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Query: UMLS:C0019045 (hemoglobinopathies)
 2,704 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.
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PMID:An in vitro model of human red blood cell production from hematopoietic progenitor cells. 953 74

This report describes in detail the procedures for growing human erythroid cells in liquid culture for evaluating the potential of pharmacological agents to affect hemoglobin production. The procedure consists of two phases: an erythropoietin-independent phase in which peripheral blood mononuclear cells are first cultured in the presence of a combination of hemopoietic growth factors, but in the absence of erythropoietin, where early erythroid committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an erythropoietin-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. This procedure produces large cultures of relatively pure and synchronized erythroid cell populations derived from normal donors or patients with beta hemoglobinopathies. The cultured cells recapitulate many aspects of erythropoiesis in vivo, including the donor's pattern of hemoglobin production (types and proportions). Tested compounds, at different concentrations, are added at different stages of the culture. The various types of hemoglobins and globin chains produced can be measured by high performance liquid chromatographic techniques and their cellular distribution analyzed by flow cytometry using fluorescently labeled antibodies against specific hemoglobins. This approach provides a screening system for compounds with potential therapeutic efficacy in patients with beta hemoglobinopathies.
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PMID:Techniques for studying stimulation of fetal hemoglobin production in human erythroid cultures. 985 28

Proteins involved in repression of the human beta-globin gene may be useful in the treatment of sickle cell anemia, in conjunction with therapy to reactivate fetal globin genes. If there is a reciprocal elevation of gamma-globin expression upon repression, this approach could be useful in additional hemoglobinopathies. We previously showed that repression of the beta-globin gene appears to be mediated through two DNA sequences, silencers I and II, and identified a protein termed BP1 which binds to both silencer sequences. In this study, we cloned two cDNAs encoding proteins which bind to an oligonucleotide in silencer I containing a BP1 binding site. These cDNAs correspond to HMG-I and HMG-Y, isoforms regarded as architectural proteins. We demonstrate that binding of HMG-I(Y) to this oligonucleotide causes bending/flexure of the DNA. HMG-I(Y) also binds to a second oligonucleotide containing a BP1 binding site located in a negative control region upstream of the delta-globin gene, suggesting a role for HMG-I(Y) in repression of adult globin genes. Expression studies revealed that HMG-I(Y) is ubiquitously expressed in human tissues that do not express beta-globin, being present in 48 of 50 tissues and six hematopoietic cell lines examined. Furthermore, HMG-I(Y) expression is down-regulated during differentiation of primary erythroid cells. We present a model in which HMG-I(Y) alters DNA conformation to allow binding of repressor proteins, and in which the relative amount of HMG-I(Y) helps to determine the repressive state of the beta-globin gene.
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PMID:Binding of HMG-I(Y) elicits structural changes in a silencer of the human beta-globin gene. 988 3

Iron deficiency is the most frequently encountered cause of suboptimal response to recombinant human erythropoietin (rHuEPO). Carefully assessing iron status is of paramount importance in chronic renal failure patients prior to or during rHuEPO therapy. Because there is great need for iron in the EPO-stimulated erythroid progenitors, it is essential that serum ferritin and transferrin saturation levels should be maintained over 300 microg/liter and 30%, respectively. Investigators have shown that oral iron is unlikely to keep pace with the iron demand for an optimal rHuEPO response in uremics. Therefore, patients with iron deficiency will always require intravenous iron therapy. The early and prompt iron supplementation can lead to reductions in rHuEPO dose and hence cost. After the iron deficiency has been corrected or excluded, we must remember all of the possible causes of hyporesponsiveness in every rHuEPO-treated patient. As dose requirements vary, it is not clear which dose of rHuEPO causes this hyporesponsiveness. However, if the patient with iron repletion does not respond well after the induction period, the major causes blunting the response to rHuEPO should be investigated. Most factors are reversible and remediable, except resistant anemia associated with hemoglobinopathy or bone marrow fibrosis, which requires a further increase in the rHuEPO dose. By means of early detection and correction of the possible causes, the goal of increasing therapeutic efficacy can be achieved. Iron overload may lead to an enhanced risk for infection, cardiovascular complication, and cancer. Over-treatment with iron should be avoided in dialysis patients, despite the fact that the safe upper limit of serum ferritin to avoid iron overload is not clearly defined. On the other hand, functional iron deficiency may develop even when serum ferritin levels are increased. Controversy remains as to whether intravenous iron therapy can overcome this form of hyporesponsiveness in iron-overloaded patients. Moreover, a treatment option of iron supplementation is not warranted in these patients, as the potential hazards of iron overload will be worsened. We demonstrated that the mean hematocrit significantly increased from 25.1+/-0.9% to 31+/-1.2% after eight weeks of intravenous ascorbate therapy (300 mg three times a week) in 12 hemodialysis patients with serum ferritin levels of more than 500 microg/liter. The enhanced erythropoiesis paralleled with a rise in transferrin saturation (27.8+/-2.5% vs. 44.8+/-9.5%, P < 0.05) and reductions in erythrocyte zinc protoporphyrin (130+/-32 vs. 72+/-19 micromol/mol heme, P < 0.05) and monthly rHuEPO dose (24.2+/-4.5 vs. 16.8+/-3.4 x 10(3) units, P < 0.05) at the end of study. It is speculated that ascorbate supplementation not only facilitates the iron release from storage sites and its delivery to hematopoietic tissues, but also increases iron utilization in erythroid cells. Our study provides a more complete understanding of the pathogenesis of iron overload-related anemia and the development of an adjuvant therapy, intravenous ascorbic acid, to the existing treatments.
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PMID:Erythropoietin hyporesponsiveness: from iron deficiency to iron overload. 1008 94

