Fetal Hemoglobin Synthesis Research Articles

Fetal Hemoglobin Synthesis Determined by γ-mRNA/γ-mRNA + β-mRNA Quantitation in Infants at Risk for Sudden Infant Death Syndrome Being Monitored at Home for Apnea

Fetal hemoglobin (HbF) levels in the hemolysates obtained from infants who died from sudden infant death syndrome (SIDS) are reported to be markedly increased compared with controls. This finding could have been explained by increased HbF synthesis caused by episodes of hypoxemia in the SIDS infants. A prospective study in a group of infants being monitored at home after an apparent life-threatening event (ALTE) and considered at increased risk for SIDS was conducted with an improved ribonuclease protection assay. The ribonuclease protection assay allowed for the quantitation of [gamma/(gamma+beta)]-globin mRNAs, which has a highly significant correlation with the levels of HbF synthesis. Thirty-five infants who were admitted for an ALTE were included in the study. All infants were at home under surveillance with a cardiorespiratory monitor and followed in an apnea clinic with monthly appointments. Seventy-three blood samples were obtained between 38 and 61 weeks of postconceptional age. For control purposes, a similar group of 37 normal infants (99 samples) whose HbF synthesis was previously determined were included. Mean [gamma/(gamma+beta)]-globin mRNAs were increased in the ALTE group at 42 to 45 and 46 to 49 weeks of postconceptional age (mean: 55.2 +/- 17.4% and 33.9 +/- 14%) in comparison with HbF synthesis in controls (mean: 42.6 +/- 13.7% and 23.6 +/- 9.8%). The data obtained in this report from infants who were considered at risk for SIDS show that HbF synthesis is increased between 42 and 49 weeks of postconceptional age. Determining HbF synthesis as described in this study may have value as a marker for episodes of hypoxemia for certain infants who are at risk for SIDS.

Effect of hydroxyurea on G gamma chain fetal hemoglobin synthesis by sickle-cell disease patients.

Hydroxyurea is used for sickle-cell disease patients in order to increase fetal hemoglobin synthesis and consequently decrease the severity of pain episodes. Fetal hemoglobin, which is formed by gamma-globin chains A and G, is present in a constant composition throughout fetal development: about 75% of Ggamma and 25% of Agamma. In contrast, adult red cells contain about 40% of Ggamma and 60% of Agamma. In the present study, we analyzed the effect of hydroxyurea induction on the gamma chain composition of fetal hemoglobin in 31 sickle-cell disease patients treated with hydroxyurea. The control group was composed of 30 sickle-cell disease patients not treated with hydroxyurea in clinical steady state. The patients were older than 13 years and were not matched for age. All patients were seen at Hemocentro/UNICAMP and Boldrini Infantile Center, Campinas, SP, Brazil. The levels of total hemoglobin were significantly higher in patients treated with hydroxyurea (mean +/- SD, 9.6+/-2.16 g/dl) than in untreated patients (8.07+/-0.91 g/dl). Fetal hemoglobin levels were also higher in treated patients (14.16+/-8.31%) than in untreated patients (8.8+/-4.09%), as was the Ggamma/Agamma ratio (1.45+/-0.78 vs 0.98+/-0.4, P < 0.005). The increase in the Ggamma/Agamma ratio in patients treated with hydroxyurea suggests the prevalence of a pattern of fetal hemoglobin synthesis, whereas patients not treated with hydroxyurea maintain the adult pattern of fetal hemoglobin synthesis. Because no correlation was observed between the Ggamma/Agamma ratio and total hemoglobin or fetal hemoglobin levels, the increase in Ggamma chain synthesis may not imply a higher production of hemoglobin.

