from less number of painful crises episodes when compared to children with low HbF. But the requirement of blood transfusion was similar in both groups.

The First Two Years of Life in Sickle Cell Anemia Infants: Results of a Comprehensive Longitudinal Study

Background and hypothesis: Infancy is a critical time during which the first complications of sickle cell anemia (SCA) emerge. Very early prognostic factors of severity are needed to select high-risk children to offer precisely tailored therapy. Our hypothesis was that clinical, biological or genetic parameters measurable soon after birth (3 to 6 months of age) could predict severe outcomes in the first two years of life in SCA infants.Methods: Infants with SCA were offered to participate in a prospective study (ClinicalTrials.gov: NCT01207037) that included clinical and laboratory assessment at enrollment (3-6 months) and at 12, 18 and 24 months of age. The prognostic performance of conventional and SCA-specific biomarkers (notably erythroid adhesion markers, dense cells, ektacytometry indices) on disease severity was assessed using Cox's proportional hazard regression models. The disease severity was defined as time to first occurrence of either acute splenic sequestration (ASS), vaso occlusive (VOC) event requiring hospitalization, transfusion, conditional or abnormal Trans Cranial Doppler, acute chest syndrome and death. Hazard ratios (HRs) were calculated with 95% Cis.Results: Fifty-seven infants (55 SS; 2 Sβ°; 54.4% males), diagnosed through neonatal screening, were included in the study at a mean age of 4.4 ± 1 months and longitudinally assessed with a median follow-up of 19.4 months (range 3.1-23.2). Only 1 of 57 infants had experienced a SCA-related event prior to enrolment (dactilytis). At inclusion, clinical examinations were unremarkable except for pallor found in 15 (26,3%) and none of the infants had an enlarged or palpable spleen. Growth parameters were normal. Main biological characteristics were a mild hemolytic anemia (Hb 9.3 ±1.3 g/dL; reticulocytes count: 151.2 ±76 x 109/L), increased HbF level (3.8 ±1.3 g/dL. or 41.4 ±11.7%) and presence of dense red cells (23.1 % ±10.8). Genetic analysis showed one alpha globin deletion in 18 (32.1%) and a balanced distribution of beta globin haplotypes: 16 (30.8%), 18 (34.6%) and 18 (34.6%) in the favorable (SEN/SEN, BEN/SEN, CAM/SEN or SEN/ATYP), unfavorable (CAR/CAR, CAM/CAR, CAR/ATYP or BEN/CAR) and intermediate category (other) respectively.During the 2 years duration of the study, 44 of 57 (77%) infants required 157 hospital admissions, median (range): 2 (1-12) per patient. Infection was a leading cause of hospitalization although no serious adverse event related to pneumococcal infection was noted. Eight (14%) children experienced an episode of ASS at a median (range) age of 13.4 months (7.8- 15.9) while 13 infants (22.8%) experienced at least one VOC event at a median age of 12.7 months (7-22.5), with 6 experiencing ≥ 2 episodes. Nineteen infants (33.3%) required at least one transfusion with 10 (17.6%) requiring more than 2. Altogether, 22 (38.6%) infants of this cohort experienced a SCA-related severe clinical event by 24 months of age. The Kaplan Meier estimate of the 24-month event-free rate was 54.4% (95% CI, 39.7 to 74.5%) (Figure).Univariate analysis of potential prognostic markers at inclusion showed that higher HbF % and concentration were the strongest protective parameters for ASS in particular and for all severe outcomes, except for VOC (p < 0.001). Unfavorable haplotypes were also associated with severe outcome (HR (95% CI) 4.73 (1.31-17.01), p=0.017). Protective factors for VOC were higher Hb level (threshold > 8 g/dL) and low % of circulating dense cells (p=0.04 and 0.02, respectively). In a multivariate analysis, Hb level ≥ 8 g/dL and HbF ≥ 2.8 g/dL proved to be two independent prognostic factors of a SCA-related severe event (adjusted HRs (95% CI) 0.27 (0.11-0.73) and 0.16 (0.06-0.43) respectively). Interestingly, absolute neutrophil or reticulocyte counts, level of expression of known potentially pathogenic erythroid adhesion markers (CD36, Lu/B-CAM, ICAM-4/LW), % of red dense cell, or deformability parameters failed to be prognostic factors of specific complications or overall severity in this very young cohort.Conclusion: HbF and Hb levels measured between 3 and 6 months of age in SCA infants predict the risk of subsequent severe clinical outcome in the next 2 years. These events are frequent even in a high-income setting where neonatal screening is implemented. These findings further support the early use of HbF inducers such as hydroxyurea in high-risk infants to sustain a protective HbF level. [Display omitted] DisclosuresBrousse:Add Medica: Membership on an entity's Board of Directors or advisory committees. De Montalembert:Novartis: Consultancy, Honoraria, Research Funding; Addmedica: Consultancy, Honoraria, Research Funding.

