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Sample records for adult globin genes

  1. Molecular mechanisms of human hemoglobin switching: selective undermethylation and expression of globin genes in embryonic, fetal, and adult erythroblasts.

    PubMed Central

    Mavilio, F; Giampaolo, A; Carè, A; Migliaccio, G; Calandrini, M; Russo, G; Pagliardi, G L; Mastroberardino, G; Marinucci, M; Peschle, C

    1983-01-01

    The globin chain synthetic pattern and the extent of DNA methylation within embryonic, fetal, and adult beta-like globin gene domains were evaluated in greater than or equal to 90% purified human erythroblasts from yolk sacs and fetal livers in the 6- to 12-wk gestational period as well as from adult marrows. The 6-wk erythroblasts produce essentially embryonic epsilon chains, whereas the 12-wk erythroblasts synthesize largely fetal gamma globin and the adult marrow erythroblasts synthesize almost exclusively adult beta chains. In all phases of ontogenic development, a strong correlation exists between DNA hypomethylation in the close flanking sequences of globin genes and their expression. These results suggest that modulation of the methylation pattern may represent a key mechanism for regulating expression of human globin genes during embryonic leads to fetal and fetal leads to adult Hb switches in humans. In ontogenic development this mechanism might in turn correlate with a gradual modification of chromatin structure in the non-alpha gene cluster, thus leading to a 5' leads to 3' activation of globin genes in a balanced fashion. Images PMID:6316333

  2. [Main regulatory element (MRE) of the Danio rerio α/β-globin gene domain exerts enhancer activity toward the promoters of the embryonic-larval and adult globin genes].

    PubMed

    Kovina, A P; Petrova, N V; Razin, S V; Yarovaia, O V

    2016-01-01

    In warm-blooded vertebrates, the α- and β-globin genes are organized in domains of different types and are regulated in different fashion. In cold-blooded vertebrates and, in particular, the tropical fish Danio rerio, the α- and β-globin genes form two gene clusters. A major D. rerio globin gene cluster is in chromosome 3 and includes the α- and β-globin genes of embryonic-larval and adult types. The region upstream of the cluster contains c16orf35, harbors the main regulatory element (MRE) of the α-globin gene domain in warm-blooded vertebrates. In this study, transient transfection of erythroid cells with genetic constructs containing a reporter gene under the control of potential regulatory elements of the domain was performed to characterize the promoters of the embryonic-larval and adult α- and β-globin genes of the major cluster. Also, in the 5th intron of c16orf35 in Danio reriowas detected a functional analog of the warm-blooded vertebrate MRE. This enhancer stimulated activity of the promoters of both adult and embryonic-larval α- and β-globin genes.

  3. Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

    SciTech Connect

    Miller, B.A.; Perrine, S.P.; Antognetti, G.; Perlmutter, D.H.; Emerson, S.G.; Sieff, C.; Faller, D.V.

    1987-06-01

    The effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes.

  4. A long non-coding RNA promotes full activation of adult gene expression in the chicken α-globin domain

    PubMed Central

    Arriaga-Canon, Cristian; Fonseca-Guzmán, Yael; Valdes-Quezada, Christian; Arzate-Mejía, Rodrigo; Guerrero, Georgina; Recillas-Targa, Félix

    2014-01-01

    Long non-coding RNAs (lncRNAs) were recently shown to regulate chromatin remodelling activities. Their function in regulating gene expression switching during specific developmental stages is poorly understood. Here we describe a nuclear, non-coding transcript responsive for the stage-specific activation of the chicken adult αD globin gene. This non-coding transcript, named α-globin transcript long non-coding RNA (lncRNA-αGT) is transcriptionally upregulated in late stages of chicken development, when active chromatin marks the adult αD gene promoter. Accordingly, the lncRNA-αGT promoter drives erythroid-specific transcription. Furthermore, loss of function experiments showed that lncRNA-αGT is required for full activation of the αD adult gene and maintenance of transcriptionally active chromatin. These findings uncovered lncRNA-αGT as an important part of the switching from embryonic to adult α-globin gene expression, and suggest a function of lncRNA-αGT in contributing to the maintenance of adult α-globin gene expression by promoting an active chromatin structure. PMID:24196393

  5. A long non-coding RNA promotes full activation of adult gene expression in the chicken α-globin domain.

    PubMed

    Arriaga-Canon, Cristian; Fonseca-Guzmán, Yael; Valdes-Quezada, Christian; Arzate-Mejía, Rodrigo; Guerrero, Georgina; Recillas-Targa, Félix

    2014-01-01

    Long non-coding RNAs (lncRNAs) were recently shown to regulate chromatin remodelling activities. Their function in regulating gene expression switching during specific developmental stages is poorly understood. Here we describe a nuclear, non-coding transcript responsive for the stage-specific activation of the chicken adult α(D) globin gene. This non-coding transcript, named α-globin transcript long non-coding RNA (lncRNA-αGT) is transcriptionally upregulated in late stages of chicken development, when active chromatin marks the adult α(D) gene promoter. Accordingly, the lncRNA-αGT promoter drives erythroid-specific transcription. Furthermore, loss of function experiments showed that lncRNA-αGT is required for full activation of the α(D) adult gene and maintenance of transcriptionally active chromatin. These findings uncovered lncRNA-αGT as an important part of the switching from embryonic to adult α-globin gene expression, and suggest a function of lncRNA-αGT in contributing to the maintenance of adult α-globin gene expression by promoting an active chromatin structure.

  6. Characterization of the 5'-to-5'linked adult alpha- and beta-globin genes from three sciaenid fish species (Pseudosciaena crocea, Sciaenops ocellatus, Nibea miichthioides).

    PubMed

    Chu, Wuying; Wei, Yongwei; Qian, Ronghua; Yu, Xiameng; Yu, Lian

    2006-09-01

    Recently, we cloned the adult alpha-globin genes from large yellow croaker Pseudosciaena crocea, cuneate drum Nibea miichthioides and red drum Sciaenops ocellatus. All these alpha-globins have a unique Gly insertion at the 47th residue. In this paper, the three sciaenid globin complexes were identified and compared in detail. Linkage analysis indicated that the sciaenid alpha- and beta-globin genes were oriented head-to-head relative to each other. The sciaenid intergenic regions between the linked alpha- and beta-globin genes were the smallest in reported fish globin gene complexes to date. Classical promoter elements were condensed and the CCAAT box unstable duplication was found in these regions. The promoter function of the intergenic region from large yellow croaker was tested by transient expression of EGFP in Vero cells. We also described a method for studying luciferase reporter gene transient expression in primary fish erythrocytes. We used the method to assess the promoter strength of the three intergenic regions between the sciaenid alpha- and beta-globin genes.

  7. Genomic organization and characterization of a three-gene rat adult beta-globin haplotype.

    PubMed

    Au, D M; Wong, W M; Tam, J W; Cheng, L Y; Lam, V M

    1995-11-20

    The isolation and detailed characterization of a three-beta-globin gene (GloB) haplotype in the Sprague-Dawley (S-D) rat is described. An enriched library, lambda SDHelib, was screened with a human GloB probe, humbg44, and from which a beta minor gene, Rathbbz, was isolated, sequenced and characterized. A S-D rat GloB-specific probe, Ratbgze12, derived from the Rathbbz gene, was then used to screen a S-D rat genomic library, lambda SDglib. The clone T1510 was isolated and identified to include the entire Rathbbz gene and part of another GloB gene, Rathbby, which was 5' upstream from Rathbbz. Chromosomal walking upstream using the riboprobe, rnaT71, led to the isolation of an overlapping clone, Ta49, which was shown to include two full-length GloB genes; the most 5' was Rathbbx followed by Rathbby. Sequence data suggests that Rathbbx is a beta major gene, whereas Rathbby is a hybrid gene of Rathbbx and Rathbbz. Genomic hybridization confirmed this particular three-gene haplotype in the S-D rat. This haplotype, a1, may be the prototype of the GloB cluster in rat.

  8. Reactivation of developmentally silenced globin genes by forced chromatin looping

    PubMed Central

    Krivega, Ivan; Breda, Laura; Motta, Irene; Jahn, Kristen S.; Reik, Andreas; Gregory, Philip D.; Rivella, Stefano; Dean, Ann; Blobel, Gerd A.

    2014-01-01

    Summary Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggered its transcriptional reactivation. This activity depended on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting SA to the fetal γ-globin promoter in primary adult human erythroblasts increased γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications. PMID:25126789

  9. Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals.

    PubMed

    Patel, Vidushi S; Cooper, Steven J B; Deakin, Janine E; Fulton, Bob; Graves, Tina; Warren, Wesley C; Wilson, Richard K; Graves, Jennifer A M

    2008-07-25

    Vertebrate alpha (alpha)- and beta (beta)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the alpha- and beta-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil beta-globin gene (omega) in the marsupial alpha-cluster, however, suggested that duplication of the alpha-beta cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous alpha- and beta-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation. The platypus alpha-globin cluster (chromosome 21) contains embryonic and adult alpha- globin genes, a beta-like omega-globin gene, and the GBY globin gene with homology to cytoglobin, arranged as 5'-zeta-zeta'-alphaD-alpha3-alpha2-alpha1-omega-GBY-3'. The platypus beta-globin cluster (chromosome 2) contains single embryonic and adult globin genes arranged as 5'-epsilon-beta-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate alpha-globin clusters are flanked by MPG-C16orf35 and LUC7L, whereas all bird and mammal beta-globin clusters are embedded in olfactory genes. Thus, the mammalian alpha- and beta-globin clusters are orthologous to the bird alpha- and beta-globin clusters respectively. We propose that alpha- and beta-globin clusters evolved from an ancient MPG-C16orf35-alpha-beta-GBY-LUC7L arrangement 410 million years ago. A copy of the original beta (represented by omega in marsupials and monotremes) was inserted into an array of olfactory genes before the amniote radiation (>315 million years ago), then duplicated and diverged to form orthologous clusters of beta-globin genes with different expression

  10. Chicken alpha-globin switching depends on autonomous silencing of the embryonic pi globin gene by epigenetics mechanisms.

    PubMed

    Rincón-Arano, Héctor; Guerrero, Georgina; Valdes-Quezada, Christian; Recillas-Targa, Félix

    2009-10-15

    Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

  11. Genomic evidence for independent origins of beta-like globin genes in monotremes and therian mammals.

    PubMed

    Opazo, Juan C; Hoffmann, Federico G; Storz, Jay F

    2008-02-05

    Phylogenetic reconstructions of the beta-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult beta-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic beta-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the "epsilon-globin" gene in birds is not orthologous to the epsilon-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto beta-globin gene. Here, we report evidence that the early and late expressed beta-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto beta-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the epsilon- and beta-globin genes of therian mammals arose via duplication of a proto beta-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of beta-like globin genes that originated via duplication of a proto beta-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto beta-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development.

  12. Butyrate Infusions in the Ovine Fetus Delay the Biologic Clock for Globin Gene Switching

    NASA Astrophysics Data System (ADS)

    Perrine, Susan P.; Rudolph, Abraham; Faller, Douglas V.; Roman, Christine; Cohen, Ruth A.; Chen, Shao-Jing; Kan, Yuet Wai

    1988-11-01

    The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal β -globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switching.

  13. Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts

    PubMed Central

    Byrnes, Colleen; Kaushal, Megha; Rabel, Antoinette; Tumburu, Laxminath; Allwardt, Joshua M.; Miller, Jeffery L.

    2015-01-01

    Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30–40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts. PMID:26675483

  14. Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts.

    PubMed

    Lee, Y Terry; de Vasconcellos, Jaira F; Byrnes, Colleen; Kaushal, Megha; Rabel, Antoinette; Tumburu, Laxminath; Allwardt, Joshua M; Miller, Jeffery L

    2015-01-01

    Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30-40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.

  15. The globin gene family of the cephalochordate amphioxus: implications for chordate globin evolution

    PubMed Central

    2010-01-01

    Background The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire. Results We show that B. floridae harbors at least fifteen paralogous globin genes, all of which reveal evidence of gene expression. The protein sequences of twelve globins display the conserved characteristics of a functional globin fold. In phylogenetic analyses, the amphioxus globin BflGb4 forms a common clade with vertebrate neuroglobins, indicating the presence of this nerve globin in cephalochordates. Orthology is corroborated by conserved syntenic linkage of BflGb4 and flanking genes. The kinetics of ligand binding of recombinantly expressed BflGb4 reveals that this globin is hexacoordinated with a high oxygen association rate, thus strongly resembling vertebrate neuroglobin. In addition, possible amphioxus orthologs of the vertebrate globin X lineage and of the myoglobin/cytoglobin/hemoglobin lineage can be identified, including one gene as a candidate for being expressed in notochord tissue. Genomic analyses identify conserved synteny between amphioxus globin-containing regions and the vertebrate β-globin locus, possibly arguing against a late transpositional origin of the β-globin cluster in vertebrates. Some amphioxus globin gene structures exhibit minisatellite-like tandem duplications of intron-exon boundaries ("mirages"), which may serve to explain the creation of novel intron positions within the globin genes. Conclusions The identification

  16. The chromosomal arrangement of human alpha-like globin genes: sequence homology and alpha-globin gene deletions.

    PubMed

    Lauer, J; Shen, C K; Maniatis, T

    1980-05-01

    We report the isolation of a cluster of four alpha-like globin genes from a bacteriophage lambda library of human DNA (Lawn et al., 1978). Analysis of the cloned DNA confirms the linkage arrangement of the two adult alpha-globin genes (alpha 1 and alpha 2) previously derived from genomic blotting experiments (Orkin, 1978) and identifies two additional closely linked alpha-like genes. The nucleotide sequence of a portion of each of these alpha-like genes was determined. One of these sequences is tentatively identified as an embryonic zeta-globin gene (zeta 1) by comparison with structural data derived from purified zeta-globin protein (J. Clegg, personal communication), while the other sequence cannot be matched with any known alpha-like polypeptide sequence (we designate this sequence phi alpha 1). Localization of the four alpha-like sequences on a restriction map of the gene cluster indicates that the genes have the same transcriptional orientation and are arranged in the order 5'-zeta 1-phi alpha 1-alpha 2-alpha 1-3'. Genomic blotting experiments identified a second, nonallelic zeta-like globin gene (phi 2) located 10-12 kb 5' to the cloned zeta-globin gene. Comparison of the locations of restriction sites within alpha 1 and alpha 2 and heteroduplex studies reveal extensive sequence homology within and flanking the two genes. The homologous sequences, which are interrupted by two blocks of nonhomology, span a region of approximately 4 kb. This extensive sequence homology between two genes which are thought to be the products of an ancient duplication event suggests the existence of a mechanism for sequence matching during evolution. One consequence of this arrangement of homologous sequences is the occurrence of two types of deletions in recombinant phage DNA during propagation in E. coli. The locations and sizes of the two types of deletions are indistinguishable from those of the two types of deletions associated with alpha-thalassemia 2 (Embury et al., 1979

  17. A zinc-finger transcriptional activator designed to interact with the γ-globin gene promoters enhances fetal hemoglobin production in primary human adult erythroblasts

    PubMed Central

    Wilber, Andrew; Tschulena, Ulrich; Hargrove, Phillip W.; Kim, Yoon-Sang; Persons, Derek A.; Barbas, Carlos F.

    2010-01-01

    Fetal hemoglobin (HbF) is a potent genetic modifier of the severity of β-thalassemia and sickle cell anemia. We used an in vitro culture model of human erythropoiesis in which late-stage erythroblasts are derived directly from human CD34+ hematopoietic cells to evaluate HbF production. This system recapitulates expression of globin genes according to the developmental stage of the originating cell source. When cytokine-mobilized peripheral blood CD34+ cells from adults were cultured, background levels of HbF were 2% or less. Cultured cells were readily transduced with lentiviral vectors when exposed to vector particles between 48 and 72 hours. Among the genetic elements that may enhance fetal hemoglobin production is an artificial zinc-finger transcription factor, GG1-VP64, designed to interact with the proximal γ-globin gene promoters. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of relatively weak erythroid-specific promoters induced significant amounts of HbF (up to 20%) in erythroblasts derived from adult CD34+ cells without altering their capacity for erythroid maturation and only modestly reducing the total numbers of cells that accumulate in culture after transduction. These observations demonstrate the potential for sequence-specific enhancement of HbF in patients with β-thalassemia or sickle cell anemia. PMID:20190190

  18. Thalidomide induces γ-globin gene expression through increased reactive oxygen species–mediated p38 MAPK signaling and histone H4 acetylation in adult erythropoiesis

    PubMed Central

    Aerbajinai, Wulin; Zhu, Jianqiong; Gao, Zhigang; Chin, Kyung

    2007-01-01

    Although thalidomide has been shown to improve anemia in some patients with myelodysplastic syndromes and stimulates erythropoietin in patients with multiple myeloma, thalidomide's specific effects on γ-globin gene expression during erythroid differentiation have not been studied. Here, we investigated the effects of thalidomide on γ-globin gene expression and the involved signaling pathway using an ex vivo culture system of primary human CD34+ cells. We found that thalidomide induced γ-globin mRNA expression in a dose-dependent manner, but had no effect on β-globin expression. We also demonstrated that intracellular reactive oxygen species (ROS) levels were increased by treatment with thalidomide for 48 hours (from day 3 to day 5). Western blot analysis demonstrated that thalidomide activated the p38 mitogen-activated protein kinase (MAPK) signaling pathway in a time- and dose-dependent manner and increased histone H4 acetylation. Pretreatment of cells with the antioxidant enzyme catalase and the intracellular hydroxyl scavenger dimethylthiourea (DMTU) abrogated the thalidomide-induced p38 MAPK activation and histone H4 acetylation. Moreover, pretreatment with catalase and DMTU diminished thalidomide-induced γ-globin gene expression. These data indicate that thalidomide induces increased expression of the γ-globin gene via ROS-dependent activation of the p38 MAPK signaling pathway and histone H4 acetylation. PMID:17620452

  19. Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation.

    PubMed

    Huang, Xiaosong; Wang, Ying; Yan, Wei; Smith, Cory; Ye, Zhaohui; Wang, Jing; Gao, Yongxing; Mendelsohn, Laurel; Cheng, Linzhao

    2015-05-01

    Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479.

  20. Ruminant globin gene structures suggest an evolutionary role for Alu-type repeats.

    PubMed Central

    Schimenti, J C; Duncan, C H

    1984-01-01

    Bovine fetal and adult globin genes were cloned and subjected to DNA sequence analysis. Both of these genes contained insertions of Alu-type repetitive DNA within their introns. Comparison of cow and goat beta-type globin genes indicates that intragenic DNA insertions played a role in their evolution. These data support the theory that Alu-type repeats maintain genetic diversity by inhibiting gene conversion. PMID:6322113

  1. Globin and globin gene structure of the nerve myoglobin of Aphrodite aculeata.

    PubMed

    Dewilde, S; Blaxter, M; Van Hauwaert, M L; Vanfleteren, J; Esmans, E L; Marden, M; Griffon, N; Moens, L

    1996-08-16

    The globin of the nerve cord of the polychaete annelid Aphrodite aculeata was isolated and purified to homogeneity. The native molecule has a pI of 6.3 and acts as a dimer of two identical Mr 15, 644.5 polypeptide chains as determined by electrospray mass spectrometry. It has an average affinity for oxygen (P50 = 1.24 torr) resulting from fast association (kon = 170 X 10(6) M-1 . s-1) and dissociation rates (koff = 360 s-1). The partial primary structure of this nerve globin was determined at the protein level and completed and confirmed by translation of the cDNA sequence. The globin chain has 150 amino acid residues and a calculated Mr of 15, 602.69 strongly suggesting that the amino terminus is acetylated. The absence of a leader sequence and the lack of Cys at the positions NA2 and H9 needed for the formation of the high Mr complexes found in extracellular annelid globins classify the Aphrodite globin with the cellular globin species. The Aphrodite nerve globin is unlikely to represent a separate globin family, as cDNA derived primers detect globin messenger RNA in muscle, gut, and pharynx tissue as well. The gene encoding this globin species is interrupted by a single intron, inserted at position G7.0. Comparison to other globin gene structures strongly suggest that introns can be lost independently, rather than simultaneously as a result of a single conversion event as suggested previously (Lewin, R. (1984) Science 226, 328).

  2. The nucleotide sequence of the human beta-globin gene.

    PubMed

    Lawn, R M; Efstratiadis, A; O'Connell, C; Maniatis, T

    1980-10-01

    We report the complete nucleotide sequence of the human beta-globin gene. The purpose of this study is to obtain information necessary to study the evolutionary relationships between members of the human beta-like globin gene family and to provide the basis for comparing normal beta-globin genes with those obtained from the DNA of individuals with genetic defects in hemoglobin expression.

  3. Mutation screening in the human epsilon-globin gene using single-strand conformation polymorphism analysis.

    PubMed

    Papachatzopoulou, Adamantia; Menounos, Panagiotis G; Kolonelou, Christina; Patrinos, George P

    2006-02-01

    The human epsilon-globin gene is necessary for primitive human erythropoiesis in the yolk sac. Herein we report a non-radioactive single-strand conformation polymorphism (SSCP) approach to screen the human epsilon-globin gene and its regulatory regions for possible mutations and single-nucleotide polymorphisms in normal adult subjects, in order to determine those genomic regions, which are not necessary for its proper regulation and function. We identified no sequence variations apart from the expected 5'epsilon /HincII polymorphism in the fragments analyzed, suggesting that genomic alterations in the epsilon-globin gene are most likely incompatible with normal erythropoiesis and proper embryonic development.

  4. A newly discovered human α-globin gene

    PubMed Central

    Goh, Sung-Ho; Lee, Y. Terry; Bhanu, Natarajan V.; Cam, Margaret C.; Desper, Richard; Martin, Brian M.; Moharram, Ramy; Gherman, Robert B.; Miller, Jeffery L.

    2005-01-01

    A previously undefined transcript with significant homology to the pseudo-α2 region of the α-globin locus on human chromosome 16 was detected as part of an effort to better define the transcriptional profiles of human reticulocytes. Cloning and sequencing of that transcript (GenBank AY698022; named μ-globin) revealed an insert with a 423-nucleotide open reading frame. BLASTP and ClustalW and phylogenetic analyses of the predicted protein demonstrated a high level of homology with the avian α-D globin. In addition, the heme- and globin-binding amino acids of μ-globin and avian α-D globin are largely conserved. Using quantitative real-time polymerase chain reaction (PCR), μ-globin was detected at a level of approximately 0.1% that measured for α-globin in erythroid tissues. Erythroid-specific expression was detected by Northern blot analysis, and maximal expression during the erythroblast terminal differentiation was also detected. Despite this highly regulated pattern of μ-globin gene transcription, μ-globin protein was not detected by mass spectrometry. These results suggest the human genome encodes a previously unrecognized globin member of the avian α-D family that is transcribed in a highly regulated pattern in erythroid cells. (Blood. 2005;106:1466-1472) PMID:15855277

  5. Globin gene structure in a reptile supports the transpositional model for amniote α- and β-globin gene evolution.

    PubMed

    Patel, Vidushi S; Ezaz, Tariq; Deakin, Janine E; Graves, Jennifer A Marshall

    2010-12-01

    The haemoglobin protein, required for oxygen transportation in the body, is encoded by α- and β-globin genes that are arranged in clusters. The transpositional model for the evolution of distinct α-globin and β-globin clusters in amniotes is much simpler than the previously proposed whole genome duplication model. According to this model, all jawed vertebrates share one ancient region containing α- and β-globin genes and several flanking genes in the order MPG-C16orf35-(α-β)-GBY-LUC7L that has been conserved for more than 410 million years, whereas amniotes evolved a distinct β-globin cluster by insertion of a transposed β-globin gene from this ancient region into a cluster of olfactory receptors flanked by CCKBR and RRM1. It could not be determined whether this organisation is conserved in all amniotes because of the paucity of information from non-avian reptiles. To fill in this gap, we examined globin gene organisation in a squamate reptile, the Australian bearded dragon lizard, Pogona vitticeps (Agamidae). We report here that the α-globin cluster (HBK, HBA) is flanked by C16orf35 and GBY and is located on a pair of microchromosomes, whereas the β-globin cluster is flanked by RRM1 on the 3' end and is located on the long arm of chromosome 3. However, the CCKBR gene that flanks the β-globin cluster on the 5' end in other amniotes is located on the short arm of chromosome 5 in P. vitticeps, indicating that a chromosomal break between the β-globin cluster and CCKBR occurred at least in the agamid lineage. Our data from a reptile species provide further evidence to support the transpositional model for the evolution of β-globin gene cluster in amniotes.

  6. Molecular Characterization and Expression of α-Globin and β-Globin Genes in the Euryhaline Flounder (Platichthys flesus).

    PubMed

    Lu, Weiqun; Mayolle, Aurelie; Cui, Guoqiang; Luo, Lei; Balment, Richard J

    2011-01-01

    In order to understand the possible role of globin genes in fish salinity adaptation, we report the molecular characterization and expression of all four subunits of haemoglobin, and their response to salinity challenge in flounder. The entire open reading frames of α1-globin and α2-globin genes were 432 and 435 bp long, respectively, whereas the β1-globin and β2-globin genes were both 447 bp. Although the head kidney (pronephros) is the predicted major site of haematopoiesis, real-time PCR revealed that expression of α-globin and β-globin in kidney (mesonephros) was 1.5 times higher than in head kidney. Notably, the α1-globin and β1-globin mRNA expression was higher than α2-globin and β2-globin in kidney. Expression levels of all four globin subunits were higher in freshwater- (FW-) than in seawater- (SW-)adapted fish kidney. If globins do play a role in salinity adaptation, this is likely to be more important in combating the hemodilution faced by fish in FW than the dehydration and salt loading which occur in SW.

  7. Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

    PubMed

    Lee, M H; Shroff, R; Cooper, S J; Hope, R

    1999-07-01

    Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.

  8. Retroviral transfer of a human beta-globin/delta-globin hybrid gene linked to beta locus control region hypersensitive site 2 aimed at the gene therapy of sickle cell disease.

    PubMed Central

    Takekoshi, K J; Oh, Y H; Westerman, K W; London, I M; Leboulch, P

    1995-01-01

    Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I hypersensitive site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients. Images Fig. 4 Fig. 5 PMID:7708766

  9. A Limited Number of Globin Genes in Human DNA

    PubMed Central

    Gambino, Roberto; Kacian, Daniel; O'Donnell, Joyce; Ramirez, Francesco; Marks, Paul A.; Bank, Arthur

    1974-01-01

    The number of globin genes in human cells was determined by hybridizing DNA from human spleens to 3H-labeled DNA complementary to human globin mRNA. Assuming the rates of reannealing of complementary DNA and cellular DNA are similar, the extent of hybridization of complementary DNA at various ratios of cellular DNA to complementary DNA indicate that there are fewer than 10 globin gene copies per haploid human genome. An alternative analysis of the data, which introduces no assumptions concerning the relative rates of reaction of complementary DNA and cellular DNA, indicates fewer than 20 globin gene copies are present. DNA isolated from the spleen of a patient with β+ thalassemia contained a number of globin gene copies similar to that of normal DNA. PMID:4530276

  10. The role of EKLF in human beta-globin gene competition.

    PubMed

    Wijgerde, M; Gribnau, J; Trimborn, T; Nuez, B; Philipsen, S; Grosveld, F; Fraser, P

    1996-11-15

    We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

  11. Phylogenetic Diversification of the Globin Gene Superfamily in Chordates

    PubMed Central

    Storz, Jay F.; Opazo, Juan C.; Hoffmann, Federico G.

