Quantitative analysis of erythrocytes containing fetal hemoglobin (F cells) in children with sickle cell disease

Variation in the level of fetal hemoglobin (HbF) accounts for much of the clinical heterogeneity observed in patients with sickle cell disease (SCD). The HbF level has emerged as an important prognostic factor in both sickle cell pain and mortality, and a % HbF of 10–20% has been suggested as a threshold level for diminished clinical severity. The number of erythrocytes that contain HbF (termed F cells) may also be critically important, as F cells resist intravascular sickling and have preferential in vivo survival. Since F cells can be enumerated with high accuracy using flow cytometry methods, we prospectively studied a cohort of 242 children with SCD. Children with HbS and hereditary persistence of fetal hemoglobin (S/HPFH) had essentially 100% F cells. In contrast, children with homozygous sickle cell anemia (HbSS), HbS/β⁰ thalassemia, or HbS/β⁺ thalassemia had significantly lower mean % F cell values (55.9, 61.6, and 51.3%, respectively; P < 0.001), and children with HbSC had even fewer F cells (27.0%; P < 0.001). There was a highly significant correlation between the % F cells and the log (% HbF), which was observed for the total population of children (r = 0.95, P < 0.001), as well as for each of the individual subgroups of children with HbSS (r = 0.94, P < 0.001), HbSC (r = 0.89, P < 0.001), or HbS/β⁰ thalassemia and HbS/β⁺ thalassemia (r = 0.95, P <0.001). This logarithmic correlation between % F cells and % HbF has not been previously described and has important implications for the pharmacologic manipulation of HbF in patients with SCD. Am. J. Hematol. 54:40–46, 1997 © 1997 Wiley-Liss, Inc.     

Molecular identification of hereditary persistence of fetal hemoglobin type 2 (HPFH type 2) in patients from Brazil

The HPFH deletion type 2 was first described in a patient from Ghana and is characterized by a large deletion of approximately 105 kb extension. We report here the results obtained in studying a black Brazilian patient who presented an association of -thalassemia and HPFH type 2, using a PCR strategy for detection of the breakpoint region. This procedure allows a rapid molecular identification of this condition and is a reliable procedure for screening patients with a hematological picture of HPFH deletion types.     

Novel valproic acid derivatives with hemoglobin F inducing activity

Pharmacological induction of hemoglobin F expression may be a promising approach for the treatment of beta-thalassemia and sickle cell disease. Valproic acid, a drug frequently used for the treatment of seizure disorders, has been shown to enhance fetal hemoglobin synthesis in erythroid cells. However, this effect is only modest and requires relative high concentrations. Therefore, the drug appears not to be applicable for the treatment of beta-globin chain disorders. Here, we describe the identification of novel valproic acid derivatives with potent hemoglobin F inducing activities at concentrations that presumably can be obtained in vivo.     

Histone deacetylase inhibitor FK228 is a potent inducer of human fetal hemoglobin

We investigated the induction of the human fetal globin gene using five potent histone deacetylase (HDAC) inhibitors: FK-228, HC-Toxin, Trichostatin, MS-275, and Apicidin, using in vitro assays and cultures of primary human erythroblasts. The results showed that FK228 is the most potent inducer of fetal hemoglobin and exhibits its effects in picomolar concentrations. FK228 should be considered as a potential therapeutic for induction of fetal hemoglobin in patients with beta chain hemoglobinopathies.     

Quantitation of hemoglobin biosynthesis with agarose isoelectric focusing

Isoelectric focusing in polyacrylamide gels has been widely used to separate fetal (HbF) and adult (HbA) hemoglobins, and the relative synthesis of HbA and HbF has been estimated by fluorography or autography of dried gels. In order to measure the absolute synthesis of hemoglobins, we developed a system that utilizes isoelectric focusing in agarose and quantitates the total radioactivity of separated hemoglobins. After isoelectric focusing, the protein bands are individually eluted from the agarose and the radioactivity measured by liquid scintillation counting. We used this technique to study the synthetic capabilities of erythroid precursors at sequential times in culture. As previously reported, the relative ratio of HbF decreased over time in culture. However, our results clearly revealed that the absolute synthesis of HbF did not decrease until there was a parallel decrease in hemoglobin A, and that changes in the relative ratio occur because of disproportionate increases in HbA. This methodology, allowing independent evaluation of the radioactivity in synthesized hemoglobin, enabled us to gain new insight into the biosynthetic capabilities of erythroid precursors in clonal cell culture.     

