Prospects for research in hematologic disorders: sickle cell disease and thalassemia.

Sickle cell anemia and thalassemia constitute the most common genetic diseases in the world. Affected patients carry a heavy burden of morbidity and early mortality. With improved understanding of the pathophysiology and molecular basis of these diseases, treatment is evolving from management of symptoms to more effective strategies that aim to modify diseased red blood cells or replace them with normal cells. Available treatment options include red blood cell transfusions, pharmacologic interventions to increase fetal hemoglobin levels, and stem cell transplantation. Improvements in these approaches or the development of means to replace defective genes with normal ones using techniques of gene transfer offer hope for the future.

Bone marrow transplantation for beta-thalassemia: the University of California San Francisco experience.

We report the results of allogeneic HLA-matched family donor stem cell transplantation in 17 North American children with beta-thalassemia major or hemoglobin E/beta-thalassemia who received transplants at the University of California San Francisco. Pretransplantation conditioning was with busulfan, cyclophosphamide, and antithymocyte globulin and graft-versus-host prophylaxis used cyclosporine, usually with added methotrexate. Twelve children are alive, well, and free of the clinical manifestations of thalassemia. Four experienced graft rejection and autologous recovery, and one died from complications shortly after transplantation. Overall survival was 94% and event-free survival 71%, results similar to those obtained by other transplantation centers. Improved conditioning regimes and enlargement of the donor pool will be needed to improve the outcome of transplantation and to make this treatment available to more children with thalassemia.

Bone marrow transplantation for hemoglobinopathies.

In patients with hemoglobinopathy who are treated by allogeneic matched sibling bone marrow transplantation before the onset of disease-associated organ damage, long term, disease-free survival currently stands at approximately 90%, and transplant-associated mortality is 5% or less. Less toxic nonmyeloablative conditioning regimens that have the potential to reduce procedure-related mortality to even lower levels are under active investigation. Expansion of the donor pool by use of unrelated matched donors awaits improvement in HLA-typing methodology.

Impact of bone marrow transplantation for symptomatic sickle cell disease: an interim report. Multicenter investigation of bone marrow transplantation for sickle cell disease.

Fifty children who had symptomatic sickle cell disease received matched sibling marrow allografts between September 1991 and March 1999, with Kaplan-Meier probabilities of survival and event-free survival of 94% and 84%, respectively. Twenty-six patients (16 male, 10 female) had at least 2 years of follow-up after transplantation and were evaluated for late effects of transplantation and for its impact on sickle cell-related central nervous system (CNS) and pulmonary disease. Patients ranged between 3.3 and 14.0 (median, 9. 4) years of age and had a median follow-up of 57.9 (range 38-95) months after transplantation. Among 22 of 26 patients who had stable donor engraftment, complications related to sickle cell disease resolved, and none experienced further episodes of pain, stroke, or acute chest syndrome. All 10 engrafted patients with a prior history of stroke had stable or improved cerebral magnetic resonance imaging results. Pulmonary function tests were stable in 22 of the 26 patients, worse in two, and not studied in two. Seven of eight patients transplanted for recurrent acute chest syndrome had stable pulmonary function. Linear growth measured by median height standard deviation score improved from -0.7 before transplantation to -0.2 after transplantation. An adverse effect of busulfan conditioning on ovarian function was demonstrated in five of seven evaluable females who are currently at least 13 years of age. None of the four males tested had elevated serum gonadotropin levels. These data confirm that allogenic bone marrow transplantation establishes normal erythropoiesis and is associated with improved growth and stable CNS imaging and pulmonary function in most patients. (Blood. 2000;95:1918-1924)

Stomatocytosis is absent in "stomatin"-deficient murine red blood cells.

To examine the relationship between erythrocyte membrane protein 7. 2b deficiency and the hemolytic anemia of human hereditary stomatocytosis, we created 7.2b knock-out mice by standard gene targeting approaches. Immunoblots showed that homozygous knock-out mice completely lacked erythrocyte protein 7.2b. Despite the absence of protein 7.2b, there was no hemolytic anemia and mouse red blood cells (RBCs) were normal in morphology, cell indices, hydration status, monovalent cation content, and ability to translocate lipids. The absence of the phenotype of hereditary stomatocytosis implies that protein 7.2b deficiency plays no direct role in the etiology of this disorder and casts doubt on the previously proposed role of this protein as a mediator of cation transport in RBC.