    2015-01-01

    Summary Phylogenetic reconstructions provide a means of inferring the branching relationships among members of multigene families that have diversified via successive rounds of gene duplication and divergence. Such reconstructions can illuminate the pathways by which particular expression patterns and protein functions evolved. For example, phylogenetic analyses can reveal cases in which similar expression patterns or functional properties evolved independently in different lineages, either through convergence, parallelism, or evolutionary reversals. The purpose of this paper is to provide a robust phylogenetic framework for interpreting experimental data and for generating hypotheses about the functional evolution of globin proteins in chordate animals. To do this we present a consensus phylogeny of the chordate globin gene superfamily. We document the relative roles of gene duplication and whole-genome duplication in fueling the functional diversification of vertebrate globins, and we unravel patterns of shared ancestry among globin genes from representatives of the three chordate subphyla (Craniata, Urochordata, and Cephalochordata). Our results demonstrate the value of integrating phylogenetic analyses with genomic analyses of conserved synteny to infer the duplicative origins and evolutionary histories of globin genes. We also discuss a number of case studies that illustrate the importance of phylogenetic information when making inferences about the evolution of globin gene expression and protein function. Finally, we discuss why the globin gene superfamily presents special challenges for phylogenetic analysis, and we describe methodological approaches that can be used to meet those challenges. PMID:21557448

  12. Murine erythroleukemia cell line GM979 contains factors that can activate silent chromosomal human. gamma. -globin genes

    SciTech Connect

    Zitnik, G.; Hines, P.; Stamatoyannopoulos, G.; Papayannopoulou, T. )

    1991-03-15

    The authors introduced a normal chromosome 11 into GM979 murine erythroleukemia cells by fusing them with Epstein-Barr virus-transformed lymphocytes from a normal individual. In contrast to precious data obtained with other murine erythroleukemia cells, they detected activation of human chromosomal {gamma}-globin genes in GM979 cells. GM979, unlike previously used murine erythroleukemia cell lines, expresses murine embryonic globin in addition to adult globin. While all the hybrids expressed {gamma}- and {beta}-globin, they displayed a wide range of {gamma}-globin expression in relation to that of {beta}-globin. No correlation, however, was found in quantitative expression between murine embryonic globin and human {gamma}-globin in these hybrids, suggesting that the two globins are regulated independently, at least in this cell line. These data indicate that {gamma}-globin genes from normal, nonerythroid chromosomes are not irreversibly silenced, and they can be activated by a positive trans factor(s) present in GM979 cells.

  13. Gene Turnover in the Avian Globin Gene Families and Evolutionary Changes in Hemoglobin Isoform Expression

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Natarajan, Chandrasekhar; Witt, Christopher C.; Berenbrink, Michael; Storz, Jay F.

    2015-01-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the αD-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the αA-globin gene), recurrent losses of αD-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa. PMID:25502940

  14. Gene turnover in the avian globin gene families and evolutionary changes in hemoglobin isoform expression.

    PubMed

    Opazo, Juan C; Hoffmann, Federico G; Natarajan, Chandrasekhar; Witt, Christopher C; Berenbrink, Michael; Storz, Jay F

    2015-04-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the α(D)-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the α(A)-globin gene), recurrent losses of α(D)-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa.

  15. Evolution of the Genome 3D Organization: Comparison of Fused and Segregated Globin Gene Clusters.

    PubMed

    Kovina, Anastasia P; Petrova, Natalia V; Gushchanskaya, Ekaterina S; Dolgushin, Konstantin V; Gerasimov, Evgeny S; Galitsyna, Aleksandra A; Penin, Alexey A; Flyamer, Ilya M; Ioudinkova, Elena S; Gavrilov, Alexey A; Vassetzky, Yegor S; Ulianov, Sergey V; Iarovaia, Olga V; Razin, Sergey V

    2017-06-01

    The genomes are folded in a complex three-dimensional (3D) structure. Some features of this organization are common for all eukaryotes, but little is known about its evolution. Here, we have studied the 3D organization and regulation of zebrafish globin gene domain and compared its organization and regulation with those of other vertebrate species. In birds and mammals, the α- and β-globin genes are segregated into separate clusters located on different chromosomes and organized into chromatin domains of different types, whereas in cold-blooded vertebrates, including Danio rerio, α- and β-globin genes are organized into common clusters. The major globin gene locus of Danio rerio is of particular interest as it is located in a genomic area that is syntenic in vertebrates and is controlled by a conserved enhancer. We have found that the major globin gene locus of Danio rerio is structurally and functionally segregated into two spatially distinct subloci harboring either adult or embryo-larval globin genes. These subloci demonstrate different organization at the level of chromatin domains and different modes of spatial organization, which appears to be due to selective interaction of the upstream enhancer with the sublocus harboring globin genes of the adult type. These data are discussed in terms of evolution of linear and 3D organization of gene clusters in vertebrates. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Interaction of five globin gene abnormalities in a Cambodian family.

    PubMed

    Simpkins, H; Hill, A V; Derry, S; Clegg, J B; Weatherall, D J

    1986-05-01

    Members of a Cambodian family with an undiagnosed hypochromic, microcytic anaemia were found by haemoglobin and DNA analysis to have five interacting globin gene abnormalities. One child has Hb E and typical Hb H disease, while his mother has the form of Hb H disease associated with Hb Constant Spring interacting with Hb E. Quantitation of Hbs E and A2 by globin chain separation and triton/urea gel electrophoresis support the concept that Hb H/Constant Spring disease is a more severe form of alpha thalassaemia than Hb H disease. This family illustrates how the remarkably high prevalence of globin gene abnormalities in Southeast Asians can give rise to a series of atypical thalassaemic phenotypes, and how they can be defined by direct globin gene analysis.

  17. The structure of the human zeta-globin gene and a closely linked, nearly identical pseudogene.

    PubMed

    Proudfoot, N J; Gil, A; Maniatis, T

    1982-12-01

    DNA sequencing studies indicate that only one of two closely linked human embryonic alpha-like globin genes, zeta (zeta), encodes a functional polypeptide. The other is a pseudogene (psi zeta) that differs by only 3 bp in the protein coding sequence, one of which converts the codon for amino acid 6 into a chain termination codon. Both zeta-globin genes differ from all other alpha-like genes thus far reported in that they contain large introns consisting, in part, of simple repeat sequences. Intron 1 of each gene contains a variation of the repeat sequence ACAGTGGGGAGGGG, while intron 2 contains the repeat sequence CGGGG. Comparison of the human zeta- and alpha-globin gene sequences reveals that the embryonic and adult alpha-like genes began to diverge from each other relatively early in vertebrate evolution (400 million years ago). In contrast, the beta-like embryonic globin gene, epsilon (epsilon), is the product of a much more recent evolutionary event (200 million years ago). Thus, even though the temporal and quantitative expression of zeta- and epsilon-globin genes must be coordinately controlled during development, their evolutionary histories are clearly distinct.

  18. The linkage arrangement of four rabbit beta-like globin genes.

    PubMed

    Lacy, E; Hardison, R C; Quon, D; Maniatis, T

    1979-12-01

    Four different regions of rabbit beta-like globin gene sequences designated beta 1, beta 2, beta 3 and beta 4 were identified in a set of clones isolated from a bacteriophage lambda library of chromosomal DNA fragments (Maniatis et al., 1978). Restriction mapping and blot hybridization (Southern, 1975) studies indicate that a subset of these clones containing beta 1 and beta 2 hybridizes to an adult beta-globin cDNA clone (Maniatis et al., 1976) more efficiently than to a human gamma-globin cDNA clone (Wilson et al., 1978), while another subset containing beta 3 and beta 4 displays the converse hybridization specificity. beta 1 was identified as the adult beta-globin gene, while beta 2, beta 3 and beta 4 have not been identified with any known rabbit globin polypeptides. Cross-hybridization and transcriptional orientation experiments indicate that the set of beta-like gene clones contains overlapping restriction fragments encompassing 44 kb of rabbit chromosomal DNA. In addition, all four genes have the same transcriptional orientation and are arranged in the order 5'-beta 4-beta 3-beta 2-beta 1-3'.

  19. Distinctive Patterns of Evolution of the δ-Globin Gene (HBD) in Primates

    PubMed Central

    Moleirinho, Ana; Lopes, Alexandra M.; Seixas, Susana; Morales-Hojas, Ramiro; Prata, Maria J.; Amorim, António

    2015-01-01

    In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψβ(HBBP1), and the late expressed genes, δ (HBD) and β (HBB). While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a

  20. Molecular cloning and characterization of the human beta-like globin gene cluster.

    PubMed

    Fritsch, E F; Lawn, R M; Maniatis, T

    1980-04-01

    The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

  1. Fetal Globin Gene Inducers: Novel Agents & New Potential

    PubMed Central

    Perrine, Susan P.; Castaneda, Serguei A.; Chui, David H.; Faller, Douglas V.; Berenson, Ronald J.; Fucharoen, Suthat

    2013-01-01

    Inducing expression of endogenous fetal globin (γ-globin) gene expression to 60-70% of alpha globin synthesis produces β-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced γ-globin expression in beta thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as 7 years. However, prior generations were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic sodium 2,2-dimethylbutyrate is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of 3 major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used world-wide. PMID:20712788

  2. Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum.

    PubMed

    Shishikura, Fumio; Takeuchi, Hiro-aki; Nagai, Takatoshi

    2005-11-01

    Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.

  3. Conservation of globin genes in the "living fossil" Latimeria chalumnae and reconstruction of the evolution of the vertebrate globin family.

    PubMed

    Schwarze, Kim; Burmester, Thorsten

    2013-09-01

    The (hemo-)globins are among the best-investigated proteins in biomedical sciences. These small heme-proteins play an important role in oxygen supply, but may also have other functions. In addition to well known hemoglobin and myoglobin, six other vertebrate globin types have been identified in recent years: neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Analyses of the genome of the "living fossil" Latimeria chalumnae show that the coelacanth is the only known vertebrate that includes all eight globin types. Thus, Latimeria can also be considered as a "globin fossil". Analyses of gene synteny and phylogenetic reconstructions allow us to trace the evolution and the functional changes of the vertebrate globin family. Neuroglobin and globin X diverged from the other globin types before the separation of Protostomia and Deuterostomia. The cytoglobins, which are unlikely to be involved in O2 supply, form the earliest globin branch within the jawed vertebrates (Gnathostomata), but do not group with the agnathan hemoglobins, as it has been proposed before. There is strong evidence from phylogenetic reconstructions and gene synteny that the eye-specific globin E and muscle-specific myoglobin constitute a common clade, suggesting a similar role in intracellular O2 supply. Latimeria possesses two α- and two β-hemoglobin chains, of which one α-chain emerged prior to the divergence of Actinopterygii and Sarcopterygii, but has been retained only in the coelacanth. Notably, the embryonic hemoglobin α-chains of Gnathostomata derive from a common ancestor, while the embryonic β-chains - with the exception of a more complex pattern in the coelacanth and amphibians - display a clade-specific evolution. Globin Y is associated with the hemoglobin gene cluster, but its phylogenetic position is not resolved. Our data show an early divergence of distinct globin types in the vertebrate evolution before the emergence of tetrapods. The subsequent loss of

  4. Repeated Evolution of Chimeric Fusion Genes in the β-Globin Gene Family of Laurasiatherian Mammals

    PubMed Central

    Gaudry, Michael J.; Storz, Jay F.; Butts, Gary Tyler; Campbell, Kevin L.; Hoffmann, Federico G.

    2014-01-01

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB “Lepore” deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived “anti-Lepore” duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20–100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion. PMID:24814285

  5. Repeated evolution of chimeric fusion genes in the β-globin gene family of laurasiatherian mammals.

    PubMed

    Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler; Campbell, Kevin L; Hoffmann, Federico G

    2014-05-09

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  6. Analysis of the human [alpha]-globin gene cluster in transgenic mice

    SciTech Connect

    Sharpe, J.A.; Vyas, P.; Higgs, D.R.; Wood, W.G. ); Wells, D.J. ); Whitelaw, E. )

    1993-11-15

    A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS -40), upstream of the [alpha]-globin gene cluster, has been identified as the major tissue-specific regulator of the [alpha]-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of [alpha] gene expression, the authors have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human [zeta]- and [alpha]-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human [alpha]-globin genes is not equivalent to that upstream of the [beta] locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.

  7. Retroviral-mediated transfer of genomic globin genes leads to regulated production of RNA and protein

    SciTech Connect

    Karlsson, S.; Papayannopoulou, T.; Schweiger, S.G.; Stamatoyannopoulos, G.; Nienhuis, A.W.

    1987-04-01

    A high-titer amphotropic retroviral vector containing the neomycin resistance gene and a hybrid ..gamma..-..beta.. genomic human globin gene has been constructed. Mouse erythroleukemia cells infected with this virus were found to contain the full transcriptional unit of the transferred human globin gene by Southern blot analysis. These cells contain normally initiated, spliced, and terminated human globin mRNA. The human globin mRNA level increased 5- to 10-fold upon induction of the mouse erythroleukemia cells. Human globin chains were produced but only in a fraction of the cells as detected by immunofluorescent staining. A similar retrovirus containing a human ..beta..-globin gene was used to transduce mouse erythroleukemia cells resulting in much higher levels of human globin synthesis than detected in mouse erythroleukemia cells transduced with the ..gamma..-..beta.. globin virus.

  8. Identification of patients with defects in the globin genes

    PubMed Central

    Dell’Edera, Domenico; Epifania, Annunziata Anna; Milazzo, Giusi Natalia; Leo, Manuela; Santacesaria, Carmela; Allegretti, Arianna; Mazzone, Eleonora; Panetta, Paolo; Iammarino, Giovanna; Lupo, Maria Giovanna; Barbieri, Rocchina; Lioi, Maria Brigida

    2013-01-01

    Summary Introduction hemoglobinopathies constitute a major health problem worldwide. These disorders are characterized by a clinical and hematological phenotypic heterogeneity. The increase of HbA2 is an invaluable hematological marker of the beta-thalassemia heterozygosis and of double heterozygosis for the alleles of delta and alpha globin genes or for the alleles of delta and beta globin genes which can cause the increase of HbA2 up to normal or borderline values. Case Report we report the case of a 30-year-old woman (first pregnant) who was admitted to our Unit at 12 weeks for a screening for thalassemia. The outcomes of the biochemical and haematological exams (MCV, MCH, HbA2, HbF) highlighted that the patient was a carrier of a beta-thalassemic trait. Molecular analysis of the beta globin genes highlighted a β039C>T heterozygous mutation. Biochemical and hematological parameters of the husband (MCV, MCH, HbA2, HbF) were normal except for the level of HbA2 (3,6%). The molecular analysis of the beta globin genes highlighted a IVS2 nt844 C>G heterozygous mutation. Furthermore, the heterozygous mutation δ+cod.27G>T was detected in his δ globin gene. For this reason, he was diagnosed a δ+β Thal. Conclusions the aim of this paper is to highlight that biochemical diagnosis could not exhaustive and a molecular diagnostic widening is required to detect the genetic deficiency causing the thalassemic trait. PMID:24611095

  9. The hematopoietic regulator TAL1 is required for chromatin looping between the β-globin LCR and human γ-globin genes to activate transcription

    PubMed Central

    Yun, Won Ju; Kim, Yea Woon; Kang, Yujin; Lee, Jungbae; Dean, Ann; Kim, AeRi

    2014-01-01

    TAL1 is a key hematopoietic transcription factor that binds to regulatory regions of a large cohort of erythroid genes as part of a complex with GATA-1, LMO2 and Ldb1. The complex mediates long-range interaction between the β-globin locus control region (LCR) and active globin genes, and although TAL1 is one of the two DNA-binding complex members, its role is unclear. To explore the role of TAL1 in transcription activation of the human γ-globin genes, we reduced the expression of TAL1 in erythroid K562 cells using lentiviral short hairpin RNA, compromising its association in the β-globin locus. In the TAL1 knockdown cells, the γ-globin transcription was reduced to 35% and chromatin looping of the Gγ-globin gene with the LCR was disrupted with decreased occupancy of the complex member Ldb1 and LMO2 in the locus. However, GATA-1 binding, DNase I hypersensitive site formation and several histone modifications were largely maintained across the β-globin locus. In addition, overexpression of TAL1 increased the γ-globin transcription and increased interaction frequency between the Gγ-globin gene and LCR. These results indicate that TAL1 plays a critical role in chromatin loop formation between the γ-globin genes and LCR, which is a critical step for the transcription of the γ-globin genes. PMID:24470145

  10. Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

    NASA Astrophysics Data System (ADS)

    Lau, Yun-Fai; Kan, Yuet Wai

    1983-09-01

    We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

  11. Presence of tadpole and adult globin RNA sequences in oocytes of Xenopus laevis

    PubMed Central

    Perlman, S. M.; Ford, P. J.; Rosbash, M. M.

    1977-01-01

    Complementary DNA transcribed from adult Xenopus laevis globin mRNA was used to assay ovary RNA from Xenopus for the presence of globin sequences by RNA·cDNA hybridization. These sequences are present at approximately the same concentration as the majority of poly(A)-containing ovary sequences. The sequences are also found at approximately 200,000 copies per cell in poly(A)-containing RNA extracted from mature oocytes. To rule out contamination of the oocytes with somatic cells, two additional experiments were performed. First, RNA isolated from ovulated unfertilized eggs, which are devoid of somatic cells, was also shown to contain the globin sequences. Second, globin mRNA was isolated from Xenopus tadpoles. Adult globin mRNA is free of the tadpole sequence and no homology was detected between adult and tadpoles globin RNA. The ovary was shown to contain tadpole globin RNA at nearly the same concentration as the adult sequences. Thus, the results cannot be explained by contamination with erythroid cells which should contain only the adult sequence. The swimming tadpole, which possesses an active circulatory system, was also assayed for the tadpole and adult globin sequences. Whereas the adult sequences are present at approximately the same concentration as in the mature oocyte, the concentration of the tadpole sequences increases at least 300-fold in the first 3 days following fertilization. PMID:269434

  12. Structure and in vitro transcription of human globin genes.

    PubMed

    Proudfoot, N J; Shander, M H; Manley, J L; Gefter, M L; Maniatis, T

    1980-09-19

    The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.

  13. Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

    SciTech Connect

    Miller, A.D.; Bender, M.A.; Harris, E.A.S.; Kaleko, M.; Gelinas, R.E.

    1988-11-01

    Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.

  14. α-Globin gene mutations in Isfahan Province, Iran.

    PubMed

    Karamzade, Arezo; Mirzapour, Hadi; Hoseinzade, Majid; Asadi, Sara; Gholamrezapour, Tahere; Tavakoli, Parvaneh; Salehi, Mansoor; Selebi, Mansoor

    2014-01-01

    α-Thalassemia (α-thal) encompasses a spectrum of mutations including deletion and point mutations on the α-globin chains that is characterized by a reduction or complete absence of α-globin genes. Most of the α-thal cases are deletions involving one (α(+)) or both (α(0)) α-globin genes, although point mutations (α(T)α or αα(T)) are found as well. In this study, 314 individuals with low hematological values, normal Hb A2 who were not affected with β-thal or iron deficiency, were investigated for the presence of α-thal mutations. The most common deletion was -α(3.7) (rightward) with a frequency of 70.7%, followed by α(-5 nt) (-TGAGG) (8.7%), -α(4.2) (leftward) (4.7%), the polyadenylation signal (polyA2) site (AATAAA > AATGAA) (4.2%), -(α)(20.5) (3.8%), Hb Constant Spring [Hb CS, α142, Stop→Gln; HBA2: c.427T > C] (2.9%), polyA1 (AATAAA > AATAAG) and α(codon 19) (GCG > GC-, α2) (16%), and - -(MED) (0.9%). The results of this study may be valuable for designing a plan for carrier screening, premarital genetic counseling, prenatal diagnosis (PND) and reducing excessive health care costs to an affordable level in Isfahan Province, Iran.

  15. The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells.

    PubMed

    Chao, M V; Mellon, P; Charnay, P; Maniatis, T; Axel, R

    1983-02-01

    We have introduced a hybrid mouse-human beta-globin gene as well as the intact human beta-globin gene into murine erythroleukemia (MEL) cells and have demonstrated that these genes are appropriately regulated during differentiation of the MEL cell in culture. The addition of chemical inducers to cotransformed cells results in a 5 to 50 fold increase in the level of mRNA transcribed from the exogenous globin gene. S1 nuclease and primer extension analyses demonstrate that these mRNAs initiate and terminate correctly. Nuclear transcription experiments indicate that induction of hybrid mRNA results at least in part from the increase in the rate of globin gene transcription. Furthermore, the induction appears to be specific for globin genes within an erythroid cell. These results permit the study of expression of the globin gene during erythroid differentiation and suggest that the specific induction of the globin gene is an inherent property of DNA sequences within or flanking the beta-globin genes. Moreover, the fact that the human and hybrid globin genes are both inducible in MEL cells suggests that these regulatory sequences are conserved between mouse and human cells.

  16. New Genes Originated via Multiple Recombinational Pathways in the β-Globin Gene Family of Rodents

    PubMed Central

    Hoffmann, Federico G.; Opazo, Juan C.; Storz, Jay F.

    2008-01-01

    Species differences in the size or membership composition of multigene families can be attributed to lineage-specific additions of new genes via duplication, losses of genes via deletion or inactivation, and the creation of chimeric genes via domain shuffling or gene fusion. In principle, it should be possible to infer the recombinational pathways responsible for each of these different types of genomic change by conducting detailed comparative analyses of genomic sequence data. Here, we report an attempt to unravel the complex evolutionary history of the β-globin gene family in a taxonomically diverse set of rodent species. The main objectives were: 1) to characterize the genomic structure of the β-globin gene cluster of rodents; 2) to assign orthologous and paralogous relationships among duplicate copies of β-like globin genes; and 3) to infer the specific recombinational pathways responsible for gene duplications, gene deletions, and the creation of chimeric fusion genes. Results of our comparative genomic analyses revealed that variation in gene family size among rodent species is mainly attributable to the differential gain and loss of later expressed β-globin genes via unequal crossing-over. However, two distinct recombinational mechanisms were implicated in the creation of chimeric fusion genes. In muroid rodents, a chimeric γ/ε fusion gene was created by unequal crossing-over between the embryonic ε- and γ-globin genes. Interestingly, this γ/ε fusion gene was generated in the same fashion as the “anti-Lepore” 5′-δ-(β/δ)-β-3′ duplication mutant in humans (the reciprocal exchange product of the pathological hemoglobin Lepore deletion mutant). By contrast, in the house mouse, Mus musculus, a chimeric β/δ fusion pseudogene was created by a β-globin → δ-globin gene conversion event. Although the γ/ε and β/δ fusion genes share a similar chimeric gene structure, they originated via completely different recombinational pathways. PMID

  17. Characterization of the enhancer element of the Danio rerio minor globin gene locus.

    PubMed

    Nefedochkina, Anastasia V; Petrova, Natalia V; Ioudinkova, Elena S; Kovina, Anastasia P; Iarovaia, Olga V; Razin, Sergey V

    2016-04-01

    In Danio rerio, the alpha- and beta-globin genes are present in two clusters: a major cluster located on chromosome 3 and a minor cluster located on chromosome 12. In contrast to the segregated alpha- and beta-globin gene domains of warm-blooded animals, in Danio rerio, each cluster contains both alpha- and beta-globin genes. Expression of globin genes present in the major cluster is controlled by an erythroid-specific enhancer similar to the major regulatory element of mammalian and avian alpha-globin gene domains. The enhancer controlling expression of the globin genes present in the minor locus has not been identified yet. Based on the distribution of epigenetic marks, we have selected two genomic regions that might harbor an enhancer of the minor locus. Using transient transfection of constructs with a reporter gene, we have demonstrated that a ~500-bp DNA fragment located ~1.7 Kb upstream of the αe4 gene possesses an erythroid-specific enhancer active with respect to promoters present in both the major and the minor globin gene loci of Danio rerio. The identified enhancer element harbors clustered binding sites for GATA-1, NF-E2, and EKLF similar to the enhancer of the major globin locus on chromosome 3. Both enhancers appear to have emerged as a result of independent evolution of a duplicated regulatory element present in an ancestral single alpha-/beta-globin locus that existed before teleost-specific genome duplication.

  18. Expression of human. alpha. -globin and mouse/human hybrid. beta. -globin genes in murine hemopoietic stem cells transduced by recombinant retroviruses

    SciTech Connect

    Li, C.L.; Dwarki, V.J.; Verma, I.M. )

    1990-06-01

    Murine cell lines releasing helper-free recombinant retroviruses containing human {alpha}-globin and mouse/human hybrid {beta}-globin genes were generated. The expression of the hybrid {beta}-globin gene but not the human {alpha}-globin gene was regulated appropriately in infected mouse erythroid leukemia (MEL) cells. Murine bone marrow cells were infected by coculture with virus-producing cells and transplanted into lethally irradiated syngeneic recipients. Greater than 90% of the spleen colonies (12-15 days), which are derived from hemopoietic multipotential stem cells, showed proviral integration. Various levels of expression of the transduced globin genes were detected in all of the provirus-positive spleen colonies. Proviral sequences and transcripts from the transduced globin genes could also be detected in a few long-term reconstituted recipients in an observation period of 10 months after transplantation.

  19. The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification

    PubMed Central

    2008-01-01

    Background Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles. Results In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection. Conclusion Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying

  20. The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification.

    PubMed

    Hoogewijs, David; De Henau, Sasha; Dewilde, Sylvia; Moens, Luc; Couvreur, Marjolein; Borgonie, Gaetan; Vinogradov, Serge N; Roy, Scott W; Vanfleteren, Jacques R

    2008-10-09

    Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles. In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection. Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying selection subsequently

  1. Sardinian delta beta zero-thalassemia: a further example of a C to T substitution at position -196 of the A gamma globin gene promoter.

    PubMed

    Ottolenghi, S; Giglioni, B; Pulazzini, A; Comi, P; Camaschella, C; Serra, A; Guerrasio, A; Saglio, G

    1987-04-01

    Selective overexpression (50- to 100-fold) in adult erythroid cells of either G gamma or A gamma fetal globin gene is observed in hereditary conditions known as delta beta zero-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Recently, a C----T change at position -196 of an overexpressed A gamma globin gene from an Italian HPFH was hypothesized, on the basis of indirect evidence, to represent the cause of the functional defect. We now show that the same mutation is present in a different overexpressed A gamma-globin gene from a Sardinian patient with a different syndrome (delta beta zero-thalassemia). The Sardinian A gamma globin gene differs from both the HPFH and the normal A gamma globin gene at nucleotide 1,560 in the noncoding portion of the third exon, where an A is deleted. In addition, the mutant -196 A gamma-globin gene is linked to a normal beta globin gene in HPFH, and to a beta-thalassemic gene (beta 39CAG----TAG) in delta beta zero-thalassemia. These data strengthen the suggestion that -196 mutation is causally linked to the abnormal phenotype and raise the question of whether the same or multiple mutational events are responsible for the appearance of the -196 mutation in different syndromes.

  2. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

    PubMed

    Green, M R; Treisman, R; Maniatis, T

    1983-11-01

    When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

  3. Genetic recombination as a major cause of mutagenesis in the human globin gene clusters.

    PubMed

    Borg, Joseph; Georgitsi, Marianthi; Aleporou-Marinou, Vassiliki; Kollia, Panagoula; Patrinos, George P

    2009-12-01

    Homologous recombination is a frequent phenomenon in multigene families and as such it occurs several times in both the alpha- and beta-like globin gene families. In numerous occasions, genetic recombination has been previously implicated as a major mechanism that drives mutagenesis in the human globin gene clusters, either in the form of unequal crossover or gene conversion. Unequal crossover results in the increase or decrease of the human globin gene copies, accompanied in the majority of cases with minor phenotypic consequences, while gene conversion contributes either to maintaining sequence homogeneity or generating sequence diversity. The role of genetic recombination, particularly gene conversion in the evolution of the human globin gene families has been discussed elsewhere. Here, we summarize our current knowledge and review existing experimental evidence outlining the role of genetic recombination in the mutagenic process in the human globin gene families.

