BCL11A enhancer haplotypes and fetal hemoglobin in sickle cell anemia.

BACKGROUND: Fetal hemoglobin (HbF) levels in sickle cell anemia patients vary. We genotyped polymorphisms in the erythroid-specific enhancer of BCL11A to see if they might account for the very high HbF associated with the Arab-Indian (AI) haplotype and Benin haplotype of sickle cell anemia. METHODS AND RESULTS: Six BCL112A enhancer SNPs and their haplotypes were studied in Saudi Arabs from the Eastern Province and Indian patients with AI haplotype (HbF ~20%), African Americans (HbF ~7%), and Saudi Arabs from the Southwestern Province (HbF ~12%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) and their haplotypes were consistently associated with HbF levels. The distributions of haplotypes differ in the 3 cohorts but not their genetic effects: the haplotype TCAG was associated with the lowest HbF level and the haplotype GTAC was associated with the highest HbF level and differences in HbF levels between carriers of these haplotypes in all cohorts were approximately 6%. CONCLUSIONS: Common HbF BCL11A enhancer haplotypes in patients with African origin and AI sickle cell anemia have similar effects on HbF but they do not explain their differences in HbF.

Hemoglobin Parchman: double crossover within a single human gene.

Structural analysis of a new variant hemoglobin revealed tryptic peptides with the amino acid composition of normal delta-globin, except for two internal peptides, which had the compositions of normal beta-globin. The most likely explanation for these findings is that a double, nonhomologous crossover between the delta-and beta-globin genes had occurred.

Spontaneous oxygen radical generation by sickle erythrocytes.

Since the various membrane abnormalities of sickle erythrocytes might result from excessive accumulation of oxidant damage, we have measured the generation of superoxide, peroxide, and hydroxyl radical by normal and sickle erythrocytes using assays involving reduction of cytochrome c, aminotriazole inhibition of catalase, and methane evolution from dimethyl sulfoxide, respectively. Compared with normal erythrocytes, sickle erythrocytes spontaneously generate approximately twice as much superoxide, peroxide, and hydroxyl radical. One possible source of hydroxyl radical generation was identified as hemichrome, excessive amounts of which are bound to sickle erythrocyte membranes. Hemichrome did not generate hydroxyl radical when exposed to superoxide alone or peroxide alone. However, in the presence of both superoxide and peroxide, hemichrome greatly facilitated hydroxyl radical generation. Supporting this, normal erythrocyte membranes induced to acquire sickle hemichrome concomitantly acquired an enhanced ability to mediate hydroxyl radical generation. Finally, sickle erythrocyte membranes greatly enhanced superoxide/peroxide-driven hydroxyl radical generation as compared with normal erythrocyte membranes. These data suggest that an excessive accumulation of oxidant damage in sickle erythrocyte membranes might contribute to the accelerated membrane senescence of these cells. They further indicate that accumulation of oxidant damage could be a determinant of normal erythrocyte membrane senescence.

Two missense mutations in the beta-globin gene can cause severe beta thalassemia. Hemoglobin Medicine Lake (beta 32[B14]leucine-->glutamine; 98 [FG5] valine-->methionine).

We studied the molecular basis of transfusion-dependent hemolytic anemia in an infant who rapidly developed the phenotype of beta thalassemia major. DNA sequence of one beta-globin gene of the proband revealed two mutations, one for the moderately unstable hemoglobin (Hb) Köln and another for a novel codon 32 cytosine-thymidine-guanine-->cytosine-adenine-guanine transversion encoding a leucine-->glutamine mutation. A hydrophilic glutamine residue at beta 32 has an uncharged polar side chain that could potentially distort the B helix and provoke further molecular instability. This new hemoglobin was called Hb Medicine Lake. Biosynthesis studies showed a deficit of beta-globin synthesis with early loss of beta-globin chains. An abnormal unstable hemoglobin, globin chain, or tryptic globin peptide was not present, demonstrating the extreme lability of this novel globin. Hb Medicine Lake mRNA was present, but an aberrantly spliced message was not. Absence of an abnormal beta-globin gene in the mother makes it likely that a de novo mutation occurred in the proband. The molecular pathogenesis of Hb Medicine Lake illustrates a mechanism whereby the phenotype of a genetic disorder, like the mild hemolytic anemia associated with a hemoglobinopathy, can be modulated by a coincident mutation in the same gene.

Molecular basis for nondeletion alpha-thalassemia in American blacks. Alpha 2(116GAG----UAG).