Elevated resting energy expenditure in adolescents with sickle cell anemia.

OBJECTIVE: To assess the reliability of standard prediction equations in estimating resting energy expenditure (REE) values in adolescents with sickle cell anemia. SUBJECTS/DESIGN: Body composition and metabolic measurements were performed in 8 adolescents, aged 11 to 18 years, with homozygous sickle cell anemia. REE was measured by indirect calorimetry under standard conditions, and measurements were compared with 4 prediction formulas (Harris-Benedict, Schofield, Mayo Clinic, and Food and Agriculture Organization/World Health Organization/United Nations University). Fat-free mass was measured to assess REE per unit of actively metabolizing tissue. Fat-free mass was expressed as a mean of values obtained by densitometry, deuterium dilution, 40K-counting, and total body electrical conductivity. STATISTICAL ANALYSES: Repeated measures analysis of variance was performed to determine whether measured REE values and predicted values differed. The Fischer test was used to identify which predicted values differed significantly from the measured REE. RESULTS: All 4 prediction formulas significantly underestimated REE. Group mean values for the prediction formulas ranged from 83% to 89% of the measured value. REE averaged 47.7 +/- 10.0 kcal/kg fat-free mass per day, which is 30% to 50% higher than reported values in healthy adolescent populations. CONCLUSIONS: These data suggest that REE is elevated in adolescents with sickle cell anemia. Standard equations used to predict REE are unreliable in these patients. APPLICATIONS: REE in patients with sickle cell anemia is best determined by indirect or direct measurement of energy expenditure. Clinically useful formulas to estimate REE should be developed for patients with conditions, including sickle cell anemia, where the metabolic rate may be altered.

Collaborative multicenter investigation of marrow transplantation for sickle cell disease: current results and future directions.

We present updated results of a multicenter collaborative investigation of bone marrow transplantation for sickle cell disease. Between September 1991 and April 1997, thirty-four children less than 16 years of age with severe sickle cell disease received marrow allografts from HLA-identical siblings. Indications for transplantation included a history of stroke (n = 17), recurrent acute chest syndrome or sickle pulmonary disease (n = 10), and recurrent vaso-occlusive crises (n = 7). Twenty-one patients received regular red blood cell (RBC) transfusions to prevent complications of sickle cell disease. Patients were prepared for transplantation with busulfan, cyclophosphamide, and antithymocyte globulin or CAMPATH (Cambridge Pathology) antibody. Thirty-two of the 34 patients survived, with a median follow-up of 26.5 months (range, 0.2-66.9 months); and 28 patients demonstrated stable engraftment of donor hematopoietic cells. Graft rejection or recurrence of sickle cell disease occurred in four patients, and two patients died of intracranial hemorrhage or graft-vs.-host disease. In the group of 34 children with symptoms of advanced sickle cell disease, current Kaplan-Meier estimates of survival and event-free survival are 93% and 79%, respectively.

Bone marrow transplantation for sickle cell disease.

BACKGROUND: We investigated the risks and benefits of allogeneic bone marrow transplantation in children with complications of sickle cell disease. METHODS: Twenty-two children less than 16 years of age who had symptomatic sickle cell disease received marrow allografts from HLA-identical siblings between September 1991 and April 1995. The indications for transplantation included a history of stroke (n = 12), recurrent acute chest syndrome (n = 5), and recurrent painful crises (n = 5). Patients were prepared for transplantation with busulfan, cyclophosphamide, and antithymocyte globulin. RESULTS: Twenty of the 22 patients survived, with a median follow-up of 23.9 months (range, 10.1 to 51.0), and 16 patients had stable engraftment of donor hematopoietic cells. In three patients the graft was rejected and sickle cell disease recurred; in a fourth patient graft rejection was accompanied by marrow aplasia. In 1 of the 16 patients with engraftment, there was stable mixed chimerism. Two patients died of central nervous system hemorrhage or graft-versus-host disease. Kaplan-Meier estimates of survival and event-free survival at four years were 91 percent and 73 percent, respectively. Among patients with a history of acute chest syndrome, lung function stabilized; among patients with prior central nervous system vasculopathy who had engraftment, stabilization of cerebrovascular disease was documented by magnetic resonance imaging. CONCLUSIONS: Allogeneic stem-cell transplantation can be curative in young patients with symptomatic sickle cell disease.