Flow cytometric analysis of fetal hemoglobin in erythroid precursors of β‐thalassemia

Fetal hemoglobin (HbF), the major hemoglobin species in fetal life, drops to <1% in normal adults, where it is restricted to a few ‘F‐cells’, which may increase in various acquired and genetic conditions, including thalassemia. Using flow cytometry, we studied the percentage of HbF‐containing cells and their HbF content in RBC, reticulocytes (retics) and normoblasts (NRBC) present in the peripheral blood of patients with β‐thalassemia. Thiazol orange, a nucleic acid‐specific dye, and anti‐CD45 antibodies identified the various blood cells and antihuman HbF antibodies quantitated HbF. The results indicated that F‐RBC were more numerous in β‐thalassemic (both transfused and nontransfused) patients than in normal donors, but, in most cases, their HbF content was comparable, suggesting that increased HbF in thalassemia is mainly due to higher %F‐cells rather than an increased HbF per cell. Among the retics, the %F‐cells and their HbF content were highest in immature retics and decreased with maturation to levels of RBC. This may reflect preferential maturation of F‐retics into RBC in the circulation. The NRBC population contained the lowest %F‐cells. This could be due to preferential maturation of F‐NRBC, having more normal phenotype than non‐F‐NRBC, in the bone marrow into F‐retics, while non‐F‐NRBC enter the circulation.     

Acquired hemoglobin H disease in idiopathic myelofibrosis.

A 68-year-old male, diagnosed 1 year previously as having myelofibrosis, developed hemolysis, red cell inclusions, and 37% Hb H. The alpha/beta globin synthetic ratio for circulating reticulocytes, determined by 3H-leucine incorporation and globin chain separation by carboxymethylcellulose chromatography in urea, was 0.049. When total RNA was purified from peripheral blood cells and translated in a wheat germ cell-free translation system, the alpha/beta ratio of the translation products was 0.26, indicating mRNA as a major cause of the globin synthetic imbalance. This study demonstrates that myelofibrosis is one setting in which acquired Hb H disease occurs; that the synthetic imbalance may be extreme; and that it can be associated with an imbalance in the activities of specific globin mRNAs.     

5-Azacytidine and fetal hemoglobin

The evidence that 5-azacytidine stimulates the production of Hb F and F cells in baboon and man is reviewed. The mechanism of this effect is not entirely clear, but 5-azacytidine produces hypomethylation of the gamma gene at certain sites, and gene expression and DNA hypomethylation are related phenomena in many other systems. Other mechanisms have been postulated by other investigators. The therapeutic significance of increased Hb F levels in homozygous beta thalassemia and sickle cell anemia is exemplified. The potential risk of carcinogenicity has delayed more extensive clinical trials.     

Hemoglobin aggregation and pseudosickling in vitro of hemoglobin setif-containing erythrocytes

Erythrocytes from individuals heterozygous for hemoglobin Setif (α⁹⁴ Asp→Tyr) sickle in vitro without deoxygenation when incubated in chloride buffer due to hemoglobin aggregation. We now report quantitative studies of hemoglobin polymerization and deformability in these cells. Hemoglobin polymer gradually increased in intact cells during a 24 h incubation period at 24°C. After 24 hr, about 80% of the cells in 290 mosm sodium chloride buffer contained polymer which appeared as short rods compared to >99% containing polymer at 450 mOsm. Similar proportions of cells were morphologically sickled. Deformability of erythrocytes with 40% hemoglobin Setif incubated in 290 mOsm buffer at 37°C decreased to 80% of normal by 210 min but in 450 mOsm decreased to 50% after only 30 min as measured by the ektacytometer. However, at 4°C deformability remained normal even in 450 mOsm buffer. The solubility of gelled hemolysate containing 40% hemoglobin Setif was 24 g/dl and 21 g/dl at 290 and 459 mOsm buffer respectively. The gel persisted at 4°C with a solubility of 25 g/dl, but melted when dialyzed into sodium phosphate or potassium phosphate buffer. These data suggest that hemoglobin polymerization, reduced deformability, and sickling of hemoglobin Setif-containing erythrocytes are related to reduced hemoglobin solubility. The rate and extent of intracellular polymerization in vitro are considerably reduced (as in the case of sickle trait) compared with erythrocytes from individuals with sickle cell anemia. Hence, the slower kinetics of hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin polymerization and abnormal rhelogy.     

Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin

Sickle trait, the heterozygous state of normal hemoglobin A (HbA) and sickle hemoglobin S (HbS), confers protection against malaria in Africa. AS children infected with Plasmodium falciparum are less likely than AA children to suffer the symptoms or severe manifestations of malaria, and they often carry lower parasite densities than AA children. The mechanisms by which sickle trait might confer such malaria protection remain unclear. We have compared the cytoadherence properties of parasitized AS and AA erythrocytes, because it is by these properties that parasitized erythrocytes can sequester in postcapillary microvessels of critical tissues such as the brain and cause the life-threatening complications of malaria. Our results show that the binding of parasitized AS erythrocytes to microvascular endothelial cells and blood monocytes is significantly reduced relative to the binding of parasitized AA erythrocytes. Reduced binding correlates with the altered display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface. These findings identify a mechanism of protection for HbS that has features in common with that of hemoglobin C (HbC). Coinherited hemoglobin polymorphisms and naturally acquired antibodies to PfEMP-1 may influence the degree of malaria protection in AS children by further weakening cytoadherence interactions.     