An American black woman was found to have the phenotype of moderately severe alpha-thalassemia normally associated with the loss of two to three alpha-globin genes despite an alpha-globin gene map that demonstrated the loss of only a single alpha-globin gene (-alpha/alpha alpha). Several individuals in her kindred with normal alpha-globin gene mapping studies (alpha alpha/alpha alpha) had mild alpha-thalassemia hematologic values consistent with the loss of one to two alpha-globin genes. These data suggested the presence of a nondeletion alpha-thalassemia defect in this family which segregates with the intact alpha alpha gene cluster. An abnormally migrating and highly unstable alpha-globin gene product was demonstrated by in vitro translation of the reticulocyte mRNA from the proposita and this mutant alpha-globin protein was mapped to the alpha 2-globin gene by hybrid-selected translation. The abnormal alpha 2-globin gene was cloned and sequenced. A single base mutation that results in a premature termination codon was identified at codon 116 (GAG----UAG). The defined alpha-globin genotype of the proposita (-alpha/alpha 116UAG alpha) and the positioning of this nonsense mutation at the alpha 2-globin gene locus are fully consistent with the observed alpha-thalassemia phenotype.

Hemoglobin Mississippi (beta 44ser----cys). Studies of the thalassemic phenotype in a mixed heterozygote with beta +-thalassemia.

Hemoglobin Mississippi (HbMS: beta 44ser----cys) has anomalous properties that include disulfide linkages with normal beta-, delta-, gamma-, and alpha-chains, and the formation of high molecular weight multimers. While heterozygotes for HbMS are clinically and hematologically normal and carriers of the beta +-thalassemia gene in our family had mild microcytic anemia, the proband with HbMS-beta +-thalassemia had a hemoglobin level of 7 g/dl, mean corpuscular volume (MCV) of 68 fl, reticulocytes of 2-6%, HbF of 18%, marked anisocytosis and poikilocytosis, and splenomegaly, all features of thalassemia intermedia. With oxidant stress, her erythrocytes developed multiple dispersed Heinz bodies, but HbMS was only mildly unstable. HbMS was susceptible to proteolytic degradation in the presence of ATP. The unexpectedly severe clinical findings in HbMS-beta +-thalassemia may result from the proteolytic digestion of HbMS, as well as the excessive alpha-chains characteristic of beta +-thalassemia, which combined provide the increment of cellular damage that results in the phenotype of thalassemia intermedia.

Hemoglobin Indianapolis (beta 112[G14] arginine). An unstable beta-chain variant producing the phenotype of severe beta-thalassemia.

Hemoglobin (Hb) Indianapolis is an extremely labile beta-chain variant, present in such small amounts that it was undetectable by usual techniques. Clinically, it produces the phenotype of severe beta-thalassemia. Biosynthetic studies showed a beta:alpha ratio of 0.5 in reticulocytes and about 1.0 in marrow after a 1-h incubation. These results, similar to those seen in typical heterozygous beta-thalassemia, suggested that betaIndianapolis was destroyed so rapidly that its net synthesis was essentially zero. To examine the kinetics of globin synthesis, reticulocyte incubations of 1.25--20 min were performed with [3H]leucine. The betaIndianapolis:beta A ratio at 1.25 min was 0.80 suggesting that beta Indianapolis was synthesized at a near normal rate. At 20 min, this ratio was 0.46 reflecting rapid turnover of beta Indianapolis. The erythrocyte ghosts from these incubations contained only betaIndianapolis and alpha-chains, and the proportion of betaIndianapolis decreased with time, indicating loss of betaIndianapolis. Pulse-chase studies showed little change in beta A:alpha ratio and decreasing betaIndianapolis:alpha and betaIndianapolis:beta A with time. The half-life of betaIndianapolis in the soluble hemoglobin was approximately equal to 7 min. There was also rapid loss of beta Indianapolis from the erythrocyte membrane. From these results, it may be inferred that betaIndianapolis is rapidly precipitated from the soluble cell phase to the membrane, where it is catabolized. Heterozygotes for beta 0-thalassemia usually have minimal hematologic abnormalities, whereas heterozygotes with betaIndianapolis, having a similar net content of beta-chain, have severe disease. The extremely rapid precipitation and catabolism of betaIndianapolis and the resulting excess of alpha-chains, both causing membrane damage, may be responsible for the severe clinical manifestations associated with this variant. It seems likely that other, similar disturbances in the primary sequence of globin polypeptide chains may produce clinical findings similar to those seen with hemoglobin Indianapolis and thus produce the phenotype of severe beta-thalassemia.

Effects of alpha-thalassemia and sickle polymerization tendency on the urine-concentrating defect of individuals with sickle cell trait.