In Vivo Effects of LSD1 Inhibition By Small Chemical Inhibitors in Sickle Cell Mice

For the past decades the principal strategy for treating the β-globinopathies [sickle cell disease (SCD) and β-thalassemia] has been to devise methods for increasing the levels of fetal hemoglobin (HbF) in adult erythroid cells, as a strong clinical literature has demonstrated that high HbF levels ameliorate disease pathophysiology.We originally reported the lysine-specific histone demethylase 1 (LSD1) as a new protein involved in the regulation of the fetal γ-globin genes. Inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine in primary human erythroid progenitor cells induces HbF to therapeutic levels. Thus, inhibition of LSD1 activity presents a novel exploratory avenue as a therapeutic strategy to treat SCD. Subsequent studies using another inhibitor RN-1 showed that RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with sickle cell disease.Here we report studies designed to examine the in vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 on HbF synthesis and erythroid physiology in SCD mice. In control SCD mice treated with DMSO only, the number of HbF positive cells was about 2.5% of total in RBC, while in SCD mice exposed to GSK-LSD1, or OG-L002, there was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment (up to 8%, or 6% respectively). Furthermore, sickled RBCs and reticulocytes are significantly reduced while the number of mature erythrocytes increased markedly in the peripheral blood of GSK-LSD1, or OG-L002 treated SCD mice. These findings suggest that GSK-LSD1 and/or OG-L002 could be potentially new HbF inducers based on LSD1 inhibition. We believe that these experiments will be of wide general interest and will be the foundation for development of small molecule therapeutics with which to treat hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

The presence of F cells with a fetal phenotype in adults with hemoglobinopathies limits the utility of flow cytometry for quantitation of fetomaternal hemorrhage.

Detection and quantitation of fetomaternal hemorrhage (FMH) can be difficult in patients with pre-existing elevations of HbF, such as those with hemoglobinopathies. The aim of this study was to evaluate the utility of dual-color flow cytometry with the Fetal Cell Count Kit (FCCK) in differentiating adult and fetal HbF in this population, as compared to flow cytometry (FC) using HbF alone. Peripheral blood was obtained from normal adults and patients with hemoglobinopathies (β-thalassemia and sickle cell disease), including a small number of pregnant females. Cord blood was used to spike some samples with 5% fetal cells. Analysis by single color (HbF) and dual-color (HbF and carbonic anhydrase) FC was performed on these samples. Fetal cells were defined as those with high HbF fluorescence on single-color FC, and those that were HbF + CA- using the FCCK. The quantity of fetal cells detected by each technique was compared. Forty-six adult patients were included. In non-pregnant adults with hemoglobinopathies, a population of red cells with a fetal cell phenotype were detected by both techniques. The dual-color method reported lower quantities of these cells. In nineteen samples spiked with cord blood the FCCK consistently underestimated the quantity of fetal cells. Patients with β-thalassemia and sickle cell disease have a population of HbF-containing cells which are phenotypically similar to fetal cells. Even with dual-color flow cytometry (FCCK), the detection and quantification of FMH by flow cytometry in this population remains difficult. © 2017 International Clinical Cytometry Society.

