Fetal and embryonic hemoglobins in erythroblasts from fetal blood and fetal cells enriched from maternal blood in fetal anemia.
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BACKGROUND AND OBJECTIVES: To determine whether there is a delay or reversal in switch mechanisms from embryonic (e and z) to fetal (g) hemoglobins accompanying the erythroblastosis of anemic fetuses and whether an increased erythroblast count in fetal blood is associated with an increase in feto-maternal cell trafficking. DESIGN AND METHODS: Fetal and maternal blood samples were obtained from 10 cases with rhesus isoimmunization and 2 cases with maternal Parvo-B19 virus at 19-33 weeks' gestation. Blood samples were also taken as controls from 61 fetuses and 86 mothers. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody. Fluorescent antibodies were used to immuno-stain for zeta (z), epsilon (e) and gamma (g) hemoglobin chains. In the maternal samples, fluorescence in situ hybridization (FISH) for X and Y chromosomes was also carried out to confirm the presence and proportion of the enriched fetal cells from maternal blood. RESULTS: In both fetal and maternal blood the percentage of erythroblasts positive for g-globin chain was significantly higher in the anemic fetuses compared to the controls (fetal blood, p<0.001, R=0.91; maternal blood, p<0.001, R=0.56), but there was no significant difference in expression of the e and z-chains. The percentage of cells with Y-signals was also higher in the maternal samples of anemic fetuses compared to normal controls (p<0.001, R=0.56). INTERPRETATION AND CONCLUSIONS: These findings suggest that the erythroblastosis of anemic fetuses is not accompanied by a delay or a reversal in switch from embryonic to fetal hemoglobin chains. Severe fetal anemia is associated with an increase in feto-maternal cell trafficking. (+info)
N-terminal contributions of the gamma-subunit of fetal hemoglobin to its tetramer strength: remote effects at subunit contacts.
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The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70-fold smaller tetramer-dimer dissociation constant. This property has been shown previously to be only partially caused by the 5-amino-acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18-amino-acid N-terminal A helix of the gamma-subunit of fetal hemoglobin, which differs from the beta-subunit of adult hemoglobin at eight amino acid residues. This long-distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the gamma-subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the beta-subunit by the corresponding residues in the gamma-subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (gamma) of HbF with its high pK(a) of 8.1 compared with a 7.1 value for Val 1 (beta) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the gamma-subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (beta) by Glu 5 (gamma) promotes tetramer strengthening. The Glu --> Asp replacement at position 7 enhances this effect because of the lower pK(a) of Asp but the Val --> Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen-binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer-dimer allosteric interface, oxygen-binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface. (+info)
A new hematologic syndrome with a distinct karyotype: the 5 q--chromosome.
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Five patients, four women and one man, age 32-8- yr, all whites, had refractory anemia with the same abnormal bone marrow karyotype, i.e., a partial deletion of the long arm of the No. 5 chromosome. The hematologic syndrome was practically the same in these five cases. Examination of the blood revealed a moderate to severe, generally macrocytic anemia with slight leukopenia but normal or elevated platelet count. The bone marrow showed a depressed erythroid series and some abnormalities of the granulocytic series with an occasional excess of myeloblasts. Most of the megakaryocytes had a nonlobulated nucleus. These features, as well as cytogenetic, electron microscopic, isotopic, platelet function, and immunologic studies, are described in detail. The relationship of this newly established syndrome to other hematologic diseases is discussed. The syndrome constitutes another example of the association between a specific abnormal chromosome and a distinct hematologic disorder. (+info)
A new form of hereditary persistence of fetal hemoglobin in blacks and its association with sickle cell trait.
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A new form of hereditary persistence of fetal hemoglobin (HPFH) producing 3%-8% Hb F in heterozygotes and an elevation of F-cell counts as measured by both the Kleihauer test and an antibody fluorescent procedure was found during the study of a black family. Individuals with this anomaly also had sickle cell trait. A sickle cell homozygote who had apparently inherited the HPFH determinant had 20.3% Hb F. Both types of gamma-chains were present in equal proportions in the Hb F of these individuals. A population study revealed other AS individuals with increased Hb F synthesis, three of whom were sibs. The presence of this previously unrecognized form of HPFH might explain the mild clinical manifestations and the hemoglobin phenotypes of sickle cell homozygotes with unusual elevations of Hb F. (+info)
Predictors of fetal hemoglobin response in children with sickle cell anemia receiving hydroxyurea therapy.