Retrovirus vectors for A gamma-globin are being developed for the treatment of beta chain hemoglobinopathies. Toward the goal of achieving therapeutic expression levels, core elements of the beta-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhancer activity in erythroid MEL and K562 cell lines using a drug-resistant colony assay. When used alone, core elements of HS1, HS3, and HS4 showed no activity and a fragment for HS2 showed only modest activity in the colony assay. However, a 1.1 kb combination of fragments for HS2, HS3, and HS4 (termed a nLCR) enhanced colony formation 17-fold in K562 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhanced nLCR activity only modestly in MEL cells. When linked to a beta-globin gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per copy of mouse alpha-globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a beta-globin promoter and various A gamma-globin gene expression cassettes resulted in extreme genetic instability and reduced titers. Specific deletions were abrogated by removing homologous sequences, but random recombinations were still observed at significant frequencies. In MEL cells containing intact provirus, A gamma-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse alpha-globin) compared to the same vector without the nLCR. These data suggest that vector elements detract from the ability of the nLCR to enhance expression of the beta pr.A gamma cassettes.
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PMID:Development of a condensed locus control region cassette and testing in retrovirus vectors for A gamma-globin. 1008 91

Progress toward gene therapy of beta-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the Agamma- and beta-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that the Agamma-globin promoter could be deleted to -159 without affecting expression, while deleting the beta-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal beta-globin gene promoter was consistently higher than that from the optimal Agamma-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the beta-globin locus control region (termed a muLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the alpha-globin locus. All three enhancers increased expression from the beta-globin gene to roughly the same extent, while the HS-40 element was notably less effective with the Agamma-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Agamma-globin gene linked to the beta-globin promoter. Inclusion of extended 3' sequences from either the beta-globin or the Agamma-globin genes had no significant effect on expression. A 714-bp internal deletion of Agamma-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 beta-globin promoter, an Agamma-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, gamma-globin expression averaged 166% of murine alpha-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine alpha-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for gamma-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.
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PMID:Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: optimization of a gamma-globin gene expression cassette. 1009 Sep 29

Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.
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PMID:Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells. 1023 79

In Papua New Guinea (PNG), numerous blood group polymorphisms and hemoglobinopathies characterize the human population. Human genetic polymorphisms of this nature are common in malarious regions, and all four human malaria parasites are holoendemic below 1500 meters in PNG. At this elevation, a prominent condition characterizing Melanesians is alpha(+)-thalassemia. Interestingly, recent epidemiological surveys have demonstrated that alpha(+)-thalassemia is associated with increased susceptibility to uncomplicated malaria among young children. It is further proposed that alpha(+)-thalassemia may facilitate so-called "benign" Plasmodium vivax infection to act later in life as a "natural vaccine" against severe Plasmodium falciparum malaria. Here, in a P. vivax-endemic region of PNG where the resident Abelam-speaking population is characterized by a frequency of alpha(+)-thalassemia >/=0.98, we have discovered the mutation responsible for erythrocyte Duffy antigen-negativity (Fy[a-b-]) on the FY*A allele. In this study population there were 23 heterozygous and no homozygous individuals bearing this new allele (allele frequency, 23/1062 = 0.022). Flow cytometric analysis illustrated a 2-fold difference in erythroid-specific Fy-antigen expression between heterozygous (FY*A/FY*A(null)) and homozygous (FY*A/FY*A) individuals, suggesting a gene-dosage effect. In further comparisons, we observed a higher prevalence of P. vivax infection in FY*A/FY*A (83/508 = 0.163) compared with FY*A/FY*A(null) (2/23 = 0.087) individuals (odds ratio = 2.05, 95% confidence interval = 0.47-8.91). Emergence of FY*A(null) in this population suggests that P. vivax is involved in selection of this erythroid polymorphism. This mutation would ultimately compromise alpha(+)-thalassemia/P. vivax-mediated protection against severe P. falciparum malaria.
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PMID:Emergence of FY*A(null) in a Plasmodium vivax-endemic region of Papua New Guinea. 1057 Jan 83