The ‑α3.7 deletion in α‑globin genes increases the concentration of fetal hemoglobin and hemoglobin A2 in a Saudi Arabian population

The regions of Al‑Qatif and Al‑Ahssa in the Eastern Province of Saudi Arabia are known for their high prevalence of hemoglobinopathies, including β‑thalassemia and sickle cell anemia. Previously, the α‑gene deletion has been demonstrated as highly prevalent among populations residing in these two regions. The present study was conducted in order to investigate the implications of the α‑globin gene deletion on fetal hemoglobin (HbF) and hemoglobin α2 (HbA2) concentrations in patients with transfusion‑dependent β‑thalassemia. A total of 166 Saudi patients with transfusion‑dependent β‑thalassemia and 337 healthy Saudi patients were included in the study. The ‑α3.7, ‑α4.2, -‑FIL, -‑SEA, -‑MED and -‑(20.5) gene deletions were identified using multiplex α‑globin deletion polymerase chain reaction. The present study revealed that the ‑α3.7 gene deletion is the most prevalent (43.5%) in the Saudi populations that were analyzed and is characterized by the deletion of 3,804 base pairs. Numerous genotypes, namely ‑3.7α2/α1α2, ‑3.7α2/α1α12, ‑3.7α2/‑3.7α2, ‑3.7α2HphI/α1α2HphI, ‑3.7α2/α1‑4.2, ‑3.7α2/α1polyA‑1α2, ‑3.7α12/α1α12, ‑‑FIL/‑3.7α2 and ‑3.7α2/‑3.7α2Hb Villiers le Bel were also identified in the investigated population. Furthermore, a gradual increase in the concentration of HbF and HbA2 in patients with β‑thalassemia and the number of α‑gene deletions was demonstrated; whereas in healthy patients the level of HbA2 was demonstrated to decrease as the number of α‑gene deletions increased. Therefore, it can be concluded that the high HbF concentration in the present study is predominantly associated with other mutations associated with β‑thalassemia rather than α‑globin deletions. Furthermore, the results of the present study also revealed novel α‑gene deletion genotypes prevalent in the population studied, namely α1α2/α1α2HphI, α1α2HphI/α1α2HphI, α1α2/α1α2Hb Handsworth, ‑3.7α2HphI/α1α2HphI, ‑3.7α2/‑3.7α2Hb Villiers le Bel and ‑-MED/α1α2HphI.

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.

Alterations on high HbF levels may be associated with KLF1 gene mutations.

The KLF1 gene synthesizes a transcription factor in the zinc finger structure that regulates the transcription of β-, γ-globin, and Foxm1 genes. This factor plays an important role in the erythropoiesis mechanism by modifying the chromatin structure and is involved in the regulation of transcription in the opening of the β-globin gene. β-globin gene expression could be disrupted by a mutation, which may be a possible cause of a disruption in regulation of the promotor of the β-globin gene where the KLF1 transcription factor binds. This can lead to an inherited high fetal hemoglobin (HbF) ratio in people. Therefore, the main aim of this study was to determine the effects of KLF1 mutations on these high levels of HbF. In this study, in order to determine the relationship between the KLF1 mutations and the high HbF levels three exons along with the 5'-UTR and 3'-UTR regions of the KLF1 gene were sequenced of 53 volunteers. In this study, 3 variations in the non-coding regions of the KLF1 gene were not associated with a high level of HbF. Five variations were detected in the second exon of KLF1 gene. One of these is a frame shift that occurs when GG bases are inserted between the 59-60 codons, and the other four variations occur as a base substitution variations. No correlation was found between high HbF levels and neutral variants. Only polar-nonpolar amino acid changes were found at two points. At one of them, a significant drop in the high HbF levels was observed, while the other was observed to be high near to the critical limit. These findings suggested that variations in function of the KLF1 gene can alter the HbF levels.