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In the phase I/II pediatric hydroxyurea safety trial (HUG-KIDS), school-aged children with sickle cell anemia receiving hydroxyurea at the maximally tolerated dose (MTD) had variable increases in the percentage of fetal hemoglobin (%HbF). To identify predictors of the HbF response to hydroxyurea therapy, baseline clinical and laboratory values (age, sex, hemoglobin concentration, %HbF, reticulocytes, white blood cell [WBC], platelets, and serum chemistries), as well as treatment variables (number of toxicities, noncompliance, MTD dose, and MTD blood counts) were analyzed in 53 HUG-KIDS children who achieved MTD. Baseline %HbF values (P =.001), baseline hemoglobin concentration (P =.01), MTD dose (P =.02), and compliance (P =.02) were significantly associated with a higher %HbF at MTD; in contrast, age, sex, number of toxicities, and other baseline hematologic parameters were not. After adjusting for variations in baseline %HbF, the baseline reticulocyte count (P =.05) and baseline WBC count (P =.05) were also significantly associated with a higher %HbF at MTD. Hydroxyurea-induced increases in the hemoglobin concentration and mean corpuscular volume (both higher absolute values at MTD and larger positive changes from baseline values), as well as hydroxyurea-induced decreases in reticulocytes and WBC count, were significantly associated with a higher %HbF at MTD. These data suggest that selected baseline laboratory parameters, a higher MTD dose with attention to compliance, and greater therapy-related changes in blood counts may predict the HbF response to hydroxyurea therapy for children with sickle cell anemia. The HbF response to hydroxyurea is variable and complex, however, and even children with low baseline %HbF values can develop substantial increases in %HbF at MTD. (+info)
Evidence of genetic interaction between the beta-globin complex and chromosome 8q in the expression of fetal hemoglobin.
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During human development, the switch from fetal to adult hemoglobin (Hb) is not complete with the residual gamma-globin expression being restricted to a subset of erythrocytes termed "F cells" (FC). Statistical analyses have shown the FC trait to be influenced by a common sequence variant (C-->T) at position -158 upstream of the Ggamma-globin gene, termed the "XmnI-Ggamma polymorphism." The XmnI-Ggamma site is believed to be involved in the expression of the Ggamma-globin gene through interaction with transcription factors, and polymorphisms in the transcription factors could be influencing fetal Hb expression, conditional on the XmnI-Ggamma site. Using a two-locus model, in which the second locus was the known quantitative-trait locus (QTL) at the XmnI-Ggamma site, we showed suggestive linkage to chromosome 8q. A maximum single-point LOD score of 4.33 and a multipoint LOD score of 4.75 were found in a 15-20 cM region of chromosome 8q. A single-locus analysis failed to show linkage of FC to the region when the XmnI-Ggamma site was accounted for by removing its effects from the data or including it as a covariate. Results of the single-locus analysis were significant when the effects of the XmnI-Ggamma site were not accounted for in any way. The results of analysis in a large Indian kindred indicate that there is an interaction between the XmnI-Ggamma site and a QTL on chromosome 8q that is influencing the production of fetal Hb. (+info)
Assembly of human hemoglobin (Hb) beta- and gamma-globin chains expressed in a cell-free system with alpha-globin chains to form Hb A and Hb F.
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Rates of in vitro synthesis of radiolabeled gamma and beta chains made in a cell-free transcription/translation system were similar, but expressed globin chains were unstable. The addition of unlabeled beta or gamma chains at the start of chain synthesis generated radiolabeled beta(4) or gamma(2) and gamma(4) chains, respectively. If unlabeled alpha-globin chains were added at the start of chain synthesis, then approximately equal amounts of radiolabeled alphabeta or alphagamma bands were generated. If unlabeled Hb A or Hb F was added to reactions containing radiolabeled alphabeta or alphagamma prior to electrophoresis, then radiolabeled Hb A or Hb F tetramers, respectively, were generated. If alpha chains were added after synthesis of radiolabeled gamma chains made in the presence of unlabeled gamma chains, then little radiolabeled alphagamma formed. In contrast, if alpha chains were added after synthesis of radiolabeled beta chains made in the presence of unlabeled beta chains, then radiolabeled alpha(2)beta(2) formed. These findings suggest that beta and gamma chains associate with alpha chains during or soon after translation. This would prevent the formation of unstable monomers as well as stable gamma(2) dimers and suggests that alpha chains may bind to nascent non-alpha chains, acting as folding catalysts to promote functional tetrameric hemoglobin formation in vivo. (+info)
Hemoglobin F synthesis is not restricted to fetal erythropoietic organs during extramedullary hematopoiesis.
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We investigated whether the anatomic distribution of hematopoietic cells determines the type of hemoglobin produced in patients with extramedullary hematopoiesis (EMH). Fetal hemoglobin (HbF) production is not restricted to fetal erythropoietic organs during EMH. A shift of erythropoiesis to fetal hematopoietic organs in EMH does not necessarily induce HbF synthesis in adulthood. (+info)