Association in cis of beta +-thalassemia and hemoglobin S

A Moroccan woman was investigated because of a typical beta-thalassemia trait associated with a low-percentage (11%) hemoglobin (Hb) variant. The beta-thalassemia trait was manifested by a microcytosis, a high HbA2 (above 6%), and an increase of the alpha/beta biosynthetic ratio (1.31). The variant was identified to HbS by amino acid analysis of the abnormal peptide (beta T1) and by DNA mapping with Sau I (Mst II) restriction endonuclease. No additional amino acid substitution was recorded in the beta s-chain. The reduction of beta-globin synthesis occurred exclusively at the expense of the beta s-chain. These results are consistent with the existence of a beta s mutation and a beta +-thalassemia in cis.     

HbS/βdel-thalassemia associated with high levels of hemoglobins A2 and F in a Turkish family

β-thalassemia and sickle cell disease (SCD) are common disorders in Turkey. Compound heterozygosity for these two disorders (β^S/β-thalassemia) is encountered frequently. In this report we present hematological and molecular data of two Turkish siblings with β^S/β^del-thalassemia caused by a 290 base pair (bp) deletion and associated with increased levels of hemoglobin A₂ (HbA₂) and hemoglobin F (HbF). Clinical analysis of the two patients showed a mild course of the disease. Haplotypic factors involved in increasing the levels of HbF were analyzed. The two patients showed no changes from the normal sequences at the XmnI site of Gγ-globin promoter and the (AT)_xT_y microsatellite 5′ to the β-globin mRNA cap site. The removal of the region between positions −125 to +78 relative to the β-globin gene mRNA cap site by the 290 bp deletion is thought to allow the β-locus control region to interact with the promoters of the δ- and γ-globin genes, leading to increased HbA₂ and HbF levels. Am. J. Hematol. 59:83–86, 1998. © 1998 Wiley-Liss, Inc.     

Ten novel mutations in the erythroid transcription factor KLF1 gene associated with increased fetal hemoglobin levels in adults

We investigated whether mutations in the KLF1 gene are associated with increased Hb F levels in ethnically diverse patients referred to our laboratory for hemoglobinopathy investigation. Functionally effective KLF1 mutations were identified in 11 out of 131 adult samples with an elevated Hb F level (1.5-25.0%). Eleven different mutations were identified, 9 of which were previously unreported. KLF1 mutations were not identified in a matched cohort of 121 samples with normal Hb F levels (<1.0%). A further novel KLF1 mutation was also found in a sickle cell disease patient with a Hb F level of 20.3% who had a particularly mild phenotype. Our results indicate KLF1 mutations could make a significant contribution to Hb F variance in malarial regions where hemogobinopathies are common. All the mutations identified were heterozygous providing further in vivo evidence that a single altered KLF1 allele is sufficient to increase Hb F levels.     

Intracranial extramedullary hematopoiesis in β°-thalassemia/hemoglobin E disease

A 27-year-old woman with β-thalassemia/hemoglobin E disease presented with an intracranial mass of 10 × 9 × 1.2 cm firmly attached to the dura and the falx cerebri extending over the central part of both hemispheres, with pressure atrophy of the underlying brain. Histopathologic examination revealed that the mass consisted entirely of hematopoietic cells. We have seen 1,315 cases with β-thalassemia/hemoglobin E, but this was the first case with intracranial extramedullary hematopoietic mass.     

Hypocholesterolemia in adult patients with thalassemia: a link with the severity of genotype in thalassemia intermedia patients

Objectives: Hypocholesterolemia has been previously described in patients affected by thalassemia. In this study we retrospectively evaluated the cholesterol level in two groups of patients affected by either thalassemia major (TM) or thalassemia intermedia (TI), with the aim of establishing factors correlated to hypocholesterolemia within both populations. Methods: All patients referring to our Unit of Thalassemia were considered. Observation time, defined as the interval between the first and last set of laboratory test, was 2 yr (January 2005–December 2006). Results: We found that patients with TI had significantly lower cholesterol and hemoglobin level as compared with TM patients. In addition there was no correlation between groups with identical genotype and cholesterol levels in both populations. However, within the TI group, lower values of cholesterol were found in patients with more severe genotype and lower body mass index. Conclusions: Our data support the hypothesis that, independently from single genotype involved, the reduced level of cholesterol in patients with TI are sustained by active erythropoiesis which increases cholesterol requirement.