Alterations on high HbF levels may be associated with KLF1 gene mutations

Alterations on high HbF levels may be associated with KLF1 gene mutations

An A\u03b3-globin G->A gene polymorphism associated with \u03b2039 thalassemia globin gene and high fetal hemoglobin production

BackgroundIncrease of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in β-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify β-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea).MethodsAγ-globin gene sequencing was performed on genomic DNA isolated from a total of 75 β-thalassemia patients, including 31 β039/β039, 33 β039/β+IVSI-110, 9 β+IVSI-110/β+IVSI-110, one β0IVSI-1/β+IVSI-6 and one β039/β+IVSI-6.ResultsThe results show that the rs368698783 polymorphism is present in β-thalassemia patients in the 5’UTR sequence (+25) of the Aγ-globin gene, known to affect the LYAR (human homologue of mouse Ly-1 antibody reactive clone) binding site 5′-GGTTAT-3′. This Aγ(+25 G->A) polymorphism is associated with the Gγ-globin-XmnI polymorphism and both are linked with the β039-globin gene, but not with the β+IVSI-110-globin gene. In agreement with the expectation that this mutation alters the LYAR binding activity, we found that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are associated with high HbF in erythroid precursor cells isolated from β039/β039 thalassemia patients.ConclusionsAs a potential explanation of our findings, we hypothesize that in β-thalassemia the Gγ-globin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with β0-thalassemia mutations, but not with the β+-thalassemia mutation here studied (i.e. β+IVSI-110) and that this genetic combination has been selected within the population of β0-thalassemia patients, due to functional association with high HbF. Here we describe the characterization of the rs368698783 (+25 G->A) polymorphism of the Aγ-globin gene associated in β039 thalassemia patients with high HbF in erythroid precursor cells.

Fetal hemoglobin in sickle cell anemia: The Arab-Indian haplotype and new therapeutic agents.

Fetal hemoglobin (HbF) has well-known tempering effects on the symptoms of sickle cell disease and its levels vary among patients with different haplotypes of the sickle hemoglobin gene. Compared with sickle cell anemia haplotypes found in patients of African descent, HbF levels in Saudi and Indian patients with the Arab-Indian (AI) haplotype exceed that in any other haplotype by nearly twofold. Genetic association studies have identified some loci associated with high HbF in the AI haplotype but these observations require functional confirmation. Saudi patients with the Benin haplotype have HbF levels almost twice as high as African patients with this haplotype but this difference is unexplained. Hydroxyurea is still the only FDA approved drug for HbF induction in sickle cell disease. While most patients treated with hydroxyurea have an increase in HbF and some clinical improvement, 10 to 20% of adults show little response to this agent. We review the genetic basis of HbF regulation focusing on sickle cell anemia in Saudi Arabia and discuss new drugs that can induce increased levels of HbF.

KLF1 drives the expression of fetal hemoglobin in British HPFH

Abstractβ-Hemoglobinopathies are among the most common single-locus inherited diseases. In this condition, high fetal hemoglobin (HbF) levels have been found to be beneficial, and boosting HbF expression is seen as an attractive therapy. Naturally occurring mutations in the fetal globin promoter can result in high HbF persisting into adulthood in a benign condition known as hereditary persistence of fetal hemoglobin (HPFH). Individuals with one form of HPFH, British HPFH, carry a T to C substitution at position −198 of the fetal globin gene promoter. These individuals exhibit HbF levels of up to 20%, enough to ameliorate the symptoms of β-hemoglobinopathies. Here, we use clustered regularly interspaced short palindromic repeat–mediated genome editing to introduce the −198 substitution into human erythroid HUDEP-2 cells and show that this mutation is sufficient to substantially elevate expression of HbF. We also examined the molecular mechanism underlying the increase in fetal globin expression. Through a combination of in vitro and in vivo studies, we demonstrate that the mutation creates a de novo binding site for the important erythroid gene activator Krüppel-like factor 1 (KLF1/erythroid KLF). Our results indicate that introducing this single naturally occurring mutation leads to significantly boosted HbF levels.