Genomic organization and 5'-flanking DNA sequence of the murine stomatin gene (Epb72).

Stomatin is a poorly understood integral membrane protein that is absent from the erythrocyte membranes of many patients with hereditary stomatocytosis. This report describes the cloning of the murine stomatin chromosomal gene, determination of its genomic structure, and characterization of the 5'-flanking genomic DNA sequences. The stomatin gene is encoded by seven exons spread over approximately 25 kb of genomic DNA. There is no concordance between the exon structure of the stomatin gene and the locations of three domains predicted on the basis of protein structure. Inspection of the 5'-flanking DNA sequences reveals features of a TATA-less housekeeping gene promoter and consensus sequences for a number of potential DNA-binding proteins.

Barriers to bone marrow transplantation for sickle cell anemia.

While allogeneic marrow transplantation is curative therapy for patients with sickle cell anemia, only a small fraction of patients in the United States receive this treatment. We surveyed participants in our multicenter study of marrow transplantation for sickle cell anemia to determine reasons for not proceeding to transplantation. Among the 4848 patients less than 16 years of age with sickle cell anemia that were followed in 22 collaborating centers, 315 (6.5%) patients were reported to meet protocol entry criteria for transplantation, although there was wide variation among the institutions (0.9-36%). Among the 315 patients eligible for transplantation, 128 (41%) had human leukocyte antigen (HLA) typing performed, and of these 44 (14% of those meeting entry criteria) had an HLA-identical sibling. Common reasons for not proceeding with HLA typing in the remaining 187 patients included lack of a candidate sibling donor (76 patients, 24% of those meeting criteria) and lack of financial or psychosocial support (33, 10.5%). Parental refusal (30, 9.5%), physician refusal (13, 4%), history of medical noncompliance (2, < 1%), and other reasons (33, 10.5%) were less frequently cited. To date, 25 patients have been transplanted. Of the remaining 19 patients with HLA-matched donors, seven did not proceed to transplantation because of parental refusal, while the others anticipate a future transplantation (6), have experienced symptomatic improvement (4), or have relocated abroad (2). We conclude that the major barrier to marrow transplantation for sickle cell anemia is lack of an HLA-identical donor. But since only 6.5% of all children with sickle cell disease were considered eligible for transplantation, it is possible that other significant obstacles remain to be identified. For patients reported to meet eligibility criteria, parental refusal and limited financial or psychosocial support were infrequent barriers to transplantation.

Thrombo-embolic disease after splenectomy for hereditary stomatocytosis.

Nine cases of hereditary stomatocytosis (HSt) are presented which show documented thrombotic complications after splenectomy. In three cases, patients became severely ill with pulmonary hypertension and a fourth developed portal hypertension. One unsplenectomized affected adult relative had suspected but unconfirmed thrombotic pathology; the six other affected unsplenectomized adults did not. Since splenectomy is of only limited therapeutic benefit in stomatocytosis, it should not be performed without careful consideration. A tendency to iron overload, even without hypertransfusion and irrespective of splenectomy, is evident in many of these patients.

Nontraumatic fat embolism syndrome in sickle cell anemia.

A 14-year-old girl with sickle cell disease and nephrotic syndrome developed bone pain, followed by pulmonary edema, seizures, coma, and bilateral flaccid paralysis. Fat embolism syndrome was diagnosed by cranial magnetic resonance imaging and an exchange transfusion was performed. Within 3 months, all symptoms had resolved. It is concluded that fat embolism syndrome must be considered as a possible cause of acute neurologic deterioration in patients with sickle cell anemia.

Recombinant human erythropoietin stimulates erythropoiesis and reduces erythrocyte transfusions in very low birth weight preterm infants.

