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Beta(0)-thalassaemia intermedia (beta(0)-TI) describes patients who lack beta-globin synthesis yet manifest a non-transfusion-dependent form of beta-thalassaemia. Co-inheritance of alpha-thalassaemia, certain variants of the beta-like globin gene cluster and elevated fetal haemoglobin (HbF) production are all associated with beta(0)-TI. However, the mild phenotypes of many beta(0)-TI patients are unexplained. Genetically determined HbF levels in beta-thalassaemia are difficult to assess because erythrocytes containing HbF (F cells) preferentially survive over erythrocytes without HbF. To evaluate the importance of genetically elevated HbF in beta-thalassaemia, F-cell levels of 19 TI patients' relatives were compared with relatives of transfusion-dependent beta-thalassaemia major patients and those of beta-globin genotype-matched controls. The beta-globin and alpha-globin genotypes, as well as their Ggamma promoter were also examined. Using this approach, in all but one patient the mild phenotype was attributable to either alpha-globin genotype, gamma-globin promoter polymorphism or inherited elevated F-cell levels. The findings of this study establish the F-cell levels required to modify the degree of disease severity significantly and demonstrate that F-cell level is a crucial parameter in the understanding of phenotypic variation in beta-thalassaemia.

Sodium phenylbutyrate (PB) is an aromatic fatty acid with cytostatic and differentiating activity against malignant myeloid cells (ID(50), 1-2 mM). Higher doses induce apoptosis. Patients with myelodysplasia (n = 11) and acute myeloid leukemia (n = 16) were treated with PB as a 7-day continuous infusion repeated every 28 days in a Phase I dose escalation study. The maximum tolerated dose was 375 mg/kg/day; higher doses led to dose-limiting reversible neurocortical toxicity. At the maximum tolerated dose, PB was extremely well tolerated, with no significant toxicities; median steady-state plasma concentration at this dose was 0.29 +/- 0.16 mM. Although no patients achieved complete or partial remission, four patients achieved hematological improvement (neutrophils in three, platelet transfusion-independence in one). Other patients developed transient increases in neutrophils or platelets and decrements in circulating blasts. Monitoring of the percentage of clonal cells using centromere fluorescence in situ hybridization over the course of PB administration showed that hematopoiesis remained clonal. Hematological response was often associated with increases in both colony-forming units-granulocyte-macrophage and leukemic colony-forming units. PB administration was also associated with increases in fetal erythrocytes. These data document the safety of continuous infusion PB and provide preliminary evidence of clinical activity in patients with myeloid malignancies.

Background: Iron deficiency anemia (IDA) in young children is important to identify because of its adverse effects on behavior and development. Because of costs and inconvenience associated with blood test screening and the decline in prevalence of IDA, the Institute of Medicine and the Centers for Disease Control and Prevention recommend that blood test screening for IDA be targeted to children first identified by dietary and health history., Objective: To evaluate a parent-completed dietary and health history as the first stage of 2-stage screening for IDA., Design and Methods: A cross-sectional study was conducted in inner-city clinics in children 9 to 30 months old having routine anemia screening as part of a scheduled visit. Parents completed a questionnaire and children had venous blood sampling for complete blood count and ferritin. Anemia was defined as Hb <11.0 g/dL. Iron deficiency (ID) was defined as ferritin <10 microg/L or mean corpuscular volume <70 fL and red cell distribution width >14.5%. Children were categorized into 1 of 4 groups: iron-sufficient, not anemic (ISNA); iron-sufficient, anemic (ISA); iron-deficient, not anemic (IDNA); and iron-deficient anemic (IDA). The questionnaire consisted of 15 dietary items in domains of infant diet, intake of solid food, intake of beverages, and participation in the Special Supplemental Nutrition Program for Women, Infants, and Children together with 14 historical items in domains of birth history, recent illness, chronic medical conditions, history of anemia, and maternal history. Analysis was performed on individual items, domains, and combinations of selected items., Results: In the 282 study subjects, the prevalence of anemia (35%), IDNA (7%), and IDA (8%) did not vary significantly by age. Among individual historical and dietary questions, maternal history of anemia and drinking >2 glasses of juice per day identified the highest proportion of children with IDA: 50% sensitivity (95% confidence interval [CI]: 16,81) and 77% sensitivity (95% CI: 54,89), respectively. However, specificities for these questions were 60% (95% CI: 55,65) and 22% (95% CI: 17,27), respectively. Domains of questions with the highest sensitivity for IDA were beverage intake (91%; 95% CI: 68,99) and intake of solid food (91%; 95% CI: 68,99). However, specificities of the domains were only 14% (95% CI: 10,18) and 29% (95% CI: 24,35), respectively. The dietary items used by Boutry and Needlman were 95% (95% CI: 77, 99) sensitive but only 15% (95% CI: 11,19) specific for IDA. The recommendations of the Centers for Disease Control and Prevention for health and dietary screening were 73% (95% CI: 56,92) sensitive and 29% (95% CI: 24,35) specific for IDA. The individual questions, domains of questions, and interdomain groups of questions had similar sensitivity and specificity for anemia and ID (IDA + IDNA)., Conclusion: In this high-risk population, neither individual nor combinations of parental answers to dietary and health questions were able to predict IDA, anemia, or ID well enough to serve as a first-stage screening test.