  4. Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin −198 Mutation in the γ-Globin Gene Promoter*

    PubMed Central

    Olave, Ivan A.; Doneanu, Catalin; Fang, Xiangdong; Stamatoyannopoulos, George; Li, Qiliang

    2010-01-01

    Expression of the γ-globin gene is silenced in adult humans. However, certain point mutations in the γ-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T → C point mutation at position −198 of the Aγ-globin gene promoter results in 4–10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH −198 γ-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH −198 γ-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of γ-globin gene expression in the adult transcriptional environment is discussed. PMID:17114178

  5. Molecular nature of alpha-globin genes in the Saudi population

    PubMed Central

    Borgio, J. Francis

    2015-01-01

    Alpha-thalassemia (α-thal) is a disorder caused by the deletion of single or double α-globin genes, and/or point mutations in the α-globin genes. There are 2 common types of α-globin genes; HBA2 and HBA1. Recently, it has been discovered that the HBA2 gene is replaced by a unique HBA12 gene convert in 5.7% of the Saudi population. The α-globin genes have been emerging as a molecular target for the treatment of β-thalassemia (β-thal). Hence, it is essential to understand the molecular nature of α-globin genes to treat the most prevalent hemoglobin disorders, such as sickle cell disease, α-thal, and β-thal prevalent in the Kingdom of Saudi Arabia. Thirty-two different α-globin genotypes have been observed in the Saudi population. This review outlines the classification of the α-globin genes on the basis of their molecular nature and complex combinations of α-globin genes, and their variants predominant in Saudis. PMID:26593158

  6. Rate constancy of globin gene evolution in placental mammals.

    PubMed Central

    Easteal, S

    1988-01-01

    The molecular clock hypothesis is investigated by comparison of the rates of nucleotide substitution in globin genes of mice, cows and goats, humans, and rabbits, using the relative rate test. These comparisons are based on a branching order of genes and species established by cladistic analysis of nucleotide sequences. The species branching order is shown to be mouse, cow/goat, human, and rabbit. Relative rate tests involving paralogous and orthologous genes provide no evidence of heterogeneity, among species, in the rate of evolution of the genes. This result is discrepant with the conclusions of most other recent, similar studies. By comparison with previous studies, the present study is based on a sound phylogeny and involves a larger sample of species, genes, and genic regions. The result provides strong support for the neutral theory of molecular evolution and demonstrates that molecular evolutionary rate does not depend on generation time. PMID:3174656

  7. An insulator embedded in the chicken α-globin locus regulates chromatin domain configuration and differential gene expression.

    PubMed

    Furlan-Magaril, Mayra; Rebollar, Eria; Guerrero, Georgina; Fernández, Almudena; Moltó, Eduardo; González-Buendía, Edgar; Cantero, Marta; Montoliu, Lluís; Recillas-Targa, Félix

    2011-01-01

    Genome organization into transcriptionally active domains denotes one of the first levels of gene expression regulation. Although the chromatin domain concept is generally accepted, only little is known on how domain organization impacts the regulation of differential gene expression. Insulators might hold answers to address this issue as they delimit and organize chromatin domains. We have previously identified a CTCF-dependent insulator with enhancer-blocking activity embedded in the 5' non-coding region of the chicken α-globin domain. Here, we demonstrate that this element, called the αEHS-1.4 insulator, protects a transgene against chromosomal position effects in stably transfected cell lines and transgenic mice. We found that this insulator can create a regulated chromatin environment that coincides with the onset of adult α-globin gene expression. Furthermore, such activity is in part dependent on the in vivo regulated occupancy of CTCF at the αEHS-1.4 element. Insulator function is also regulated by CTCF poly(ADP-ribosyl)ation. Our results suggest that the αEHS-1.4 insulator contributes in organizing the chromatin structure of the α-globin gene domain and prevents activation of adult α-globin gene expression at the erythroblast stage via CTCF.

  8. An insulator embedded in the chicken α-globin locus regulates chromatin domain configuration and differential gene expression

    PubMed Central

    Furlan-Magaril, Mayra; Rebollar, Eria; Guerrero, Georgina; Fernández, Almudena; Moltó, Eduardo; González-Buendía, Edgar; Cantero, Marta; Montoliu, Lluís; Recillas-Targa, Félix

    2011-01-01

    Genome organization into transcriptionally active domains denotes one of the first levels of gene expression regulation. Although the chromatin domain concept is generally accepted, only little is known on how domain organization impacts the regulation of differential gene expression. Insulators might hold answers to address this issue as they delimit and organize chromatin domains. We have previously identified a CTCF-dependent insulator with enhancer-blocking activity embedded in the 5′ non-coding region of the chicken α-globin domain. Here, we demonstrate that this element, called the αEHS-1.4 insulator, protects a transgene against chromosomal position effects in stably transfected cell lines and transgenic mice. We found that this insulator can create a regulated chromatin environment that coincides with the onset of adult α-globin gene expression. Furthermore, such activity is in part dependent on the in vivo regulated occupancy of CTCF at the αEHS-1.4 element. Insulator function is also regulated by CTCF poly(ADP-ribosyl)ation. Our results suggest that the αEHS-1.4 insulator contributes in organizing the chromatin structure of the α-globin gene domain and prevents activation of adult α-globin gene expression at the erythroblast stage via CTCF. PMID:20813760

  9. [Joint locus of a/b-globin genes in Danio rerio is segregated into structural subdomains active at different stages of development].

    PubMed

    Dolgushin, K V; Petrova, N V; Iudinkova, E S; Razin, S V; Iarovaia, O V

    2015-01-01

    In the domain model of eukaryotic genome organization, the functional unit of the genome, along with the relevant regulatory elements, is considered to be a gene or a gene family. In hot-blooded vertebrate animals, the domains of a- and b-globin genes are positioned at different chromosomes and are organized and regulated in different fashion. In cold-blooded animals, in particular in tropical fish Danio rerio, a- and b globin genes are located in a common gene cluster. However, the joint a/b-globin gene cluster is subdivided into two development stage-specific subdomains, the adult one and the embryonic-larval one. In an attempt to find out whether this functional segregation correlates with structural segregation of the domain we compared the DNase I sensitivity and profiles of histone modifications of adult and embryonic-larval segments of the domain in cultured D. rerio fibroblasts. We have demonstrated that, in these nonerythroid cells, adult and embryonic- larval subdomains possess different DNase I sensitivities and different profiles of H3K27me3, a histone modification introduced by PRC2 complex. These observations suggest that joint a/b globin gene domain of Danio rerio is segregated into two structural subdomain harboring adult and embryonic-larval globin genes.

  10. Organization of a β and α globin gene set in the teleost Atlantic cod, Gadus morhua.

    PubMed

    Halldórsdóttir, Katrín; Árnason, Einar

    2009-12-01

    Developmental globin gene expression and gene switching in vertebrates have been extensively studied. Globin gene regions have been characterized in some fish species and show linked α and β loci. Understanding coordinated expression between α and β globin genes in fish is of importance for further insights into globin gene regulation in teleosts and higher vertebrates. We characterize linked β and α globin genes in Atlantic cod, pulled from the Atlantic cod genome with a PCR research strategy, by screening a genomic λ library and primer walking. The genes are oriented tail-to-head (5'-3'), differing from the head-to-head orientation in transcriptional polarity characteristic of teleostean globin genes. Four tandem repeats are found in an intergenic region of 1500 base pairs. One microsatellite, which consists primarily of atg tandem repeats, has an open reading frame. The globin genes and open reading frame have a CCAAT promoter element and TATA boxes. The promoters of the open reading frame and the β gene share an 89-bp block (with 100% identity) that probably regulates transcription.

  11. Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase-neo cassette located between the murine beta-major and beta-minor globin genes.

    PubMed

    Kaufman, R M; Lu, Z H; Behl, R; Holt, J M; Ackers, G K; Ley, T J

    2001-07-01

    For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked beta-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant beta-major and beta-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the beta(6I) mRNA was indistinguishable from that of the wild-type counterpart in bone marrow cells. Similarly, the output from the beta-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult beta-globin genes. (Blood. 2001;98:65-73)

  12. Evolution of the globin gene family in deuterostomes: lineage-specific patterns of diversification and attrition.

    PubMed

    Hoffmann, Federico G; Opazo, Juan C; Hoogewijs, David; Hankeln, Thomas; Ebner, Bettina; Vinogradov, Serge N; Bailly, Xavier; Storz, Jay F

    2012-07-01

    In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available

  13. Mutations on the α2-Globin Gene That May Trigger α(+)-Thalassemia.

    PubMed

    Farashi, Samaneh; Vakili, Shadi; Garous, Negin F; Ashki, Mehri; Imanian, Hashem; Azarkeivan, Azita; Najmabadi, Hossein

    2015-01-01

    In the present study, a total of 11 individuals with hypochromic microcytic anemia who did not reveal the most common α-thalassemia (α-thal) deletions or mutations, were subjected to more investigations by DNA sequencing of the α-globin genes. Seven novel nondeletional α-thal mutations localized on the α2-globin gene in the heterozygous state were identified. These mutations either corrupted regulatory splice sites and consequently affected RNA processing or created unstable hemoglobin (Hb) variants. The mutations described here produced globin gene variants that lead to amino acid changes in critical regions of the globin chain. The clinical presentation of most patients was a persistent mild microcytic anemia similar to an α(+)-thal. In the last decade, numerous α-globin mutations have been observed leading to an α-thal phenotype and these studies have been considered to be important as discussed here.

  14. Origin and Ascendancy of a Chimeric Fusion Gene: The β/δ-Globin Gene of Paenungulate Mammals

    PubMed Central

    Opazo, Juan C.; Sloan, Angela M.; Campbell, Kevin L.

    2009-01-01

    The δ-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked β-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric β/δ fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the β/δ fusion gene in elephants is structurally similar to the “anti-Lepore” duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric β/δ fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the β-chain subunits of adult hemoglobin. A phylogenetic survey of β-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric β/δ fusion gene and the concomitant inactivation of the HBB gene predated the radiation of “Paenungulata,” a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived β/δ fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal δ/β fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin. PMID:19332641

  15. Eos Negatively Regulates Human γ-globin Gene Transcription during Erythroid Differentiation

    PubMed Central

    Yu, Hai-Chuan; Zhao, Hua-Lu; Wu, Zhi-Kui; Zhang, Jun-Wu

    2011-01-01

    Background Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4), a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. Methodology/Principal Findings Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs). DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3) of the β-globin locus control region (LCR), the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C) assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. Conclusions/Significance Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation. PMID:21829552

  16. Eos negatively regulates human γ-globin gene transcription during erythroid differentiation.

    PubMed

    Yu, Hai-Chuan; Zhao, Hua-Lu; Wu, Zhi-Kui; Zhang, Jun-Wu

    2011-01-01

    Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4), a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs). DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3) of the β-globin locus control region (LCR), the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C) assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation.

  17. Genomic organization and differential signature of positive selection in the alpha and beta globin gene clusters in two cetacean species.

    PubMed

    Nery, Mariana F; Arroyo, José Ignacio; Opazo, Juan C

    2013-01-01

    The hemoglobin of jawed vertebrates is a heterotetramer protein that contains two α- and two β-chains, which are encoded by members of α- and β-globin gene families. Given the hemoglobin role in mediating an adaptive response to chronic hypoxia, it is likely that this molecule may have experienced a selective pressure during the evolution of cetaceans, which have to deal with hypoxia tolerance during prolonged diving. This selective pressure could have generated a complex history of gene turnover in these clusters and/or changes in protein structure themselves. Accordingly, we aimed to characterize the genomic organization of α- and β-globin gene clusters in two cetacean species and to detect a possible role of positive selection on them using a phylogenetic framework. Maximum likelihood and Bayesian phylogeny reconstructions revealed that both cetacean species had retained a similar complement of putatively functional genes. For the α-globin gene cluster, the killer whale presents a complement of genes composed of HBZ, HBK, and two functional copies of HBA and HBQ genes, whereas the dolphin possesses HBZ, HBK, HBA and HBQ genes, and one HBA pseudogene. For the β-globin gene cluster, both species retained a complement of four genes, two early expressed genes-HBE and HBH-and two adult expressed genes-HBD and HBB. Our natural selection analysis detected two positively selected sites in the HBB gene (56 and 62) and four in HBA (15, 21, 49, 120). Interestingly, only the genes that are expressed during the adulthood showed the signature of positive selection.

  18. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.

    PubMed

    Dever, Daniel P; Bak, Rasmus O; Reinisch, Andreas; Camarena, Joab; Washington, Gabriel; Nicolas, Carmencita E; Pavel-Dinu, Mara; Saxena, Nivi; Wilkens, Alec B; Mantri, Sruthi; Uchida, Nobuko; Hendel, Ayal; Narla, Anupama; Majeti, Ravindra; Weinberg, Kenneth I; Porteus, Matthew H

    2016-11-17

    The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

  19. cis and trans activation of globin gene transcription in transient assays.

    PubMed

    Treisman, R; Green, M R; Maniatis, T

    1983-12-01

    We examined the effects of the simian virus 40 enhancer sequence on transcription of cloned human alpha- and beta-globin genes shortly after their introduction into cultured mammalian cells. We find that (i) detectable transcription of the beta-globin gene but not the alpha-globin gene requires linkage to the enhancer; (ii) the enhancer increases the amount of beta-globin RNA at least 100-fold but results in only a 5- to 10-fold increase in the amount of alpha-globin RNA; (iii) plasmid replication does not increase the level of beta-globin RNA, regardless of linkage to the enhancer, but does result in an approximately equal to 50-fold increase in the level of alpha-globin RNA; (iv) the enhancer is not required for and does not increase transcription of either gene in 293 cells, an adenovirus 5-transformed human kidney cell line. We also show that an enhancer sequence is not required for activity of the normally enhancer-dependent simian virus 40 early promoter in 293 cells, indicating that these cells contain a trans-acting factor(s) that circumvents the requirement for the enhancer sequence.

  20. Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients

    PubMed Central

    Salvatori, Francesca; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Brognara, Eleonora; Lampronti, Ilaria; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2013-01-01

    In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations. PMID:19810011

  1. Alpha-globin gene markers identify genetic differences between Australian aborigines and Melanesians.

    PubMed Central

    Tsintsof, A S; Hertzberg, M S; Prior, J F; Mickleson, K N; Trent, R J

    1990-01-01

    Australian aborigines exhibit a number of alpha-globin cluster rearrangements involving both alpha- and zeta-globin genes. alpha+-Thalassemia (-alpha/) in this population is heterogeneous and includes the 3.7 types I, II, and III gene deletions. The alpha alpha alpha/ and zeta zeta zeta/ rearrangements are each found in association with two haplotypes, indicating origins from at least two separate DNA crossover events. Differences in alpha-globin cluster rearrangements and in haplotypes between Australian aborigines, Papua New Guinea highlanders and island Melanesians, are consistent with multiple colonizing events into Australia. PMID:2294746

  2. Organisation of the Hb 1 genes of the Antarctic skate Bathyraja eatonii: new insights into the evolution of globin genes.

    PubMed

    Marino, Katia; Boschetto, Loredana; de Pascale, Donatella; Cocca, Ennio

    2007-12-30

    An extensive investigation of the organisation of globin genes has greatly contributed to the understanding of universal mechanisms of gene evolution and expression. Cartilaginous fish are the first organisms that have evolved the tetrameric form of hemoglobin (Hb). So far, there has been absolute lack of data about globin genes in chondrichthyans. Bathyraja is the dominant rajid south of 60 degrees S. In the framework of the investigations on globin genes of Antarctic red-blooded and Hb-less fish we obtained the cloning of the alpha- and beta-globin cDNAs of the main Hb (Hb 1) of the skate Bathyraja eatonii. Then, a genomic fragment of 6.2 kb was isolated where the Hb 1 alpha and beta genes are linked in a tail-to-head (3' to 5') orientation. The beta-globin gene promoter region and the chromosomal organisation of the Hb 1 genes of B. eatonii have been compared to their homologues in other vertebrates. The finding of a tail-to-head linkage of the Hb 1 alpha- and beta-globin genes in B. eatonii is the first characterisation of the organisation of globin genes in chondrichthyes; such finding offers a novel contribution to the understanding of the evolution of this class of genes. Moreover, the characterisation of chondrichthyan genes is very important for gaining insight into the ancestral state of vertebrate genomes.

  3. Mutations in the paralogous human alpha-globin genes yielding identical hemoglobin variants.

    PubMed

    Moradkhani, Kamran; Préhu, Claude; Old, John; Henderson, Shirley; Balamitsa, Vera; Luo, Hong-Yuan; Poon, Man-Chiu; Chui, David H K; Wajcman, Henri; Patrinos, George P

    2009-06-01

    The human alpha-globin genes are paralogues, sharing a high degree of DNA sequence similarity and producing an identical alpha-globin chain. Over half of the alpha-globin structural variants reported to date are only characterized at the amino acid level. It is likely that a fraction of these variants, with phenotypes differing from one observation to another, may be due to the same mutation but on a different alpha-globin gene. There have been very few previous examples of hemoglobin variants that can be found at both HBA1 and HBA2 genes. Here, we report the results of a systematic multicenter study in a large multiethnic population to identify such variants and to analyze their differences from a functional and evolutionary perspective. We identified 14 different Hb variants resulting from identical mutations on either one of the two human alpha-globin paralogue genes. We also showed that the average percentage of hemoglobin variants due to a HBA2 gene mutation (alpha2) is higher than the percentage of hemoglobin variants due to the same HBA1 gene mutation (alpha1) and that the alpha2/alpha1 ratio varied between variants. These alpha-globin chain variants have most likely occurred via recurrent mutations, gene conversion events, or both. Based on these data, we propose a nomenclature for hemoglobin variants that fall into this category.

  4. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

    PubMed Central

    Cone, R D; Weber-Benarous, A; Baorto, D; Mulligan, R C

    1987-01-01

    We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element. Images PMID:3029570

  5. Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

    PubMed Central

    Calzolari, R; McMorrow, T; Yannoutsos, N; Langeveld, A; Grosveld, F

    1999-01-01

    The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions. PMID:10022837

  6. Alpha thalassaemia and extended alpha globin genes in Sri Lanka.

    PubMed

    Suresh, Sasikala; Fisher, Christopher; Ayyub, Helena; Premawardhena, Anuja; Allen, Angela; Perera, Ashok; Bandara, Dayananda; Olivieri, Nancy; Weatherall, David

    2013-02-01

    The α-globin genes were studied in nine families with unexplained hypochromic anaemia and in 167 patients with HbE β thalassaemia in Sri Lanka. As well as the common deletion forms of α(+) thalassaemia three families from an ethnic minority were found to carry a novel form of α(0) thalassaemia, one family carried a previously reported form of α(0) thalassaemia, --(THAI), and five families had different forms of non-deletional thalassaemia. The patients with HbE β thalassaemia who had co-inherited α thalassaemia all showed an extremely mild phenotype and reduced levels of HbF and there was a highly significant paucity of α(+) thalassaemia in these patients compared with the normal population. Extended α gene arrangements, including ααα, αααα and ααααα, occurred at a low frequency and were commoner in the more severe phenotypes of HbE β thalassaemia. As well as emphasising the ameliorating effect of α thalassaemia on HbE β thalassaemia the finding of a novel form of α(0) thalassaemia in an ethnic minority, together with an unexpected diversity of forms of non-deletion α thalassaemia in Sri Lanka, further emphasises the critical importance of micro-mapping populations for determining the frequency of clinically important forms of the disease. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  7. Understanding α-globin gene regulation and implications for the treatment of β-thalassemia.

    PubMed

    Mettananda, Sachith; Gibbons, Richard J; Higgs, Douglas R

    2016-03-01

    Over the past three decades, a vast amount of new information has been uncovered describing how the globin genes are regulated. This knowledge has provided significant insights into the general understanding of the regulation of human genes. It is now known that molecular defects within and around the α- and β-globin genes, as well as in the distant regulatory elements, can cause thalassemia. Unbalanced production of globin chains owing to defective synthesis of one, and the continued unopposed synthesis of another, is the central causative factor in the cellular pathology and pathophysiology of thalassemia. A large body of clinical, genetic, and experimental evidence suggests that altering globin chain imbalance by reducing the production of α-globin synthesis ameliorates the disease severity in patients with β-thalassemia. With the development of new genetic-based therapeutic tools that have a potential to decrease the expression of a selected gene in a tissue-specific manner, the possibility of decreasing expression of the α-globin gene to improve the clinical severity of β-thalassemia could become a reality. © 2015 New York Academy of Sciences.

  8. Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome

    PubMed Central

    Opazo, Juan C.; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F.

    2015-01-01

    Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about

  9. Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome.

    PubMed

    Opazo, Juan C; Lee, Alison P; Hoffmann, Federico G; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F

    2015-07-01

    Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about

  10. β-globin gene cluster haplotypes in ethnic minority populations of southwest China

    PubMed Central

    Sun, Hao; Liu, Hongxian; Huang, Kai; Lin, Keqin; Huang, Xiaoqin; Chu, Jiayou; Ma, Shaohui; Yang, Zhaoqing

    2017-01-01

    The genetic diversity and relationships among ethnic minority populations of southwest China were investigated using seven polymorphic restriction enzyme sites in the β-globin gene cluster. The haplotypes of 1392 chromosomes from ten ethnic populations living in southwest China were determined. Linkage equilibrium and recombination hotspot were found between the 5′ sites and 3′ sites of the β-globin gene cluster. 5′ haplotypes 2 (+−−−), 6 (−++−+), 9 (−++++) and 3′ haplotype FW3 (−+) were the predominant haplotypes. Notably, haplotype 9 frequency was significantly high in the southwest populations, indicating their difference with other Chinese. The interpopulation differentiation of southwest Chinese minority populations is less than those in populations of northern China and other continents. Phylogenetic analysis shows that populations sharing same ethnic origin or language clustered to each other, indicating current β-globin cluster diversity in the Chinese populations reflects their ethnic origin and linguistic affiliations to a great extent. This study characterizes β-globin gene cluster haplotypes in southwest Chinese minorities for the first time, and reveals the genetic variability and affinity of these populations using β-globin cluster haplotype frequencies. The results suggest that ethnic origin plays an important role in shaping variations of the β-globin gene cluster in the southwestern ethnic populations of China. PMID:28205625

  11. Correction of human. beta. sup S -globin gene by gene targeting

    SciTech Connect

    Shesely, E.G.; Hyungsuk Kim; Shehee, W.R.; Smithies, O. ); Papayannopoulou, T. ); Popovich, B.W. )

    1991-05-15

    As a step toward using gene targeting for gene therapy, the authors have corrected a human {beta}{sup S}-globin gene to the normal {beta}{sup A} allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the {beta}{sup S}-globin allele. A {beta}{sup A}-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total {approx}29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the {beta}{sup S} allele to {beta}{sup A} was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5{prime} to the human {beta}{sup A}-globin gene. Thus gene targeting can correct a {beta}{sup S} allele to {beta}{sup A}, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

  12. The primary transcription unit of the human alpha 2 globin gene defined by quantitative RT/PCR.

    PubMed Central

    Owczarek, C M; Enriquez-Harris, P; Proudfoot, N J

    1992-01-01

    We have set up an experimental system to map the primary transcription unit of the human alpha 2 globin gene. The duplicated human alpha globin genes (alpha 2-alpha 1) were linked to the alpha globin locus Positive Regulatory Element (PRE) and stably transfected into murine erythroleukaemia cells. We then developed a quantitative reverse transcriptase, polymerase chain reaction assay to map alpha 2 primary transcripts using primer pairs derived from different parts of the alpha 2 globin gene and its 3' flanking region. This approach has revealed the presence of steady state nuclear RNA past the poly(A) site of the alpha 2 globin gene at approximately 40% of the level of unspliced intron transcript. Furthermore, these 3' flanking transcripts diminish 500 bp into the 3' flanking region, identifying this part of the alpha 2 globin gene as the principal region of termination of transcription. Images PMID:1371868

  13. Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.

    PubMed Central

    Curtin, P T; Liu, D P; Liu, W; Chang, J C; Kan, Y W

    1989-01-01

    Several lines of evidence suggest that erythroid-specific DNase I hypersensitive sites (HS) located far upstream of the human beta-globin gene are important in regulating beta-globin gene expression. We used the polymerase chain reaction technique to amplify and clone an 882-base-pair DNA fragment spanning one of these HS, designated HSII, which is located 54 kilobases upstream of the beta-globin gene. The cloned HSII fragment was linked to a human beta-globin gene in either the genomic (HSII-beta) or antigenomic (HSII-beta) orientation. These two constructs and a beta-globin gene alone (beta) were injected into fertilized mouse eggs, and expression was analyzed in liver and brain from day-16 transgenic fetuses. Five of 7 beta-transgenic fetuses expressed human beta-globin mRNA, but the level of expression per gene copy was low, ranging from 0.93 to 22.4% of mouse alpha-globin mRNA (average 9.9%). In contrast, 11 of 12 HSII-beta transgenic fetuses expressed beta-globin mRNA at levels per gene copy ranging from 31.3 to 336.6% of mouse alpha-globin mRNA (average 139.5%). Only three fetuses containing intact copies of the HSII-beta construct were produced. Two of three expressed human beta-globin mRNA at levels per gene copy of 179.2 and 387.1%. Expression of human beta-globin mRNA was tissue-specific in all three types of transgenic fetuses. These studies demonstrate that a small DNA fragment containing a single erythroid-specific HS can stimulate high-level human beta-globin gene expression in transgenic mice. Images PMID:2780563

  14. CP2 binding to the promoter is essential for the enhanced transcription of globin genes in erythroid cells.

    PubMed

    Chae, Ji Hyung; Kim, Chul Geun

    2003-02-28

    We have previously reported that the reduced level of CP2 suppresses the mouse alpha- and beta-globin gene expression and hemoglobin synthesis during terminal differentiation of mouse erythroleukemia (MEL) cells in vitro [Chae et al. (1999)]. As an extension of this study, we demonstrated that human alpha-, epsilon-, and gamma- globin genes were also suppressed by the reduced expression of CP2 in K562 cells. To address how much CP2 contributes in the regulation of globin gene expression, we measured transcriptional activities of the wild type alpha-globin promoter and its various factor-binding sites mutants in erythroid and nonerythroid cells. Interestingly, CP2 site dependent transcriptional activation occurred in an erythroid-cell specific manner, even though CP2 is ubiquitously expressed. In addition, CP2 site mutation within the alpha-promoter severely suppressed promoter activity in differentiated, but not in undifferentiated MEL cells, suggesting that the CP2 binding site is needed for the enhanced transcription of globin genes during erythroid differentiation. When the human beta-globin locus control region was linked to the alpha-promoter, suppression was more severe in the CP2 site mutant in differentiated MEL cells. Overall data indicate that CP2 is a major factor in the regulation of globin expression in human and mouse erythroid cells, and CP2 binding to the globin gene promoter is essential for the enhanced transcription of globin genes in erythroid differentiation.

  15. Gene duplication, genome duplication, and the functional diversification of vertebrate globins

    PubMed Central

    Storz, Jay F.; Opazo, Juan C.; Hoffmann, Federico G.

    2015-01-01

    The functional diversification of the vertebrate globin gene superfamily provides an especially vivid illustration of the role of gene duplication and whole-genome duplication in promoting evolutionary innovation. For example, key globin proteins that evolved specialized functions in various aspects of oxidative metabolism and oxygen signaling pathways (hemoglobin [Hb], myoglobin [Mb], and cytoglobin [Cygb]) trace their origins to two whole-genome duplication events in the stem lineage of vertebrates. The retention of the proto-Hb and Mb genes in the ancestor of jawed vertebrates permitted a physiological division of labor between the oxygen-carrier function of Hb and the oxygen-storage function of Mb. In the Hb gene lineage, a subsequent tandem gene duplication gave rise to the proto α- and β-globin genes, which permitted the formation of multimeric Hbs composed of unlike subunits (α2β2). The evolution of this heteromeric quaternary structure was central to the emergence of Hb as a specialized oxygen-transport protein because it provided a mechanism for cooperative oxygen-binding and allosteric regulatory control. Subsequent rounds of duplication and divergence have produced diverse repertoires of α- and β-like globin genes that are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different stages of prenatal development and postnatal life. In the ancestor of jawless fishes, the proto Mb and Hb genes appear to have been secondarily lost, and the Cygb homolog evolved a specialized respiratory function in blood-oxygen transport. Phylogenetic and comparative genomic analyses of the vertebrate globin gene superfamily have revealed numerous instances in which paralogous globins have convergently evolved similar expression patterns and/or similar functional specializations in different organismal lineages. PMID:22846683

  16. Gene duplication, genome duplication, and the functional diversification of vertebrate globins.

    PubMed

    Storz, Jay F; Opazo, Juan C; Hoffmann, Federico G

    2013-02-01

    The functional diversification of the vertebrate globin gene superfamily provides an especially vivid illustration of the role of gene duplication and whole-genome duplication in promoting evolutionary innovation. For example, key globin proteins that evolved specialized functions in various aspects of oxidative metabolism and oxygen signaling pathways (hemoglobin [Hb], myoglobin [Mb], and cytoglobin [Cygb]) trace their origins to two whole-genome duplication events in the stem lineage of vertebrates. The retention of the proto-Hb and Mb genes in the ancestor of jawed vertebrates permitted a physiological division of labor between the oxygen-carrier function of Hb and the oxygen-storage function of Mb. In the Hb gene lineage, a subsequent tandem gene duplication gave rise to the proto α- and β-globin genes, which permitted the formation of multimeric Hbs composed of unlike subunits (α(2)β(2)). The evolution of this heteromeric quaternary structure was central to the emergence of Hb as a specialized oxygen-transport protein because it provided a mechanism for cooperative oxygen-binding and allosteric regulatory control. Subsequent rounds of duplication and divergence have produced diverse repertoires of α- and β-like globin genes that are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different stages of prenatal development and postnatal life. In the ancestor of jawless fishes, the proto Mb and Hb genes appear to have been secondarily lost, and the Cygb homolog evolved a specialized respiratory function in blood-oxygen transport. Phylogenetic and comparative genomic analyses of the vertebrate globin gene superfamily have revealed numerous instances in which paralogous globins have convergently evolved similar expression patterns and/or similar functional specializations in different organismal lineages.