Increase of Fetal Hemoglobin Synthesis Indicating Differentiation Induction in Children Receiving Valproic Acid

Differentiation induction is a distinct concept in the treatment of malignant diseases, considering that malignant cells share many features with immature progenitor cells that are capable of terminal differentiation. Treatment of tumor cells with short-chain fatty acids leads to cytostasis and differentiation induction both in vitro and in vivo. Similarly, short-chain fatty acid treatment of erythroid progenitors in vitro and in vivo induces cellular differentiation resulting in n -globin, i.e., fetal hemoglobin synthesis. Valproic acid (VPA) is a branched-chain fatty acid that is able to inhibit growth of human and rodent tumor cells and to induce a mature phenotype. The antitumoral effects observed in preclinical studies were reached at concentrations that are readily achieved in patients treated with VPA for epilepsy. Hypothesizing that anticonvulsive VPA levels may be used for antitumoral differentiation induction therapy of pediatric malignant tumors, the authors studied fetal hemoglobin-inducing capacity of VPA in children treated with VPA for epilepsy. Fetal hemoglobin was significantly increased in 30 children with epilepsy treated with VPA monotherapy for at least 3 months when compared to untreated control patients. Furthermore, fetal hemoglobin levels correlated with VPA serum levels. The study confirms the dose-dependent stimulating effect of VPA on fetal hemoglobin synthesis at anticonvulsive doses. The results suggest that nontoxic VPA levels reached in pediatric epilepsy patients should be capable of inducing cellular differentiation of pediatric malignant tumors for therapeutic purposes. Broad clinical experience with VPA and its low toxicity further encourage the evaluation of VPA in pediatric oncology for differentiation induction therapy.

Increase of Fetal Hemoglobin Synthesis Indicating Differentiation Induction in Children Receiving Valproic Acid

Increase of Fetal Hemoglobin Synthesis Indicating Differentiation Induction in Children Receiving Valproic Acid

Induction of fetal hemoglobin synthesis by valproate: modulation of MAP kinase pathways.

Valproate has been found to stimulate fetal hemoglobin (HbF) synthesis in patients with sickle cell disease. In accordance with these clinical observations, we found a moderate induction of HbF synthesis in K562 erythroid cells in vitro. Investigation of the role of the mitogen-activated protein kinase (MAPK) pathways by Western blot analysis and use of specific kinase inhibitors suggests that inhibition of ERK pathway and activation of the p38 pathway may contribute to the HbF-inducing activity of valproate.

Induction of Fetal Hemoglobin by Propionic and Butyric Acid Derivatives: Correlations between Chemical Structure and Potency of Hb F Induction

ABSTRACT Short-chain fatty acids (C2-C9) induce fetal hemoglobin synthesis in primary cell cultures, primates, and patients. We carried out experiments to test whether relationships exist between chemical structure and the Hb F-inducing potential of several short-chain fatty acid derivatives. BFUe cultures were performed in the presence of propionic and butyric congeners, covering the full spectrum of substitutions of these molecules, including polar and non-polar groups, esters, and double bonds. We found that the fetal hemoglobin inducibility is related to the chemical structure of the inducing compound. This structure–activity relation depends on the length of carbon chain, the nature of the substitutions, and the position of more potent substitutions on the carbon chain. It appears that substitutions enhancing the inducibility of these compounds are (with decreasing potency): methyl > phenyl > hydroxy ⪢ amino groups. Placement of these substitutions at a position distal to the carboxyl group enhances γ-globin inducibility. Presence of the carboxyl group is prerequisite for γ-globin inducibility.