In this project we have prospectively studied the erythropoietic activity in patients with sickle cell anaemia (SS) before and after treatment with hydroxyurea (HU). Some of the patients were enrolled in a double-blind placebo controlled trial of HU in patients with SS and others were enrolled in an open label study. Determinants of erythropoietic activity included the reticulocyte count, red blood cell (RBC) survival by the 51Cr method, plasma 59Fe clearance, plasma iron turnover (PIT), erythron transferrin uptake (ETU), RBC production/destruction rate, and RBC Fe utilization. Therapy with HU increased the mean corpuscular volume (MCV), haemoglobin (Hb)F, RBC survival and t1/2 59Fe clearance; it decreased the reticulocyte count, the white blood cell (WBC) count, ETU, and the PIT. Most of the changes in parameters of erythropoiesis could be explained by the increase in 51Cr RBC survival after therapy with HU. Together the data showed that in selected patients the net effect of HU on Hb level was a function of the difference between the suppressive effect of HU (decreased RBC production) and the increase in RBC survival. In the majority of patients who responded to HU, there was a preferential effect on RBC survival.

High levels of fetal hemoglobin (Hb F) protect from many of the complications of sickle cell disease and lead to improved survival. Butyrate and other short chain fatty acids were previously shown to increase Hb F production in erythroid cells in vitro and in animal models in vivo. However, butyrates are also known to inhibit the proliferation of many cell types, including erythroid cells. Experience with the use of butyrate in animal models and in early clinical trials demonstrated that the Hb F response may be lost after prolonged administration of high doses of butyrate. We hypothesized that this loss of response may be a result of the antiproliferative effects of butyrate. We designed a regimen consisting of intermittent or pulse therapy in which butyrate was administered for 4 days followed by 10 to 24 days with no drug exposure. This pulse regimen induced fetal globin gene expression in 9 of 11 patients. The mean Hb F in this group increased from 7.2% to 21.0% (P <.002) after intermittent butyrate therapy for a mean duration of 29.9 weeks. This was associated with a parallel increase in the number of F cells and F reticulocytes. The total hemoglobin levels also increased from a mean of 7.8 g/dL to a mean of 8.8 g/dL (P <.006). The increased levels of Hb F were sustained in all responders, including 1 patient who has been on pulse butyrate therapy for more than 28 months. This regimen, which resulted in a marked and sustained increase in Hb F levels in more than two thirds of the adult sickle cell patients enrolled in this study, was well tolerated without adverse side effects. These encouraging results require confirmation along with an appropriate evaluation of clinical outcomes in a larger number of patients with sickle cell disease.

Purpose: To anticipate the clinical challenges and financial risks facing physicians and managed care organizations who care for children with chronic illnesses, such as sickle cell anemia (SCA), under capitated managed care arrangements., Patients and Methods: A cross-sectional study based on claims data from the Washington State Medicaid Program (WSMP) and the Federal Employees Health Benefits Program (FEP). Expenditure patterns were compared for children 18 years of age or younger for whom a claim with a diagnosis of SCA was submitted and paid in the State of Washington during fiscal year 1993 (FY1993) or by the FEP during FY1992 to expenditure patterns for all children., Results: Children with SCA had mean expenditures 8.8 times the mean expenditures for all children in WSMP. There was wide variation in the annual expenditures among children with SCA; the most expensive 10% of children accounted for 56% of total expenditures. Ninety-seven percent of the expenditures were concentrated in four broad categories: 72% for inpatient care, 11% for outpatient care, 11% for physician payments, and 3% for prescription drugs. Examination of expenditure and utilization patterns for children with sickle cell anemia enrolled in the FEP yielded similar results., Conclusions: Unless managed care organizations and capitated pediatricians receive payment rates that reflect the higher expected expenditures of caring for these children, access to and quality of care may suffer. Analyses of practice guidelines and utilization patterns suggest that newborn screening, regular access to specialty facilities, and comprehensive education programs are critical areas that are vulnerable to reductions under capitation.

Homozygous beta thalassemia affects thousands of people around the world. Current management of this condition includes regular transfusion of red cells, which leads to transfusional iron overload requiring chelation therapy: increasing hemoglobin levels while decreasing or eliminating iron overload is therefore a major therapeutic goal in the treatment of thalassemia. Bone marrow transplantation may achieve this goal, but it is not an option for most patients. This study reports on efforts to increase gamma-globin transcription and HbF production using sodium phenylbutyrate (SPB) and hydroxyurea (HU). It was found that 36% (4/11) of all patients or 50% (4/8) of non-transfused patients responded to SPB (increase in Hb levels of 1 g/dL). A positive correlation between baseline serum erythropoietin level and likelihood of response to SPB was observed. Since HU may also increase HbF production, evaluation of combination therapy with these drugs is underway and preliminary results are reported.