Genotyping of BCL11A and HBS1L-MYB Single Nucleotide Polymorphisms in β-thalassemia/HbE and Homozygous HbE Subjects with Low and High Levels of HbF

Whether multiple single nucleotide polymorphisms (SNPs) of BCL11A and HBS1L-MYB genes affect hemoglobin (Hb)F production and hematological parameter variation in β-thalassemia/HbE and homozygous HbE in Thai subjects with low and high HbF levels are still unclear. Three SNPs of BCL11A gene (rs1427407, rs10189857 and rs11886868) and 3 SNPs of HBS1L-MYB gene (rs4895441, rs9399137 and rs28384513) were analyzed in 45 β-thalassemia/HbE patients who had HbF levels lower and higher than 15 %, and in 50 homozygous HbE who had HbF levels lower and higher than 5 %. Their hematological parameters were measured using automated blood counter. The HbF level was analyzed using high performance liquid chromatography (HPLC). There were no statistical significant differences of allele and genotype frequencies of 3 SNPs in the BCL11A gene between the groups of β-thalassemia/HbE patients and homozygous HbE subjects with low and high HbF levels. Significant differences in the allele frequencies in HBS1L-MYB SNP rs4895441 (p = 0.041) and rs9399137 (p = 0.048) were observed in homozygous HbE subjects with HbF £ 5 % and &gt; 5 %. Moreover, significant differences in MCV (p = 0.005) and trends towards significant differences in MCH (p = 0.057) and HbF levels (p = 0.051) were found in HBS1L-MYB SNP rs9399137 of homozygous HbE subjects. Therefore, the HBS11L-MYB SNPs especially rs9399137 had an effect on HbF production and the variation of hematological parameters in homozygous HbE subjects, but not in β-thalassemia/HbE patients.

Genome-wide analysis of aberrantly expressed lncRNAs and miRNAs with associated co-expression and ceRNA networks in β-thalassemia and hereditary persistence of fetal hemoglobin.

The implications of lncRNAs regarding fetal hemoglobin (HbF) induction in hemoglobin disorders remain poorly understood. In this study, microarray analysis was performed to profile lncRNAs, miRNAs and mRNAs in individuals with hereditary persistence of fetal hemoglobin (HPFH), β-thalassemia carriers with high HbF levels and healthy controls. The results show aberrant expression of 862 lncRNAs, 568 mRNAs and 63 miRNAs in the high-HbF group compared with the control group. Altered NR_001589, NR_120526, T315543, miR-486-3p, miR-19b-1-5p and miR-20a-3p expression was confirmed by quantitative reverse transcription-polymerase chain reaction, and Spearman correlation coefficients revealed significant positive correlations with HbF. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed the hematopoietic cell lineage and apoptosis to be most significantly dysregulated in HbF induction. We analyzed coding genes near the lncRNAs and constructed a coding-noncoding co-expression network. Based on the results, lncRNAs likely contribute to increased HbF levels by activating expression of HBE1 and hematopoietic cell lineage-inducible molecules and by inhibiting that of apoptosis-inducible molecules. Finally, through construction of a competing endogenous RNA network, we found that 6 lncRNAs could bind competitively with miR-486-3p, resulting in increased HbF levels. Taken together, our findings provide new insights into the mechanisms of HbF induction and potentially provide new targets for the treatment of β-thalassemia major.

MiR‐16 Mediated MYB Gene Silencing Induces Fetal Hemoglobin Expression

Sickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in the β‐globin gene affecting about 100,000 people in the United States and millions worldwide. Individuals living with this disease suffer from hemolytic anemia, progressive organ damage, and acute painful episodes. Fetal hemoglobin (HbF), a major genetic modifier of clinical presentation and progression of SCD, is of great interest due to its ability to inhibit sickle hemoglobin (HbS) polymerization and reducing erythrocyte damage. Variation in HbF levels among sickle cell patients is highly heritable and has been linked to the presence of single nucleotide polymorphisms (SNPs) in several genetic regulatory regions. In this study, DNA isolated from two cohorts of African American SCD patients with high and low HbF levels were analyzed by PCR‐based SNP analysis in the HBB, BCL11A, and HBS1L‐MYB (HMIP) gene regions. The presence of SNP alleles was then correlated with their clinical phenotype data to identify those associated with increased levels of HbF. Three SNPs in the BCL11A and HBB gene regions were identified in the high HbF patient cohort that may contribute to milder phenotypes in African Americans. To complement the SNP studies, genome wide microRNA (miRNA) data was generated which showed increased miR‐16 levels associated with high HbF. Furthermore, MYB, the negative regulator of γ‐globin expression, is silenced by miR‐16 through binding the 3′‐untranslated region. RT‐qPCR and western blot were used to measure γ‐globin mRNA and HbF protein levels respectively after transfection of miR‐16 mimic into KU812 cells. Preliminary data revealed dose dependent miR‐16 mediated MYB suppression and γ‐globin activation. The discovery of regulatory regions that modulate HbF expression and miRNA mediated suppression of negative regulators of γ‐globin, provides expanded understanding of gene regulation, which can be used to define novel treatment options for individuals with SCD.Support or Funding InformationSponsored by the Medical College of Georgia Medical Scholars Program

