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

Molecular analysis of normal and abnormal human globin genes and their gene products has recently provided information on the precise genetic events that result in hemoglobinopathies. In the case of structurally abnormal hemoglobins, the following mechanisms can be invoked: single nucleotide base substitutions leading to amino acid replacement or chain termination variants; nucleotide deletions (or additions) leading to deletion and frameshift variants; and nonhomologous crossing over leading to the production of fused globin chains. The molecular basis of the thalassemia syndromes, disorders characterized by absent or decreased synthesis of alpha- or beta-globin chains, is quite heterogeneous. In some cases globin gene deletions have been demonstrated; whereas in others there is probably either a defect in globin gene transcription or a defect in nuclear globin messenger RNA (mRNA) processing, mRNA transport or globin mRNA stability. In one form of beta(0)-thalassemia a nonsense mutation has recently been demonstrated, and other cases are also associated with some as yet undetermined functional abnormality of beta-globin mRNA.
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PMID:Molecular genetics of human hemoglobin synthesis. 38 60

Formamide gel electrophoresis separates the mRNA fraction from reticulocyte polyribosomes of adult humans into two major RNA species with migratory rates identical to those of the alpha- and beta-globin mRNAs of the rabbit. That these two RNAs of human origin are the globin mRNAs is further supported by the deficiency of the presumed beta mRNA in reticulocyte polyribosomes of fetuses and premature infants, whose cells make gamma chains in preference to beta chains. The globin mRNAs of reticulocyte polyribosomes from patients with hematological disorders were estimated by scanning the stained formamide gels. In contrast to individuals with either hemolytic anemia without hemoglobinopathy or sickle cell anemia who had beta mRNA to alpha mRNA ratios of approximately one, a patient with Hb S-beta-thalassemia had a ratio of beta mRNA to alpha mRNA of 0.75 while two subjects with homozygous beta-thalassemia had severe deficiencies of beta mRNA. Conversely, a patient with alpha-thalassemia (Hb H disease) had a ratio of beta mRNA to alpha mRNA on reticulocyte polyribosomes of 6. These data provide further evidence of a quantitative deficiency of chain-specific globin mRNA in patients with the thalassemia syndromes.
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PMID:Further evidence of a quantitative deficiency of chain-specific globin mRNA in the thalassemia syndromes. 105 38

Gene therapy of severe hemoglobinopathies will require high-level expression of a transferred globin gene in erythroid cells. Distant regulatory elements flanking the beta-globin gene cluster, the locus control region, are needed for appropriate expression. We have explored the use of a human parvovirus, the adeno-associated virus (AAV), for globin gene transfer. The human A gamma-globin gene, linked to hypersensitivity site 2 from the locus control region of the beta-globin gene cluster, was subcloned into a plasmid (psub201) containing the AAV inverted terminal repeats. This construct was cotransfected with a helper plasmid containing trans-acting AAV genes into human 293 cells that had been infected with adenovirus. The recombinant AAV vector containing hypersensitivity site 2 stably introduced on average one or two unrearranged proviral copies into human K562 erythroleukemia cells. The transferred globin gene exhibited normal regulation upon hemin induction of erythroid maturation and was expressed at a level equivalent to a native chromosomal A gamma-globin gene.
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PMID:Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector. 132 31

Towards a goal of using adeno-associated viruses (AAV), the human parvovirus, as the gene transfer vector for gene therapy of hemoglobinopathies, the human beta-globin (h beta G) cDNA was ligated downstream of the P40 promoter of AAV type 2 (AAV2) genome. Transfection via electroporation of the construct into human 293 cells (embryonal kidney cell line) resulted in expression of the cloned h beta G cDNA, as evidenced by the synthesis of transcripts hybridizable to h beta G probe. The transfection led to the recombinant genome to be excised out of the plasmid and replicate in the cell, followed by production of the recombinant AAV that harbors h beta G cDNA.
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PMID:The recombinant human parvoviruses for gene therapy of hemoglobinopathies. 136 18

Towards a goal of using recombinant adeno-associated viruses (AAV) for the gene therapy of hemoglobinopathies we had previously constructed plasmid pAV h beta G psi 1, which contained a human beta-globin-encoding cDNA (HBB) downstream from the P40 promoter of AAV2 DNA [Ohi et al., Gene 89 (1990) 279-282]. Transfection of the plasmid into human 293 cells (embryonal kidney cell line) resulted in the expression of HBB at the mRNA level as well as rescue and replication of the recombinant AAV genome (Ohi et al., ibid.). The present study demonstrates that the replicated recombinant DNA was packaged into an intact virion by transcomplementation with pAV2 or the defective helpers, pAV delta Bam or pAVXB. The recombinant virus could be isolated by equilibrium CsCl density gradient, the density of which was about 1.4 g/cm3. The defective helpers are used to produce wild-type AAV-free recombinant AAV. The recombinant AAV were infectious and expressed chimeric mRNAs containing the HBB sequence in virus-infected 293, KB (oral epidermoid carcinoma cell line) and K562 (human erythroleukemia cell line) cells. The importance of the infectivity and expression of the recombinant AAV in hematopoietic cells is discussed in the context of gene therapy of hemoglobinopathies.
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PMID:Construction and expression of a recombinant adeno-associated virus that harbors a human beta-globin-encoding cDNA. 165 80