Purpose: We describe a pediatric patient with sickle cell disease and life-threatening acute chest syndrome who was successfully treated with venovenous extracorporeal membrane oxygenation (ECMO)., Patient and Methods: An 8-year-old boy with sickle cell disease presented with vaso-occlusive crisis, which progressed to fulminant acute chest syndrome requiring a partial exchange transfusion and mechanical ventilation. Despite very high ventilator settings and significant barotrauma, hypoxia persisted and circulatory failure occurred. He was then successfully treated with venovenous ECMO for 11 days. One month after decannulation he had a seizure associated with abnormalities on magnetic resonance images (MRIs). His disease has been managed with a chronic transfusion program since then. Follow-up after 5 years reveals normal pulmonary function tests, a normal magnetic resonance angiogram (MRA), and above-average cognitive skills., Conclusion: This is the first report of a pediatric patient with acute chest syndrome successfully managed with venovenous ECMO. His course was complicated by a seizure associated with MRI abnormalities, although the outcome has been excellent. This case suggests that treatment with venovenous ECMO should be strongly considered for sickle cell patients with life-threatening acute chest syndrome, despite maximal conventional support.

Five factors have been hypothesized to influence the 20-fold variation in fetal haemoglobin (Hb F) levels in sickle cell anaemia (SS): age sex, alpha-globin gene number, beta-globin haplotype, and the X-linked F-cell production locus (FCP) that regulates the production of Hb F containing erythrocytes (F cells). We analysed the association of these factors with Hb F levels in 112 SS patients living in France who are homozygous for the three common African beta-globin haplotypes (Benin, Bantu or Central African Republic and Senegal). We found that: (1) FCP accounts for about 40% of the overall variation in Hb F levels, (2) when the FCP influence is removed, beta-globin haplotype is associated with 14% of the remaining Hb F variation, and (3) the other factors have little influence. Comparison with our previous study of SS individuals in Jamaica leads to the following conclusions: (1) the X-linked FCP locus is a major determinant of Hb F levels in SS disease, (2) factors linked to the beta-globin haplotype have only a small effect on the variation in Hb F levels, in either the homozygous or heterozygous state, and (3)approximately half of the variation in Hb F levels still remains to be explained.

Hydroxyurea (HU) can increase fetal hemoglobin (HbF) in sickle cell anemia (HbSS). To identify determinants of the HbF response, we studied 150 HU-treated patients grouped by quartiles of change in HbF from baseline to 2 years. Half of the HU-assigned patients had long-term increments in HbF. In the top two quartiles, HbF increased to 18.1% and 8.8%. These patients had the highest baseline neutrophil and reticulocyte counts, and largest treatment-associated decrements in these counts. In the lower two quartiles, 2-year HbF levels (4.2% and 3.9%) and blood counts changed little from baseline. In the highest HbF response quartile, myelosuppression developed in less than 6 months, compliance was best, and final doses of HU were 15 to 22.5 mg/kg. All four quartiles had substantial increases of F cells in the first year. This was maintained for 2 years only in the top three quartiles. Leukocyte and reticulocyte counts decreased initially in all quartiles, but drifted back toward baseline levels in the lowest HbF response quartile. Initial HbF level and phenotype of the F-cell production (FCP) locus were not associated with HbF response, but absence of a Central African Republic (CAR) haplotype was. Bone marrow ability to withstand HU treatment may be important for sustained HbF increases during HU treatment of HbSS.

Background: Enhanced production of fetal hemoglobin lessens the severity of beta-thalassemia and sickle cell disease. Intravenous infusion of arginine butyrate can increase the number of reticulocytes containing fetal hemoglobin in patients with these disorders, and it has induced a substantial increase in hemoglobin in one patient with thalassemia. We therefore tested the efficacy of this agent in patients with beta-hemoglobinopathies., Methods: We treated 10 patients with severe beta-thalassemia or sickle cell disease with arginine butyrate at an initial dose of 500 mg per kilogram of body weight per day (final dose, 2000 mg per kilogram per day), 6 days per week, for a mean (+/- SD) of 10 +/- 1.2 weeks (range, 9 to 13). A hematologic response was defined as an increase in the hemoglobin concentration of at least 2 g per deciliter in patients with thalassemia and as a twofold increase in the fetal hemoglobin concentration in patients with sickle cell disease., Results: Increase in gamma-globin messenger RNA and in reticulocytes containing fetal hemoglobin but not in hemoglobin were observed in the patients with thalassemia. A small, unsustained increase in fetal hemoglobin was observed in two patients with sickle cell disease. Drug toxicity was minimal at standard doses. One patient had a grand mal seizure after inadvertently receiving 2000 mg of arginine butyrate per kilogram over a period of six hours., Conclusions: Ten weeks of intravenous arginine butyrate did not produce a hematologic response in 10 patients with either severe beta-thalassemia or sickle cell disease.