  17. Differential loss of embryonic globin genes during the radiation of placental mammals

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Storz, Jay F.

    2008-01-01

    The differential gain and loss of genes from homologous gene families represents an important source of functional variation among the genomes of different species. Differences in gene content between species are primarily attributable to lineage-specific gene gains via duplication and lineage-specific losses via deletion or inactivation. Here, we use a comparative genomic approach to investigate this process of gene turnover in the β-globin gene family of placental mammals. By analyzing genomic sequence data from representatives of each of the main superordinal clades of placental mammals, we were able to reconstruct pathways of gene family evolution during the basal radiation of this physiologically and morphologically diverse vertebrate group. Our analysis revealed that an initial expansion of the nonadult portion of the β-globin gene cluster in the ancestor of placental mammals was followed by the differential loss and retention of ancestral gene lineages, thereby generating variation in the complement of embryonic globin genes among contemporary species. The sorting of ε-, γ-, and η-globin gene lineages among the basal clades of placental mammals has produced species differences in the functional types of hemoglobin isoforms that can be synthesized during the course of embryonic development. PMID:18755893

  18. Differential loss of embryonic globin genes during the radiation of placental mammals.

    PubMed

    Opazo, Juan C; Hoffmann, Federico G; Storz, Jay F

    2008-09-02

    The differential gain and loss of genes from homologous gene families represents an important source of functional variation among the genomes of different species. Differences in gene content between species are primarily attributable to lineage-specific gene gains via duplication and lineage-specific losses via deletion or inactivation. Here, we use a comparative genomic approach to investigate this process of gene turnover in the beta-globin gene family of placental mammals. By analyzing genomic sequence data from representatives of each of the main superordinal clades of placental mammals, we were able to reconstruct pathways of gene family evolution during the basal radiation of this physiologically and morphologically diverse vertebrate group. Our analysis revealed that an initial expansion of the nonadult portion of the beta-globin gene cluster in the ancestor of placental mammals was followed by the differential loss and retention of ancestral gene lineages, thereby generating variation in the complement of embryonic globin genes among contemporary species. The sorting of epsilon-, gamma-, and eta-globin gene lineages among the basal clades of placental mammals has produced species differences in the functional types of hemoglobin isoforms that can be synthesized during the course of embryonic development.

  19. Lineage-Specific Patterns of Functional Diversification in the α- and β-Globin Gene Families of Tetrapod Vertebrates

    PubMed Central

    Storz, Jay F.; Gorr, Thomas A.; Opazo, Juan C.

    2010-01-01

    The α- and β-globin gene families of jawed vertebrates have diversified with respect to both gene function and the developmental timing of gene expression. Phylogenetic reconstructions of globin gene family evolution have provided suggestive evidence that the developmental regulation of hemoglobin synthesis has evolved independently in multiple vertebrate lineages. For example, the embryonic β-like globin genes of birds and placental mammals are not 1:1 orthologs. Despite the similarity in developmental expression profiles, the genes are independently derived from lineage-specific duplications of a β-globin pro-ortholog. This suggests the possibility that other vertebrate taxa may also possess distinct repertoires of globin genes that were produced by repeated rounds of lineage-specific gene duplication and divergence. Until recently, investigations into this possibility have been hindered by the dearth of genomic sequence data from nonmammalian vertebrates. Here, we report new insights into globin gene family evolution that were provided by a phylogenetic analysis of vertebrate globins combined with a comparative genomic analysis of three key sauropsid taxa: a squamate reptile (anole lizard, Anolis carolinensis), a passeriform bird (zebra finch, Taeniopygia guttata), and a galliform bird (chicken, Gallus gallus). The main objectives of this study were 1) to characterize evolutionary changes in the size and membership composition of the α- and β-globin gene families of tetrapod vertebrates and 2) to test whether functional diversification of the globin gene clusters occurred independently in different tetrapod lineages. Results of our comparative genomic analysis revealed several intriguing patterns of gene turnover in the globin gene clusters of different taxa. Lineage-specific differences in gene content were especially pronounced in the β-globin gene family, as phylogenetic reconstructions revealed that amphibians, lepidosaurs (as represented by anole

  20. [A novel mutation in β-globin gene of a patient with β-thalassemia].

    PubMed

    Peng, Yun-Sheng; Sun, Shun-Chang; Chen, Qun-Rong; Wang, Qing; Mo, Bao-Mei

    2012-04-01

    This study was aimed to analyze the β-globin gene mutations in a patient with β-thalassemia minor. Genomic DNA was extracted from peripheral blood cells of the patient. The full-length DNA sequence coding for β-globin was amplified by polymerase chain reaction, and the gene mutation was determined by DNA sequencing. The results indicated that a heterogeneous A→G mutation was found at position 129 in intron 1 of the β-thalassemia minor patient. It is concluded that the IVS-I-129(A→G) mutation is a splicing site mutation leading to a splicing error in immature messenger RNA and a protein translation error for the β-globin gene. Thus, the IVS-I-129(A→G) is a novel mutation.

  1. The phylogenetic and evolutionary history of a novel alpha-globin-type gene in orangutans (Pongo pygmaeus).

    PubMed

    Steiper, Michael E; Wolfe, Nathan D; Karesh, William B; Kilbourn, Annelisa M; Bosi, Edwin J; Ruvolo, Maryellen

    2006-07-01

    The alpha-globin genes are implicated in human resistance to malaria, a disease caused by Plasmodium parasites. This study is the first to analyze DNA sequences from a novel alpha-globin-type gene in orangutans, a species affected by Plasmodium. Phylogenetic methods show that the gene is a duplication of an alpha-globin gene and is located 5' of alpha-2 globin. The alpha-globin-type gene is notable for having four amino acid replacements relative to the orangutan's alpha-1 and alpha-2 globin genes, with no synonymous differences. Pairwise K(a)/K(s) methods and likelihood ratio tests (LRTs) revealed that the evolutionary history of the alpha-globin-type gene has been marked by either neutral or positive evolution, but not purifying selection. A comparative analysis of the amino acid replacements of the alpha-globin-type gene with human hemoglobinopathies and hemoglobin structure showed that two of the four replaced sites are members of the same molecular bond, one that is crucial to the proper functioning of the hemoglobin molecule. This suggested an adaptive evolutionary change. Functionally, this locus may result in a thalassemia-like phenotype in orangutans, possibly as an adaptation to combat Plasmodium.

  2. The Globin Gene Repertoire of Lampreys: Convergent Evolution of Hemoglobin and Myoglobin in Jawed and Jawless Vertebrates

    PubMed Central

    Schwarze, Kim; Campbell, Kevin L.; Hankeln, Thomas; Storz, Jay F.; Hoffmann, Federico G.; Burmester, Thorsten

    2014-01-01

    Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in

  3. Human globin gene analysis for a patient with beta-o/delta beta-thalassemia.

    PubMed Central

    Ottolenghi, S; Lanyon, W G; Williamson, R; Weatherall, D J; Clegg, J B; Pitcher, C S

    1975-01-01

    Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experimenta with total mRNA from circulating cells from a patient with heterozygous beta/heterozygous beta-delta-o thalassemia (beta-o/delta beta-o-thalassemia) demonstrated no detectable mRNA for beta-globin. cDNA enriched in sequences homologous to beta-globin mRNA was prepared by hydroxylapatite fractionation of hybrids formed between beta-o/delta beta-o-thalassemic mRNA and cDNA made from mRNA from a patient with alpha-thalassemia (hemoglobin H disease). The rate of annealing of this beta-enriched cDNA to normal human nuclear DNA was that of a sequence present as only a single copy per haploid genome. The beta-enriched cDNA annealed to the beta-o-delta beta-o-thalassemia total DNA with approximately the same kinetics as to normal DNA, indicating that no total gene deletion of beta-globin genes from the diploid genome has occurred, although the accuracy of the technique could not exclude with certainty a partial deletion or a deletion of a beta-globin gene from only one of the haploid genomes. This demonstrates that at least one of the beta-o- or the delta beta-o-thalassemia haploid genomes in this case contains a substantially intact beta-globin gene. PMID:49057

  4. Expression of the human BETA-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells

    SciTech Connect

    Bender, M.A.; Miller, A.D.; Gelinas, R.E.

    1988-04-01

    Replication-defective amphotropic retrovirus vectors containing either the human ..beta..-globin gene with introns or an intronless ..beta..-globin minigene were constructed and used to study ..beta..-globin expression following gene transfer into hematopoietic cells. The ..beta..-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the ..beta..-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human ..beta..-globin RNA. Introduction of a virus containing the ..beta..-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human ..beta..-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human ..beta..-globin gene was 6 to 110% as active as the endogenous mouse ..beta../sup maj/-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced ..beta..-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

  5. Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

    PubMed Central

    Bender, M A; Miller, A D; Gelinas, R E

    1988-01-01

    Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy. Images PMID:3288863

  6. Position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator.

    PubMed

    Inoue, T; Yamaza, H; Sakai, Y; Mizuno, S; Ohno, M; Hamasaki, N; Fukumaki, Y

    1999-01-01

    The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta maj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse beta maj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse beta maj-globin

  7. Inter-MAR association contributes to transcriptionally active looping events in human beta-globin gene cluster.

    PubMed

    Wang, Li; Di, Li-Jun; Lv, Xiang; Zheng, Wei; Xue, Zheng; Guo, Zhi-Chen; Liu, De-Pei; Liang, Chi-Chuan

    2009-01-01

    Matrix attachment regions (MARs) are important in chromatin organization and gene regulation. Although it is known that there are a number of MAR elements in the beta-globin gene cluster, it is unclear that how these MAR elements are involved in regulating beta-globin genes expression. Here, we report the identification of a new MAR element at the LCR (locus control region) of human beta-globin gene cluster and the detection of the inter-MAR association within the beta-globin gene cluster. Also, we demonstrate that SATB1, a protein factor that has been implicated in the formation of network like higher order chromatin structures at some gene loci, takes part in beta-globin specific inter-MAR association through binding the specific MARs. Knocking down of SATB1 obviously reduces the binding of SATB1 to the MARs and diminishes the frequency of the inter-MAR association. As a result, the ACH establishment and the alpha-like globin genes and beta-like globin genes expressions are affected either. In summary, our results suggest that SATB1 is a regulatory factor of hemoglobin genes, especially the early differentiation genes at least through affecting the higher order chromatin structure.

  8. Mutations in Kruppel-like factor 1 cause transfusion-dependent hemolytic anemia and persistence of embryonic globin gene expression.

    PubMed

    Viprakasit, Vip; Ekwattanakit, Supachai; Riolueang, Suchada; Chalaow, Nipon; Fisher, Chris; Lower, Karen; Kanno, Hitoshi; Tachavanich, Kalaya; Bejrachandra, Sasithorn; Saipin, Jariya; Juntharaniyom, Monthana; Sanpakit, Kleebsabai; Tanphaichitr, Voravarn S; Songdej, Duantida; Babbs, Christian; Gibbons, Richard J; Philipsen, Sjaak; Higgs, Douglas R

    2014-03-06

    In this study, we report on 8 compound heterozygotes for mutations in the key erythroid transcription factor Krüppel-like factor 1 in patients who presented with severe, transfusion-dependent hemolytic anemia. In most cases, the red cells were hypochromic and microcytic, consistent with abnormalities in hemoglobin synthesis. In addition, in many cases, the red cells resembled those seen in patients with membrane defects or enzymopathies, known as chronic nonspherocytic hemolytic anemia (CNSHA). Analysis of RNA and protein in primary erythroid cells from these individuals provided evidence of abnormal globin synthesis, with persistent expression of fetal hemoglobin and, most remarkably, expression of large quantities of embryonic globins in postnatal life. The red cell membranes were abnormal, most notably expressing reduced amounts of CD44 and, consequently, manifesting the rare In(Lu) blood group. Finally, all tested patients showed abnormally low levels of the red cell enzyme pyruvate kinase, a known cause of CNSHA. These patients define a new type of severe, transfusion-dependent CNSHA caused by mutations in a trans-acting factor (Krüppel-like factor 1) and reveal an important pathway regulating embryonic globin gene expression in adult humans.

  9. Role of the duplicated CCAAT box region in gamma-globin gene regulation and hereditary persistence of fetal haemoglobin.

    PubMed Central

    Ronchi, A; Berry, M; Raguz, S; Imam, A; Yannoutsos, N; Ottolenghi, S; Grosveld, F; Dillon, N

    1996-01-01

    Hereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly alleviate the symptoms of thalassaemia or sickle cell anaemia when co-inherited with these disorders. We have examined structure-function relationships in the -117 HPFH gamma promoter by analysing the effect of mutating specific promoter elements on the functioning of the wild-type and HPFH promoters. We find that CCAAT box mutants dramatically affect expression from the HPFH promoter in adult blood but have little effect on embryonic/fetal expression from the wild-type promoter. Our results suggest that there are substantial differences in the structure of the wild-type gamma promoter expressed early in development and the adult HPFH promoter. Together with previous results, this suggests that gamma silencing is a complex multifactorial phenomenon rather than being the result of a simple repressor binding to the promoter. We present a model for gamma-globin gene silencing that has significant implications for attempts to reactivate the gamma promoters in human adults by pharmacological means. Images PMID:8598197

  10. Erythroid cell-specific alpha-globin gene regulation by the CP2 transcription factor family.

    PubMed

    Kang, Ho Chul; Chae, Ji Hyung; Lee, Yeon Ho; Park, Mi-Ae; Shin, June Ho; Kim, Sung-Hyun; Ye, Sang-Kyu; Cho, Yoon Shin; Fiering, Steven; Kim, Chul Geun

    2005-07-01

    We previously demonstrated that ubiquitously expressed CP2c exerts potent erythroid-specific transactivation of alpha-globin through an unknown mechanism. This mechanism is reported here to involve specific CP2 splice variants and protein inhibitor of activated STAT1 (PIAS1). We identify a novel murine splice isoform of CP2, CP2b, which is identical to CP2a except that it has an additional 36 amino acids encoded by an extra exon. CP2b has an erythroid cell-specific transcriptional activation domain, which requires the extra exon and can form heteromeric complexes with other CP2 isoforms, but lacks the DNA binding activity found in CP2a and CP2c. Transcriptional activation of alpha-globin occurred following dimerization between CP2b and CP2c in erythroid K562 and MEL cells, but this dimerization did not activate the alpha-globin promoter in nonerythroid 293T cells, indicating that an additional erythroid factor is missing in 293T cells. PIAS1 was confirmed as a CP2 binding protein by the yeast two-hybrid screen, and expression of CP2b, CP2c, and PIAS1 in 293T cell induced alpha-globin promoter activation. These results show that ubiquitously expressed CP2b exerts potent erythroid cell-specific alpha-globin gene expression by complexing with CP2c and PIAS1.

  11. Retroviral-mediated transfer and expression of human. beta. -globin genes in cultured murine and human erythroid cells

    SciTech Connect

    Weber-Benarous, A.; Cone, R.D.; London, I.M.; Mulligan, R.C.

    1988-05-05

    The authors cloned human ..beta..-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 ..beta..-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the ..beta..-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human ..beta..-globin gene transferred into mouse erythroleukemia cells in the same way. They have also introduced the normal human ..beta..-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal ..beta..-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, they suggest that the lack of expression of the endogenous ..beta..-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for ..beta..-lobin gene transcription. Retroviral-mediated transfer of the human ..beta..-globin gene may, however, uniquely influence expression of the gene K562 cells.

  12. β-Thalassemia due to intronic LINE-1 insertion in the β-globin gene (HBB): molecular mechanisms underlying reduced transcript levels of the β-globin(L1) allele.

    PubMed

    Lanikova, Lucie; Kucerova, Jana; Indrak, Karel; Divoka, Martina; Issa, Jean-Pierre; Papayannopoulou, Thalia; Prchal, Josef T; Divoky, Vladimir

    2013-10-01

    We describe the molecular etiology of β(+)-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the β-globin gene (HBB). The transcript level of the affected β-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed β-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the β-globin(L1) allele also resulted from a reduced rate of transcription and decreased production of full-length β-globin primary transcripts. The promoter and enhancer sequences of the β-globin(L1) allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β-globin(L1) transcription despite permanent β-globin(L1) promoter CpG methylation. This result indicates that the decreased rate of transcription from the β-globin(L1) allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β-globin gene represents a novel etiology of β-thalassemia.

  13. A phylogenomic profile of globins

    PubMed Central

    Vinogradov, Serge N; Hoogewijs, David; Bailly, Xavier; Arredondo-Peter, Raúl; Gough, Julian; Dewilde, Sylvia; Moens, Luc; Vanfleteren, Jacques R

    2006-01-01

    Background Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold. Results A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. Conclusion Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals. PMID:16600051

  14. Identification and molecular characterization of four new large deletions in the beta-globin gene cluster.

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Couprie, Nicole; Francina, Alain

    2009-01-01

    Despite the fact that mutations in the human beta-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the beta-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire beta-globin gene, including its promoter, and is associated with a typical beta-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the delta and beta-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5' and 3' breakpoints gives new insights about the differences between HPFH and (deltabeta)(0)-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed.

  15. Molecular cloning and expression of α-globin and β-globin genes from crocodile (Crocodylus siamensis).

    PubMed

    Anwised, Preeyanan; Kabbua, Thai; Temsiripong, Theeranan; Dhiravisit, Apisak; Jitrapakdee, Sarawut; Araki, Tomohiro; Yoneda, Kazunari; Thammasirirak, Sompong

    2013-03-01

    The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.

  16. Molecular evolution of globin genes in Gymnotiform electric fishes: relation to hypoxia tolerance.

    PubMed

    Tian, Ran; Losilla, Mauricio; Lu, Ying; Yang, Guang; Zakon, Harold

    2017-02-13

    Nocturnally active gymnotiform weakly electric fish generate electric signals for communication and navigation, which can be energetically taxing. These fish mainly inhabit the Amazon basin, where some species prefer well-oxygenated waters and others live in oxygen-poor, stagnant habitats. The latter species show morphological, physiological, and behavioral adaptations for hypoxia-tolerance. However, there have been no studies of hypoxia tolerance on the molecular level. Globins are classic respiratory proteins. They function principally in oxygen-binding and -delivery in various tissues and organs. Here, we investigate the molecular evolution of alpha and beta hemoglobins, myoglobin, and neuroglobin in 12 gymnotiforms compared with other teleost fish. The present study identified positively selected sites (PSS) on hemoglobin (Hb) and myoglobin (Mb) genes using different maximum likelihood (ML) methods; some PSS fall in structurally important protein regions. This evidence for the positive selection of globin genes suggests that the adaptive evolution of these genes has helped to enhance the capacity for oxygen storage and transport. Interestingly, a substitution of a Cys at a key site in the obligate air-breathing electric eel (Electrophorus electricus) is predicted to enhance oxygen storage of Mb and contribute to NO delivery during hypoxia. A parallel Cys substitution was also noted in an air-breathing African electric fish (Gymnarchus niloticus). Moreover, the expected pattern under normoxic conditions of high expression of myoglobin in heart and neuroglobin in the brain in two hypoxia-tolerant species suggests that the main effect of selection on these globin genes is on their sequence rather than their basal expression patterns. Results indicate a clear signature of positive selection in the globin genes of most hypoxia-tolerant gymnotiform fishes, which are obligate or facultative air breathers. These findings highlight the critical role of globin genes in

  17. A Dual Reporter Mouse Model of the Human β-Globin Locus: Applications and Limitations

    PubMed Central

    Papadopoulos, Petros; Gutiérrez, Laura; van der Linden, Reinier; Kong-A-San, John; Maas, Alex; Drabek, Dubravka; Patrinos, George P.; Philipsen, Sjaak; Grosveld, Frank

    2012-01-01

    The human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative defects of the globins, based on a number of genome variations found in the globin gene clusters. Hereditary persistence of fetal hemoglobin (HPFH) also caused by similar types of genomic alterations can compensate for the loss of adult hemoglobin. Understanding the regulation of the human γ-globin gene expression is a challenge for the treatment of thalassemia. A mouse model that facilitates high-throughput assays would simplify such studies. We have generated a transgenic dual reporter mouse model by tagging the γ- and β-globin genes with GFP and DsRed fluorescent proteins respectively in the endogenous human β-globin locus. Erythroid cell lines derived from this mouse model were tested for their capacity to reactivate the γ-globin gene. Here, we discuss the applications and limitations of this fluorescent reporter model to study the genetic basis of red blood cell disorders and the potential use of such model systems in high-throughput screens for hemoglobinopathies therapeutics. PMID:23272095

  18. Cell-cycle specific association of transcription factors and RNA polymerase II with the human β-globin gene locus†

    PubMed Central

    Lin, I-Ju; Liang, Shermi Y; Bungert, Jörg

    2013-01-01

    The human β-globin genes are regulated by a locus control region (LCR) and are expressed at extremely high levels in erythroid cells. How transcriptional fidelity of highly expressed genes is regulated and maintained during the cell cycle is not completely understood. Here, we analyzed the association of transcription factor USF, the co-activator CBP, topoisomerase I (Topo I), basal transcription factor TFIIB, and RNA polymerase II (Pol II) with the β-globin gene locus at specific cell-cycle stages. The data demonstrate that while association of Pol II with globin locus associated chromatin decreased in mitotically arrested cells, it remained bound at lower levels at the γ-globin gene promoter. During early S-phase, association of CBP, USF and Pol II with the globin gene locus decreased. The reassociation of CBP and USF2 with the LCR preceded reassociation of Pol II, suggesting that these proteins together mediate recruitment of Pol II to the β-globin gene locus during S-phase. Finally, we analyzed the association of Topo I with the globin gene locus during late S-phase. In general, Topo I association correlated with the binding of Pol II. Inhibition of Topo I activity reduced Pol II binding at the LCR and intergenic regions but not at the γ-globin gene promoter. The data demonstrate dynamic associations of transcription factors with the globin gene locus during the cell cycle and support previous results showing that specific components of transcription complexes remain associated with highly transcribed genes during mitosis. PMID:23519692

  19. Mutations in two regions upstream of the A gamma globin gene canonical promoter affect gene expression.

    PubMed Central

    Lloyd, J A; Lee, R F; Lingrel, J B

    1989-01-01

    Two regions upstream of the human fetal (A gamma) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position -290 to -267 bp upstream of the transcription initiation site, the other (site II) is at -182 to -168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T----C) at -175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A gamma globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at -175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives gamma-CAT activity well below wild-type, suggesting that this factor is a positive regulator. Images PMID:2472607

  20. Recent advances in globin research using genome-wide association studies and gene editing.

    PubMed

    Orkin, Stuart H

    2016-03-01

    A long-sought goal in the hemoglobin field has been an improved understanding of the mechanisms that regulate the switch from fetal (HbF) to adult (HbA) hemoglobin during development. With such knowledge, the hope is that strategies for directed reactivation of HbF in adults could be devised as an approach to therapy for the β-hemoglobinopathies thalassemia and sickle cell disease. Recent genome-wide association studies (GWAS) led to identification of three loci (BCL11A, HBS1L-MYB, and the β-globin cluster itself) in which natural genetic variation is correlated with different HbF levels in populations. Here, the central role of BCL11A in control of HbF is reviewed from the perspective of how findings may be translated to gene therapy in the not-too-distant future. This summary traces the evolution of recent studies from the initial recognition of BCL11A through GWAS to identification of critical sequences in an enhancer required for its erythroid-specific expression, thereby highlighting an Achilles heel for genome editing.

  1. Recent advances in globin research using genome-wide association studies and gene editing

    PubMed Central

    Orkin, Stuart H.

    2015-01-01

    A long-sought goal in the hemoglobin field has been an improved understanding of the mechanisms that regulate the switch from fetal (HbF) to adult (HbA) hemoglobin during development. With such knowledge, the hope is that strategies for directed reactivation of HbF in adults could be devised as an approach to therapy for the β-hemoglobinopathies thalassemia and sickle cell disease. Recent genome-wide association studies led to identification of three loci (BCL11A, HBS1L-Myb, and the β-globin cluster itself) in which natural genetic variation is correlated with different HbF levels in populations. Here, the central role of BCL11A in control of HbF is reviewed from the perspective of how findings may be translated to gene therapy in the not-too-distant future. This summary traces the evolution of recent studies from the initial recognition of BCL11A through GWAS to identification of critical sequences in an enhancer required for its erythroid-specific expression, thereby highlighting an Achilles heel for genome editing. PMID:26866328

  2. Phenotype of mutations in the promoter region of the β-globin gene.

    PubMed

    Ropero, Paloma; Erquiaga, Sara; Arrizabalaga, Beatriz; Pérez, Germán; de la Iglesia, Silvia; Torrejón, María José; Gil, Celia; Elena, Cela; Tenorio, María; Nieto, Jorge M; de la Fuente-Gonzalo, Félix; Villegas, Ana; González Fernández, Fernando-Ataúlfo; Martínez, Rafael

    2017-10-01

    β(+)-Thalassaemia is characterised by reduced production of β chains, which decrease can be caused by mutations in the promoter region (CACCC or TATA box), and is classified as mild or silent depending on the extent of β-globin chain reduction. In both cases, homozygotes or compound heterozygotes for these mutations usually have thalassaemia intermedia. Frequently the diagnosis is made in adulthood or even in old age. A total of 37 alterations in the promoter region have been described so far. In this report we describe the mutations found in the promoter region of the β-globin gene in a single hospital in Madrid. Between 1998 and 2015, more than 9000 blood samples were analysed for full blood count and underwent haemoglobin electrophoresis and high performance liquid chromatography. Genetic analysis of the β and Gγ-globin genes was carried out by automatic sequencing and, in the case of α genes, by multiplex PCR. 35 samples showed mutation in the promoter region of the β-globin gene, with a total of six different mutations identified: one in the distal CACCC box, two in the proximal CACCC box, three in the ATA box. Any alterations in the proximal CACCC and TATA boxes lead to a moderate decrease in synthesis of the β-globin chain, which has been demonstrated in cases of thalassaemia intermedia that have presented in the second decade of life with a moderate clinical course. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  3. Whole-Genome Duplications Spurred the Functional Diversification of the Globin Gene Superfamily in Vertebrates

    PubMed Central

    Hoffmann, Federico G.; Opazo, Juan C.; Storz, Jay F.