A defect in urine concentrating ability occurs in individuals with sickle cell trait (HbAS). This may result from intracellular polymerization of sickle hemoglobin (HbS) in erythrocytes, leading to microvascular occlusion, in the vasa recta of the renal medulla. To test the hypothesis that the severity of the concentrating defect is related to the percentage of sickle hemoglobin present in erythrocytes, urinary concentrating ability was examined after overnight water deprivation, and intranasal desmopressin acetate (dDAVP) in 27 individuals with HbAS. The HbAS individuals were separated into those who had a normal alpha-globin genotype (alpha alpha/alpha alpha), and those who were either heterozygous (-alpha/alpha alpha) or homozygous (-alpha/-alpha) for gene-deletion alpha-thalassemia, because alpha-thalassemia modulates the HbS concentration in HbAS. The urinary concentrating ability was less in the alpha alpha/alpha alpha genotype than in the -alpha/alpha alpha or -alpha/-alpha genotypes (P less than 0.05). After dDAVP, the urine osmolality was greater in patients with the -alpha/-alpha genotype than with the -alpha/alpha alpha genotype (882 +/- 37 vs. 672 +/- 38 mOsm/kg H2O) (P less than 0.05); patients with the -alpha/alpha alpha genotype had greater concentrating ability than individuals with a normal alpha-globin gene arrangement. There was an inverse linear correlation between urinary osmolality after dDAVP and the percentage HbS in all patients studied (r = -0.654; P less than 0.05). A linear correlation also existed for urine concentrating ability and the calculated polymerization tendencies for an oxygen saturation of 0.4 and O (r = -0.62 and 0.69, respectively). We conclude that the severity of hyposthenuria in HbAS is heterogeneous. It is determined by the amount of HbS polymer, that in turn is dependent upon the percentage HbS, which is itself related to the alpha-globin genotype.

Dominantly inherited beta thalassaemia intermedia caused by a new single nucleotide deletion in exon 2 of the beta globin gene: Hb morgantown (beta91 CTG>CG).

Family members in multiple generations of an Irish-American family were investigated for moderate to severe microcytic anaemia, inherited in an autosomal dominant fashion. A novel frameshift mutation of the beta globin gene was discovered. This study highlights the importance of considering dominantly inherited beta thalassemia in the investigation of anaemia, even in patients with ethnic backgrounds not usually associated with beta thalassaemia.

Is HIV/AIDS a primary-care disease? Appropriate levels of outpatient care for patients with HIV/AIDS.

OBJECTIVE: To estimate the proportion of outpatient visits that could be managed at a primary-care level, by World Health Organization (WHO) clinical staging. DESIGN: Prospective, descriptive study. Six medical doctors in a tertiary hospital HIV ambulatory clinic recorded clinical diagnoses, WHO clinical staging and their recommendation regarding the appropriate level of care for each outpatient seen. SETTING AND STUDY POPULATION: All HIV-infected patients attending a public-sector, urban, South African, referral and teaching hospital HIV outpatient clinic between September and November 1992. PARTICIPANTS: There were 238 visits by 148 patients during the study period. RESULTS: Of 238 visits, 165 (69.3%) were deemed suitable for treatment at the primary-care level. After allowing for contradictory responses, at least 141 visits (59.2%) could be appropriately treated at the primary-care level. Although all six doctors assessed more than half of their visits as suitable for primary care, there were significant differences among them. In total, 83 visits (34.8%) needed a medical specialist, and 45 (18.9%) required tertiary-care facilities. Of all the visits, 58 (24.9%), 51 (21.9%), 60 (25.8%) and 64 (27.4%) were classified as WHO stages 1, 2, 3 and 4, respectively. For these stages, 55 (94.8%), 38 (74.5%), 42 (70.0%) and 26 (40.5%) visits, respectively, were suitable for treatment at a primary-care facility. CONCLUSIONS: Many of the outpatient visits to this outpatient specialist clinic could have been safely cared for at a primary-care level. As the severity of the disease increases, there is a decrease in the proportion of patients that can be treated at a primary-care level.

Determinants of fetal hemoglobin response to hydroxyurea.