Background: In a previous open-label study of hydroxyurea therapy, the synthesis of fetal hemoglobin increased in most patients with sickle cell anemia, with only mild myelotoxicity. By inhibiting sickling, increased levels of fetal hemoglobin might decrease the frequency of painful crises., Methods: In a double-blind, randomized clinical trial, we tested the efficacy of hydroxyurea in reducing the frequency of painful crises in adults with a history of three or more such crises per year. The trial was stopped after a mean follow-up of 21 months., Results: Among 148 men and 151 women studied at 21 clinics, the 152 patients assigned to hydroxyurea treatment had lower annual rates of crises than the 147 patients given placebo (median, 2.5 vs. 4.5 crises per year, P < 0.001). The median times to the first crisis (3.0 vs. 1.5 months, P = 0.01) and the second crisis (8.8 vs. 4.6 months, P < 0.001) were longer with hydroxyurea treatment. Fewer patients assigned to hydroxyurea had chest syndrome (25 vs. 51, P < 0.001), and fewer underwent transfusions (48 vs. 73, P = 0.001). At the end of the study, the doses of hydroxyurea ranged from 0 to 35 mg per kilogram of body weight per day. Treatment with hydroxyurea did not cause any important adverse effects., Conclusions: Hydroxyurea therapy can ameliorate the clinical course of sickle cell anemia in some adults with three or more painful crises per year. Maximal tolerated doses of hydroxyurea may not be necessary to achieve a therapeutic effect. The beneficial effects of hydroxyurea do not become manifest for several months, and its use must be carefully monitored. The long-term safety of hydroxyurea in patients with sickle cell anemia is uncertain.

Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < .05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40% of Hb F variation. beta-Globin haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).

Butyrate analogues have been shown to increase fetal hemoglobin (HbF) production in vitro and in vivo. Sodium phenylbutyrate (SPB), an oral agent used to treat individuals with urea-cycle disorders, has been shown to increase HbF in nonanemic individuals and in individuals with sickle cell disease. We have treated eleven patients with homozygous beta thalassemia (three transfusion dependent) and one sickle-beta-thalassemia patient with 20 g/d (forty 500-mg tablets) of SPB for 41 to 460 days. All patients showed an increase in the percent of F reticulocytes associated with treatment, but only four patients responded by increasing their Hb levels by greater than 1 g/dL (mean increase, 2.1 g/dL; range, 1.2 to 2.8 g/dL). None of the transfusion-dependent thalassemia subjects responded. Increase in Hb was associated with an increase in red blood cell number (mean increase, 0.62 x 10(12)/L), and mean corpuscular volume (mean increase, 6 fL). Changes in percent HbF, absolute HbF levels, or alpha- to non-alpha-globin ratios as measured by levels of mRNA and globin protein in peripheral blood did not correlate with response to treatment. Response to treatment was not associated with the type of beta-globin mutation, but baseline erythropoietin levels of greater than 120 mU/mL was seen in all responders and only two of eight nonresponders to SPB. Compliance with treatment was greater than 90% as measured by pill counts. Side effects of the drug included weight gain and/or edema caused by increase salt load in 2/12, transient epigastric discomfort in 7/12, and abnormal body odor in 3/12 subjects. Two splenectomized patients who were not on prophylactic antibiotics developed sepsis while on treatment. We conclude that SPB increases Hb in some patients with thalassemia, but the precise mechanism of action is unknown.

Intravenous arginine butyrate has been shown to increase fetal hemoglobin (HbF) in sickle cell and thalassemia patients. Recently, we observed that sodium 4-phenylbutyrate, a drug administered orally to treat urea cycle disorders, increases HbF production in nonanemic children and adults. We treated six subjects with sickle cell disease over a period of 14 to 179 days. All subjects received their initial therapy of 9 to 13 g/m2/day as 0.5-g tablets of sodium 4-phenylbutyrate as inpatients. All subjects showed a rapid increase in the percentage of F-reticulocytes (pretreatment, 1% to 20%; posttreatment, 10% to 44%). Four subjects were treated only 11 to 25 days as inpatients. Two of these four subjects failed to respond to the outpatient component because of their inability to maintain an intake of 30 to 40 tablets per day. One subject (C) developed a rash at day 10 and discontinued treatment at day 14. Another subject (B) was transfused for a painful crisis on day 25. Subject A, treated for 179 days, has an increased percentage of F cells, from 54% to 77%, and increased HbF levels, from 10.6% to 18%. Subject F, treated for 154 days, has an increased percentage of F cells, from 59% to 73%, and an increased percentage of HbF, from 10.4% to 16%. All subjects showed some increase in weight. Subject A developed mild transient ankle edema. Myelotoxicity was not seen in any treated patient. Oral administration of sodium 4-phenylbutyrate rapidly increases F-cell production in sickle cell disease.

The prevalence of impaired growth was evaluated in 63 patients with homozygous sickle cell anemia. Twenty-five percent of the children studied were less than the fifth percentile of National Center for Health Statistics growth standards for either height/age, weight/age, or weight/height. Significantly more 11- to 18-year-old children were less than the fifth percentile for weight/height compared with earlier age ranges. No differences due to gender were found. Nutritional intervention may improve growth in selected children.