    2012-01-01

    It has been hypothesized that two successive rounds of whole-genome duplication (WGD) in the stem lineage of vertebrates provided genetic raw materials for the evolutionary innovation of many vertebrate-specific features. However, it has seldom been possible to trace such innovations to specific functional differences between paralogous gene products that derive from a WGD event. Here, we report genomic evidence for a direct link between WGD and key physiological innovations in the vertebrate oxygen transport system. Specifically, we demonstrate that key globin proteins that evolved specialized functions in different aspects of oxidative metabolism (hemoglobin, myoglobin, and cytoglobin) represent paralogous products of two WGD events in the vertebrate common ancestor. Analysis of conserved macrosynteny between the genomes of vertebrates and amphioxus (subphylum Cephalochordata) revealed that homologous chromosomal segments defined by myoglobin + globin-E, cytoglobin, and the α-globin gene cluster each descend from the same linkage group in the reconstructed proto-karyotype of the chordate common ancestor. The physiological division of labor between the oxygen transport function of hemoglobin and the oxygen storage function of myoglobin played a pivotal role in the evolution of aerobic energy metabolism, supporting the hypothesis that WGDs helped fuel key innovations in vertebrate evolution. PMID:21965344

  4. Whole-genome duplications spurred the functional diversification of the globin gene superfamily in vertebrates.

    PubMed

    Hoffmann, Federico G; Opazo, Juan C; Storz, Jay F

    2012-01-01

    It has been hypothesized that two successive rounds of whole-genome duplication (WGD) in the stem lineage of vertebrates provided genetic raw materials for the evolutionary innovation of many vertebrate-specific features. However, it has seldom been possible to trace such innovations to specific functional differences between paralogous gene products that derive from a WGD event. Here, we report genomic evidence for a direct link between WGD and key physiological innovations in the vertebrate oxygen transport system. Specifically, we demonstrate that key globin proteins that evolved specialized functions in different aspects of oxidative metabolism (hemoglobin, myoglobin, and cytoglobin) represent paralogous products of two WGD events in the vertebrate common ancestor. Analysis of conserved macrosynteny between the genomes of vertebrates and amphioxus (subphylum Cephalochordata) revealed that homologous chromosomal segments defined by myoglobin + globin-E, cytoglobin, and the α-globin gene cluster each descend from the same linkage group in the reconstructed proto-karyotype of the chordate common ancestor. The physiological division of labor between the oxygen transport function of hemoglobin and the oxygen storage function of myoglobin played a pivotal role in the evolution of aerobic energy metabolism, supporting the hypothesis that WGDs helped fuel key innovations in vertebrate evolution.

  5. Nonrandom association of polymorphic restriction sites in the beta-globin gene cluster.

    PubMed

    Antonarakis, S E; Boehm, C D; Giardina, P J; Kazazian, H H

    1982-01-01

    By using probes for epsilon-, Psibeta(1)-, and beta-globin genes, we found four additional polymorphic restriction sites that have frequencies >0.1 in persons of Mediterranean area origin, Asian Indians, and American Blacks. Three of these (HincII sites) and the two previously described polymorphic HindIII sites [one in intervening sequence (IVS) II of each gamma-globin gene] are distributed over 32 kilobases (kb) of DNA located 5' to the delta-globin gene. This region of DNA comprises two-thirds of the beta-globin gene cluster. Since each of these five polymorphic sites can be present (+) or absent (-), in theory there exist 32 possible combinations of sites (haplotypes). However, in Italians, Greeks, Indians, and Turks, 3 of the 32 haplotypes, (+----), (-+-++), and (-++-+), account for 92% of 89 beta(A) chromosomes examined. The observed frequencies for these haplotypes are 0.64, 0.15, and 0.13 in the populations studied, in contrast to expected frequencies (based on the observed gene frequencies at each of the five sites) of 0.20, 0.006, and 0.005, respectively. In American Blacks, a fourth haplotype, (----+), which is rare in non-Black populations, has a frequency of 0.37 in contrast to its expected frequency of 0.05. These results suggest a nonrandom association of DNA sequences over 32 kb 5' to the delta-globin gene in all populations studied. Two other polymorphic sites 3' to the delta gene (the newly discovered Ava II site in IVS II of the beta-globin gene and the BamHI site 3' to it) are nonrandomly associated with each other but randomly distributed with respect to the above haplotypes. This suggests that randomization of sequences has occurred within 12 kb of DNA between these two nonrandomly associated sequence clusters. Nonrandom association of polymorphic restriction sites has practical consequences in that it limits the usefulness of these additional HincII sites for prenatal diagnosis of hemoglobinopathies by linkage analysis. These sites provide

  6. Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells

    PubMed Central

    Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen; Mead, Paul E; Smeltzer, Matthew P; Weiss, Mitchell J; Wilber, Andrew; Persons, Derek A

    2015-01-01

    Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans. PMID:26665131

  7. Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells.

    PubMed

    Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen; Mead, Paul E; Smeltzer, Matthew P; Weiss, Mitchell J; Wilber, Andrew; Persons, Derek A

    2015-01-01

    Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans.

  8. Globin genes in Micronesia: origins and affinities of Pacific Island peoples.

    PubMed Central

    O'Shaughnessy, D F; Hill, A V; Bowden, D K; Weatherall, D J; Clegg, J B

    1990-01-01

    DNA polymorphisms and copy-number variants of alpha-, zeta-, and gamma-globin genes have been studied in seven Micronesian island populations and have been compared with those in populations from Southeast Asia, Melanesia, and Polynesia. Micronesians are not significantly different from Polynesians at these loci and appear to be intermediate between Southeast Asians and Melanesians. There is evidence of significant Melanesian input into the Micronesian gene pool and of substantial proto-Polynesian contact with Melanesia. PMID:1967206

  9. Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors.

    PubMed

    Dulmovits, Brian M; Appiah-Kubi, Abena O; Papoin, Julien; Hale, John; He, Mingzhu; Al-Abed, Yousef; Didier, Sebastien; Gould, Michael; Husain-Krautter, Sehba; Singh, Sharon A; Chan, Kyle W H; Vlachos, Adrianna; Allen, Steven L; Taylor, Naomi; Marambaud, Philippe; An, Xiuli; Gallagher, Patrick G; Mohandas, Narla; Lipton, Jeffrey M; Liu, Johnson M; Blanc, Lionel

    2016-03-17

    Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with β-hemoglobinopathies.

  10. Expression of the human. beta. -globin gene following retroviral-mediated transfer into multipotential hematopoietic progenitors of mice

    SciTech Connect

    Karlsson, S.; Bodine, D.M.; Perry, L.; Papayannopoulou, T.; Nienhuis, A.W. )

    1988-08-01

    Efficient transfer of the {beta}-globin gene into primitive hematopoietic progenitors was achieved with consistent and significant expression in the progeny of those cells. Retroviral vectors containing the intact genomic human {beta}-globin gene and the neomycin (G418)-resistance (neo{sup R}) gene were constructed. These gave titers of 10{sup 6} or more neo{sup R} colony-forming units/ml when packaged in {psi}2 cells. Mouse bone marrow cells were infected by coculture with producer cells and injected into lethally irradiated animals. Several parameters were varied to enhance infection frequency of colony-forming units, spleen (CFU-S); overall 41% of 116 foci studied contained an intact proviral genome. The human {beta}-globin gene was expressed in 31 of 35 CFU-S-derived spleen colonies that contained the intact vector genome at levels ranging from 1% to 5% of that of the mouse {beta}-globin genes. Infected bone marrow cells were also injected into genetically anemic W/W{sup v} recipients without prior irradiation. Human {beta}-globin chains were detected in circulating erythrocytes by immunofluorescent staining with a specific monoclonal antibody. All animals injected with donor cells that had been cultured in G418 (1 mg/ml) for 48 hr after retroviral infection had circulating erythrocytes containing human {beta}-globin chains between 3 and 8 weeks after transplantation.

  11. Analysis of delta-globin gene alleles in the Sicilian population: identification of five new mutations.

    PubMed

    Giambona, Antonino; Passarello, Cristina; Ruggeri, Gaetano; Renda, Disma; Teresi, Pietro; Anzà, Maurizio; Maggio, Aurelio

    2006-12-01

    Although delta-globin gene (HBD MIM#142000) mutations have no clinical implications, co-inheritance of beta- and delta-thalassemia may lead to misdiagnosis. Among 7,153 samples studied for beta-thalassemia, 205 samples with lower than expected HbA2 levels were selected for our analysis and 183 samples (2.5%) were positive for delta-globin gene mutations. Twelve different mutations were detected, and among these five have not been not previously described (HbA2-Catania HBD c.8A-->T, HbA2-Corleone HBD c.41C-->A, HbA2-Ventimiglia HBD c.212C-->G, HbA2-Montechiaro HBD c.260C-->A, and HbA2-Bagheria HBD c.422C-->T). This study suggests that delta-globin gene defects are very common in Sicily. Thus, these mutations need to be considered during beta-thalassemia screening to avoid false negative results in the detection of at-risk couples.

  12. The globin gene repertoire of lampreys: convergent evolution of hemoglobin and myoglobin in jawed and jawless vertebrates.

    PubMed

    Schwarze, Kim; Campbell, Kevin L; Hankeln, Thomas; Storz, Jay F; Hoffmann, Federico G; Burmester, Thorsten

    2014-10-01

    Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in

  13. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the βA(T87Q)-Globin Gene

    PubMed Central

    Negre, Olivier; Eggimann, Anne-Virginie; Beuzard, Yves; Ribeil, Jean-Antoine; Bourget, Philippe; Borwornpinyo, Suparerk; Hongeng, Suradej; Hacein-Bey, Salima; Cavazzana, Marina; Leboulch, Philippe; Payen, Emmanuel

    2016-01-01

    β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (βAT87Q-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. βAT87Q-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease. PMID:26886832

  14. beta(S)-Globin gene cluster haplotypes in the West Bank of Palestine.

    PubMed

    Samarah, Fekri; Ayesh, Suhail; Athanasiou, Miranda; Christakis, John; Vavatsi, Norma

    2009-01-01

    Sickle cell disease is an inherited autosomal recessive disorder of the beta-globin chain. In Palestine it is accompanied by a low level of Hb F (mean 5.14%) and a severe clinical presentation. In this study, 59 Palestinian patients, homozygotes for Hb S were studied for their haplotype background. Eight polymorphic sites in the beta-globin gene cluster were examined. The Benin haplotype was predominant with a frequency of 88.1%, followed by a frequency of 5.1% for the Bantu haplotype. One chromosome was found to carry the Cameroon haplotype (0.85%). Three atypical haplotypes were also found (5.95%). Heterogeneity was observed in Hb F production, ranging between 1.5 and 17.0%, whereas the (G)gamma ratio was homogeneous among all haplotypes with a normal amount of about 41%. Our results are in agreement with previous reports of the Benin haplotype origin in the Mediterranean.

  15. Total alpha-globin gene cluster deletion has high frequency in Filipinos

    SciTech Connect

    Hunt, J.A.; Haruyama, A.Z.; Chu, B.M.

    1994-09-01

    Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms; {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).

  16. Effect of beta-globin gene cluster haplotype on the hematological and clinical features of sickle cell anemia.

    PubMed

    Rieder, R F; Safaya, S; Gillette, P; Fryd, S; Hsu, H; Adams, J G; Steinberg, M H

    1991-03-01

    In 113 black American adults with sickle cell anemia (HbSS), we examined nine polymorphic restriction sites, including the Xmnl site 5' to the G gamma gene, to see whether haplotype is related to the level of HbF and the proportion of G gamma chains or if it influences the hematological and clinical features of the disease. Seventy-five percent of the patients were homozygous or heterozygous for the Benin (no. 19) or Central African Republic (Bantu, no. 20) haplotypes; 13.3% were homozygous or heterozygous for the Senegal (no. 3) haplotype, while 11.5% had other genotypes. Of the subjects, 14.2% were either homozygous or heterozygous for the Xmnl restriction site 5' to the G gamma gene. We found no effect of haplotype on HbF levels. The level of G gamma chains was 60.5% +/- 17.0% in individuals heterozygous or homozygous for haplotype no. 3 and was 46.9% +/- 11.6% in individuals with other haplotypes. Subjects with the Xmnl site 5' to the G gamma gene had G gamma globin levels of 59.5% +/- 16.7% while those lacking that site had an average of 47.2% +/- 12.1%. There were no significant differences among these groups in hemoglobin concentration, packed cell volume, mean cell volume, or clinical indicators of vaso-occlusive severity, including crises, hospitalizations per year, aseptic bone necrosis, acute chest syndrome, or leg ulcers. While the presence of haplotype 3 and the 5' G gamma Xmnl site were associated with increased G gamma chains, there was no effect on HbF level or other hematological and clinical features that might reflect disease severity. It is likely that determinants unrelated to haplotype, linked or unlinked to the beta-globin gene cluster, are the major effectors of differences in the levels of HbF in American patients with sickle cell anemia.

  17. The DNA deletion in an Indian delta beta-thalassaemia begins one kilobase from the A gamma globin gene and ends in an L1 repetitive sequence.

    PubMed

    Mishima, N; Landman, H; Huisman, T H; Gilman, J G

    1989-11-01

    High fetal haemoglobin levels of 5-15% are present in adult heterozygotes for delta beta-thalassaemia as the result of large deletions of DNA. We have cloned DNA spanning the deletion breakpoint for a new Indian delta beta-thalassaemia associated with mild anaemia. The 5' breakpoint is at 42151 of GenBank file HUMHBB, which is about 1 kb 3' of the A gamma globin gene poly A site at 41003. On the 3' side of the breakpoint, the sequence is homologous to L1 (KpnI) repetitive DNA located 3.6-10 kb 3' of the beta-globin gene: Indian delta beta-thalassaemia DNA is 74% homologous to the inverted complement of HUMHBB from 69849 to 70020, followed by a region 78% homologous to the direct sequence of HUMHBB from 70534 to 71010. The precise location of the 3' endpoint of this deletion has not been determined, but it is within L1 sequences located more than 10 kb 3' of the beta-globin gene.

  18. Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus

    PubMed Central

    Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

    2015-01-01

    Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer–promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them. PMID:25588787

  19. Globins in Caenorhabditis elegans.

    PubMed

    Tilleman, Lesley; Germani, Francesca; De Henau, Sasha; Geuens, Eva; Hoogewijs, David; Braeckman, Bart P; Vanfleteren, Jacques R; Moens, Luc; Dewilde, Sylvia

    2011-03-01

    Extensive in silico search of the genome of Caenorhabditis elegans revealed the presence of 33 genes coding for globins that are all transcribed. These globins are very diverse in gene and protein structure and are localized in a variety of cells, mostly neurons. The large number of C. elegans globin genes is assumed to be the result of multiple evolutionary duplication and radiation events. Processes of subfunctionalization and diversification probably led to their cell-specific expression patterns and fixation into the genome. To date, four globins (GLB-1, GLB-5, GLB-6, and GLB-26) have been partially characterized physicochemically, and the crystallographic structure of two of them (GLB-1 and GLB-6) was solved. In this article, a three-dimensional model was designed for the other two globins (GLB-5 and GLB-26), and overlays of the globins were constructed to highlight the structural diversity among them. It is clear that although they all share the globin fold, small variations in the three-dimensional structure have major implications on their ligand-binding properties and possibly their function. We also review here all the information available so far on the globin family of C. elegans and suggest potential functions. Copyright © 2011 Wiley Periodicals, Inc.

  20. Beta globin gene cluster haplotypes of the beta thalassemia mutations observed in Denizli province of Turkey.

    PubMed

    Bahadır, Anzel; Öztürk, Onur; Atalay, Ayfer; Atalay, Erol Ömer

    2009-09-05

    Our aim is to identify the beta globin gene cluster haplotypes for the beta thalassemia mutations in Turkey at regional basis. Beta thalassemia mutations included in this study were IVS-I-110 (G>A), FSC 8/9 (+G), IVS-II-1 (G>A), IVS-I-5 (G>C), IVS-I-1 (G>A), IVS-I-6 (T>C) and FSC 8 (-AA). We studied 22 unrelated patients with β-thalassemia major and 72 unrelated healthy subjects from our Department's DNA bank. Haplotype analysis was done by polymerase chain reaction (PCR)-based restriction enzyme digestion for the beta globin gene cluster of the following polymorphic restriction sites: Hinc II 5' to ε, Hind III 5' to Gγ, Hind III in the IVS-II 5' to Aγ, Hinc II in pseudo β, Hinc II 3' to pseudo β, Ava II in β, Hinf I 3' to β. Associated haplotypes for the normal control samples (72 individuals, 144 chromosomes) were determined by Arlequin 3.1 software with unknown gametic phase. According to the results obtained, the most frequent beta globin gene cluster haplotypes in the normal population are (+----++), (+----+-), (-+-++++), (+-----+) with the frequencies of 28.6 %, 17.2 %, 9.8 % and 8.3 % respectively. IVS-I-110 mutation is linked with the haplotypes (+----++) and (+-----+). Observed haplotypes are (+----++) for FSC 8/9 (+G), (-+-+++-) for IVS-II-1 (G>A), (-+-++-+ and -+-++++) for IVS-I-5 (G>C), (+----+- and +------) for IVS-I-1 (G>A), (-++---+) for IVS-I-6 (T>C) and (+-----+) for FSC 8 (-AA). In conclusion, our region shows the Mediterranean character for the beta thalassemia mutations. According to the obtained results, IVS-I-110 (G>A) mutation linked with haplotype VII (+-----+), IVS-I-5 (G>C) mutation with haplotype IV (-+-++-+), codon 8/9 (+G) linked with haplotype I (+----++) were shown for the first time in Turkish population. The linkage of haplotype (+------) with the IVS-I-1 (G>A) mutation is reported for the first time in the published literature. In Denizli province of Turkey, beta globin gene cluster haplotypes of the normal population are

  1. Phylogenetic relationships within the genus Equus and the evolution of alpha and theta globin genes.

    PubMed

    Oakenfull, E A; Clegg, J B

    1998-12-01

    Sequences of the alpha1, alpha2 and theta globin genes from six equid species have been determined to investigate relationships within the genus Equus. Analyses using standard phylogenetic methods, or an approach designed to account for the effects of gene conversion between the alpha genes, gave broadly similar results and show that the horses diverged from the zebra/ass ancestor approximately 2.4 million years ago and that the zebra and ass species arose in a rapid radiation approximately 0.9 million years ago. These results from the alpha genes are corroborated by theta gene data and are in contrast to mitochondrial DNA studies of the phylogeny of this genus, which suggest a more gradual set of speciation events.

  2. Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of β(E) pre-mRNA splicing determine disease severity.

    PubMed

    Tubsuwan, Alisa; Munkongdee, Thongperm; Jearawiriyapaisarn, Natee; Boonchoy, Chanikarn; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

    2011-09-01

    Thalassaemia is characterized by the reduced or absent production of globins in the haemoglobin molecule leading to imbalanced α-globin/non α-globin chains. HbE, the result of a G to A mutation in codon 26 of the HBB (β-globin) gene, activates a cryptic 5' splice site in codon 25 leading to a reduction of correctly spliced β(E) -globin (HBB:c.79G>A) mRNA and consequently β(+) -thalassaemia. A wide range of clinical severities in bothα- and β-thalassaemia syndromes, from nearly asymptomatic to transfusion-dependent, has been observed. The correlation between clinical heterogeneity in various genotypes of thalassaemia and the levels of globin gene expression and β(E) -globin pre-mRNA splicing were examined using multiplex quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and allele-specific RT-qPCR. The α-globin/non α-globin mRNA ratio was demonstrated to be a good indicator for disease severity among different thalassaemia disorders. However, the α-globin/non α-globin mRNA ratio ranged widely in β-thalassaemia/HbE patients, with no significant difference between mild and severe phenotypes. Interestingly, the correctly to aberrantly spliced β(E) -globin mRNA ratio in 30% of mild β-thalassaemia/HbE patients was higher than that of the severe patients. The splicing process of β(E) -globin pre-mRNA differs among β-thalassaemia/HbE patients and serves as one of the modifying factors for disease severity. © 2011 Blackwell Publishing Ltd.

  3. Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells.

    PubMed

    Urbinati, Fabrizia; Hargrove, Phillip W; Geiger, Sabine; Romero, Zulema; Wherley, Jennifer; Kaufman, Michael L; Hollis, Roger P; Chambers, Christopher B; Persons, Derek A; Kohn, Donald B; Wilber, Andrew

    2015-05-01

    Sickle cell disease (SCD) can be cured by allogeneic hematopoietic stem cell transplant. However, this is only possible when a matched donor is available, making the development of gene therapy using autologous hematopoietic stem cells a highly desirable alternative. We used a culture model of human erythropoiesis to directly compare two insulated, self-inactivating, and erythroid-specific lentiviral vectors, encoding for γ-globin (V5m3-400) or a modified β-globin (βAS3-FB) for production of antisickling hemoglobin (Hb) and correction of red cell deformability after deoxygenation. Bone marrow CD34+ cells from three SCD patients were transduced using V5m3-400 or βAS3-FB and compared with mock-transduced SCD or healthy donor CD34+ cells. Lentiviral transduction did not impair cell growth or differentiation, as gauged by proliferation and acquisition of erythroid markers. Vector copy number averaged approximately one copy per cell, and corrective globin mRNA levels were increased more than sevenfold over mock-transduced controls. Erythroblasts derived from healthy donor and mock-transduced SCD cells produced a low level of fetal Hb that was increased to 23.6 ± 4.1% per vector copy for cells transduced with V5m3-400. Equivalent levels of modified normal adult Hb of 17.6 ± 3.8% per vector copy were detected for SCD cells transduced with βAS3-FB. These levels of antisickling Hb production were sufficient to reduce sickling of terminal-stage red blood cells upon deoxygenation. We concluded that the achieved levels of fetal Hb and modified normal adult Hb would likely prove therapeutic to SCD patients who lack matched donors. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  4. The beta-globin gene in Sardinian delta beta 0-thalassemia carries a C----T nonsense mutation at codon 39.

    PubMed

    Guida, S; Giglioni, B; Comi, P; Ottolenghi, S; Camaschella, C; Saglio, G

    1984-04-01

    Sardinian delta beta 0-thalassemia is an inherited syndrome characterized by the inactivity of the beta-globin gene and the persistent activity of the fetal gamma-globin genes, particularly the A gamma-globin gene. Previous mapping studies with restriction enzymes failed to show any abnormality in the non-alpha globin gene cluster. We have now examined the possibility that this syndrome might result from a single rather than two different defects. Restriction enzyme polymorphisms linked to the delta beta 0-thalassemic non-alpha globin fragments were defined providing the basis for cloning the delta beta 0-thalassemic beta-globin gene from the DNA of a heterozygous patient. This gene appears to carry a C----T single mutation causing the appearance of a stop codon at amino acid position 39 of the beta-globin gene. This mutation was previously reported in beta 0-thalassemic patients, in linkage with different haplotypes. We conclude that Sardinian delta beta 0-thalassemia is the result of two separate mutations, the former one (unknown) responsible for persistent expression of gamma-globin genes, the latter for beta 0-thalassemia.

  5. Severe fetal and neonatal hemolytic anemia due to a 198 kb deletion removing the complete β-globin gene cluster.

    PubMed

    Verhovsek, Madeleine; Shah, Nirmish R; Wilcox, Ibifiri; Koenig, Sara C; Barros, Tiago; Thornburg, Courtney D; Steinberg, Martin H; Luo, Hong-yuan; Chui, David H K

    2012-11-01

    Fetal and neonatal hemolytic anemia can be caused by (γδβ)(0)-thalassemia deletions of the β-globin gene cluster. Many of these deletions have not been well characterized, and diagnostic tests are not readily available, thus hampering carrier detection, family counseling, and antenatal diagnosis. We report and define a 198 kb deletion removing the entire β-globin gene cluster, which was found in members of a multigeneration family of Irish/Scottish descent. The proband had life-threatening fetal and neonatal hemolytic anemia which subsided by 1 year of age. Copyright © 2012 Wiley Periodicals, Inc.

  6. Structure of cloned delta-globin genes from a normal subject and a patient with delta-thalassemia; sequence polymorphisms found in the delta-globin gene region of Japanese individuals.

    PubMed

    Kimura, A; Matsunaga, E; Ohta, Y; Fujiyoshi, T; Matsuo, T; Nakamura, T; Imamura, T; Yanase, T; Takagi, Y

    1982-10-11

    The delta-globin genes of a normal Japanese and a Japanese patient with homozygous delta-thalassemia were cloned, and the nucleotide sequence of a region including the gene was determined. Comparison of the nucleotide sequences of these two individuals with that of pH delta 1, delta-globin clone from the gene library constructed by Maniatis et al., showed differences in the large intervening sequence (IVS 2), at positions 137, 151, 186, 188, 291, 292 and 540 as one base substitutions, at 339 and 823 as one base additions, at 548 as a one base deletion, and a 9 bp duplication between positions 651 and 659, and differences in the 3'-flanking sequence at 51 and 98 nucleotides 3' to the AATAAA sequence. However, in the region studied, no differences was observed in the nucleotide sequences of the normal subject and the patient with delta-thalassemia. Therefore, these differences may represent polymorphisms of the delta-globin gene present in Japanese individuals. These data suggest that IVS 2 is more divergent than other regions, and that a DNA region(s) other than the globin gene may affect expression of the gene.

  7. Conservation of position and sequence of a novel, widely expressed gene containing the major human {alpha}-globin regulatory element

    SciTech Connect

    Vyas, P.; Vickers, M.A.; Picketts, D.J.; Higgs, D.R.

    1995-10-10

    We have determined the cDNA and genomic structure of a gene (-14 gene) that lies adjacent to the human {alpha}-globin cluster. Although it is expressed in a wide range of cell lines and tissues, a previously described erythroid-specific regulatory element that controls expression of the {alpha}-globin genes lies within intron 5 of this gene. Analysis of the -14 gene promoter shows that it is GC rich and associated with a constitutively expressed DNase 1 hypersensitive site; unlike the {alpha}-globin promoter, it does not contain a TATA or CCAAT box. These and other differences in promoter structure may explain why the erythroid regulatory element interacts specifically with the {alpha}-globin promoters and not the -14 gene promoter, which lies between the {alpha} promoters and their regulatory element. Interspecies comparisons demonstrate that the sequence and location of the -14 gene adjacent to the a cluster have been maintained since the bird/mammal divergence, 270 million years ago. 38 refs., 6 figs.

  8. Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

    PubMed Central

    Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

    1995-01-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. Images Figure 2 Figure 3 PMID:8533755

  9. Human {beta}-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old

    SciTech Connect

    Beraud-Colomb, E. |; Maroc, N.; Roubin, R.

    1995-12-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the P-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for P-globin frameworks by sequencing through two variable positions and for a polymorphic (AT){sub x}(T){sub y} microsatellite 500 bp upstream of the P-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. 34 refs., 3 figs., 2 tabs.

  10. Beta-globin gene evolution in the ruminants: evidence for an ancient origin of sheep haplotype B.

    PubMed

    Jiang, Y; Wang, X; Kijas, J W; Dalrymple, B P

    2015-10-01

    Domestic sheep (Ovis aries) can be divided into two groups with significantly different responses to hypoxic environments, determined by two allelic beta-globin haplotypes. Haplotype A is very similar to the goat beta-globin locus, whereas haplotype B has a deletion spanning four globin genes, including beta-C globin, which encodes a globin with high oxygen affinity. We surveyed the beta-globin locus using resequencing data from 70 domestic sheep from 42 worldwide breeds and three Ovis canadensis and two Ovis dalli individuals. Haplotype B has an allele frequency of 71.4% in O. aries and was homozygous (BB) in all five wild sheep. This shared ancestry indicates haplotype B is at least 2-3 million years old. Approximately 40 kb of the sequence flanking the ~37-kb haplotype B deletion had unexpectedly low identity between haplotypes A and B. Phylogenetic analysis showed that the divergent region of sheep haplotype B is remarkably distinct from the beta-globin loci in goat and cattle but still groups with the Ruminantia. We hypothesize that this divergent ~40-kb region in haplotype B may be from an unknown ancestral ruminant and was maintained in the lineage to O. aries, but not other Bovidae, evolving independently of haplotype A. Alternatively, the ~40-kb sequence in haplotype B was more recently acquired by an ancestor of sheep from an unknown non-Bovidae ruminant, replacing part of haplotype A. Haplotype B has a lower nucleotide diversity than does haplotype A, suggesting a recent bottleneck, whereas the higher frequency of haplotype B suggests a subsequent spread through the global population of O. aries. © 2015 Stichting International Foundation for Animal Genetics.

  11. Multiple linked β and α globin genes in Atlantic cod: A PCR based strategy of genomic exploration.

    PubMed

    Halldórsdóttir, Katrín; Arnason, Einar

    2009-01-01

    Allozyme variation in Atlantic cod hemoglobins shows various signs of natural selection. We report a genomic exploration of globin genes in this non-model organism. Applying a PCR based strategy with a strict criterion of phylogenetically informative sites we estimate the number of linked β and α globin genes. We estimate PCR error rate by PCR of cloned DNA and recloning and by analysis of singleton variable sites among clones. Based on the error rate we exclude variable sites so that the remaining variation meets successively stricter criteria of doubleton and triplet variable site. Applying these criteria we find ten clusters of linked β/α globin genes in the genome of Atlantic cod. Six variable amino acid changes in both genes were found in linkage disequilibrium with silent nucleotide substitutions. A phylogenetic tree, based on our strictly phylogenetically informative sites among 57 clones from 19 individuals, is split into two major branches by an amino acid change in a β gene. This change is supported by extensive linkage disequilibrium between the amino acid change and numerous other phylogenetically informative silent nucleotide sites. The different gene sets in the genome may represent different loci encoding different globins and/or allelic variation at some loci.