Does the Novel KLF1 Gene Mutation Lead to a Delay in Fetal Hemoglobin Switch?

The Kruppel-like factor 1 (KLF1) gene is an essential transcription factor that is required for the proper maturation of the erythroid cells. Recent studies have reported that KLF1 variations are associated with increased fetal hemoglobin (HbF) levels. Here we report a novel KLF1 gene variation codon 211 A→G (c.632 A>G) in a family who was referred for hemoglobinopathy screening. Both parents were classical β-thalassemia trait (mother: HbA2 4.1%, HbF 8.6%; father: HbA2 5.5%, HbF 0.6%) codon 15 G→A heterozygous, and the child was β-thalassemia homozygous. Because the mother showed a high HbF level, the genetic determinants for raised HbF were screened. We detected a novel KLF1 gene variant in the mother and the child in the heterozygous state. The co-inheritance of this novel KLF1 variant might have increased the HbF levels in the mother and may have ameliorated the clinical manifestations of the 6-year-old untransfused β-thalassemia homozygous child. Identification of KLF1 gene variants may act as a novel target for increasing HbF levels in patients with β-hemoglobinopathies.

The association between four SNPs (rs7482144, rs4671393, rs28384513 and rs4895441) and fetal hemoglobin levels in Chinese Zhuang β-thalassemia intermedia patients

Four SNPs (rs7482144, rs4671393, rs28384513 and rs4895441) associated with HbF levels have been identified in different populations worldwide. To explore whether these SNPs modulate HbF expression in Chinese Zhuang population, 436 Chinese Zhuang β-thalassemia intermedia (β-TI) patients were divided into high HbF level group (mean HbF=25.5%, n=218) and low group (mean HbF=6.51%, n=218) for genotyping using PCR-HRM method. Results demonstrated that there was a significantly higher minor allele frequency (MAF=34.2%) of rs4895441 (G) in HMIP in high HbF level group than that in low group (MAF=19.8%) (P=0.001, OR=1.73, 95% CI: 1.24–2.57). The cumulative effects of risk genotypes of these loci for patients carrying any combination of 1, 2 or 3 risk genotype had a gradually increased risk of high HbF level phenotype compared to those without the risk genotypes (OR=1.50–9.06, P=0.0008); Gene-gene interaction of rs7842144 and rs4895441 showed the best model with the smallest prediction error (0.4259) and the greatest consistency of coefficient of variation (P=0.01). We concluded that rs4895441, G on HMIP might be a high-risk modifier variant for high HbF level expression, and HBG2, BCL11A and HMIP genes, as HbF quantitative trait loci (QTL) could have a synergistic effect on increasing the HbF level in Chinese Zhuang β-TI patients.

Existence of HbF Enhancer Haplotypes at HBS1L-MYB Intergenic Region in Transfusion-Dependent Saudi β-Thalassemia Patients.

