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Query: UMLS:C0039730 (thalassemia)
 10,305 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan-Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.
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PMID:Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) 226 55

To study the feasibility of a therapy for thalassemia based on addition of a correctly functioning globin gene to bone marrow stem cells, we have developed retroviral vectors that can transfer the human beta-globin gene into pluripotent hematopoietic stem cells of the mouse. Mice reconstituted with virus-infected bone marrow cells showed long-term tissue-specific expression of human beta-globin RNA and protein. Recently, we have redesigned the retroviral vector to improve the efficiency of stem cell infection and to raise the level of globin expression obtained from the virally transduced gene. Removal of a portion of the second intron of the beta-globin gene resulted in the accumulation of a higher level of full-length viral RNA in retrovirus packaging cell lines, and these cell lines produced beta-globin virus particles at substantially higher titers. Addition of fragments from the locus activation region (LAR) of the beta-like globin gene cluster to the retroviral vectors increased beta-globin expression in infected murine erythroleukemia (MEL) cells. Fragments from the -18 and -10.9 kbp DNase I-hypersensitive sites of the LAR increased human beta-globin RNA levels to 35% and 132% of the endogenous mouse beta maj-globin RNA level, respectively. Increased expression was also found for neomycin phosphotransferase RNA, which was transcribed from the retroviral long terminal repeat (LTR), showing that the LAR fragments also activated expression from a nearby heterologous promoter. These results are discussed in the context of the efficacy and safety of gene therapy for chronic anemia in humans.
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PMID:Retroviral vectors for the beta-globin gene that demonstrate improved titer and expression. 229 69

Over the past five years, several new defects in the beta-thalassemias have been described from this laboratory using both restriction enzyme and sequencing analyses of cloned beta-thalassemia genes. The enzyme HphI has been shown to recognize a single nucleotide change at the 5' end of beta-IVS 2, and, using restriction enzyme analysis, demonstrated for the first time a specific defect associated with beta(0)-thalassemia. Cloning and sequencing of a beta-thalassemia gene have identified a single base change within IVS 2 at a position 705 nucleotides from the 5' end of IVS 2 that results in a beta(0)-thalassemia phenotype; no normal splicing occurs in this gene despite the fact that both the 5' and 3' ends of IVS 2 are unchanged. A unique and strong cryptic 3' acceptor splice site present in the normal gene at a position 580 nucleotides from the 5' end is used extensively in the mutant gene. Studies of this gene have indicated that there are sequences within IVS that are responsible for optimal expression of this gene; changes in these sequences can lead to markedly abnormal patterns of splicing. In addition, beta-globin gene expression has been evaluated in human erythroleukemia cells, K562 cells, and, although stable transformants with integrated beta-globin genes have been obtained, none of these transformants expressed the added beta-globin genes. This is presumably due to trans-acting factors or distal cis-acting effects that suppress the expression of these added beta-globin genes. In addition, a low epsilon-producing cell line, Bos cells, was used as a recipient for an exogenous epsilon-globin gene. A neomycin resistance gene was cotransfected into these cells, and a neomycin analogue (G418) was used to select cells containing both the neomycin resistance and epsilon-globin genes. Using Southern blotting, 10 of 11 stably transformed G418-resistant lines, which contain intact epsilon-globin genes, express epsilon-globin mRNA at much higher levels than the Bos cells into which they were transfected. Two of these lines express the epsilon-globin genes at a level comparable to that of wild-type K562 cells. These results indicate that the transfer and expression of human globin genes in human erythroid cells is feasible, and can occur at a high level.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Abnormal globin gene structure and expression in beta-thalassemia. 299 Feb 98

A 27 year old male with aplastic anemia developed a high fetal hemoglobin, a low hemoglobin A2, a decreased beta/alpha synthetic ratio, and an increased G gamma/A gamma synthetic ratio. This acquired hemoglobinopathy resembling delta beta-thalassemia was recognized at the onset of acute erythroleukemia. Certain features of this abnormal globin synthetic pattern resemble those of the normal fetus and thus appear to provide another example of gene expression by malignant cells resembling that of an earlier stage of the organism's development.
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PMID:Erythroleukemia manifesting delta beta-thalassemia. 618 18

A human beta-globin gene derived from an established human lymphoblast cell line was introduced into murine erythroleukemia (MEL) cells by cell fusion. The globin genes in MEL cells are inducible by dimethyl sulfoxide (Me2SO); induction leads to the accumulation of mouse globin mRNA and hemoglobin. Globin mRNA was not detected in the cytoplasm of the human lymphoblast cells, even at low levels, whether or not these cells were treated with Me2SO. In cell hybrids that had retained the lymphoblast-derived beta-globin gene, human beta-globin mRNA was induced by Me2SO. Poly(A)-containing 10S human beta-globin mRNA was detected in the cytoplasm of the hybrid cells. Karyologic and isozymic analyses of a series of hybrids and subclones showed that human beta-globin gene expression occurred only in hybrids that had retained human chromosome 11. Analysis of one hybrid bearing a deletion of both the beta-globin and lactate dehydrogenase A genes indicated that the beta-globin gene is located on the short arm of human chromosome 11. No other human chromosomes are required for human beta-globin gene expression in MEL cell hybrids. We conclude that the restricted expression of a globin gene in a human nonerythroid cell can be reversed. Furthermore, all components required for the transcription, processing, and transport to the cytoplasm of a human globin mRNA appear to be present in mouse erythroleukemia cells. Thus cell fusion with MEL cells provides a way to isolate permanent cell lines with functioning human globin genes. The technique should be useful for studying the biochemical basis for abnormal function of mutant globin genes, such as those present in individuals with the thalassemia syndromes.
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PMID:Activation of human beta-globin genes from nonerythroid cells by fusion with murine erythroleukemia cells. 693 30