Fetal hemoglobin (HbF) modulates the phenotypic diversity of sickle cell anemia (HbSS), and the prevalence of many disease complications is related to the level of HbF. Hydroxyurea (HU) increases HbF levels in many patients. Adults with HbSS have HbF levels that may differ by two orders of magnitude, suggesting the possibility of genetic modulation of gamma-globin gene expression. In HbSS, the beta-globin gene haplotype is associated with HbF. Polymorphisms present in cis-acting elements are usually linked to the haplotype, suggesting that within a haplotype, mutations in these areas are uncommon mechanisms of HbF modulation in patients with untreated HbSS. In a multicenter trial of HU in HbSS, we examined genetic elements, patient characteristics, and follow-up features to determine their association with changes in HbF. Following treatment, F cells increased in HU-treated patients compared with controls. The increases in the HbF level at 2 years were greatest in patients who had the highest baseline counts of reticulocytes and neutrophils, two or more episodes of study-defined myelotoxicity, and absence of a Bantu haplotype. However, after 2 years, half of the patients had no or only trivial increments in HbF. The ability to respond to HU may depend on bone marrow reserve, or the capacity of the marrow to withstand moderate doses of HU with acceptable myelotoxicity. Baseline reticulocyte and neutrophil counts may be a measure of this marrow reserve. Sustained HbF increases during HU treatment can occur in individuals with bone marrow reserve sufficient to respond to the myelotoxicity of this agent with the evolution of a population of high HbF-producing erythroblasts.

Pathophysiology of sickle cell disease: role of cellular and genetic modifiers.

Sickle hemoglobin (HbS), caused by a point mutation in the beta-globin gene of hemoglobin, polymerizes when deoxygenated. The pathophysiology of sickle cell disease results from cellular defects caused directly by the hemoglobin mutation interacting with the environment and many other gene products--a few known, but most yet unidentified--a typical example of epistasis. How normal tissue perfusion is interrupted is complex and why the phenotype of sickle cell disease differs from patient to patient is poorly understood. We review the "classic" aspects of the pathophysiology of sickle cell disease and focus on known and potential modulators of the phenotype of this disorder.

Hydroxyurea and sickle cell anemia. Clinical utility of a myelosuppressive "switching" agent. The Multicenter Study of Hydroxyurea in Sickle Cell Anemia.

Painful crises in patients with sickle cell anemia are caused by vaso-occlusion and infarction. Occlusion of blood vessels depends on (at least) their diameter, the deformability of red cells, and the adhesion of blood cells to endothelium. Deoxygenated sickle cells are rigid because they contain linear polymers of hemoglobin S (Hb S); polymerization is highly concentration dependent, and dilution of Hb S by a nonsickling hemoglobin such as fetal hemoglobin (Hb F) would be expected to lead ultimately to a decrease in the frequency of painful crises. It might also be expected to decrease the severity of anemia, although the pathogenesis of anemia in sickle cell anemia (SS disease) is not clearly understood. Reversion to production of fetal rather than adult hemoglobin became practical with the discovery that HU was an orally effective and relatively safe "switching agent." Preliminary dose-ranging studies led to a double-blind randomized controlled clinical trial, the Multicenter Study of Hydroxyurea in Sickle Cell Anemia (MSH), designed to test whether patients treated with HU would have fewer crises than patients treated with placebo. The MSH was not designed to assess the mechanism(s) by which a beneficial effect might be achieved, but it was hoped that observations made during the study might illuminate that question. The 2 MSH treatment groups were similar to each other and were representative of African-American patients with relatively severe disease. The trial was closed earlier than expected, after demonstration that median crisis rate was reduced by almost 50% (2.5 versus 4.5 crises per year) in patients assigned to HU therapy. Hospitalizations, episodes of chest syndrome, and numbers of transfusions were also lower in patients treated with HU. Eight patients died during the trial, and treatment was stopped in 53. There were no instances of alarming toxicity. Patients varied widely in their maximum tolerated doses, but it was not clear that all were taking their prescribed treatments. When crisis frequency was compared with various clinical and laboratory measurements, pretreatment crisis rate and treatment with HU were clearly related to crisis rate during treatment. Pretreatment laboratory measurements were not associated with crisis rates during the study in either treatment group. It was not clear that clinical improvement was associated with an increase in Hb F. Crisis rates of the 2 treatment groups became different within 3 months. Mean corpuscular volumes (MCVs) and the proportion of Hb F containing red cells (F cells) rose, and neutrophil and reticulocyte counts fell, within 7 weeks. When patients were compared on the basis of 2-year crisis rates, those with lower crisis rates had higher F-cell counts and MCVs and lower neutrophil counts. Neutrophil, monocyte, reticulocyte, and platelet counts were directly associated, and F cells and MCV were inversely associated, with crisis rates in 3-month periods. In multivariable analyses, there was strong evidence of independent association of lower neutrophil counts with lower crisis rates. F-cell counts were associated with crisis rate only in the first 3 months of treatment; MCV showed an association over longer periods of time. Overall, the evidence that decreased neutrophil counts played a role in reducing crisis rates was strong. Increased F cells or MCV and evidence of cytoreduction by HU were also associated with decreased crisis rates, but no definitive statement can be made regarding the mechanism of action of HU because the study was not designed to address that question. Future studies should be designed to explore the mechanism of action of HU, to identify the optimal dosage regimen, and to study the effect of HU when combined with other antisickling agents.