Human erythroblasts are a logical target for studies of expression of transferred globin genes because high-level expression is a prerequisite for gene therapy of hemoglobinopathies. Early erythroid progenitors (erythroid burst-forming units, BFU-E) are readily available from human peripheral blood and can be cultured to produce erythroblasts. However, conditions for efficient transfer into these normal progenitors have not been previously described. Here we demonstrate efficient transfer of the neomycin resistance gene into human peripheral blood BFU-E using the retrovirus vector, N2. We show that liquid culture of mononuclear cells from peripheral blood for 18-24 h prior to retroviral infection leads to increased transfer efficiency of N2 as determined by G418 resistance, and we are able to detect viral DNA by polymerase chain reaction (PCR) analysis. In addition, a second retrovirus, beta(gamma)-SVX, prepared with a human beta-globin gene containing a gamma-globin second exon to facilitate transcript detection and the 3'-enhancer sequence, was also used to determine whether similar results could be obtained when more than one gene is transferred. Using the beta(gamma)-SVX virus, increased transfer efficiency into BFU-E was similarly found after liquid culture for up to 4 days. Expression of the transferred globin gene was also detected by PCR analysis of cDNA made from erythroblast RNA. The human peripheral blood BFU-E system described should allow determination of sequences required for high-level expression of transferred globin and other erythroid genes.
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PMID:Retroviral transfer of genes into erythroid progenitors derived from human peripheral blood. 169 73

This report describes a patient with thalassemia intermedia-like phenotype born to normal parents in whom globin gene sequencing detected a novel abnormal hemoglobin (Hb) due to a T to A substitution at codon 60 of the beta-globin gene arising as a de novo mutation. Normal sequences were detected at the homologous beta-globin locus. This mutation results in the substitution of a polar (glutamic acid) for a nonpolar (valine) residue near the corner of the heme pocket of the beta-globin chain. The novel variant has been designated Hb Cagliari, from the place of birth of the propositus. Kinetics of globin synthesis performed following splenectomy suggest that this new Hb variant is synthesized at a near normal rate but undergoes rapid breakdown. The extreme lability of the variant explains the clinical and hematologic picture characterized by marked ineffective erythropoiesis, thalassemia-like bone changes, iron overload, high proportion of Hb F in the peripheral blood, reduced beta/alpha-globin chain synthesis ratio in peripheral blood reticulocytes, and absence of the abnormal Hb in peripheral blood at extensive protein structural analysis before splenectomy. This case indicates that a thalassemic hemoglobinopathy should be suspected in the presence of a patient with a thalassemia intermedia-like phenotype born to normal parents, even when protein structural analysis fails to detect an abnormal Hb. DNA sequencing may allow to define the mutation, thus making the proper diagnosis.
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PMID:Hemoglobin Cagliari (beta 60 [E4] Val----Glu): a novel unstable thalassemic hemoglobinopathy. 198 2

With the goal of using adeno-associated viruses (AAV) as the gene-transfer vector for gene therapy of hemoglobinopathies, human beta-globin cDNA was ligated downstream from the P40 promoter of the AAV type-2 (AAV2) genome. To circumvent difficulties of cloning DNA containing palindromic sequences, two of which exist in the termini of AAV genome, a step-wise approach handling one palindrome at a time was devised to construct the chimeric expression vector. Electroporation of the construct into human 293 cells (embryonal kidney cell line) resulted in expression of the cloned human beta-globin cDNA, as evidenced by the synthesis of transcripts hybridizable to human beta-globin cDNA probe. Addition of the 3'-end region of AAV DNA that contains both the transcription termination signal and origin of DNA replication for AAV to the construct permitted the recombinant AAV genome to be rescued and replicate in the cell.
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PMID:Construction and replication of an adeno-associated virus expression vector that contains human beta-globin cDNA. 216 23

A region of DNA located far upstream of the human beta-globin locus is critically involved in the regulation of the beta-globin gene family. Recent experiments in transgenic mice suggest that switching from fetal to adult globin gene expression during human development results from competition among individual globin gene family members for interaction with sequences in this region. The phenotypes of patients with defined hemoglobinopathies support this hypothesis.
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PMID:Human globin locus activation region (LAR): role in temporal control. 220 10

A major challenge in genetics is identifying the basis of human heritable disease. We describe an "exon scanning" technique which surveys exons in genomic DNA for sequence alterations. By hybridizing genomic DNA to RNA probes derived from cDNAs, we can use RNase A to survey entire coding regions, comprising exons spread across extensive regions of genomic DNA, for mutations associated with genetic disease. Exon scanning of the beta-globin locus in the DNA of patients with 12 different hemoglobinopathies detected all of the culpable single base substitutions and deletions, but not single base insertions. Our analysis also revealed unsuspected polymorphisms and corrected a diagnosis originally based on hemoglobin electrophoresis. Exon scanning of the ornithine aminotransferase gene in a gyrate atrophy patient detected and localized a mutation in the sixth exon. Subsequent PCR amplification and sequencing characterized this as a missense mutation (proline----glutamine). Exon scanning of genomic DNA for sequence alterations, in combination with PCR amplification and sequencing, should be a generally useful strategy for evaluating suspect genes in disorders of unknown etiology, as well as for clinical diagnosis.
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PMID:Detection of point mutations associated with genetic diseases by an exon scanning technique. 227 38


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