The globin chains of human embryonic, fetal, and adult hemoglobins can be separated by electrophoresis on gels containing polyacrylamide, acid, urea, and Triton X-100. Whole hemolysates are used, and only microgram quantities are required. The order of the major human erythrocyte proteins, from anode to cathode, is zeta, epsilon, carbonic anhydrase, A gamma, delta and G gamma together, beta, and alpha. Protein composition can be measured on Coomassie blue-stained disc gels, and protein synthesis on fluorograms of slab gels containing 3H-leucine-labelled material. These gels have been used to examine the ratio of G gamma to A gamma in blood from fetuses and newborn infants, and to suggest that the switch from A gamma to G gamma during ontogeny may not be linked to the switch from gamma to beta production. beta/gamma synthetic ratios were determined in fetuses at risk for thalassemia. Embryonic and fetal globin synthesis ratios were measured in hemin-induced human erythroleukemia cells K562 in tissue culture. Fetal globin synthesis and the proportion that was of the "fetal" type (G gamma approximately 70%) was studied in erythroid colonies grown in plasma clot cultures from adult, newborn, and 6 month infant specimens. The gels provide a rapid, simple, and inexpensive approach to many problems of globin composition and synthesis.
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PMID:Gel electrophoretic separation of globin chains. 727 69

The human alpha-globin-like embryonic zeta-globin chains are present in abundance during the first 5 to 6 weeks of gestation. Subsequently, zeta-globin chains are present in fetal blood at a very low level, which is supplanted by the expression of alpha-globin chains. Adult individuals who are carriers of the (--SEA/) alpha-thalassemia deletion, in contrast to normal adults, have low levels of embryonic zeta-globin chains in their circulating erythrocytes. In this investigation, we constructed stable mouse-human hybrid cells with murine erythroleukemia cells bearing human chromosome 16, with either the normal alpha-globin gene cluster (alpha alpha/) or the (--SEA/) type of alpha-thalassemia deletion. The results on the human zeta-globin gene expression in these hybrid cells indicate that murine adult erythroid transcription factors can induce the expression of human embryonic zeta-globin gene is cis to the (--SEA/) deletion, in parallel with the endogenous mouse alpha-globin gene expression. These data also show the importance of the DNA sequences within the (--SEA) deletion in regulating the expression of zeta-globin gene in cis during normal human hemoglobin ontogeny.
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PMID:Human embryonic zeta-globin gene expression in mouse-human hybrid erythroid cell lines. 762 Jan 74

Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.
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PMID:Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene. 762 11

Gene therapy approaches for beta-thalassemia and sickle cell anemia focus on the transfer of a human beta-globin gene into the patient's hematopoietic stem cells (HSC). Expression of the transferred sequences should be erythroid specific and match the expression of the endogenous alpha-globin genes in adult erythropoiesis. Here we explore the potential of recombinant adeno-associated virus (AAV) vectors for human beta-globin gene transfer. We have constructed a recombinant AAV-vector containing a human beta-globin gene together with the DNasel hypersensitive sites 4, 3 and 2 of the human beta-globin locus control region. The vector replicates to high titers and can efficiently transduce hematopoietic and non-hematopoietic cells. In transduced and G418 selected murine erythroleukemia (MEL) cell clones, human beta-globin gene expression was regulated and reached levels comparable to endogenous murine beta maj. These data show that AAV-vectors are promising tools in gene therapy approaches for the haemoglobinopathies.
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PMID:Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adeno-associated virus-mediated gene transfer. 767 Nov 9

We have used the gel retardation assay to investigate the binding of nuclear proteins to the duplicated CACCC boxes in the beta-globin gene promoter region. The effect of beta-thalassemia mutations affecting both of these consensus sequences (the -88 C-->T and -101 C-->T mutations) were studied by using appropriate mutant oligonucleotides. Upon incubation with nuclear proteins from human erythroleukemia cells, the synthetic oligonucleotides containing single and/or duplicated CACCC box(es) generated five retarded bands. Bands B1 and B2 appeared to be highly specific complexes as determined by competition experiments whereas bands B3, B4 and B5 were nonspecific. The binding of the specific trans-acting factors was stable and could be competed out only by relatively high concentrations of competitor DNA. The proximal CACCC box (as in the -88N probe) appeared to be essential in the binding of nuclear proteins and a mutation at nt -88 (C-->T) abolished binding completely. In contrast, the distal CACCC box showed no binding activity either as normal (-101N) or as mutant (-101M). A full competition with the Sp1 oligonucleotide, even at low concentrations, suggested a high affinity binding to the Sp1 consensus sequence. The close similarities in the binding patterns of the labelled -88N, -88N/-101N and that of the labelled Sp1 probe add further credence to the possibility that bands B1 and B2 may be due to the Sp1 protein, a ubiquitously expressed trans-activator.
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PMID:Binding of nuclear factors to the proximal and distal CACCC motifs of the beta-globin gene promoter: implications for the -101 (C-->T) 'silent' beta-thalassemia mutation. 790 40


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