Purpose: Stimulating expression of the normal fetal globin genes is a preferred method of ameliorating sickle cell disease and beta-thalassemia for the majority of patients in North America who do not have appropriate bone marrow donors., Patients and Methods: Due to increased survival of red blood cells that contain both hemoglobin S and hemoglobin F, as little as 4-8% fetal globin synthesis in the bone marrow can produce levels of hemoglobin F of approximately 20% in the peripheral circulation. Some success has been achieved in stimulating hemoglobin F using chemotherapeutic agents (such as hydroxyurea and 5-azacytidine) and growth factors (erythropoietin) that alter erythroid growth kinetics. However, there is reluctance to treat children with chemotherapeutic agents because of possible undesirable long-term side effects., Results: Butyric acid and butyrate derivatives are generally safe compounds that stimulate the promoters of individual fetal and embryonic globin genes and thus provide a more specific therapy. An initial trial with the parent compound, given as arginine butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that can ameliorate these conditions. Phase I trials of an oral butyrate derivative with a long plasma half-life have begun., Conclusions: These agents may provide a new and specific approach for ameliorating the clinical manifestations of sickle cell disease and beta-thalassemia.

Background: Hydroxyurea increases the production of fetal hemoglobin in patients with sickle cell anemia, inhibiting the polymerization of hemoglobin S and potentially improving vaso-occlusive manifestations and hemolysis. Recombinant erythropoietin increases the number of reticulocytes containing fetal hemoglobin in laboratory animals and in humans. We studied whether hydroxyurea and erythropoietin might have a potentiating effect on the production of fetal hemoglobin in patients with sickle cell disease., Methods: We treated four patients who were receiving hydroxyurea for sickle cell disease (three who were homozygous for sickle cell anemia and one with sickle beta zero-thalassemia) with escalating doses of intravenous erythropoietin for seven weeks, along with oral iron sulfate. Doses of hydroxyurea on four consecutive days were alternated with doses of erythropoietin on three consecutive days., Results: There was a 28 percent increase in the number of reticulocytes containing fetal hemoglobin and a 48 percent increase in the percentage of fetal hemoglobin, as compared with the maximal values obtained with hydroxyurea alone. The percentage of erythrocytes containing fetal hemoglobin (F cells) increased from 64 to 78 percent. As compared with hydroxyurea alone, treatment with hydroxyurea and erythropoietin decreased the mean (+/- SD) serum indirect bilirubin level from 0.8 +/- 0.2 to 0.5 +/- 0.1 mg per deciliter (13.3 +/- 2.9 to 8.9 +/- 2.2 mumol per liter) (P = 0.02), suggesting a further decrease in hemolysis. Red-cell filterability improved., Conclusions: Intravenous recombinant erythropoietin with iron supplementation alternating with hydroxyurea elevates fetal-hemoglobin and F-cell levels more than hydroxyurea alone. Such increases decrease intracellular polymerization of hemoglobin S and improve the overall rheologic characteristics of erythrocytes. A reduced dosage of hydroxyurea alternating with erythropoietin may prove less myelotoxic than hydroxyurea given daily or in pulsed-dose regimens. It may also increase levels of fetal hemoglobin in patients with sickle cell disease who have not been helped by hydroxyurea alone.

Hydroxyurea, a cell-cycle-specific cytotoxic agent, has been shown to increase fetal hemoglobin (HbF) production. This property makes it an attractive drug for treatment of sickle cell disease and severe beta thalassemia. Its potential efficacy is limited because of a variable and often suboptimal response. Combinations of hydroxyurea and other drugs may induce more clinically significant increases in HbF. We have utilized chronically phlebotomized rhesus monkeys, treated with oral hydroxyurea, to investigate the capacity of several other agents to further augment HbF synthesis. Recombinant human erythropoietin, in super-pharmacologic doses, increased F-reticulocyte production when given on a weekly sequential schedule (3 of 7 days) with hydroxyurea (4 of 7 days), but it was less effective on an alternate day schedule when hydroxyurea was given daily. Neither recombinant human interleukin 3 (IL-3) nor recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), when infused individually, increased F-reticulocytes in animals receiving daily hydroxyurea. Sequential, overlapping infusions of IL-3 and GM-CSF produced a small but statistically significant increase in F-reticulocytes in one of two hydroxyurea-treated animals. Infusions of sodium butyrate produced a substantial augmentation in F-reticulocyte production in animals chronically treated with hydroxyurea. Thus, our studies have identified several agents that may prove useful in combination with hydroxyurea to achieve clinically beneficial levels of HbF.

Fetal hemoglobin (Hb F) production in sickle cell (SS) disease and in normal individuals varies over a 20-fold range and is under genetic control. Previous studies suggested that variant Hb F levels might be controlled by genetic loci separate from the beta-globin complex on chromosome 11. Using microscopic radial immunodiffusion and flow cytometric immunofluorescent assays to determine the percentage of F reticulocytes and F cells in SS and nonanemic individuals, we observed that F-cell levels were significantly higher in nonanemic females than males (mean +/- SD, 3.8% +/- 3.2% v 2.7% +/- 2.3%). F-cell production as determined by F reticulocyte levels in SS females was also higher than in SS males (17% +/- 10% v 13% +/- 8%). We tested the hypothesis that F-cell production in both normal and anemic SS individuals was controlled by an X-linked locus with two alleles, high (H) and low (L). Using an algorithm to determine the 99.8% confidence interval of a normal distribution in nonanemic individuals, we estimated that males and females with at least one H allele had greater than 3.3% F cells. Comparisons of male-male or female-female SS sib pairs with discordant F reticulocyte levels distinguished two phenotypes in SS males (L, less than 12%; H, greater than 12%) and three phenotypes in SS females (LL, less than 12%; HL, 12% to 24%, HH greater than 24%). Linkage analysis using polymorphic restriction sites along the X chromosome in eight SS and one AA family localized the F-cell production (FCP) locus to Xp22.2, with a maximum lod score (logarithm of odds of linkage v independent assortment) of 4.6 at a recombination fraction of 0.04.