The promoter region of adult beta globin genes in humans and other mammals contains conserved regions of pivotal importance for their regulated tissue specific expression. These include the CACCC and CAAT motifs. The CACCC motif is duplicated in humans and other mammals. The human delta-globin gene lacks these conserved regions and its expression in normal individuals is about 3% that of the beta globin gene. Previous studies have shown that the introduction of the beta-globin CACCC or CAAT can activate the delta-globin gene promoter, but the effect of the distal CACCC element has not yet been tested. In the present study, using site-specific mutagenesis, we have introduced the consensus sequence for the distal and proximal CACCC motif and the CAAT box alone or in combination in the wild-type delta-globin gene promoter. The resulting mutants, as well as the wild type (wt) delta- and beta-globin gene promoters, have been analyzed in a transient expression assay in Cos7, K562, and MEL cell lines. The results show that the CACCC boxes can increase the transcription efficiency of the delta-globin gene promoter in both erythroid and non-erythroid cell systems. The contribution of the two CACCC elements is almost equal in the non-erythroid (Cos7) and erythroid embryonic-fetal cell lines (K562), while the proximal CACCC element is more active in adult erythroid cells (MEL). Nonetheless, duplication of this element does not appear to affect the efficiency of the promoter synergistically. Furthermore, to assess the competitive ability of the delta globin promoter containing the proximal or distal CACCC consensus sequences over the wt beta globin gene promoter, we have carried out transient expression experiments using DNA constructs in which the delta and beta globin gene promoters are linked in cis and are sharing a single enhancer (competitive transient expression). The results show that both CACCC elements are able to activate the delta globin gene promoter in Cos7 and K562 cells, although to a different extent, whereas only the proximal CACCC element is effective in increasing the transcription efficiency in MEL cells. These findings are in agreement with the more severe clinical phenotype produced by the beta-thalassemia mutations affecting the proximal CACCC box as compared with those within the distal CACCC box. The Erythroid Kruppel Like Factor (EKLF) is a nuclear protein restricted to erythroid cells which specifically bind the CACCC box sequence and activate the beta-globin gene. In the present study we carried out transactivation experiments of the mutagenized delta-globin gene promoter by introducing an EKLF expressing construct in erythroid cells. Constructs containing the proximal but not those bearing the distal CACCC element are transactivated. Our results indicate that the proximal CACCC box and, to a lesser extent, also the distal box have a role in the regulated stage specific expression of a beta-like globin gene, and show that the insertion of a single CACCC motif in the delta-globin gene promoter is sufficient to increase its activity. Nevertheless only the delta globin gene promoter containing the proximal CACCC element is able to compete with the wt beta globin gene promoter in the adult erythroid environment. These findings have potential relevance for the future prospective treatment of inherited hemoglobinopathies based on the conversion of the low functioning delta-globin gene into a high functioning beta-like globin gene.
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PMID:Activation of the delta-globin gene by the beta-globin gene CACCC motif. 1057 45

One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the alpha-globin gene (alpha-thalassemia trait) and that 6% to 17% are beta-thalassemia carriers. In this population, a single mutation of beta-globin gene (Q39X, beta(0) 39) accounts for >95% of beta-thalassemia cases. Because previous studies have shown that Sardinian beta-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the beta-thalassemia trait was also present in subjects with familial hypercholesterolemia (FH). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g>a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g>a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76+/-1.08 mmol/L in subjects with beta(0)-thalassemia trait and 8.25+/-1.66 mmol/L in subjects without this trait (P<0.001). This LDL-lowering effect was confirmed in an FH heterozygote of the same cluster who had beta(0)-thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of beta(0)-thalassemia trait emerged also when we pooled the data from all FH subjects with and without beta(0)-thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of beta(0)-thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B-containing lipoproteins. The observation that our FH subjects with beta(0)-thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of beta(0)-thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.
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PMID:Influence of beta(0)-thalassemia on the phenotypic expression of heterozygous familial hypercholesterolemia : a study of patients with familial hypercholesterolemia from Sardinia. 1063 24


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