The Challenge of Painful Crisis in Sickle Cell Disease

ACUTE PAINFUL CRISIS IN SICKLE CELL DISEASE IS A FREquent complication and considerably diminishes the quality of life of patients with this disease. The pain of sickle cell crisis is one of the most intense in medicine—it has been compared with that of a severe toothache—and affects the extremities, abdomen, lower back, or multiple sites simultaneously. The unpredictability of such painful episodes adds to their discomfort. Some major precipitating factors and aggravating circumstances that contribute to the complex pathophysiology of acute painful crisis include infection, dehydration, excessive exercise, emotional disturbances, or stressful environments. Furthermore, lack of prompt and individualized acute pain treatment increases hospitalization rates as well as the duration and intensity of painful episodes. Even though sickle cell disease was discovered in 1910 and defined at the molecular level in the 1950s, it was not until 1995 that a proven ameliorating therapy was discovered. In a randomized controlled trial, hydroxyurea was shown to decrease the incidence of painful crises. The mechanisms by which hydroxyurea provides benefit are likely to be multiple and complex, including a modest increase in fetal hemoglobin. Efforts are under way to identify a more effective “hydroxyurea-type” approach with administration of arginine butyrate, which induces fetal hemoglobin synthesis more rapidly and more effectively than hydroxyurea. Attempts to develop curative measures for sickle cell disease appear promising but, thus far, have had limited success. Bone marrow transplantation is associated with small but significant mortality, difficulty in identifying donors, and shortand long-term transplant-related complications, particularly among older patients with lifelong complications. Encouraging advances in the prospects for gene therapy for sickle cell disease that involve allogeneic instead of heterologous transplantation have been reported recently. Thus far, testing other potentially ameliorating agents has been relatively unsuccessful. Use of urea was proven to be ineffective, use of cyanate too toxic, the effect of citiedil citrate too modest, and use of methylprednisolone too controversial, with continuing doubts about its efficacy and concerns about adverse effects. In this issue of THE JOURNAL, Orringer and colleagues report the results of a multicenter investigation evaluating the use of purified poloxamer 188 (PP188) for treatment of sickle cell painful crises. Polaxamer 188 is a nonionic surfactant with hemorheological and antithrombotic properties that putatively should improve microvascular flow by reducing bulk viscosity and adhesive frictional forces. In their carefully conducted placebo-controlled trial involving 255 adults and children with painful sickle cell crisis severe enough to require narcotic analgesics and hospital admission, Orringer et al demonstrated a modest reduction in the duration of painful crises (ie, from time of randomization to time of crisis resolution) in the PP188 group (6.4% reduction; 5.5 vs 5.9 days) and report that a greater proportion of patients in the treatment group achieved the protocolspecified end point of resolution of the painful crises within 168 hours (7 days) (51.6% vs 36.6%). Results for children aged 15 years or younger (n=73) were somewhat better, with a statistically significant reduction in painful crisis duration (5.3 vs 6.2 days) and a greater proportion achieving resolution of the painful crisis within 7 days (59.5% vs 27.8%). The results reported by Orringer et al are important but modest, suggesting that it is possible that the potential rheological effects of PP188 were not optimized in this study. However, lack of clear understanding about the mechanism of action of this drug, as acknowledged by the authors, reduces the chances of optimization under the present treatment protocol. Moreover, it would be important not to optimize the untoward effects of PP188 (ie, inhibition of platelet function), particularly since adults with sickle cell disease are at risk of episodes of cerebral hemorrhage. The authors note that the overall incidence of adverse effects of the presently available drug was similar in the treatment and placebo groups and point out that there was no evidence of increased risk of bleeding among patients receiving the study drug. Other efforts directed toward the amelioration of painful sickle cell crisis, such as use of strategies to inhibit vaso-

A new anti-hemoglobin F antibody against synthetic peptides for the detection of F-cell precursors (F-blasts) in bone marrow.

To date there are few antibodies available for the detection of fetal hemoglobin (HbF)-containing erythroblasts (F-blasts) in paraffin-embedded hematopoietic tissues. Recently, we developed a new polyclonal antibody specific to F-blasts by immunization of a rabbit with synthetic peptides of human HbF. Specificity and reactivity of the antibody were confirmed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. ELISA confirmed that the antibody showed strong immunoreactivity to fetal hemoglobin but no reaction to adult hemoglobin. The antibody could detect the presence of fetal blood hemolysates (10 microg/mL) at a dilution of 10(-5). According to immunohistochemical analysis, there were strong positive reactions to fetal erythroblasts in the liver and the spleen at 29 weeks of gestation and to erythroblasts from patients with myelodysplastic syndromes and erythroleukemia but no reactions to normal adult erythroblasts in bone marrow. Fetal erythrocytes in fetal blood vessels of placental tissues were strictly distinguished from maternal erythrocytes in the same sections by their positive reactions, indicating the presence of HbF. This new antibody will be a useful tool for studying fetal hemoglobin synthesis and for detecting F-blasts in archival specimens of various hematological diseases.