Background and Objectives. β-Thalassemia and sickle cell disease are genetic disorders characterized by reduced and abnormal β-globin chain production, respectively. The elevation of fetal hemoglobin (HbF) can ameliorate the severity of these disorders. In sickle cell disease patients, the HbF level elevation is associated with three quantitative trait loci (QTLs), BCL11A, HBG2 promoter, and HBS1L-MYB intergenic region. This study elucidates the existence of the variants in these three QTLs to determine their association with HbF levels of transfusion-dependent Saudi β-thalassemia patients. Materials and Methods. A total of 174 transfusion-dependent β-thalassemia patients and 164 healthy controls from Eastern Province of Saudi Arabia were genotyped for fourteen single nucleotide polymorphisms (SNPs) from the three QTL regions using TaqMan assay on real-time PCR. Results. Genotype analysis revealed that six alleles of HBS1L-MYB QTL (rs9376090C p = 0.0009, rs9399137C p = 0.008, rs4895441G p = 0.004, rs9389269C p = 0.008, rs9402686A p = 0.008, and rs9494142C p = 0.002) were predominantly associated with β-thalassemia. In addition, haplotype analysis revealed that haplotypes of HBS1L-MYB (GCCGCAC p = 0.022) and HBG2 (GTT p = 0.009) were also predominantly associated with β-thalassemia. Furthermore, the HBS1L-MYB region also exhibited association with the high HbF cohort. Conclusion. The stimulation of HbF gene expression may provide alternative therapies for the amelioration of the disease severity of β-thalassemia.

Pre-Treatment Steady-State Rheology Predicts Response to Hydroxyurea in Patients with Sickle Cell Disease

Background: Patients with sickle cell disease (SCD) have altered blood rheology due to erythrocyte abnormalities, such as increased aggregation and RBC density. The hematocrit to viscosity ratio (HVR) is a measure of RBC oxygen carrying capacity, and is reduced in SCD. Higher percent dense red blood cells (%DRBC) and lower HVR have been associated with higher rates of disease related complications. The aim of this study was to elucidate the effects of hydroxyurea (HU), fetal hemoglobin (HbF) and transfusion therapy (TF) on the rheology of patients with SCD.Methods: We measured whole blood viscosity of samples from children with SCD at low shear (45 s-1) and high shear (225 s-1) using a Brookfield cone and plate viscometer under oxygenated conditions. Venous blood samples in EDTA were analyzed within 4 hours of phlebotomy. Laboratory values were obtained with a Siemens ADVIA120 Hematology System; Hb profiles by high performance liquid chromatography. Samples were collected at routine clinic visits from SCD patients prior to initiation of therapy (n=105 for HbSS, n=77 for HbSC), then on HU at MTD, (n=30 for HbSS, n=13 for HbSC), or at least one week after TF (n=32). A paired T-test was used to assess treatment changes; correlation/trend tests were used to test association of hematological and rheology parameters. Results: Blood viscosities at low and high shear rates were significantly higher in untreated patients with HbSC compared to untreated patients with HbSS (p<0.001), while HVR was similar between patients with HbSC or HbSS. High endogenous HbF levels were significantly associated with higher HVR 225 s-1 in patients with HbSS but not HbSC. Patients with HbSS after TF had a significant improvement (rise) in their HVR at high shear (p=0.04) but not at low shear. This TF mediated rise in HVR 225 s-1 correlated with increased Hb (r=0.56, p<0.002), and decreased WBC and ANC (r= - 0.46 and r=- 0.36 respectively, p<0.05). Patients with greatest decreases in %HbS had most improvement in their HVR 225 s-1 (r = -0.36, p=0.15). HbSS patients treated with HU also had a rise in their HVR 225 s-1 (p=0.01) but not HVR 45 s-1. This improved HVR 225 s-1 was associated with typical HU induced changes in Hb, Hct, HbF and decreases in WBC and ANC.For HbSS patients treated with either HU or TF, a baseline HVR (at either shear rate) below the group mean experienced a greater rise in HVR, while a baseline HVR above the mean experienced a decline (p<0.001; Figure 1). HbSS patients with a pre-treatment %DRBC above the mean experienced a decline in %DRBC on HU at MTD, while those with a %DRBC below the mean did not (p=0.001).A similar trend was seen in HbSC patients treated with HU to MTD, but did not reach statistical significance.Patients with HbSC had significantly higher %DRBC compared to patients with HbSS (p<0.001). HbSC patients placed on HU had significantly higher baseline %DRBC than HbSC patients not treated with HU (15.8 ±13 versus 8.2± 6.5, p=0.001). However, HbSC patients treated to HU MTD did not experience a significant reduction in %DRBC. Individuals with HbSS and high endogenous HbF had significantly lower %DRBC (r= -0.36, p=0.002), and greater production of HbF on HU also resulted in lower %DRBC (p=0.036) in HbSS (n=25), but not HbSC patients (n=13).Conclusions: In HbSS patients, high endogenous HbF levels were significantly associated with higher HVR at high shear and with lower %DRBC, but not in HbSC patients. In HbSS patients, HU and TF therapy improved HVR at high shear rates but not low shear, possibly indicating persistent red cell adhesion at low shear rates. This improvement correlated with HbF levels in HU treated patients, and HbS levels in TF treated patients. Patients with SCD and greater than average impairment of whole blood rheology at pre-treatment steady-state experienced greater rheological improvement, demonstrated clinically by a reduction in %DRBC or a rise in HVR on HU. A cohort of 13 HbSC patients treated prospectively with HU did not demonstrate statistically significant rheological improvement, either a result of small sample size or the minimal rise in HbF despite HU therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Patients with Sickle Cell Disease in Sicily Have Lower Rates of End Organ Damage, Allo-Immunization and Opioid Prescription Compared to a US Cohort