In the past 8 years, it has become apparent that some cytotoxic drugs that interfere with DNA replication can reprogram erythroid progenitors to switch from adult hemoglobin to fetal hemoglobin (HbF) production. Hydroxyurea has now been shown to substantially increase HbF in patients with sickle cell anemia. Since HbF interferes with sickle hemoglobin polymerization, hydroxyurea may become an important therapeutic agent for patients with sickle cell anemia.

Hydroxyurea has been shown to increase fetal hemoglobin (Hb F) production in patients with sickle cell disease and therefore has the potential to alleviate both the hemolytic and vaso-occlusive manifestations of the disease. Preliminary evidence indicates that recombinant human erythropoietin (rhEpo) may also induce Hb F. Three sickle cell anemia patients were treated with escalating doses of intravenous rhEpo and, subsequently, with daily oral hydroxyurea. After the optimal hydroxyurea dose was attained, rhEpo was added again. Two additional patients were treated with hydroxyurea alone. Treatment with rhEp, either alone or in combination with hydroxyurea, had no significant effect on the percentage of F reticulocytes or F cells. In contrast, hydroxyurea treatment was associated with a 1.5-fold to sevenfold increase in F cells and a 2.3- to 27-fold increase in the percentage of Hb F. In the three patients whose response reached a plateau, hydroxyurea treatment was associated with lessened hemolysis, decreased serum bilirubin and lactate dehydrogenase levels, and prolonged 51chromium-labeled RBC survival. Hydroxyurea treatment also resulted in decreased numbers of irreversibly sickled cells and in decreased sickling at partial oxygen saturation, increased oxygen affinity, increased total RBC cation content, and diminished potassium:chloride co-transport. All five patients treated with hydroxyurea experienced a decrease in severity and frequency of painful sickle crises. This study confirms that hydroxyurea therapy increases Hb F production and provides objective evidence of a significant reduction in hemolytic rate and intracellular polymerization. In contrast, rhEpo, either alone or in combination with hydroxyurea, offered no measurable benefit. Based on these encouraging preliminary data, large-scale, controlled clinical trials are warranted to study the safety and efficacy of hydroxyurea in the treatment of sickle cell disease.

Patients with sickle cell anemia were treated with daily doses of hydroxyurea, to assess pharmacokinetics, toxicity, and increase in fetal hemoglobin (Hb) production in response to the drug. Plasma hydroxyurea clearances were not a useful guide to maximum tolerated doses of the drug. The mean daily single oral dose that could be maintained for at least 16 weeks was 21 mg/kg (range, 10 to 35 mg/kg). Among 32 patients, last HbF levels were 1.9% to 26.3% (mean, 14.9%) with increases in HbF over initial values of 1.4% to 20.2% (mean, 11.2%). The most significant predictors of last HbF were last plasma hydroxyurea level, initial white blood count and initial HbF concentration. Last HbF was not related to beta globin haplotype or alpha globin gene number. No serious toxicity was encountered. Clinically significant bone marrow depression was avoided, and chromosome abnormalities after 2 years of treatment were no greater than those observed before treatment. The period of observation has been too short to evaluate the risk of carcinogenesis. Patient's red cells developed striking macrocytosis. Median red cell Hb concentrations did not change. Hb concentrations increased, on average 1.2 g/dL, but serum erythropoietin levels increased. Patients' body weights increased, and some returned to work or school, but no conclusions regarding therapeutic efficacy could be drawn from this uncontrolled open-label study.

The identification of genetically coherent populations is essential for understanding human evolution. Among the culturally uniform ethnic groups of west Africa, there are two geographically distinct populations with high frequencies of sickle-cell hemoglobin (HbS). Although the HbS mutation in each group is found on distinguishable chromosomes 11, these populations have been assumed to be parts of a single population. Analysis of mitochondrial DNA (mtDNA) in these populations demonstrated that the two populations identified by alternative chromosomes 11 bearing HbS have distinct distributions of mitochondrial genotypes, i.e., they are maternally separate. These studies also showed that, contrary to expectation, the mtDNA of some individuals is heteroplasmic. For nuclear loci, a comparison of the frequency of alternative alleles established that these populations are genetically distinct. Both the mitochondrial and nuclear data indicate that these populations have been separate for approximately 50,000 years. Although HbS in the two populations is usually attributed to recent, independent mutations, the duration of the separation and the observed geographic distribution of the population allow for the possibility of an ancient origin of HbS. Assuming an ancient mutation and considering the known biogeography, we suggest that HbS protected selected populations from malaria in rain forest refuges during the most recent ice age.