Role of ERK Activation in Growth and Erythroid Differentiation of K562 Cells

Inhibition of signaling through Ras in BCR-ABL-positive pluripotent K562 cells leads to apoptosis and spontaneous differentiation. However, Ras-induced activation of the mitogen-activated protein kinase ERK has been suggested to play a critical role in either growth or differentiation in different model systems. We studied the role of ERK activation in the growth-promoting and anti-apoptotic effect of Ras and its involvement in hemin-induced nonterminal erythroid differentiation using the BCR-ABL-positive K562 cell line as a model. K562 cells were stably transfected with ERK1 or the dominant inhibitory mutant of ERK1 (ERK1-KR). Overexpression of ERK1-KR inhibited cell growth with an approximately fourfold increase in doubling time and induced apoptosis in K562 cells. Incubation with the MEK1 inhibitor UO126 inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner as well. In the presence of exogenously added hemin, K562 cells differentiate into erythroblasts, as indicated by the production of large amounts of fetal hemoglobin. We examined the activation of MAP kinases during hemin-induced differentiation. The ERK1 and 2 activity increased within 2 h post hemin treatment and remained elevated for 24–48 h. During this time, fetal hemoglobin synthesis also increases from 0.8 to 10 pg/cell. There was no activation of JNK or p38 protein kinases. The hemin-induced accumulation of hemoglobin was inhibited in ERK1-KR overexpressing cells and was enhanced in the wild-type ERK1 transfectants. Our results suggest that ERK activation is involved in both growth and hemin-induced erythroid differentiation in the BCR-ABL-positive K562 cell line.

Pharmacologic Induction of Fetal Hemoglobin Synthesis: Cellular and Molecular Mechanisms

Pharmacologic Induction of Fetal Hemoglobin Synthesis: Cellular and Molecular Mechanisms

Pharmacologic Induction of Fetal Hemoglobin Synthesis: Cellular and Molecular Mechanisms

The switch from embryonic to fetal then to adult hemoglobin synthesis is a unique phenomenon during early human development. Fetal hemoglobin (Hb F) is known to interfere with polymerization of Hb S in erythrocytes. Several pharmacologic agents such as 5-azacytidine, myleran, hydroxyurea, erthropoietin, and butyrates enhance fetal hemoglobin production and have been used in hemoglobinopathy patients to ameliorate severe pain episodes and reduce severe anemia. Among these, hydroxyurea is the agent of choice because of its safety and ease of administration. One of the primary cellular mechanisms involved in pharmacologic induction of Hb F synthesis is rapid regeneration of erythroid precursors following the cytoreduction phase of certain pharmacologic agents. Molecular mechanisms involving changes in chromatin structure and/or transcription factor binding have been demonstrated for gamma gene induction by butyrate. Identifying the proteins involved in gamma gene activation by various compounds may offer a new strategy for gene therapy to cure hemoglobinopathy disorders.

Delivering Erythropoietin through Genetically Engineered Cells

Abstract. Erythropoietin (Epo) is a glycoprotein hormone produced by genetic engineering. Many pathologic conditions could benefit from its administration, such as chronic renal failure or hemoglobinopathies. Epo secretion from genetically modified tissued could be proposed to patients only if the protocol is low cost and low risk. For that purpose, retroviral vectors and adeno-associated vectors expressing the Epo cDNA were developed. Gene transfer was performed into skeletal muscles. To avoid polycythemia, a tetracycline-regulated system was used to control the levels of protein secretion in vivo. β-thalassemias are among diseases that could benefit from an Epo gene transfer. β-thalassemias are attributable to deficient synthesis of β-globin and accumulation of unpaired α-chains. Stimulation of fetal globin synthesis is one strategy to correct the globin chain imbalance. There is evidence that Epo could play this role. In a mouse model of β-thalassemia, an adeno-associated vector expressing the Epo cDNA was injected intramuscularly. Epo was secreted continuously during at least 1 yr. Erythropoiesis was improved in those mice by increasing the synthesis of fetal hemoglobin.