DESIGN AND METHODS: We hypothesized that treatment with recombinant human erythropoietin (r-HuEPO) would stimulate erythropoiesis and would thereby reduce the need for erythrocyte transfusions in preterm infants. We treated 157 preterm infants born at 26.9 +/- 1.6 weeks of gestation who weighed 924 +/- 183 g at birth with either subcutaneous r-HuEPO (100 U/kg/d, 5 days per week) or placebo for 6 weeks in a randomized, double-blind, controlled clinical trial. All patients received oral iron and were managed according to uniform conservative transfusion guidelines. RESULTS: Treatment with r-HuEPO was associated with fewer erythrocyte transfusions (1.1 +/- 1.5 per infant in the r-HuEPO group versus 1.6 +/- 1.7 per infant in the placebo group; P = .046) and with a reduction in the volume of packed erythrocytes transfused (16.5 +/- 23.0 mL versus 23.9 +/- 25.7 mL per infant; P = .023). Overall, 43% of the infants in the r-HuEPO group and 31% of placebo-treated infants were transfusion-free during the study (P = .18). The volume of blood removed for laboratory tests and the need for respiratory support at the start of treatment had major effects on transfusion requirements independent of r-HuEPO. Reticulocyte counts were higher during treatment in the r-HuEPO group (P = .0001), and r-HuEPO-treated infants had higher hematocrit values at the end of the study (32% versus 27.3% in the placebo group; P = .0001). We found no differences in the incidence of major complications of prematurity between the treatment groups. CONCLUSION: We conclude that treatment with r-HuEPO at a weekly dose of 500 U/kg stimulates erythropoiesis, moderates the course of anemia, is associated with a reduction in erythrocyte transfusions, and appears safe in very low birth weight preterm infants who are receiving iron supplements. Conservative transfusion criteria, minimization of phlebotomy losses, and treatment with r-HuEPO are complementary strategies to reduce erythrocyte transfusions in these infants.

Chronic pulmonary disorders in sickle cell disease: findings at thin-section CT.

PURPOSE: To characterize the non-acute abnormalities seen at computed tomography (CT) in patients with sickle cell (SC) disease and a prior history of acute chest syndrome (ACS)-pneumonia. MATERIALS AND METHODS: Twenty-nine patients with SC disease who had experienced one to more than 10 (median, six) previous episodes of ACS-pneumonia were prospectively studied with thin-section CT of the thorax. Scans were graded for interstitial disease and assigned a disease index ranging from 0 to 3. Twenty-four patients underwent pulmonary function tests (PFTs) and measurement of their blood gasses. RESULTS: Twelve of the 29 patients (41%) had significant interstitial disease that was multifocal. A correlation was found between the disease index and number of episodes of ACS-pneumonia (P = .02) but not between the disease index and PFT results. CONCLUSION: Thin-section CT demonstrates significant multifocal interstitial lung abnormalities in 41% of selected patients with SC disease. The pattern is most consistent with scarring from episodes of infarction or infection.

Prenatal diagnosis of unusual hemoglobinopathies.

While analyzing 280 hemoglobinopathy kindreds with prescribed molecular tests, 3 unusual mutations were observed that required additional characterization. In the first case, the hypervariable region flanking the alpha-globin genes generated an intermediate length 8.2 kb psi zeta-globin gene fragment on a Southeast Asian chromosome with two deleted alpha-globin genes. Rehybridization of the Southern blot with alpha-globin probe distinguished the mutation unambiguously. In the second case, restriction enzyme analysis of a PCR amplified black beta-globin gene detected a novel beta-83 point mutation adjacent to a promoter element. In the third case, which was uninformative with available allele specific oligonucleotides (ASOs), total genomic PCR amplification and sequencing identified a single basepair insertion in codon 36/37 of an Iranian beta-globin gene that shifted the reading frame and obliterated gene activity. Developing additional region-specific ASOs will further diminish the number of cases that must be characterized by genomic PCR sequencing.

Bone marrow transplantation for sickle cell disease. The United States experience.