The leukocyte composition of peripheral blood buffy coat.

To assist in the interpretation of buffy coat smears from leukopenic patients, we determined the composition of the buffy coat of the blood of normal volunteers. Leukocytes were concentrated by centrifugation in microhematocrit tubes. Stained smears of these cells showed minor differences in the proportions of neutrophils, lymphocytes, and monocytes when compared to unconcentrated peripheral blood. Granulocytes of greater immaturity than the metamyelocyte were not encountered, and their presence should suggest the possibility of marrow dysfunction. Occasional young monocytoid cells and "stimulated" lymphocytes were seen.

Management of factor XI deficiency in gynecologic and obstetric patients.

Deficiency of factor XI (plasma thromboplastin antecedent) can result in severe bleeding in women undergoing obstetric or gynecologic procedures, with the highest risk in women of Ashkenazi Jewish background. Most patients do not bleed if treated with sufficient fresh frozen plasma to maintain a factor XI level of 30% or more but occasionally patients may require higher levels. Plasma infusion should be continued for several days, even if bleeding does not seem excessive, since delayed bleeding is not uncommon. The use of hepatitis B vaccine and of plasma from a single donor, may reduce the risk of hepatitis.

The interactions of alpha-thalassemia with hemoglobinopathies.

Because of their high prevalence throughout the world, alpha-thalassemia and hemoglobinopathies often occur in the same individual. Southern blot hybridization analysis of the alpha-globin gene cluster permits the detection of the common deletion alpha-thalassemias and the definition of the phenotypes affiliated with the interactions of alpha-thalassemia and hemoglobinopathies. Important insights into the pathophysiology of Hb SS have been gained, and the nature of the perturbation of heterozygous hemoglobinopathies by alpha-thalassemia has been established.

Case report: effects of iron deficiency and the -88 C-->T mutation on HbA2 levels in beta-thalassemia.

Hemoglobin A2 levels in members of an African American family with -88 C-->T beta(+)-thalassemia were measured, and two patients in whom iron deficiency anemia developed were evaluated during treatment. Iron deficiency may diminish the level of HbA2 in healthy control subjects and in patients with heterozygous beta-thalassemia; in addition, it may reduce the amount of variant hemoglobin in certain hemoglobinopathies. Although iron deficiency appeared to be associated with a reduction in HbA2 quantity in the patient with heterozygous beta-thalassemia, the level of HbA2 did not fall below the range characteristic of beta-thalassemia. It had been proposed that mutations in the beta-globin gene promoter may be associated with higher-than-expected levels of HbA2. However, this "mild" beta-globin gene promoter mutation did not seem to result in HbA2 concentrations higher than anticipated in the heterozygous beta-thalassemias.

Sickle cell anemia and fetal hemoglobin.

Fetal hemoglobin, the predominant hemoglobin of the fetus, is good for sickle cell anemia. This hemoglobin inhibits the polymerization of sickle hemoglobin. Clinical studies have shown that at any level of fetal hemoglobin, the more that is present, the better off is the patient. We are now able to increase fetal hemoglobin levels by pharmacologic means. We should know shortly if this is associated with clinical benefit.

Thalassemia: molecular pathology and management.

Recent advances in molecular biology have allowed us to develop an almost complete picture of the molecular pathology of the thalassemia syndromes. The different classes of mutations that are responsible for the thalassemia syndromes will be discussed along with the special insights they have provided into the controls of eukaryotic gene expression. While management of these disorders has not kept pace with our understanding of their cause, there have been notable advances in treatment. Perhaps even more exciting is what the future holds, as the continued march of molecular biology is melded with novel approaches to the definitive treatment of thalassemias.

Review: sickle cell disease: present and future treatment.

Over the past few decades, the life expectancy of patients with sickle cell disease has improved. This has been because of better supportive care and greater awareness of the complications of this disorder. Recent successes of neonatal screening, childhood prophylactic penicillin, and, perhaps, hydroxyurea in adults may further extend the life of sickle cell disease patients. This review will do the following: 1) briefly highlight the major aspects of the conventional treatment of sickle cell disease, 2) address the present use of hydroxyurea in more depth, and 3) succinctly preview what the near-term future of treatment may bring.