Improvement of erythropoiesis in β-thalassemic mice by continuous erythropoietin delivery from muscle

beta-Thalassemias are highly prevalent genetic disorders that can cause severe hemolytic anemia. The main pathophysiologic feature of beta-thalassemia is the accumulation of unpaired alpha-globin chains in erythrocyte precursors and red blood cells (RBCs). This accumulation alters cell membrane function and results in early cell destruction and ineffective erythropoiesis. Correction of globin chain imbalance through the induction of fetal hemoglobin (HbF) synthesis is a tentative therapeutic approach for this class of diseases. In short-term in vitro or in vivo assays, recombinant human erythropoietin increases the frequency of erythroid precursors programmed to HbF in humans and to beta-minor globin in mice. In contrast, long-term treatment of beta-thalassemic patients did not induce HbF significantly. We took advantage of highly efficient adeno-associated virus-mediated (AAV-mediated) gene transfer into mouse muscle to induce a robust and sustained secretion of mouse erythropoietin in beta-thalassemic mice, which represent a suitable model for human beta-thalassemia intermedia. A 1-year follow-up of 12 treated animals showed a stable correction of anemia associated with improved RBC morphology, increased beta-minor globin synthesis, and decreased amounts of alpha-globin chains bound to erythrocyte membranes. More effective erythropoiesis probably accounted for a reduction of erythroid cell proliferation, as shown by decreased proportions of circulating reticulocytes and by reduced iron 59 ((59)Fe) incorporation into erythroid tissues. This study indicates that the continuous delivery of high amounts of autologous erythropoietin induced a sustained stimulation of beta-minor globin synthesis and a stable improvement of erythropoiesis in the beta-thalassemic mouse model. (Blood. 2000;95:2793-2798)

Induction of fetal hemoglobin synthesis with recombinant human erythropoietin in anemic patients with heterozygous beta‐thalassemia during pregnancy

Objective: Recombinant human erythropoietin (rhEPO) increases fetal hemoglobin synthesis in nonpregnant thalassaemic patients. We used rhEPO in 4 pregnant patients with heterozygous β-thalassemia and anemia to study its effect on erythropoiesis, F cell production, and HbF synthesis. Methods: Patients were treated with a combination therapy of rhEPO and iron. The effect on HbF synthesis was assessed by the percentage of F reticulocytes, F cells, and total HbF, erythropietis by reticulocyte count, and hemoglobin measurements and iron status by ferritin levels, transferrin saturation, and percentage of hypochromic red cells. Results: RhEPO caused an increase of F reticulocytes (1.5 to 10.5 fold), F cells (5.0 to 7.7 fold), and HbF (1.4 to 2.2 fold). All patients showed an increase of young, immature reticulocytes and had elevated reticulocytes at the end of therapy. Hemoglobin increased with a range from 0.3 to 1.5 g/dL. Transferrin saturation and ferritin levels were normal at the end of the study. There was an increase of the percentage of hypochromic red cells, indicating functional iron deficiency after rhEPO administration despite supplemental iron. Conclusions: RhEPO stimulates both HbF synthesis and erythropoiesis in pregnant patients with heterozygous β-thalassemia and anemia. Since it is known that high HbF levels ameliorate thalassemia symptoms in nonpregnant patients, use of rhEPO for the treatment of severe anemia in thalassaemic patients during pregnancy might be further evaluated. J. Matern.-Fetal Med. 1999;8:1–7. © 1999 Wiley-Liss, Inc.

Induction of fetal hemoglobin synthesis with recombinant human erythropoietin in anemic patients with heterozygous beta-thalassemia during pregnancy.

Recombinant human erythropoietin (rhEPO) increases fetal hemoglobin synthesis in nonpregnant thalassaemic patients. We used rhEPO in 4 pregnant patients with heterozygous beta-thalassemia and anemia to study its effect on erythropoiesis, F cell production, and HbF synthesis. Patients were treated with a combination therapy of rhEPO and iron. The effect on HbF synthesis was assessed by the percentage of F reticulocytes, F cells, and total HbF, erythropietis by reticulocyte count, and hemoglobin measurements and iron status by ferritin levels, transferrin saturation, and percentage of hypochromic red cells. RhEPO caused an increase of F reticulocytes (1.5 to 10.5 fold), F cells (5.0 to 7.7 fold), and HbF (1.4 to 2.2 fold). All patients showed an increase of young, immature reticulocytes and had elevated reticulocytes at the end of therapy. Hemoglobin increased with a range from 0.3 to 1.5 g/dL. Transferrin saturation and ferritin levels were normal at the end of the study. There was an increase of the percentage of hypochromic red cells, indicating functional iron deficiency after rhEPO administration despite supplemental iron. RhEPO stimulates both HbF synthesis and erythropoiesis in pregnant patients with heterozygous beta-thalassemia and anemia. Since it is known that high HbF levels ameliorate thalassemia symptoms in nonpregnant patients, use of rhEPO for the treatment of severe anemia in thalassaemic patients during pregnancy might be further evaluated.