PURPOSE: As of June 1992, five patients with sickle cell disease had been treated by matched sibling bone marrow transplantation in the United States. PATIENTS AND METHODS: Three patients underwent transplantations for complications related to sickle cell disease, two with previous cerebrovascular accidents (CVAs) and one who had had multiple severe vasoocclusive crises. Two patients had other indications for allogeneic bone marrow transplantation: one had acute myeloid leukemia and the other had Morquio's disease. The patients' ages ranged from 3 to 10 years, and four were girls. Ages of the donors ranged from 4 to 13 years; four of the donors were boys and three carried the sickle cell trait. For four patients, the preparative regimen consisted of busulfan and cyclophosphamide given either alone or combined with antithymocyte globulin (ATG). The patient with leukemia was prepared with cyclophosphamide and total body irradiation (TBI). The regimens for prophylaxis of graft-versus-host disease (GVHD) included various combinations of cyclosporine A, methotrexate, and prednisone. RESULTS: The patient with Morquio's disease failed to engraft but underwent a successful retransplantation from the same donor. All patients eventually demonstrated donor engraftment and the donor's hemoglobin electrophoretic pattern posttransplant. Two patients had moderately severe GVHD of the skin and gastrointestinal tract, which resolved with prednisone therapy. One of these patients developed transient chronic GVHD involving the skin. Other acute complications included mild venoocclusive disease of the liver, central line infection with bacteremias, uterine hemorrhage in one patient, and pseudomonas sepsis in another. CONCLUSIONS: Both patients who underwent transplantation after CVAs have experienced subsequent neurological events. However, with a median follow-up of 16 months (range 8 months to 9.3 years), all patients are surviving in good to excellent clinical condition and appear to have benefitted from treatment by bone marrow transplantation.

Availability of related donors for bone marrow transplantation in sickle cell anemia.

PURPOSE: To determine who might qualify for allogeneic bone marrow transplantation (BMT), we reviewed the medical records of all 143 patients with sickle cell anemia under the age of 16 years who were registered at our center. PATIENTS AND METHODS: A total of 135 records were complete and were used to estimate donor availability and disease severity. The mean number of siblings per patient was two, but this number decreased to 0.73 if half-siblings and siblings who had sickle cell anemia were excluded. Probability calculations indicated that a human leukocyte antigen (HLA)-matched sibling donor would be available for only 18% of patients with sickle cell disease. RESULTS: With regard to clinical severity, if only stroke and chronic debilitating pain are considered criteria for bone marrow transplantation, only 16% of sickle cell patients would qualify, but with use of the broader criteria of the National Collaborative Study, 38% of patients would qualify. However, not all parents will consent to have bone marrow transplantation for their child, and only a minority of patients (18%) will have an HLA-matched sibling donor. Thus, as few as 1-2% of the total population of children with sickle cell anemia will ultimately qualify for marrow transplantation. Increasing the number who can undergo transplantation will require increasing the size of the donor pool. CONCLUSIONS: Search for other therapies not based on marrow transplantation should continue. For the majority of patients with sickle cell disease, these nontransplant treatments offer the best chance for enabling patients to achieve greater longevity and a better quality of life.

Low number of antibody producing cells in patients with sickle cell anemia.

B cell function is impaired in patients with sickle cell anemia. Although the number of surface IgM positive cells was similar in sickle cell patients and controls, in vitro spontaneous IgM, and PWM stimulated IgA, IgM, and IgG synthesis was significantly lower in the patients than in controls. The number of PWM induced and antigen specific immunoglobulin producing cells after immunization with Pneumovax, containing 21 serotypes of Streptococcus pneumoniae, was about 100-fold lower in the patients as compared with controls. Finally, the ability of the patients' peripheral blood mononuclear cells to proliferate in response to mitogens (PWM, SAC, PHA) was diminished. Because of the observed impairments in both nonspecific and antigen specific immunoglobulin synthesis and cell proliferation assays in the patients, we determined serum concentrations of IL-4 and IL-6, two cytokines associated with antibody production. IL-4 concentrations appeared low in sickle cell patients, and correlated with that of serum IgM. We hypothesize that B cell maturation in sickle cell patients is arrested at an IL-4 dependent stage.