A rationale for clinical trials of hydroxyurea (HU) treatment in sickle cell disease is that the agent increases red blood cell (RBC) fetal hemoglobin content. However, an additional effect of HU is to raise the mean corpuscular volume (MCV). To investigate the action of HU in a species that makes no electrophoretically distinguishable fetal hemoglobin, we treated dogs with the drug and compared their response to that of five patients with sickle cell anemia. Both dogs and patients had an increase in MCV, but the effect of HU treatment on the mean corpuscular hemoglobin concentration (MCHC), density, and water content of the RBCs differed in the two species. The dog RBCs became low in MCHC, high in ion and water content, and low in mean density. Thus, HU can raise MCV and lower MCHC without influencing fetal hemoglobin synthesis. A different pattern was seen in the sickle cell patients during HU treatment. Although the MCV of their RBCs increased, there was no change in MCHC, ion content, or mean density. A notable change in the sickle cell patients' blood was that two subpopulations of cells were nearly eliminated during HU treatment; the hypodense reticulocyte fraction and the hyperdense fraction that contains irreversibly sickled cells. These findings lead us to suggest that trials of HU in sickle cell disease must recognize the possibility that any beneficial effect of this agent might be due not only to an increase in hemoglobin F alone, but perhaps also to the associated increase in MCV or the altered RBC density profile.

Single nucleotide substitutions in the promoter regions of the A gamma- and G gamma-globin genes have been associated with increased fetal hemoglobin (HbF) production. We wished to determine whether these or other unrecognized substitutions in the gamma promoter regions are responsible for the 20-fold variation in HbF production in sickle cell patients or normal adults. From a random sampling of 250 sickle cell (SS) patients and 125 normal adults, 17 individuals representing the highest and lowest HbF producers were selected for study. All three common restriction fragment length polymorphism beta-globin region haplotypes (Benin, Central African Republic, and Senegal) were found in both the highest and lowest HbF producers with SS disease. Using the polymerase chain reaction amplification and direct sequencing of the amplified DNA product, we examined the promoter regions of both the A gamma and G gamma genes from -350 bp to +50 bp of the CAP site. No mutations were found in either gamma gene promoter region. We conclude that nucleotide substitutions in the promoter regions (-350 to +50 bp) of both gamma genes are not responsible for the marked variation in HbF production among SS or normal individuals.

Background: Hydroxyurea increases the production of fetal hemoglobin (hemoglobin F) in patients with sickle cell anemia and therefore has the potential for alleviating both the hemolytic and vaso-occlusive manifestations of the disease. There is preliminary evidence that recombinant human erythropoietin may also increase hemoglobin F production., Methods and Results: We treated five patients with sickle cell disease with escalating doses of intravenous erythropoietin for eight weeks. Three of these patients were subsequently treated with daily doses of oral hydroxyurea. After the optimal dose was determined, erythropoietin was then given along with hydroxyurea for four weeks. Treatment with erythropoietin, either alone or in combination with hydroxyurea, had no significant effect on the percentage of hemoglobin F-containing reticulocytes (F reticulocytes) or red cells (F cells). In contrast, hydroxyurea treatment was associated with a 3-to-25-fold increase in F reticulocytes, a 1.6-to-7-fold increase in F cells, and a 2.3-to-16-fold increase in the percentage of hemoglobin F. In all three patients given hydroxyurea, treatment with this drug was associated with reduced hemolysis, shown by decreases in serum bilirubin and lactic dehydrogenase and prolongation of red-cell survival. Hydroxyurea treatment also resulted in a decrease in the percentage of irreversibly sickled cells and sickling at partial oxygen saturation, an increase in oxygen affinity and total red-cell cation content, and a reduction in potassium-chloride cotransport. All three patients had a decrease in the number of pain crises., Conclusions: This study confirms that hydroxyurea therapy increases hemoglobin F production and provides objective evidence that hydroxyurea reduces the rate of hemolysis and intracellular polymerization of hemoglobin S. In contrast, recombinant human erythropoietin, whether alone or in combination with hydroxyurea, offers no measurable benefit.