Induction of Fetal Hemoglobin Synthesis With Recombinant Human Erythropoietin in Anemic Patients With Heterozygous Beta-Thalassemia During Pregnancy

Induction of Fetal Hemoglobin Synthesis With Recombinant Human Erythropoietin in Anemic Patients With Heterozygous Beta-Thalassemia During Pregnancy

Heme Arginate Therapy forBeta Thalassemia:In vitro versus in vivo Effects

Hemin has a profound effect on erythroid cell maturation and promotes fetal hemoglobin synthesis in vitro. In β-thalassemia, increasing fetal hemoglobin levels can ameliorate the anemia. We administered heme arginate, a novel stable form of hemin, to 4 patients with thalassemia intermedia and studied the in vitro versus in vivo effects. In erythroid cultures, there was a marked rise in total hemoglobin and hemoglobin F. In vivo, 3 of 4 patients had a rise in hemoglobin levels (from 0.4 to 1.1 g%), which was statistically significant in 1 patient. There were no serious adverse effects. Heme arginate may be useful in the treatment of thalassemia intermedia.

Fetal gene reactivation

Reactivation of silent fetal or embryonic genes could be used for the treatment of genetic diseases caused by mutations of genes normally expressed during the adult stage of development. A paradigm of this approach is the activation of fetal hemoglobin synthesis in adult individuals and its use in the treatment of β chain hemoglobinopathies. The current understanding of the molecular control of the β globin locus is reviewed, as are the cellular and molecular basis of induction of fetal hemoglobin in the adult and the approaches used for stimulation of fetal hemoglobin synthesis in patients with β chain hemoglobinopathies.

Hemoglobin F in myelodysplastic syndrome.

Reactivation of fetal hemoglobin synthesis in adulthood can be seen in hematological disorders affecting the erythropoietic system. The objective of the present study was to evaluate the incidence and prognostic significance of increased hemoglobin F in patients with myelodysplastic syndrome. Hemoglobin F concentrations and Ggamma/Ggamma + A gamma-globin chain ratios were determined in 26 patients with primary myelodysplastic syndrome. Median age of the patients was 65 years; all FAB subtypes were included. Increased hemoglobin F concentration of up to 20% of total hemoglobin (normal: below 2%) was seen in 16 patients; ten patients had normal values. There was a significant relation between hemoglobin F concentration and the course of disease, e.g., 12 of the 16 patients with elevated hemoglobin F survived at least 1 year after the examination, in contrast to only three of the ten patients with normal hemoglobin F (p < 0.025). All of six patients with hemoglobin F above 5% survived at least 1 year. There was no significant difference in the hemoglobin F concentration between patients with and without cytogenetic anomalies. The Ggamma/Ggamma + A gamma-globin chain ratio was slightly elevated in all patients, with a weak correlation to the degree of hemoglobin F elevation. The values were not of additional prognostic significance. The data of the present study suggest that the hemoglobin F concentration may be a prognostic parameter in myelodysplastic syndrome; increased hemoglobin F concentration may indicate a better prognosis.

Valproic Acid and Augmentation of Fetal Hemoglobin in Individuals With and Without Sickle Cell Disease

To the Editor: Increased synthesis of fetal hemoglobin may ameliorate the clinical severity of sickle cell disease1; both chemotherapeutic and nonchemotherapeutic agents have been shown to augment fetal hemoglobin synthesis in selected populations of affected patients.[2][1] ,[3][2] Some studies