  12. An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age.

    PubMed

    Chang, Y C; Smith, K D; Moore, R D; Serjeant, G R; Dover, G J

    1995-02-15

    Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < .05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40% of Hb F variation. beta-Globin haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).

  13. Molecular adaptive convergence in the α-globin gene in subterranean octodontid rodents.

    PubMed

    Tomasco, Ivanna H; Boullosa, Nicolás; Hoffmann, Federico G; Lessa, Enrique P

    2017-09-10

    Tuco-tucos (Ctenomys) and related coruros (Spalacopus) are South American subterranean rodents. An energetically demanding lifestyle within the hypoxic/hypercapnic underground atmosphere may change the selective regime on genes involved in O2 transport in blood. In addition, some species of tuco-tucos may be found at high altitude, thus facing additional reductions in changes O2 availabily. We examined sequence variation in the alpha globin subunit gene of hemoglobine in these lineages, within a robust phylogenetic context. Using different approaches (classical and Bayesian maximum likelihood (PAML/Datamonkey) and alternatives methods (TreeSAAP)) we found at least 2 sites with evidence of positive selection in the basal branch of Octodontidae, but not in tuco-tucos. These results suggest some adaptive changes associated to fossoriality, but not strictly to life underground. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Total beta-globin gene deletion has high frequency in Filipinos

    SciTech Connect

    Patrick, N.; Miyakawa, F.; Hunt, J.A.

    1994-09-01

    The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5 of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].

  15. The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

    PubMed Central

    Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

    2017-01-01

    Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

  16. A mutation of the beta-globin gene initiation codon, ATG-->AAG, found in a French Caucasian man.

    PubMed

    Lacan, Philippe; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2005-01-01

    A new mutation of the beta-globin gene initiation codon, ATG-->AAG (Met-->Tyr), is reported in a man originating from the southeast of France. Typical hematological findings of beta-thalassemia (thal) trait were found. We emphasize the importance of characterizing uncommon beta-thal mutations for genetic counseling.

  17. Bergamot (Citrus bergamia Risso) fruit extracts as γ-globin gene expression inducers: phytochemical and functional perspectives.

    PubMed

    Guerrini, Alessandra; Lampronti, Ilaria; Bianchi, Nicoletta; Zuccato, Cristina; Breveglieri, Giulia; Salvatori, Francesca; Mancini, Irene; Rossi, Damiano; Potenza, Rocco; Chiavilli, Francesco; Sacchetti, Gianni; Gambari, Roberto; Borgatti, Monica

    2009-05-27

    Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia.

  18. Inter-ethnic polymorphism of the beta-globin gene locus control region (LCR) in sickle-cell anemia patients.

    PubMed

    Périchon, B; Ragusa, A; Lapouméroulie, C; Romand, A; Moi, P; Ikuta, T; Labie, D; Elion, J; Krishnamoorthy, R

    1993-06-01

    Sequence polymorphisms within the 5'HS2 segment of human locus control region is described among sickle cell anemia patients. Distinct polymorphic patterns of a simple sequence repeat are observed in strong linkage disequilibrium with each of the five major beta s haplotypes. Potential functional relevance of this polymorphic region in globin gene expression is discussed.

  19. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells

    PubMed Central

    MONTAGNER, GIULIA; GEMMO, CHIARA; FABBRI, ENRICA; MANICARDI, ALEX; ACCARDO, IGEA; BIANCHI, NICOLETTA; FINOTTI, ALESSIA; BREVEGLIERI, GIULIA; SALVATORI, FRANCESCA; BORGATTI, MONICA; LAMPRONTI, ILARIA; BRESCIANI, ALBERTO; ALTAMURA, SERGIO; CORRADINI, ROBERTO; GAMBARI, ROBERTO

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  20. Mapping of structural and transcription-related matrix attachment sites in the alpha-globin gene domain of avian erythroblasts and erythrocytes.

    PubMed Central

    Farache, G; Razin, S V; Rzeszowska-Wolny, J; Moreau, J; Targa, F R; Scherrer, K

    1990-01-01

    The positions of preferential DNA interaction with the nuclear matrix were mapped within the domain of the chicken alpha-globin genes in transcriptionally active erythroblast nuclei and inactive nuclei of mature erythrocytes. In the latter, only two major distinct attachment sites were observed, close to the A + T-rich sequences previously found at the boundaries of the domain. Sequencing of these structural matrix attachment points revealed several known DNA motifs; some of them were present on both sides of the domain. In actively transcribing erythroblast nuclei of adult animals, a large fraction of the transcribed area was represented in nuclear matrix DNA, including upstream and downstream elements. In particular, adult alpha A- and alpha D-globin genes were found in matrix DNA, while the transcribed but translationally unexpressed embryonic pi gene was underrepresented. The data are discussed in terms of the existence of stable or structural and expression-related matrix attachment sites; correlations to the origin of replication and the units of transcription of the domain are shown. Images PMID:2398893

  1. Replication of the Chicken β-Globin Locus: Early-Firing Origins at the 5′ HS4 Insulator and the ρ- and βA-Globin Genes Show Opposite Epigenetic Modifications

    PubMed Central

    Prioleau, Marie-Noëlle; Gendron, Marie-Claude; Hyrien, Olivier

    2003-01-01

    Chromatin structure is believed to exert a strong effect on replication origin function. We have studied the replication of the chicken β-globin locus, whose chromatin structure has been extensively characterized. This locus is delimited by hypersensitive sites (HSs) that mark the position of insulator elements. A stretch of condensed chromatin and another HS separate the β-globin domain from an adjacent folate receptor (FR) gene. We demonstrate here that in erythroid cells that express the FR but not the globin genes, replication initiates at four sites within the β-globin domain, one at the 5′ HS4 insulator and the other three near the ρ- and βA-globin genes. Three origins consist of G+C-rich sequences enriched in CpG dinucleotides. The fourth origin is A+T rich. Together with previous work, these data reveal that the insulator origin has unmethylated CpGs, hyperacetylated histones H3 and H4, and lysine 4-methylated histone H3. In contrast, opposite modifications are observed at the other G+C-rich origins. We also show that the whole region, including the stretch of condensed chromatin, replicates early in S phase in these cells. Therefore, different early-firing origins within the same locus may have opposite patterns of epigenetic modifications. The role of insulator elements in DNA replication is discussed. PMID:12724412

  2. Diverse phenotypes and transfusion requirements due to interaction of β-thalassemias with triplicated α-globin genes.

    PubMed

    Mehta, Pallavi R; Upadhye, Dipti S; Sawant, Pratibha M; Gorivale, Manju S; Nadkarni, Anita H; Shanmukhaiah, Chandrakala; Ghosh, Kanjaksha; Colah, Roshan B

    2015-12-01

    Co-inheritance of triplicated α-genes can alter the clinical and hematological phenotypes of β-thalassemias. We evaluated the phenotypic diversity and transfusion requirements in β-thalassemia heterozygotes, homozygotes, and normal individuals with associated α-gene triplication. Clinical and hematological evaluation was done and the β-thalassemia mutations characterized by a covalent reverse dot blot hybridization/amplification refractory mutation system. Alpha-globin gene triplication was assessed by multiplex PCR. During the last 2.5 years, 181 β-thalassemia patients and β-thalassemia carriers with an unusual clinical presentation were referred to us for screening for the presence of associated α-globin gene triplication. Twenty-nine of them had associated α-gene triplication (3 β-thalassemia homozygotes or compound heterozygotes and 26 β-thalassemia heterozygotes). One β-thalassemia compound heterozygote [IVS 1-5 (G → C) + CD 41/42 (-CTTT)] was anemic at birth and required blood transfusions unusually early by 6 weeks of age. The second patient (4.5 years) was also clinically severe and became transfusion dependent in spite of having one mild β-thalassemia mutation [Capsite +1 (A → C)]. The third case (3.5 years) who was homozygous for a mild β-gene mutation [-88 (C → T)] with α gene triplication was untransfused. The 26 β-thalassemia heterozygotes with associated triplicated α-genes presented variably, with a β-thalassemia intermedia-like presentation. While screening the family members of all these cases, we found another 10 β-thalassemia heterozygotes and 9 normal individuals with α-globin gene triplication; however, all of them were asymptomatic. Beta-thalassemia carriers, homozygotes, and compound heterozygotes with an unusual presentation should be screened for the possible presence of associated α-globin gene triplication which could influence the clinical and hematological presentation.

  3. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    PubMed Central

    Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

  4. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    PubMed

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis.

  5. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

    PubMed Central

    Miller, J L; Donahue, R E; Sellers, S E; Samulski, R J; Young, N S; Nienhuis, A W

    1994-01-01

    Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content. Images PMID:7524085

  6. Genotyping of β-globin gene mutations in single lymphocytes: a preliminary study for preimplantation genetic diagnosis of monogenic disorders.

    PubMed

    Durmaz, Burak; Ozkinay, Ferda; Onay, Huseyin; Karaca, Emin; Aydinok, Yesim; Tavmergen, Erol; Vrettou, Christina; Traeger-Synodinos, Jan; Kanavakis, Emmanuel

    2012-01-01

    Hemoglobinopathies, especially β-thalassemia (β-thal), represent an important health burden in Mediterranean countries like Turkey. Some couples prefer the option of preimplantation genetic diagnosis (PGD). However, clinical application of PGD, especially for the monogenic disorders is technically demanding. To ensure reliable results, protocols need to be robust and well standardized. Ideally PGD-PCR (polymerase chain reaction) protocols should be based on multiplex and fluorescent PCR for analysis of the disease-causing mutation(s) along with linked markers across the disease-associated locus. In this study, we aimed to constitute a protocol in single cells involving first round multiplex PCR with primers to amplify the region of the β-globin gene containing the most common mutations. Two microsatellites linked to the β-globin gene cluster (D11S4891, D11S2362) and two unlinked (D13S314, GABRB3) microsatellite markers, were used to rule out allele dropout (ADO) and contamination; followed by nested real-time PCR for genotyping the β-globin mutations. We also investigated the allele frequencies and heterozygote rates of these microsatellites in the Turkish population that have not been reported to date. This protocol was tested in 100 single lymphocytes from heterozygotes with known β-globin mutations. Amplification failure was detected in one lymphocyte (1%) and ADO was observed in two lymphocytes (2%). No contamination was detected. All results were concordant with the genotypes of the patients. Overall, this protocol was demonstrated to be sensitive, accurate, reliable and rapid for the detection of β-globin mutations in single cells and shows potential for the clinical application of PGD for hemoglobinopathies in the Turkish population.

  7. Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human. beta. -globin gene

    SciTech Connect

    Wagner, E.F.; Mintz, B.

    1982-02-01

    Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, the authors investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH..beta..1 containing the human genomic ..beta..-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human ..beta..-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.

  8. Everolimus is a potent inducer of erythroid differentiation and gamma-globin gene expression in human erythroid cells.

    PubMed

    Zuccato, Cristina; Bianchi, Nicoletta; Borgatti, Monica; Lampronti, Ilaria; Massei, Francesco; Favre, Claudio; Gambari, Roberto

    2007-01-01

    We studied the effects of everolimus on the erythroid differentiation of human leukaemic K562 cells and on the cultures of erythroid progenitors derived from the peripheral blood of beta-thalassaemia patients. A quantitative real-time reverse-transcription polymerase chain reaction assay was employed for the quantification of the accumulation of globin mRNAs. The results obtained demonstrate that everolimus is a potent inducer of the erythroid differentiation of K562 cells. Erythroid induction is associated with an increase in alpha- and gamma-globin mRNAs. In erythroid precursor cells from 4 beta-thalassaemia patients, everolimus stimulated a preferential increase (ranging from 1.8- to 7.2-fold) in gamma-globin mRNA. Only minor effects were observed on the expression of alpha-globin genes. These results, in our opinion, are of interest as this compound is already employed in clinical trials as an anti-rejection agent following kidney transplantation. These data suggest that everolimus warrants further evaluation as a potential therapeutic drug in the treatment of beta-thalassaemia. 2007 S. Karger AG, Basel

  9. Alpha-thalassemia phenotype induced by the new IVS-II-2 (T --> A) splice donor site mutation on the alpha2-globin gene.

    PubMed

    Harteveld, Cornelis L; Jebbink, Max C W; van der Lely, Nico; van Delft, Peter; Akkermans, Nicole; Arkesteyn, Sandra; Giordano, Piero C

    2006-01-01

    We present a family of North European extraction referred for a refractory non iron depleted microcytic anemia. The proband, a 36 year-old male, presented with chronic borderline anemia and microcytic hypochromic parameters. No abnormal hemoglobin (Hb) fractions were observed on high performance liquid chromatography (HPLC) or on alkaline electrophoresis. Gap-polymerase chain reaction (gap-PCR) excluded the seven common alpha-thalassemia (thal) deletion defects. However, the beta/alpha-globin chain synthesis ratio measured in vitro was unbalanced, indicating a reduced expression of the alpha-globin genes. Direct sequencing of the alpha-globin genes revealed heterozygosity for a T --> A transversion at the IVS-II-2 position of the alpha2 gene. This is the first IVS-II splice donor site mutation described on the alpha2-globin gene.

  10. Comparative genomics of canine hemoglobin genes reveals primacy of beta subunit delta in adult carnivores.

    PubMed

    Zaldívar-López, Sara; Rowell, Jennie L; Fiala, Elise M; Zapata, Isain; Couto, C Guillermo; Alvarez, Carlos E

    2017-02-08

    The main function of hemoglobin (Hb) is to transport oxygen in the circulation. It is among the most highly studied proteins due to its roles in physiology and disease, and most of our understanding derives from comparative research. There is great diversity in Hb gene evolution in placental mammals, mostly in the repertoire and regulation of the β-globin subunits. Dogs are an ideal model in which to study Hb genes because: 1) they are members of Laurasiatheria, our closest relatives outside of Euarchontoglires (including primates, rodents and rabbits), 2) dog breeds are isolated populations with their own Hb-associated genetics and diseases, and 3) their high level of health care allows for development of biomedical investigation and translation. We established that dogs have a complement of five α and five β-globin genes, all of which can be detected as spliced mRNA in adults. Strikingly, HBD, the allegedly-unnecessary adult β-globin protein in humans, is the primary adult β-globin in dogs and other carnivores; moreover, dogs have two active copies of the HBD gene. In contrast, the dominant adult β-globin of humans, HBB, has high sequence divergence and is expressed at markedly lower levels in dogs. We also showed that canine HBD and HBB genes are complex chimeras that resulted from multiple gene conversion events between them. Lastly, we showed that the strongest signal of evolutionary selection in a high-altitude breed, the Bernese Mountain Dog, lies in a haplotype block that spans the β-globin locus. We report the first molecular genetic characterization of Hb genes in dogs. We found important distinctions between adult β-globin expression in carnivores compared to other members of Laurasiatheria. Our findings are also likely to raise new questions about the significance of human HBD. The comparative genomics of dog hemoglobin genes sets the stage for diverse research and translation.

  11. A 21 Nucleotide Duplication on the α1- and α2-Globin Genes Involves a Variety of Hypochromic Microcytic Anemias, From Mild to Hb H Disease.

    PubMed

    Farashi, Samaneh; Faramarzi Garous, Negin; Zeinali, Fatemeh; Vakili, Shadi; Ashki, Mehri; Imanian, Hashem; Najmabadi, Hossein; Azarkeivan, Azita; Tamaddoni, Ahmad

    2015-01-01

    α-Thalassemia (α-thal) is a common genetic disorder in Iran and many parts of the world. Genetic defects in the α-globin gene cluster can result in α-thal that may develop into a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. Loss of one functional α gene, indicated as heterozygous α(+)-thal, shows minor hematological abnormalities. Homozygosity for α(+)- or heterozygosity for α(0)-thal have more severe hematological abnormalities due to a markedly reduced α chain output. At the molecular level, the absence of three α-globin genes resulting from the compound heterozygous state for α(0)- and α(+)-thal, lead to Hb H disease. Here we present a 21 nucleotide (nt) duplication consisting of six amino acids and 3 bp of intronic sequence at the exon-intron boundary, in both the α-globin genes, detected by direct DNA sequencing. This duplication was identified in three patients originating from two different Iranian ethnic groups and one Arab during more than 12 years. The clinical presentation of these individuals varies widely from a mild asymptomatic anemia (heterozygote in α1-globin gene) to a severely anemic state, diagnosed as an Hb H individual requiring blood transfusion (duplication on the α2-globin gene in combination with the - -(MED) double α-globin gene deletion). The third individual, who was homozygous for this nt duplication on the α1-globin gene, showed severe hypochromic microcytic anemia and splenomegaly. In the last decade, numerous α-globin mutations have demonstrated the necessity of prenatal diagnosis (PND) for α-thal, and this study has contributed another mutation as important enough that needs to be considered.

  12. Self-catalytic DNA depurination underlies human β-globin gene mutations at codon 6 that cause anemias and thalassemias.

    PubMed

    Alvarez-Dominguez, Juan R; Amosova, Olga; Fresco, Jacques R

    2013-04-19

    The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze depurination of the 5'G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and β-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to β-globin variants underlying phenotypic diversity and disease.

  13. Self-catalytic DNA Depurination Underlies Human β-Globin Gene Mutations at Codon 6 That Cause Anemias and Thalassemias*

    PubMed Central

    Alvarez-Dominguez, Juan R.; Amosova, Olga; Fresco, Jacques R.

    2013-01-01

    The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze depurination of the 5′G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and β-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to β-globin variants underlying phenotypic diversity and disease. PMID:23457306

  14. The spectrum of beta-globin gene mutations in children with beta-thalassaemia major from Kota Kinabalu, Sabah, Malaysia.

    PubMed

    Thong, M K; Soo, T L

    2005-07-01

    Beta-thalassaemia major is one of the commonest genetic disorders in South East Asia. The strategy for the community control of beta-thalassaemia major requires the characterisation of the spectrum of beta-globin gene mutations in any multi-ethnic population. There is only a single report of mutation analyses of the beta-globin gene in an isolated Kadazandusun community in Kota Belud, Sabah, Malaysia, which showed the presence of a common 45 kb deletion. To confirm the observation that this large deletion is the commonest beta-globin gene mutation among the Kadazandusun and other indigenous populations in Sabah, Malaysia, we performed polymerase chain reaction (PCR) analysis of the beta-globin gene in ten children with beta-thalassaemia major attending the Thalassaemia Centre, Queen Elizabeth Hospital, the major paediatric referral centre in Kota Kinabalu, Sabah. The 45 kb deletion was confirmed to be the commonest mutation found in the Kadazandusun, Bajau and Murut populations, whereby it was detected in 19 out of the 20 (95 percent) alleles analysed. The other mutation was due to an IVS-1 position 1 G > T mutation. This finding confirmed the deletion in the homozygous state was associated with a severe phenotype. The reason for the predominance of this mutation in Kota Kinabalu is most likely to be due to founder effects and possibly intermarriages between the various ethnic groups. Prenatal diagnosis using PCR for this common mutation is feasible in this community. Medical workers and scientists at molecular diagnostic centres serving large South East Asian populations should incorporate a diagnostic strategy for this deletion in the appropriate population. Future studies on these indigenous ethnic groups in other areas and other groups in Sabah are required.

  15. A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling.

    PubMed Central

    Gesto-Borroto, Reinier; Sánchez-Sánchez, Miriam; Arredondo-Peter, Raúl

    2015-01-01

    Globins (Glbs) are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs) and single-domain Glbs (SDgbs); the S Glbs include globin-coupled sensors (GCSs), protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs). Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts from Bradyrhizobium japonicum USDA38 and USDA58 by (reduced + carbon monoxide (CO) minus reduced) differential spectroscopy. Database searching showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator)-like promoters. Mapping analysis revealed that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized in B. japonicum USDA38 and USDA58. PMID:26594329

  16. The Full Globin Repertoire of Turtles Provides Insights into Vertebrate Globin Evolution and Functions

    PubMed Central

    Schwarze, Kim; Singh, Abhilasha; Burmester, Thorsten

    2015-01-01

    Globins are small heme proteins that play an important role in oxygen supply, but may also have other functions. Globins offer a unique opportunity to study the functional evolution of genes and proteins. We have characterized the globin repertoire of two different turtle species: the Chinese softshell turtle (Pelodiscus sinensis) and the western painted turtle (Chrysemys picta bellii). In the genomes of both species, we have identified eight distinct globin types: hemoglobin (Hb), myoglobin, neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Therefore, along with the coelacanth, turtles are so far the only known vertebrates with a full globin repertoire. This fact allows for the first time a comparative analysis of the expression of all eight globins in a single species. Phylogenetic analysis showed an early divergence of neuroglobin and globin X before the radiation of vertebrates. Among the other globins, cytoglobin diverged first, and there is a close relationship between myoglobin and globin E; the position of globin Y is not resolved. The globin E gene was selectively lost in the green anole, and the genes coding for globin X and globin Y were deleted in chicken. Quantitative real-time reverse transcription polymerase chain reaction experiments revealed that myoglobin, neuroglobin, and globin E are highly expressed with tissue-specific patterns, which are in line with their roles in the oxidative metabolism of the striated muscles, the brain, and the retina, respectively. Histochemical analyses showed high levels of globin E in the pigment epithelium of the eye. Globin E probably has a myoglobin-like role in transporting O2 across the pigment epithelium to supply in the metabolically highly active retina. PMID:26078264

  17. Spectrum of Beta Globin Gene Mutations in Egyptian Children with β-Thalassemia

    PubMed Central

    El-Shanshory, MR; Hagag, AA; Shebl, SS; Badria, IM; Abd Elhameed, AH; Abd El-Bar, ES; Al-Tonbary, Y; Mansour, A; Hassab, H; Hamdy, M; Alfy, M; Sherief, L; Sharaf, E

    2014-01-01

    Background The molecular defects resulting in a β-thalassemia phenotype, in the Egyptian population, show a clear heterogenic mutations pattern. PCR-based techniques, including direct DNA sequencing are effective on the molecular detection and characterization of these mutations. The molecular characterization of β-thalassemia is necessary for carrier screening, genetic counseling, and to offer prenatal diagnosis. The aim of the work was to evaluate the different β-globin gene mutations in two hundred β-thalassemic Egyptian children. Subjects and Methods This study was carried out on two hundred β-thalassemic Egyptian children covering most Egyptian Governorates including 158 (79%) children with thalassemia major (TM) and 42 (21%) children with thalassemia intermedia(TI). All patients were subjected to meticulous history taking, clinical examination, complete blood count, hemoglobin electrophoresis, serum ferritin and direct fluorescent DNA sequencing of the β-globin gene to detect the frequency of different mutations. Results The most common mutations among patients were IVS I-110(G>A) 48%, IVS I-6(T>C) 40%, IVS I-1(G>A) 24%, IVS I-5(G>C)10%, IVS II-848 (C>A) 9%, IVS II-745(C>G) 8%, IVS II-1(G>A) 7%, codon “Cd”39(C> T) 4%, −87(C>G) 3% and the rare mutations were: Cd37 (G>A), Cd8 (−AA), Cd29(−G), Cd5 (−CT), Cd6(−A), Cd8/9(+G), Cd 106/107(+G), Cd27(C>T), IVS II-16(G> C), Cd 28 (−C), Cap+1(A>C), −88(C>A), all of these rare mutations were present in 1%. There was a considerable variation in phenotypic severity among patients resulting from the interaction of different β∘ and β+mutations. Furthermore, no genotype-phenotype association was found both among the cases with thalassemia major and the cases with thalassemia intermedia. Conclusion Direct DNA sequencing provides insights for the frequency of different mutations in patients with β-thalassemia including rare and/or unknown ones. The most common mutations in Egyptian children with

  18. The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

    PubMed Central

    Romero, Zulema; Campo-Fernandez, Beatriz; Wherley, Jennifer; Kaufman, Michael L; Urbinati, Fabrizia; Cooper, Aaron R; Hoban, Megan D; Baldwin, Kismet M; Lumaquin, Dianne; Wang, Xiaoyan; Senadheera, Shantha; Hollis, Roger P; Kohn, Donald B

    2015-01-01

    Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies. PMID:26029723

  19. The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells.

    PubMed

    Romero, Zulema; Campo-Fernandez, Beatriz; Wherley, Jennifer; Kaufman, Michael L; Urbinati, Fabrizia; Cooper, Aaron R; Hoban, Megan D; Baldwin, Kismet M; Lumaquin, Dianne; Wang, Xiaoyan; Senadheera, Shantha; Hollis, Roger P; Kohn, Donald B

    2015-01-01

    Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

  20. Molecular characterization of an atypical beta-thalassemia caused by a large deletion in the 5' beta-globin gene region.

    PubMed Central

    Popovich, B W; Rosenblatt, D S; Kendall, A G; Nishioka, Y

    1986-01-01

    We describe a Canadian family of Czechoslovakian descent that came to our attention because of an HbA2 percentage approximately twice that of an average case of heterozygous beta-thalassemia. This unique phenotype suggested to us the possibility of a novel genetic mechanism being responsible for their beta-thalassemia. To investigate this possibility, we mapped, cloned, and sequenced the mutant beta-globin allele. This molecular analysis demonstrated the presence of a unique 4,237 base pair (bp) deletion extending from 3.3 kilobases (kb) 5' of the beta-globin mRNA cap site to approximately the middle of beta IVS-2. This truncated beta-globin gene further extends the heterogeneity of mutations known to cause beta-thalassemia and delineates new sequences involved in nonhomologous recombination events in the beta-globin gene region. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:3799598

  1. Incidence of alpha-globin gene defect in the Lebanese population: a pilot study.

    PubMed

    Farra, Chantal; Daher, Rose; Badra, Rebecca; el Rafei, Rym; Bejjany, Rachelle; Charafeddine, Lama; Yunis, Khalid

    2015-01-01

    It is well established that the Mediterranean and Arab populations are at high risk for thalassemias in general and for alpha-thalassemia in particular. Yet, reports on alpha-thalassemia in Lebanon are still lacking. In this study, we aim at assessing the incidence of alpha-thalassemia in the Lebanese population. 230 newborns' dried blood cards remaining from routine neonatal screening at the American University of Beirut Medical Center were collected for DNA extraction. Samples were screened for the 21 most common α-globin deletions and point mutations reported worldwide, through multiplex Polymerase Chain Reaction (PCR) and Reverse-Hybridization technique. Upon analyses, the carrier rate of α-thalassemia was found to be 8%. Two mutations detected the -α(3,7) single gene deletion found in 75% of cases and the nongene deletion α2 IVS1 [-5nt] in the remaining samples. This study is the first dedicated to investigate α-thalassemia trait incidence in Lebanon. Data obtained demonstrates a high carrier rate in a relatively, highly consanguineous population; it also highlighted the presence of two common mutations. These results may be of an important impact on premarital and newborn screening policies in our country.