Background: In developed countries, childhood mortality in sickle cell disease (SCD) is less than 5%, where the SCD morbidity and mortality burden has shifted to adults. As age increases, so do end organ complications like chronic pain. Environmental factors like health economics and genetic factors may influence such outcomes. Recent data from UK, showed higher than expected survival (ASH 2015) in the setting of a national health care system. The Hospital of Gela, Sicily has a well characterized Caucasian SCD population, which also has consistent access to health care via a nationalized system. We hypothesized that traditional disease severity markers including end organ damage, the rate of red blood cell (RBC) alloimmunization and opioid use would be lower in Sicily, compared to a modern US population of African ancestry with inconsistent access to healthcare.Methods: 90 SCD patients in Sicily were followed for over 9 years (2006-2014) to identify clinical complications, alloimmunization rates and survival. Demographics and sickle and alpha globin genotypes were documented. In the US, 632 SCD patients (excluding those with SC) had the same parameters collected as part of the Bethesda Sickle Cell Cohort Study. Laboratory and clinical complications were compared between populations according to phenotype groups (SS/SB0 or SB+ thalassemia). Clinical manifestations included hospitalizations for pain per year, RBC alloimmunization, hydroxyurea prescription rate. Statistical analysis utilized univariate comparisons (t and Chi square tests).Results: In Sicily, 51 patients had SS/SB0 and 28 SB+. All were of self-described Caucasian ancestry. No SC patients were identified in Sicily, therefore all comparisons were limited to SS/SB0 and SB+ thalassemia. Sicilian SS/SB0 patients were older than the US (p<0.0001), had lower AST (p=0.04) and creatinine (p=0.005), and higher HbF (p<0.0001). SB+ patients had similar characteristics (Table 1). Sixteen Sicilian patients (18%) died at a mean age of 53 years (range 31-77). As suggested by these survival data, SCD complications were less frequent for both SS/SB0 and SB+ groups in Sicily compared to the US (Table 2). More Sicilians were prescribed hydroxyurea, especially among those with SB+, although this was not a significant difference. The pain crisis hospitalizations per year was also higher in the US (p=0.008). Consistent with less frequent hospitalizations, only 4 of the Sicilian patients (3 SS/SB0, 1 SB+) were taking long acting opioids, compared to more frequent opioid use in the US. Finally, RBC alloimmunization, another surrogate measure for disease severity, was twice as common in SS/SB0 patients from the US (30%) as in Sicily (14%, p=0.01).Conclusions: These data suggest that end organ complications, like chronic pain and alloimmunization, in self-described Caucasians with uniform access to health care in Sicily, are less common than in a US cohort of adults. These differences could be attributable to access to specialized care, genetic differences in the proportion of African ancestry or environmental factors like more frequent use of preventative therapies like hydroxyurea. RBC alloimmunization in Sicily is significantly lower than reported in the US , but is still higher than observed in thalassemics in Italy. Perhaps this could be explained a matched genetic background between blood donors and recipients in Sicily, as has been suggested as a strategy to reduce RBC alloimmunization in the US. Further studies comparing unique SCD populations from different geographic regions may be helpful to elucidate genetic or environmental risk factors for end organ complications and disease severity. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.