Adult erythroid progenitors produce significantly higher fetal hemoglobin (HbF) levels in cultures containing fetal calf serum (FCS) and erythropoietin (Ep) than in vivo. The precise mechanisms for this increased HbF production in culture have not been elucidated. We examined HbF biosynthesis by enriched human progenitors in serum-free (SF) culture. We measured globin chain biosynthesis by combination of isoelectric focusing and autoradiography and examined percent nucleated erythrocytes containing HbF (%FNRBC) using microscopic immunodiffusion. CD34 (My10)-positive marrow cells from a normal subject yielded an almost negligible amount of gamma-globin in SF culture stimulated by 100 U/mL interleukin-3 (IL-3) and 2 U/mL Ep, while corresponding FCS culture revealed significant gamma-globin biosynthesis. The %FNRBC of the erythroid bursts in SF cultures derived from nine normal adults (2.0% +/- 0.9% F cells) was 3.0% +/- 3.4%, while in FCS culture, it was 25% +/- 12% (mean +/- SD). Dosages of IL-3 between 10 and 10,000 U/mL did not increase %FNRBC in FCS of SF conditions. Mean Hb contents of nucleated erythrocytes (NRBC) assayed by microdensitometry of pericellular immunoprecipitate were similar in FCS and SF cultures. The number of erythroid bursts per 2 x 10(3) CD34-positive marrow cells was 48 +/- 20 in FCS and 36 +/- 12 in SF cultures. In two experiments, progenitors grown for 7 days under SF conditions were isolated and recultured in either SF or FCS conditions for 7 days, and the resulting erythroid bursts were analyzed for FNRBC. The bursts that had been returned to FCS cultures yielded values of %FNRBC intermediate between those obtained from progenitors grown entirely in SF or FCS cultures, indicating that serum effect is not solely due to growth selection for certain subpopulations of erythroid burst-forming units. This experiment also demonstrated that the factors present in serum responsible for HbF augmentation act at both early and late stages during erythroid burst development. SF culture of peripheral blood progenitors of one subject with heterocellular hereditary persistence of fetal hemoglobin (HPFH) yielded elevated levels of FNRBC (19% +/- 5%) that accurately reflected the F cell (18%) of the circulating blood. Similarly, FNRBC in cultures of progenitors from one umbilical cord blood sample (86% F reticulocytes) was 87 +/- 3% FNRBC. The SF culture for enriched human progenitors, which nearly reflects the physiologic HbF programs of the donor, should facilitate studies of the exact mechanisms of postnatal reactivation of HbF production.

Because fetal hemoglobin contains gammaglobin chains instead of beta chains, it is not affected by the genetic defect that causes sickle cell disease. Increased levels of fetal hemoglobin decrease the tendency toward intracellular polymerization of sickle hemoglobin that characterizes this disease. Hydroxyurea is one of several cytostatic agents that have been shown to increase the production of fetal hemoglobin in some patients with sickle cell disease. We studied the effects of hydroxyurea administration in 10 hospitalized patients with sickle cell disease, each of whom was treated for three months. Seven patients responded with a 2- to 10-fold increase in fetal hemoglobin, from a mean (+/- SD) of 1.6 +/- 1.6 percent of total hemoglobin to 6.8 +/- 4.7 percent; three patients had fetal-hemoglobin levels of 10 to 15 percent of total hemoglobin. Three did not respond to treatment. Four of the patients who responded were retreated with hydroxyurea after one to four months without treatment and were found to have larger increases in fetal-hemoglobin levels. In most patients, levels were still rising at the end of the study, even after 90 days of therapy. Fetal-hemoglobin levels tended to peak at dosages of hydroxyurea that were myelosuppressive. In the patients who responded to treatment, there were significant increases in the percentage of reticulocytes and erythrocytes containing fetal hemoglobin and in the amount of fetal hemoglobin within these cells. The percentage of dense red cells decreased in the patients who responded to treatment. The tendency toward intracellular polymerization at physiologic oxygen saturation was reduced by about 33 percent in the cells containing fetal hemoglobin, whereas there was no change in the other cells. We conclude that hydroxyurea is effective in increasing the production of fetal hemoglobin, which in this study was found to be associated with a small decrease in hemolysis and an increase in hemoglobin levels despite myelosuppression. Controlled, prospective trials are necessary to establish whether these effects will lead to clinical benefit.

Approximately 1 in 500 black American births results in children who have sickle cell (SS) disease; they are rarely symptomatic until 6 months of age. The mortality rate of SS in children under age 12 is between 10 and 15 percent. Half this mortality occurs before age 2, and most is due to overwhelming infections.

Serial microscopic immunodiffusion assays of F cells, i.e., erythrocytes that contain fetal hemoglobin (HbF), in four individuals recovering from anemia demonstrate initial increases in the percentage of circulating reticulocytes that contain HbF (F reticulocytes) and subsequent increases in the percentage of mature erythrocytes that contain HbF (F erythrocytes). In one individual responding to therapy for iron-deficiency anemia, the average percentage of F reticulocytes increased from 4.8+/-1.1 to 16.0+/-2.8% (mean+/-SD), while the mean level of F erythrocytes increased from 3.5+/-0.7 to 7.2+/-0.6%. Two normal children with transient erythroblastopenia exhibited F reticulocyte percentages of 71.3+/-6.7 and 41.5+/-1.5%, respectively, when erythropoiesis resumed. With recovery these values fell to finally measured values of 33.7+/-4.7 and 12.6+/-1.1%, respectively. In an adolescent with sickle cell anemia, F-reticulocyte percentages fluctuated between 0.6+/-1.1 and 34.0+/-2.8% and paralleled the rise and fall of total reticulocytes associated with therapy for a nasopharyngeal carcinoma. Such findings suggest that first, the production of F cells and non-F cells are separately regulated. Second, F-cell production is preferentially stimulated during escape from erythropoietic suppression and selectively depressed at the start of suppression. Third, during escape from erythropoietic suppression, F-cell production in vivo resembles that reported for in vitro cultures of erythroid stem cells. Fourth, individuals with sickle cell anemia, like individuals without hemoglobinopathies, can change their relative level of F-cell production.