  2. Spectrum of alpha-globin gene mutations among premarital Baluch couples in southeastern Iran

    PubMed Central

    Miri-Moghaddam, Ebrahim; Nikravesh, Abass; Gasemzadeh, Negin; Badaksh, Mahin; Rakhshi, Nahid

    2015-01-01

    Background: Alpha thalassemia (α-thal) is one of the most common hemoglobinopathies worldwide. The aim of this study was to investigate the spectrum of α-thal mutations among premarital Baluch couples in southeastern Iran. Subjects and Methods: We assessed 1215 individuals by multiplex gap polymerase chain reaction (gap-PCR) and amplification refractory mutation system (ARMS-PCR). Results: Of the 1215 participants with mean age of 23±5.7 years, 62.3% lived in urban areas, and the rate of consanguineous marriage was 68.1%. Five mutations were identified, the most frequent one was –α3.7 (rightward) with a frequency of 76.5%, followed by α−5 nt (16.8%), α2/ Codon 19(-G) (4%), –α4.2 (leftward)(2.4%), – –MED (0.3%) among mutated alleles of the α -globin gene. Conclusion : Knowing the alpha-genotype is helpful for genetic counseling, microcytic anemia discrimination and hemoglobinopathy prevention. PMID:26261699

  3. Globin gene-associated restriction-fragment-length polymorphisms in southern African peoples.

    PubMed Central

    Ramsay, M; Jenkins, T

    1987-01-01

    The combination of polymorphic restriction-enzyme sites in the 3' region of the beta-globin gene cluster shows very little variation in southern-African Bantu-speaking black and Kalahari !Kung San populations. The sites of the 5' region, on the other hand, show marked variation, and two common haplotypes are present--the "Negro" type (- - - - +) and the "San" type (- + - - +)--in frequencies of .404 and .106, respectively, in the Bantu-speakers and .262 and .405, respectively, in the San. Twenty of 23 beta s-associated haplotypes in southern-African Bantu-speaking black subjects were the same as that found commonly in the Central African Republic (CAR)--i.e., the "Bantu" type--a finding providing the first convincing biological evidence for the common ancestry of geographically widely separated speakers of languages belonging to the Bantu family. The (-alpha) haplotype has a frequency of .21 in the Venda, .07 in both the Sotho-Tswana and the Nguni, and .06 among the !Kung San. These data are interpreted in the light of Plasmodium falciparum malaria selection and population movements in the African subcontinent. PMID:2891298

  4. Implications of the genetic epidemiology of globin haplotypes linked to the sickle cell gene in southern Iran.

    PubMed

    Rahimi, Zohreh; Merat, Ahmad; Gerard, Nathalie; Krishnamoorthy, Rajagopal; Nagel, Ronald L

    2006-12-01

    To determine the origin of sickle cell mutation in different ethnic groups living in southern Iran, we studied the haplotype background of the betaS and betaA genes in subjects from the provinces of Fars, Khuzestan, Bushehr, Hormozgan, and Kerman and from the islands of Khark and Qeshm. beta-globin gene cluster haplotypes were determined using the PCR-RFLP technique. Detection of -alpha 3.7 deletion and beta-thalassemia mutations were defined by PCR and reverse dot blot techniques, respectively. The framework of the beta-globin gene was determined using denaturing gradient gel electrophoresis. We found that the betaS mutation in southern Iran is associated with multiple mutational events. Most of the patients were from two ethnic groups: Farsi speakers (presumably Persian in origin) from Fars province and patients of Arab origin from Khuzestan province. In both ethnic groups the Arab-Indian haplotype was the most prevalent. The frequencies of the Arab-Indian and African haplotypes in sickle cell anemia patients from the provinces of Fars and Khuzestan were similar. Among betaA chromosomes the Bantu A2 haplotype was the most prevalent. The decrease in alpha-globin production in SS patients and AS individuals appeared to be related to the reduction in mean cell volume and mean cell hemoglobin. The Arab-Indian haplotype gene flow into this region of Iran can be traced to the Sassanian Empire. It is likely that the influx of betaS genes linked to the Benin and Bantu haplotypes, of African origin, must have occurred during the Arab slave trade.

  5. Analysis of 5' flanking regions of the gamma globin genes from major African haplotype backgrounds associated with sickle cell disease.

    PubMed

    Month, S R; Wood, R W; Trifillis, P T; Orchowski, P J; Sharon, B; Ballas, S K; Surrey, S; Schwartz, E

    1990-02-01

    There are at least three major African haplotype backgrounds on which the beta s mutation arises. Sequence changes in the immediate 5' flanking area of the gamma-globin genes may account for differences in fetal hemoglobin expression among the three haplotypes. We determined the sequence from -350 to 10 bp 5' of the G gamma and A gamma fetal globin genes from one beta s-containing chromosome on each of the three major haplotype backgrounds. The Senegal chromosome had a T at -158 5' to the G gamma gene; the Benin (BEN) chromosome had an A to G change at -309 5' to the G gamma gene; and the Central African Republic (CAR) chromosome had a C to T change at -271 5' to the A gamma gene. Genomic DNA from patients with sickle cell disease was analyzed using the polymerase chain reaction and radiolabeled allele-specific oligonucleotide probes. The -309 G variant 5' to the G gamma gene is associated with BEN chromosomes, and the -271 T variant 5' to A gamma with CAR. The -309 change was also found on beta A-containing chromosomes, while the -271 change was not. The -309 change may have predated the beta s mutation on the BEN chromosome.

  6. Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells.

    PubMed

    Charnay, P; Mellon, P; Maniatis, T

    1985-06-01

    We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

  7. Frequency of haplotypes in the beta globin gene cluster in a selected sample of the mexican population.

    PubMed

    Peñaloza, Rosenda; García-Carrancá, Alejandro; Ceras, Teresa; Alvarez, Carlos; Berumen, Jaime; Zavala, Carlos; Salamanca, Fabio

    1995-01-01

    Five polymorphic restriction enzyme sites in the beta globin gene cluster (HindIII Gγ-Hind III Aγ-, Ava II(INV-2) β-and Hpa I and Bam HI 3'β-globin gene) were studied in individuals from 13 families: 13 homozygote patients for sickle cell anemia, two double heterozygotes (one SC and one S/β(Thal) ), 35 AS heterozygotes (23 parents and 12 siblings), one father (A/β(Thal) ), and three normal siblings. In addition, 17 normal unrelated Mexican subjects were studied. All subjects were from the state of Veracruz on the coast of the Gulf of Mexico. The Southern blot technique was used. Fifteen haplotypes were identified in the 142 chromosomes. Five were the most frequent: two haplotypes, (+-+++) (52.4%) and (--+-+) (19.0%) were associated with β(S) chromosomes; two haplotypes, (--+++) (38.2%) and (---++) (19.7%), were linked with β(A) chromosomes, and the fifth (--++-) was present in both types of chromosomes. Haplotype (+-+++) corresponded to the Bantu or Senegal type. With Hinc II analysis after PCR amplification in both the 5' and 3' regions of the ψβ-globin gene, it was possible to distinguish between these African types, as in the former both restriction sites are absent. This analysis was done in 23 β(S) and 10 β(A) subjects. All β(S) chromosomes disclosed the Bantu type, while β(A) were similar to Caucasians. Bantu and Benin haplotypes have been found with high frequency in African populations, indicating the great influence of African genes in the population of the Mexican coasts. In addition, two previously unidentified haplotypes were found: (++--+) and (-++++). These can be explainded by crossing-over events and/or by new mutations. © 1995 Wiley-Liss, Inc. Copyright © 1995 Wiley-Liss, Inc., A Wiley Company.

  8. Phylogenetic comparisons suggest that distance from the locus control region guides developmental expression of primate β-type globin genes

    PubMed Central

    Johnson, Robert M.; Prychitko, Tom; Gumucio, Deborah; Wildman, Derek E.; Uddin, Monica; Goodman, Morris

    2006-01-01

    Phylogenetic inferences drawn from comparative data on mammalian β-globin gene clusters indicate that the ancestral primate cluster contained a locus control region (LCR) and five paralogously related β-type globin loci (5′-LCR-ε-γ-ψη-δ-β-3′), with ε and γ expressed solely during embryonic life. A γ locus tandem duplication (5′-γ1-γ2-3′) triggered γ’s evolution toward fetal expression but by a different trajectory in platyrrhines (New World monkeys) than in catarrhines (Old World monkeys and apes, including humans). In platyrrhine (e.g., Cebus) fetuses, γ1 at the ancestral distance from ε is down-regulated, whereas γ2 at increased distance is up-regulated. Catarrhine γ1 and γ2 acquired longer distances from ε (14 and 19 kb, respectively), and both are up-regulated throughout fetal life with γ1’s expression predominating over γ2’s. On enlarging the platyrrhine expression data, we find Aotus γ is embryonic, Alouatta γ is inactive at term, and in Callithrix, γ1 is down-regulated fetally, whereas γ2 is up-regulated. Of eight mammalian taxa now represented per taxon by embryonic, fetal, and postnatal β-type globin gene expression data, four taxa are primates, and data for three of these primates are from this laboratory. Our results support a model in which a short distance (<10 kb) between ε and the adjacent γ is a plesiomorphic character that allows the LCR to drive embryonic expression of both genes, whereas a longer distance (>10 kb) impedes embryonic activation of the downstream gene. PMID:16488971

  9. Correction of murine sickle cell disease using gamma-globin lentiviral vectors to mediate high-level expression of fetal hemoglobin.

    PubMed

    Pestina, Tamara I; Hargrove, Phillip W; Jay, Dennis; Gray, John T; Boyd, Kelli M; Persons, Derek A

    2009-02-01

    Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the gamma-globin gene driven by 3.1 kb of beta-globin regulatory sequences and a 130-bp beta-globin promoter. The second vector, V5m3, was identical except that the gamma-globin 3'-untranslated region (3'-UTR) was replaced with the beta-globin 3'-UTR. Adult erythroid cells have beta-globin mRNA 3'-UTR-binding proteins that enhance beta-globin mRNA stability and we postulated this design might enhance gamma-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human gamma-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of gamma-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a gamma-globin lentiviral vector.

  10. Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-level Expression of Fetal Hemoglobin

    PubMed Central

    Pestina, Tamara I; Hargrove, Phillip W; Jay, Dennis; Gray, John T; Boyd, Kelli M; Persons, Derek A

    2008-01-01

    Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, γ-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different γ-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the γ-globin gene driven by 3.1 kb of β-globin regulatory sequences and a 130-bp β-globin promoter. The second vector, V5m3, was identical except that the γ-globin 3′-untranslated region (3′-UTR) was replaced with the β-globin 3′-UTR. Adult erythroid cells have β-globin mRNA 3′-UTR-binding proteins that enhance β-globin mRNA stability and we postulated this design might enhance γ-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human γ-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of γ-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a γ-globin lentiviral vector. PMID:19050697

  11. Genotype-phenotype correlation and report of novel mutations in β-globin gene in thalassemia patients.

    PubMed

    Nagar, Rachana; Sinha, Sujata; Raman, Rajiva

    2015-06-01

    Heterogeneity in thalassemia is due to various modifying factors viz. coinheritance of α-gene defects, abnormal hemoglobin, XmnI polymorphism, variation in repeat sequences present in LCR, and silencer region of the gene. The present work on populations from eastern regions of India was undertaken to study the genetic profile of heterogeneity in thalassemia patients. Mutation analysis in 126 index families revealed the presence of 3 novel mutations: CD2 (-A) in the 1st exon, -42 (C-G), and -223 (T-C) in the promoter region of β-globin gene. The modifying effect of coexisting α-gene defects, and abnormal Hb (HbS) was clearly observed in our study, however ameliorating effect of T allele of XmnI polymorphism was not found. Analysis of the regulatory regions (LCR) exhibited new combinations (CA(15)TA(5) and CA(13)TA(8)) in HS1 region and one (AT)(10)T(3) in (AT)(x)T(y )silencer region. Thus disparate factors, when considered together, were able to explain several of the thalassemic phenotypes, otherwise not explained by the β globin mutations. However, there were still some cases in this group whose molecular origin could not be ascertained. Our findings confirm not only the extensive genotypic and clinical heterogeneity in β thalassemia but also the need to look for more modulators and modifiers to better understand the genotype-phenotype correlation in thalassemia. Copyright © 2015. Published by Elsevier Inc.

  12. A majority of mice show long-term expression of a human. beta. -globin gene after retrovirus transfer into hematopoietic stem cells

    SciTech Connect

    Bender, M.A. . Dept. of Pathology); Gelinas, R.E.; Miller )

    1989-04-01

    Murine bone marrow was infected with a high-titer retrovirus vector containing the human {beta}-globin and neomycin phosphotransferase genes. Anemic W/W/sup v/ mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human {beta}-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human {beta}-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human {beta}-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active {beta}-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.

  13. Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

    PubMed Central

    Collis, P; Antoniou, M; Grosveld, F

    1990-01-01

    The human beta-globin dominant control region (DCR) was previously identified as a region from the 5' end of the human beta-globin locus which directs high level, site of integration-independent, copy number-dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta-globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta-globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3' enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2295312

  14. The effects of old and recent migration waves in the distribution of HBB*S globin gene haplotypes

    PubMed Central

    Lindenau, Juliana D.; Wagner, Sandrine C.; de Castro, Simone M.; Hutz, Mara H.

    2016-01-01

    Abstract Sickle cell hemoglobin is the result of a mutation at the sixth amino acid position of the beta (β) globin chain. The HBB*S gene is in linkage disequilibrium with five main haplotypes in the β-globin-like gene cluster named according to their ethnic and geographic origins: Bantu (CAR), Benin (BEN), Senegal (SEN), Cameroon (CAM) and Arabian-Indian (ARAB). These haplotypes demonstrated that the sickle cell mutation arose independently at least five times in human history. The distribution of βS haplotypes among Brazilian populations showed a predominance of the CAR haplotype. American populations were clustered in two groups defined by CAR or BEN haplotype frequencies. This scenario is compatible with historical records about the slave trade in the Americas. When all world populations where the sickle cell gene occurs were analyzed, three clusters were disclosed based on CAR, BEN or ARAB haplotype predominance. These patterns may change in the next decades due to recent migrations waves. Since these haplotypes show different clinical characteristics, these recent migrations events raise the necessity to develop optimized public health programs for sickle cell disease screening and management. PMID:27706371

  15. The effects of old and recent migration waves in the distribution of HBB*S globin gene haplotypes.

    PubMed

    Lindenau, Juliana D; Wagner, Sandrine C; Castro, Simone M de; Hutz, Mara H

    2016-01-01

    Sickle cell hemoglobin is the result of a mutation at the sixth amino acid position of the beta (β) globin chain. The HBB*S gene is in linkage disequilibrium with five main haplotypes in the β-globin-like gene cluster named according to their ethnic and geographic origins: Bantu (CAR), Benin (BEN), Senegal (SEN), Cameroon (CAM) and Arabian-Indian (ARAB). These haplotypes demonstrated that the sickle cell mutation arose independently at least five times in human history. The distribution of βS haplotypes among Brazilian populations showed a predominance of the CAR haplotype. American populations were clustered in two groups defined by CAR or BEN haplotype frequencies. This scenario is compatible with historical records about the slave trade in the Americas. When all world populations where the sickle cell gene occurs were analyzed, three clusters were disclosed based on CAR, BEN or ARAB haplotype predominance. These patterns may change in the next decades due to recent migrations waves. Since these haplotypes show different clinical characteristics, these recent migrations events raise the necessity to develop optimized public health programs for sickle cell disease screening and management.

  16. A randomized phase I/II trial of HQK-1001, an oral fetal globin gene inducer, in β-thalassaemia intermedia and HbE/β-thalassaemia.

    PubMed

    Fucharoen, Suthat; Inati, Adlette; Siritanaratku, Noppadol; Thein, Swee L; Wargin, William C; Koussa, Suzanne; Taher, Ali; Chaneim, Nattawara; Boosalis, Michael; Berenson, Ronald; Perrine, Susan P

    2013-05-01

    β-thalassaemia intermedia (BTI) syndromes cause haemolytic anaemia, ineffective erythropoiesis, and widespread complications. Higher fetal globin expression within genotypes reduces globin imbalance and ameliorates anaemia. Sodium 2,2 dimethylbutyrate (HQK-1001), an orally bioavailable short-chain fatty acid derivative, induces γ-globin expression experimentally and is well-tolerated in normal subjects. Accordingly, a randomized, blinded, placebo-controlled, Phase I/II trial was performed in 21 adult BTI patients (14 with HbE/β(0) thalassaemia and seven with β(+)/β(0) thalassaemia intermedia, to determine effective doses for fetal globin induction, safety, and tolerability. HQK-1001 or placebo were administered once daily for 8 weeks at four dose levels (10, 20, 30, or 40 mg/kg per day), and subjects were monitored for laboratory and clinical events. Pharmacokinetic profiles demonstrated a t(1/2) of 10-12 h. Adverse events with HQK-1001 treatment were not significantly different from placebo treatment. The 20 mg/kg treatment doses increased median HbF above baseline levels by 6·6% and 4·4 g/l (P < 0·01) in 8/9 subjects; total haemoglobin (Hb) increased by a mean of 11 g/l in 4/9 subjects. These findings identified a safe oral therapeutic which induces fetal globin in BTI. Further investigation of HQK-1001 with longer dosing to definitively evaluate its haematological potential appears warranted. © 2013 John Wiley & Sons Ltd.

  17. Beta-globin gene cluster haplotypes and alpha-thalassemia in sickle cell disease patients from Trinidad.

    PubMed

    Jones-Lecointe, Altheia; Smith, Erskine; Romana, Marc; Gilbert, Marie-Georges; Charles, Waveney P; Saint-Martin, Christian; Kéclard, Lisiane

    2008-01-01

    In this study, we have determined the frequency of beta(S) haplotypes in 163 sickle cell disease patients from Trinidad. The alpha(3.7) globin gene deletion status was also studied with an observed gene frequency of 0.17. Among the 283 beta(S) chromosomes analyzed, the Benin haplotype was the most prevalent (61.8%) followed by Bantu (17.3%), Senegal (8.5%), Cameroon (3.5%), and Arab-Indian (3.2%), while 5.7% of them were atypical. This beta(S) haplotypes distribution differed from those previously described in other Caribbean islands (Jamaica, Guadeloupe, and Cuba), in agreement with the known involvement of the major colonial powers (Spain, France, and Great Britain) in the slave trade in Trinidad and documented an Indian origin of the beta(S) gene.

  18. Dynamics of alpha-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34(+) cells in culture.

    PubMed

    Mahajan, Milind C; Karmakar, Subhradip; Newburger, Peter E; Krause, Diane S; Weissman, Sherman M

    2009-10-01

    The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron. A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis. Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway. Our results

  19. Dynamics of α-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34+ cells in culture

    PubMed Central

    Mahajan, Milind C; Karmakar, Subhradip; Krause, Diane; Weissman, Sherman M

    2009-01-01

    Objective The aim of the present study has been to establish serum free culture conditions for the ex vivo expansion and differentiation of human CD34+ cells into erythroid lineage and to study the chromatin structure, gene expression and transcription factor recruitment at the α–globin locus in the developing erythron. Methods A basal IMDM cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for the expansion and differentiation of the CD34+ cells. Expression patterns of the alpha and beta like genes at various stages of erythropoiesis was studied by Reverse transcriptase (RT)-qPCR analysis, profile of key erythroid transcription factors was investigated by western blotting, and the chromatin structure and transcription factor recruitment at the alpha globin locus was investigated by ChIP-qPCR analysis. Results Human CD34+ cells in the serum free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the α-globin locus during erythroid differentiation of CD34+ cells. NF-E2 was present at upstream activator sites even before addition of erythropoietin (Epo), while bound GATA-1 was only detectable after Epo treatment. After seven days of erythropoietin treatment, H3K4Me2 modification uniformly increases throughout the α–globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2 and GATA-1 were restricted to certain HS sites of the enhancer and theta gene, and were conspicuously low at the α-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the α-globin locus as CD34+ cells differentiated into erythroid pathway. Conclusion Our results indicate that remodeling of the upstream elements may be the primary event in activation of α–globin gene expression. Activation of

  20. Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients.

    PubMed

    Barbosa, C G; Goncalves-Santos, N J; Souza-Ribeiro, S B; Moura-Neto, J P; Takahashi, D; Silva, D O; Hurtado-Guerrero, A F; Reis, M G; Goncalves, M S

    2010-08-01

    Fetal hemoglobin (HbF), encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their betaS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104) and an Afro-descendant one (N = 98), to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP) located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C-->T in the HBG1 promoter region were associated with the Central African Republic betaS-globin haplotype. In contrast, the -369 C-->G and 309 A-->G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

  1. The beta-globin gene cluster haplotypes in sickle cell anemia patients from Northeast Brazil: a clinical and molecular view.

    PubMed

    Adorno, Elisângela Vitória; Zanette, Angela; Lyra, Isa; Souza, Cyntia Cajado; Santos, Leandro Ferraz; Menezes, Joelma Figueiredo; Dupuit, Marie France; Almeida, Mari Ney Tavares; Reis, Mitermayer Galvão; Gonçalves, Marilda Souza

    2004-08-01

    The beta(S)-globin haplotypes were studied in 78 sickle cell Brazilian patients from Bahia, Northeast Brazil, that has a large population of African origin. Hemoglobin (Hb) profiles were developed by high-performance liquid chromatography (HPLC), and beta(S)-globin gene haplotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. We identified 44 (55.0%) patients with the CAR/Ben (Central African Republic/Benin) genotype, 16 (20.0%) Ben/Ben, 13 (16.2%) CAR/CAR and seven (8.8%) with other genotypes. Analyses of the phenotypes showed clinical differences related only to Hb F levels and blood transfusion therapy; the presence of -alpha(-3.7)-thalassemia (thal) demonstrated statistical significance when associated with hematocrit (p=0.044), MCV (p=0.0007), MCH (p=0.012) and spleen sequestration events. The haplotype diversity found in the present study can be justified by information about the origin of the slave traffic period in Bahia during the 19th century. The specific characteristics described among the Bahian sickle cell patients could be confirmed by increasing the number of patients with specific genotypes and further studies of genetic markers.

  2. A novel deletion/insertion caused by a replication error in the β-globin gene locus control region.

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Meley, Roland; Pondarré, Corinne; Francina, Alain

    2011-01-01

    Deletions in the β-globin locus control region (β-LCR) lead to (εγδβ)(0)-thalassemia [(εγδβ)(0)-thal]. In patients suffering from these rare deletions, a normal hemoglobin (Hb), phenotype is found, contrasting with a hematological thalassemic phenotype. Multiplex-ligation probe amplification (MLPA) is an efficient tool to detect β-LCR deletions combined with long-range polymerase chain reaction (PCR) and DNA sequencing to pinpoint deletion breakpoints. We present here a novel 11,155 bp β-LCR deletion found in a French Caucasian patient which removes DNase I hypersensitive site 2 (HS2) to HS4 of the β-LCR. Interestingly, a 197 bp insertion of two inverted sequences issued from the HS2-HS3 inter-region is present and suggests a complex rearrangement during replication. Carriers of this type of thalassemia can be misdiagnosed as an α-thal trait. Consequently, a complete α- and β-globin gene cluster analysis is required to prevent a potentially damaging misdiagnosis in genetic counselling.

  3. Gene localization by chromosome fractionation: globin genes are on at least two chromosomes and three estrogen-inducible genes are on three chromosomes.

    PubMed

    Hughes, S H; Stubblefield, E; Payvar, F; Engel, J D; Dodgson, J B; Spector, D; Cordell, B; Schimke, R T; Varmus, H E

    1979-03-01

    Chicken metaphase chromosomes were partially purified by rate zonal centrifugation, and DNA was prepared from each of the fractions of the sucrose gradient. The DNA was digested with various restriction enzymes and subjected to electrophoresis in agarose gels. The DNA was transferred to nitrocellulose filters (as described by Southern), and the filters were hybridized with cDNA probes. Four globin genes alpha A, alpha D, beta, and rho or epsilon are located on at least two chromosomes, and three of the estrogen-inducible genes of the hen oviduct--ovalbumin, ovomucoid, and transferrin--are on three different chromosomes. These experiments also confirm our earlier assignment of the endogenous viral sequence related to Rous-associated virus-0 to a separate (and larger) chromosome than the cellular sequence related to the transforming gene of avian sarcoma virus (cellular sarc), although it now appears that cellular sarc is on a small macrochromosome, rather than on a microchromosome.

  4. A new Frameshift mutation on the α2-globin gene causing α⁺-thalassemia: codon 43 (TTC>-TC or TTC>T-C).

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Barro, Claire; Francina, Alain

    2012-01-01

    We report a new mutation on the α2-globin gene causing α(+)-thalassemia (α(+)-thal) with a deletion of a single nucleotide (T) at amino acid residue 43 [HBA2:c.130delT or HBA2:c.131delT]. This frameshift deletion gives rise to a premature termination codon at codon 47.

  5. A novel C-->A transversion within the distal CCAAT motif of the Ggamma-globin gene in the Algerian Ggammabeta+-hereditary persistence of fetal hemoglobin.

    PubMed

    Zertal-Zidani, S; Merghoub, T; Ducrocq, R; Gerard, N; Satta, D; Krishnamoorthy, R

    1999-05-01

    Hereditary persistence of fetal hemoglobin (HPFH) is a group of genetically heterogeneous conditions characterized by the continued expression of fetal hemoglobin in adulthood. These constitute natural models for understanding the mechanism(s) of the hemoglobin switch. Many large deletions in the beta-globin gene cluster and point mutations in one of the fetal globin gene promoters have been described before. In this study we describe a novel C-->A transversion (-114) in the distal CCAAT box of the Ggamma-globin gene promoter associated with the Ggammabeta+-HPFH phenotype in an Algerian family. Individuals heterozygous for this mutation exhibit moderate raise in Hb F levels (0.6-3.5%). Much higher Hb F levels (3.8-11.2%) are observed when a beta(o)-thalassemia allele is present in trans to the hereditary persistence of fetal hemoglobin allele. This novel Algerian HPFH mutation further stresses the importance of the distal CCAAT box in the postnatal regulation of gamma-globin gene expression.

  6. Changes in expression of murine alpha- and beta-globin genes during development

    SciTech Connect

    Popp, R.A.; D'Surney, S.J.; Wawrzyniak, C.J.

    1986-09-30

    Polyacrylamide gel isoelectric focusing was used to separate the multiple embryonic and adult hemoglobins in order to determine the relative amounts of the a1 and a/sup 2/ gene products in fetal mice of the Hba/sup c/ and Hba/sup g2/ haplotypes. In addition, centrifugal elutriation was used to separte the nucleated and non-nucleated red cells in order to determine the relative amounts of the b1/sup s2/ and b2/sup s/ gene products in each subpopulation of erythryocytes in fetal mice of the Hbb/sup s2/ haplotype. 22 refs., 2 figs.

  7. A rapid single-tube multiplex polymerase chain reaction assay for the seven most prevalent alpha-thalassemia deletions and alphaalphaalpha(anti 3.7) alpha-globin gene triplication.

    PubMed

    de Mare, Arjan; Groeneger, Antoinette Heijs-Oude; Schuurman, Sander; van den Bergh, Frank A T J M; Slomp, Jennichjen

    2010-01-01

    alpha-Globin gene triplications may exacerbate the alpha chain and beta chain imbalance in beta-thalassemia (beta-thal) and may compensate for the effect of alpha-globin gene deletion in alpha-thal. Identification of an alpha-globin gene triplication is, therefore, valuable in predicting the clinical phenotype of the thalassemias. To be able to detect alpha-globin gene triplications, we have modified an existing multiplex polymerase chain reaction (PCR) assay for the seven most prevalent alpha-globin gene deletions by incorporating two triplication-specific primers and concurrently substituting one of the original primers by a newly designed primer. This modified multiplex PCR assay was evaluated by performing the assay on archival DNA samples and on peripheral blood samples from 163 suspected thalassemia cases. It was found to function properly. Our assay thereby represents the first multiplex PCR assay that can detect both the seven most prevalent alpha-globin gene deletions and the alphaalphaalpha(anti 3.7) alpha-globin gene triplication in a single-tube reaction.

  8. Detection of beta-globin gene mutations among Kelantan Malay thalassaemia patients by polymerase chain reaction restriction fragment length polymorphism.

    PubMed

    Rozitah, R; Nizam, M Z; Nur Shafawati, A R; Nor Atifah, M A; Dewi, M; Kannan, T P; Ariffin, N; Norsarwany, M; Setianingsih, I; Harahap, A; Zilfalil, B A

    2008-12-01

    Beta-thalassaemia major is an autosomal recessive disorder that results in severe microcytic, hypochromic, haemolytic anaemia among affected patients. Beta-thalassaemia has emerged as one of the most common public health problems in Malaysia, particularly among Malaysian Chinese and Malays. This study aimed to observe the spectrum of mutations found in Kelantan Malay beta-thalassaemia major patients who attended the Paediatrics Daycare Unit, Hospital Universiti Sains Malaysia, Kelantan, Malaysia, the data of which was being used in establishing the prenatal diagnosis in this Human Genome Centre. This was a cross-sectional study conducted with 35 Kelantan Malay beta-thalassaemia major patients. DNA was extracted from the blood collected from the patients and subjected to polymerase chain reaction (PCR) amplification. Six restriction enzymes were used to digest the PCR products for the detection of mutations. Five out of the six beta-globin gene defects were detected, namely, IVS-1 nt5 (G>C), IVS-1 nt1 (G>T), codon 26 (G>A), codon 41-42 (4 bp del) and codon 19 (A>G). The mutation which was not observed in this study was in codon 15 (G>A). The two most common mutations observed were codon 26 (G>A) and IVS-1 nt5 (G>C), which was detected in 26 and 17 patients, respectively. Two patients did not show any of the six mutations. Our results added to the existing data on the common beta-globin gene defects in Kelantan Malay beta-thalassaemia patients.

  9. Domains of α- and β-globin genes in the context of the structural-functional organization of the eukaryotic genome.

    PubMed

    Razin, S V; Ulianov, S V; Ioudinkova, E S; Gushchanskaya, E S; Gavrilov, A A; Iarovaia, O V

    2012-12-01

    The eukaryotic cell genome has a multilevel regulatory system of gene expression that includes stages of preliminary activation of genes or of extended genomic regions (switching them to potentially active states) and stages of final activation of promoters and maintaining their active status in cells of a certain lineage. Current views on the regulatory systems of transcription in eukaryotes have been formed based on results of systematic studies on a limited number of model systems, in particular, on the α- and β-globin gene domains of vertebrates. Unexpectedly, these genomic domains harboring genes responsible for the synthesis of different subunits of the same protein were found to have a fundamentally different organization inside chromatin. In this review, we analyze specific features of the organization of the α- and β-globin gene domains in vertebrates, as well as principles of activities of the regulatory systems in these domains. In the final part of the review, we attempt to answer the question how the evolution of α- and β-globin genes has led to segregation of these genes into two distinct types of chromatin domains situated on different chromosomes.

  10. Comparative analysis of the locus control region of the rabbit beta-like gene cluster: HS3 increases transient expression of an embryonic epsilon-globin gene.

    PubMed Central

    Hardison, R; Xu, J; Jackson, J; Mansberger, J; Selifonova, O; Grotch, B; Biesecker, J; Petrykowska, H; Miller, W

    1993-01-01

    The rabbit homolog to the locus control region (LCR) of the human beta-like globin gene cluster was isolated, and long segments containing the DNase I hypersensitive sites (HS) were sequenced. The order and spacing of HS4, HS3, HS2 and HS1 are conserved between rabbit and human. Alignment of these sequences with their homologs from human, goat, and mouse shows that very long segments of DNA match between species, for over a thousand base pairs on either side of the previously identified functional cores, indicating that some important functions are found outside the cores. The activity of rabbit HS2 and HS3 was tested by attaching each to a novel reporter gene constructed by inserting the luciferase coding region into the rabbit epsilon-globin gene. In contrast to previous reports showing no effect of human or mouse HS3 on transient expression, both the rabbit HS2 and HS3 DNA fragments separately increased transient expression from the epsilon-luciferase hybrid gene and expression from stably integrated constructs in K562 erythroleukemia cells. PMID:8464710

  11. Two novel copy number variations involving the α-globin gene cluster on chromosome 16 cause thalassemia in two Chinese families.

    PubMed

    Hu, Lingling; Shang, Xuan; Yi, Sheng; Cai, Ren; Li, Zhetao; Liu, Cuixian; Liang, Yidan; Cai, Decheng; Zhang, Feng; Xu, Xiangmin

    2016-06-01

    Copy number variations (CNVs) can cause many genetic disorders and the structure analysis of unknown CNVs is important for clinical diagnosis. The human α-globin gene cluster lies close to the telomere of the short arm on chromosome 16. Copy number variations of this region produce excessive or insufficient α-globin chains which imbalances the β-globin chains, resulting in thalassemia. However, these CNVs usually cannot be precisely defined by traditional methods. Here, we designed a technique strategy and applied it to identify two CNVs involving the α-globin gene cluster causing thalassemia in two Chinese families. A novel 282 kb duplication (αααα(282)) was identified in family A and a novel 235 kb deletion (--(235)) in family B. Proband A is a coinheritance of β(CD41-42) and αααα(282) and showed severe β-thalassemia intermedia phenotype. Proband B is a compound heterozygote of --(235)/α(CS)α genotype and was diagnosed with hemoglobin H disease. The clinical phenotypic features of the CNVs carriers were described, together with a complete picture of molecular structure of these rearrangements. Two CNVs are novel rearrangements in α-globin clusters and the αααα(282) is the first to identify the exact insert position of a duplication region from the telomere on chromosome 16. In a conclusion, successful identification and characterization of these two novel CNVs not only demonstrates the precision and effectiveness of our strategy in analyzing the structure of unknown CNVs, but also extended the spectrum of thalassemia and provide new examples for studying genomic recombination.

  12. Globin gene server: a prototype E-mail database server featuring extensive multiple alignments and data compilation for electronic genetic analysis.

    PubMed

    Hardison, R; Chao, K M; Schwartz, S; Stojanovic, N; Ganetsky, M; Miller, W

    1994-05-15

    The sequence of virtually the entire cluster of beta-like globin genes has been determined from several mammals, and many regulatory regions have been analyzed by mutagenesis, functional assays, and nuclear protein binding studies. This very large amount of sequence and functional data needs to be compiled in a readily accessible and usable manner to optimize data analysis, hypothesis testing, and model building. We report a Globin Gene Server that will provide this service in a constantly updated manner when fully implemented. The Server has two principal functions. The first (currently available) provides an annotated multiple alignment of the DNA sequences throughout the gene cluster from representatives of all species analyzed. The second compiles data on functional and protein binding assays throughout the gene cluster. A prototype of this compilation using the aligned 5' flanking region of beta-globin genes from five species shows examples of (1) well-conserved regions that have demonstrated functions, including cases in which the functional data are in apparent conflict, (2) proposed functional regions that are not well conserved, and (3) conserved regions with no currently assigned function. Such an electronic genetic analysis leads to many readily testable hypotheses that were not immediately apparent without the multiple alignment and compilation. The Server is accessible via E-mail on computer networks, and printed results can be obtained by request to the authors. This prototype will be a helpful guide for developing similar tools for many genomic loci.

  13. Possible interaction between B1 retrotransposon-containing sequences and β(major) globin gene transcriptional activation during MEL cell erythroid differentiation.

    PubMed

    Vizirianakis, Ioannis S; Tezias, Sotirios S; Amanatiadou, Elsa P; Tsiftsoglou, Asterios S

    2012-01-01

    Repetitive sequences consist of >50% of mammalian genomic DNAs and among these SINEs (short interspersed nuclear elements), e.g. B1 elements, account for 8% of the mouse genome. In an effort to delineate the molecular mechanism(s) involved in the blockade of the in vitro differentiation program of MEL (murine erythroleukaemia) cells by treatment with methylation inhibitors, we detected a DNA region of 559 bp in chromosome 7 located downstream of the 3'-end of the β(major) globin gene (designated B1-559) with unique characteristics. We have fully characterized this B1-559 region that includes a B1 element, several repeats of ATG initiation codons and consensus DNA-binding sites for erythroid-specific transcription factors NF-E2 (nuclear factor-erythroid-derived 2), GATA-1 and EKLF (erythroid Krüppel-like factor). Fragments derived from B1-559 incubated with nuclear extracts form protein complexes in both undifferentiated and differentiated MEL cells. Transient reporter-gene experiments in MEL and human erythroleukaemia K-562 cells with recombinant constructs containing B1-559 fragments linked to HS-2 (hypersensitive site-2) sequences of human β-globin gene LCR (locus control region) indicated potential cooperation upon erythropoiesis and globin gene expression. The possible interaction between the B1-559 region and β(major) globin gene transcriptional activation upon execution of erythroid MEL cell differentiation programme is discussed. © The Author(s) Journal compilation © 2012 Portland Press Limited

  14. Reduction of two functional gamma-globin genes to one: an evolutionary trend in New World monkeys (infraorder Platyrrhini).

    PubMed Central

    Chiu, C H; Schneider, H; Schneider, M P; Sampaio, I; Meireles, C; Slightom, J L; Gumucio, D L; Goodman, M

    1996-01-01

    Nucleotide sequences were determined for the gamma1- and gamma2-globin loci from representatives of the seven anciently separated clades in the three extant platyrrhine families (Atelidae, Pitheciidae, and Cebidae). These sequences revealed an evolutionary trend in New World monkeys either to inactivate the gamma1 gene or to fuse it with the gamma2 gene, i.e. to have only one functional fetally expressed gamma gene. This trend is clearly evident in six of the seven clades: (i) it occurred in atelids by deletion of most of the gamma1 gene in the basal ancestor of this clade; (ii-iv) in pitheciid titi, saki, and cebid capuchin monkeys by potentially debilitating nucleotide substitutions in the proximal CCAAT box of the gamma1 promoters and (v and vi) in cebid owl and squirrel monkeys by crossovers that fused 5' sequence from gamma1 with 3' sequence from gamma2. In the five clades with gamma1 and gamma2 loci separated by intergenic sequences (the fifth clade being the cebid marmosets), the gamma2 genes retained an unaltered proximal CCAAT motif and their gamma2 promoters accumulated fewer nucleotide substitutions than did the gamma1 promoters. Thus, phylogenetic considerations indicate that the stem platyrrhines, ancestral to all New World monkeys, had gamma2 as the primary fetally expressed gamma gene. A further inference is that when the earlier stem anthropoid gamma gene duplicated, gamma2 (at its greater downstream distance from epsilon) could evade embryonic activation by the locus control region but could be fetally activated once released by regulatory mutations from fetal repressors. PMID:8692846

  15. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations

    PubMed Central

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease’s high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics’ assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions’ setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  16. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

    PubMed

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions' setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning.

  17. Analysis of a mouse. cap alpha. -globin gene mutation induced by ethylnitrosourea

    SciTech Connect

    Popp, R.A.; Bailiff, E.G.; Skow, L.C.; Johnson, F.M.; Lewis, S.E.

    1983-09-01

    A DBA/2 mouse treated with ethylnitrosourea sired an offspring whose hemoglobin showed an extra band following starch gel electrophoresis. The variant hemoglobin migrated to a more cathodal posititon in starch gel. Isoelectric focusing indicated that chain 5 of the mutant hemoglobin migrated to a more cathodal position than the normal chain 5 from DBA/2 mice and that the other ..cap alpha..-globin, chain 1, was not affected. On focusing gels the phenotype of the mutant allele, Hba/sup y9/, was expressed without dominance to normal chain 5, and Hba/sup y9/ / Hba/sup y9/ homozygotes were fully viable in the laboratory. The molecular basis for the germinal mutation was investigated by analyzing the amino acid sequence of chain 5/sup y9/, the mutant form of ..cap alpha..-chain 5. A single amino acid substitution (His ..-->.. Leu) at position 89 was found in chain 5/sup y9/. The authors propose that ethylnitrosourea induced an A ..-->.. T transversion in the histidine codon at position 89 (CAC ..-->.. CTC). This mutation has apparently not been observed previously in humans, mice or other mammals, and its novel occurrence may be indicative of other unusual mutational events that do not ordinarily occur in the absence of specific mutagen exposure.

  18. Evaluation of Signaling Pathways Involved in γ-Globin Gene Induction Using Fetal Hemoglobin Inducer Drugs.

    PubMed

    Rahim, Fakher; Allahmoradi, Hossein; Salari, Fatemeh; Shahjahani, Mohammad; Fard, Ali Dehghani; Hosseini, Seyed Ahmad; Mousakhani, Hadi

    2013-01-01

    Potent induction of fetal hemoglobin (HbF) production results in alleviating the complications of β-thalassemia and sickle cell disease (SCD). HbF inducer agents can trigger several molecular signaling pathways critical for erythropoiesis. Janus kinase/Signal transducer and activator of transcription (JAK/STAT), mitogen activated protein kinas (MAPK) and Phosphoinositide 3-kinase (PI3K) are considered as main signaling pathways, which may play a significant role in HbF induction. All these signaling pathways are triggered by erythropoietin (EPO) as the main growth factor inducing erythroid differentiation, when it binds to its cell surface receptor, erythropoietin receptor (EPO-R) HbF inducer agents have been shown to upregulate HbF production level by triggering certain signaling pathways. As a result, understanding the pivotal signaling pathways influencing HbF induction leads to effective upregulation of HbF. In this mini review article, we try to consider the correlation between HbF inducer agents and their molecular mechanisms of γ-globin upregulation. Several studies suggest that activating P38 MAPK, RAS and STAT5 signaling pathways result in efficient HbF induction. Nevertheless, the role of other erythroid signaling pathways in HbF induction seems to be indispensible and should be emphasized.

  19. Variation in the level of fetal hemoglobin in (delta beta) (0)-thalassemia heterozygotes with different numbers of alpha-globin genes.

    PubMed

    Oner, C; Gurgey, A; Altay, C; Kutlar, F; Huisman, T H

    1990-07-01

    The Sicilian type of (delta beta) (0)-thalassemia characterized by a approximately 13 kb deletion, was present in a Turkish boy who is a homozygote and in his heterozygous parents who are first cousins. The father with approximately 21% Hb F had five alpha-globin genes (alpha alpha/alpha alpha alpha) and the mother with approximately 10% Hb F had an alpha-thal-2 heterozygosity (alpha alpha/-alpha). The difference in Hb F level is explained by a decreased formation of alpha 2 gamma 2 tetramers in the mother with an alpha-chain deficiency while the extra alpha-globin gene in the father will promote Hb F production.

  20. A Novel Mutation of the α2-Globin Gene Causing α(+)-Thalassemia: Hb Nanning (HBA2: c.369_370delinsGA).

    PubMed

    Chen, Biyan; Lin, Li; Yi, Shang; Chen, Qiuli; Wei, Hongwei; Li, Guojian; Zheng, Chenguang; He, Sheng; Qiu, XiaoXia

    2017-01-01

    We report a novel mutation on the α2-globin gene, Hb Nanning (HBA2:c.369_370delinsGA) detected in a Chinese family. This mutation gives rise to a previously undescribed hemoglobin (Hb) variant that was undetectable by various separation techniques. Both carriers of the mutation have mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) values that are below normal, as would be predicted for an α(+)-thalassemia (α(+)-thal) patient.

  1. Enhancement of β-Globin Gene Expression in Thalassemic IVS2-654 Induced Pluripotent Stem Cell-Derived Erythroid Cells by Modified U7 snRNA.

    PubMed

    Phanthong, Phetcharat; Borwornpinyo, Suparerk; Kitiyanant, Narisorn; Jearawiriyapaisarn, Natee; Nuntakarn, Lalana; Saetan, Jirawat; Nualkaew, Tiwaporn; Sa-Ngiamsuntorn, Khanit; Anurathapan, Usanarat; Dinnyes, Andras; Kitiyanant, Yindee; Hongeng, Suradej

    2017-04-01

    The therapeutic use of patient-specific induced pluripotent stem cells (iPSCs) is emerging as a potential treatment of β-thalassemia. Ideally, patient-specific iPSCs would be genetically corrected by various approaches to treat β-thalassemia including lentiviral gene transfer, lentivirus-delivered shRNA, and gene editing. These corrected iPSCs would be subsequently differentiated into hematopoietic stem cells and transplanted back into the same patient. In this article, we present a proof of principle study for disease modeling and screening using iPSCs to test the potential use of the modified U7 small nuclear (sn) RNA to correct a splice defect in IVS2-654 β-thalassemia. In this case, the aberration results from a mutation in the human β-globin intron 2 causing an aberrant splicing of β-globin pre-mRNA and preventing synthesis of functional β-globin protein. The iPSCs (derived from mesenchymal stromal cells from a patient with IVS2-654 β-thalassemia/hemoglobin (Hb) E) were transduced with a lentivirus carrying a modified U7 snRNA targeting an IVS2-654 β-globin pre-mRNA in order to restore the correct splicing. Erythroblasts differentiated from the transduced iPSCs expressed high level of correctly spliced β-globin mRNA suggesting that the modified U7 snRNA was expressed and mediated splicing correction of IVS2-654 β-globin pre-mRNA in these cells. Moreover, a less active apoptosis cascade process was observed in the corrected cells at transcription level. This study demonstrated the potential use of a genetically modified U7 snRNA with patient-specific iPSCs for the partial restoration of the aberrant splicing process of β-thalassemia. Stem Cells Translational Medicine 2017;6:1059-1069. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  2. Beta-globin gene cluster haplotypes and HbF levels are not the only modulators of sickle cell disease in Lebanon.

    PubMed

    Inati, A; Taher, A; Bou Alawi, W; Koussa, S; Kaspar, H; Shbaklo, H; Zalloua, P A

    2003-02-01

    Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the beta-globin chain. Despite the fact that all subjects with SCD have the same single base pair mutation, the severity of the clinical and hematological manifestations is extremely variable. This study examined for the first time in Lebanon the correlation between the clinical manifestation of SCD and the beta-globin gene haplotypes. The haplotypes of 50 patients diagnosed with SCD were determined using polymerase chain reaction amplification of fragments containing nine polymorphic restriction sites around and within the epsilon-Ggamma-Agamma-psibeta-delta-beta-globin gene complex. Most reported haplotypes were found in our population with the Benin haplotype as the most prevalent one. When the patients were divided according to their HbF levels into three groups (Group A: HbF < 5%, Group B: HbF between 5 and 15%, and Group C: HbF > 15%), surprisingly, the highest levels of HbF were associated with the most severe clinical cases. Our findings suggest that fetal hemoglobin levels are important but not the only parameters that affect the severity of the disease. In addition, the high levels of HbF in patients with CAR haplotypes did not seem to ameliorate the severity of symptoms, suggesting that genetic factors other than haplotypes are the major determinants of increased HbF levels in Lebanon.

  3. Beta-globin gene cluster haplotypes in Afro-Uruguayans from two geographical regions (South and North).

    PubMed

    Da Luz, Julio; Kimura, Elza Miyuki; Costa, Fernando Ferreira; Sonati, Maria de Fatima; Sans, Mónica

    2010-01-01

    The beta-globin gene cluster haplotypes were identified in 52 and 40 chromosomes from two Afro-Uruguayan populations located in the South and North of the country, respectively. In both regions, the 5' haplotype 2 (+ - - - -), characteristic of non-African populations, was the most frequent, reflecting a strong process of admixture in Afro-Uruguayans (0.355 and 0.262, respectively). The haplotypes 3 (- - - - +) and 4 (- + - - +), characteristics of African sub-Saharan populations, present inverse frequencies in North and South: whereas in the South haplotype 3 is the second most frequent (0.232), and haplotype 4 presents a low frequency (0.019), in the North haplotype 4 is the third most frequent (0.140), and haplotype 3 only reaches an intermediate frequency (0.088). The pairwise F(ST) and the exact test of differentiation show genetic heterogeneity between both regions. Nei's genetic distance show that South and North present affinities with Bantu groups, although the North present the smallest genetic distance with the Mandenka, a Senegalese population. With respect to 3' haplotypes, haplotype I was the most frequent in both populations, followed by haplotype II, characteristic of sub-Saharan Africans. The high frequencies of haplotype III-Asian could indicate admixture with Native American populations. The differences observed between both Uruguayan regions could be explained by microevolutionary events as genetic drift, founder effects, differential admixture, and/or distinct origin of the African slaves introduced in those regions.

  4. Epigenetic interplay at the β-globin locus.

    PubMed

    Lee, Wei Shern; McColl, Bradley; Maksimovic, Jovana; Vadolas, Jim

    2017-02-01

    During development, the α- and β-globin genes exhibit a highly conserved pattern of expression, giving rise to several developmental stage-specific hemoglobin variants. Networks of regulatory proteins interact with epigenetic complexes to regulate DNA accessibility and histone modifications, thereby determining appropriate patterns of globin gene expression. In this review, we focus on recent advances in the understanding of the molecular mechanisms that underpin globin gene expression, focusing on multi-subunit regulatory complexes that bind to specific regions of DNA to orchestrate globin gene transcription throughout development.

  5. Use of multiplex PCR and CE for gene dosage quantification and its biomedical applications for SMN, PMP22, and alpha-globin genes.

    PubMed

    Hung, Chia-Cheng; Chien, Shu-Chin; Lin, Chia-Yun; Chang, Chien-Hui; Chang, Yin-Fei; Jong, Yuh-Jyh; Hsieh, Sung-Tsang; Hsieh, Wu-Shiun; Liu, Ming S; Lin, Win-Li; Lee, Chien-Nan; Su, Yi-Ning

    2007-08-01

    Many genetic diseases are caused by the presence of point mutations, small insertions, and deletions in respective genes, and the number of diseases known to be caused by deletions and duplications involving large DNA genomes is increasing. These changes lead to underexpression or overexpression of the gene, according to changes in gene dosage. The methods for the detection of point mutations, small insertions, and deletions are well established, but the detection of larger genomic deletions or duplications is more difficult. Due to the lack of efficient and technically feasible protocols for gene dosage quantification, we describe a diagnostic protocol employing a combination of available methods. The efficient and accurate gene dosage quantification platform is combined with multiplex PCR and CE, and applied to detect dosages of several genes, including SMN, PMP22, and alpha-globin genes. The reliability of this novel methodology shows that it is a relatively speedy and low-cost procedure and a significant tool for genetic diagnosis. Its sensitivity and specificity for identifying deletion and duplication genotypes approach 100%. Moreover, once we establish this powerful system, we will further apply this technique to the rapid detection of trisomy syndromes and microdeletion syndromes, including trisomy 13, Down syndrome, DiGeorge syndrome, and others.

  6. A novel chromatin insulator regulates the chicken folate receptor gene from the influence of nearby constitutive heterochromatin and the β-globin locus.

    PubMed

    González-Buendía, Edgar; Escamilla-Del-Arenal, Martín; Pérez-Molina, Rosario; Tena, Juan J; Guerrero, Georgina; Suaste-Olmos, Fernando; Ayala-Ortega, Erandi; Gómez-Skarmeta, José Luis; Recillas-Targa, Félix

    2015-08-01

    The three-dimensional architecture of genomes provides new insights about genome organization and function, but many aspects remain unsolved at the local genomic scale. Here we investigate the regulation of two erythroid-specific loci, a folate receptor gene (FOLR1) and the β-globin gene cluster, which are separated by 16kb of constitutive heterochromatin. We found that in early erythroid differentiation the FOLR1 gene presents a permissive chromatin configuration that allows its expression. Once the transition to the next differentiation state occurs, the heterochromatin spreads into the FOLR1 domain, concomitant with the dissociation of CTCF from a novel binding site, thereby resulting in irreversible silencing of the FOLR1 gene. We demonstrate that the sequences surrounding the CTCF-binding site possess classical insulator properties in vitro and in vivo. In contrast, the chicken cHS4 β-globin insulator present on the other side of the heterochromatic segment is in a constitutive open chromatin configuration, with CTCF constantly bound from the early stages of erythroid differentiation. Therefore, this study demonstrates that the 16kb of constitutive heterochromatin contributes to silencing of the FOLR1 gene during erythroid differentiation.

  7. Long-term and efficient expression of human β-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system.

    PubMed

    Dormiani, K; Mir Mohammad Sadeghi, H; Sadeghi-Aliabadi, H; Ghaedi, K; Forouzanfar, M; Baharvand, H; Nasr-Esfahani, M H

    2015-08-01

    Targeted integration of a therapeutic gene at specific loci in safe genomic regions by a non-viral vector can restore the function of the damaged gene. This approach also minimizes the potential genotoxic effects of transferred DNA. In this study, we have developed a non-viral vector that functions according to site-specific recombination (SSR). The vector contained a bacterial backbone and puromycin resistance gene (pur(r)), a β-globin expressing cassette and an attB recombination site. We used phiC31 integrase to insert a copy of the vector into specific genomic locations of a human hematopoietic cell line. Site-specific integration of the vector with one or two copies in the transcriptionally active regions of the genome was confirmed. After genomic integration, we used Cre recombinase to remove the bacterial backbone and pur(r). This removal was verified by negative selection and genomic PCR screening. Following deletion of these sequences, the stable β-chain expression was continued for several months in the absence of selective pressure. Consequently, this vector may potentially be a powerful tool for ex vivo correction of β-globinopathies such as β-thalassemia through successful genomic integration of a functional copy of the globin gene into the patient's target cells.

  8. A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction.

    PubMed

    Perumbeti, Ajay; Higashimoto, Tomoyasu; Urbinati, Fabrizia; Franco, Robert; Meiselman, Herbert J; Witte, David; Malik, Punam

    2009-08-06

    We show that lentiviral delivery of human gamma-globin gene under beta-globin regulatory control elements in hematopoietic stem cells (HSCs) results in sufficient postnatal fetal hemoglobin (HbF) expression to correct sickle cell anemia (SCA) in the Berkeley "humanized" sickle mouse. Upon de-escalating the amount of transduced HSCs in transplant recipients, using reduced-intensity conditioning and varying gene transfer efficiency and vector copy number, we assessed critical parameters needed for correction. A systematic quantification of functional and hematologic red blood cell (RBC) indices, organ pathology, and life span was used to determine the minimal amount of HbF, F cells, HbF/F-cell, and gene-modified HSCs required for correcting the sickle phenotype. We show that long-term amelioration of disease occurred (1) when HbF exceeded 10%, F cells constituted two-thirds of the circulating RBCs, and HbF/F cell was one-third of the total hemoglobin in sickle RBCs; and (2) when approximately 20% gene-modified HSCs repopulated the marrow. Moreover, we show a novel model using reduced-intensity conditioning to determine genetically corrected HSC threshold that corrects a hematopoietic disease. These studies provide a strong preclinical model for what it would take to genetically correct SCA and are a foundation for the use of this vector in a human clinical trial.

  9. Non-random association of the Rsa I polymorphic site 5' to the beta-globin gene with major sickle cell haplotypes.

    PubMed

    Sharon, B; Poncz, M; Surrey, S; Schwartz, E

    1988-01-01

    There are three main African haplotypes associated with the sickle mutation on chromosome 11. We have examined an Rsa I polymorphism 550 bp 5' to the beta-globin gene to study the degree of linkage disequilibrium between this Rsa I site and the three haplotypes. This Rsa I site is contained within the 10.3 kb or less area of randomization separating the 5'- and 3'-haplotype clusters. The beta S-containing chromosomes of the Benin and Senegal haplotypes are not cut, while those of the Central African Republic are cleaved by Rsa I at this site. Possible explanations of these findings are discussed.

  10. Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

    PubMed Central

    Bruno, J; Reich, N; Lucas, J J

    1981-01-01

    The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed. Images PMID:7346715

  11. Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya

    PubMed Central

    Bilgen, Türker; Altıok Clark, Özden; Öztürk, Zeynep; Yeşilipek, M. Akif; Keser, İbrahim

    2016-01-01

    Objective: Although the calculated carrier frequency for point mutations of the β-globin gene is around 10% for Antalya Province, nothing is known about the profile of large deletional mutations involving the β-globin gene. In this study, we aimed to screen common deletional mutations in the β-globin gene cluster in patients for whom direct DNA sequencing was not able to demonstrate the mutation(s) responsible for the disease phenotype. Materials and Methods: Thirty-one index cases selected with a series of selection events among 60 cases without detected β-globin gene mutation from 580 thalassemia-related cases tested by direct sequencing over the last 4 years in our diagnostic center were screened for the most common 8 different large deletional mutations of the β-globin gene cluster by gap-PCR. Results: We detected 1 homozygous and 9 heterozygous novel unrelated cases for the Turkish inversion/deletion (δβ)0 mutation in our series of 31 cases. Our study showed that the Turkish inversion/deletion (δβ)0 mutation per se accounts for 16.6% of the unidentified causative alleles and also accounts for 1.5% of all detected mutations over the last 4 years in our laboratory. Conclusion: Since molecular diagnosis of deletional mutations in the β-globin gene cluster warrants different approaches, it deserves special attention in order to provide prenatal diagnosis and prevention opportunities to the families involved. We conclude that the Turkish inversion/deletion (δβ)0, as the most prevalent deletional mutation detected so far, has to be routinely tested for in Antalya, and the gap-PCR approach has valuable diagnostic potential in the patients at risk. PMID:26377447

  12. Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells.

    PubMed

    Levasseur, Dana N; Ryan, Thomas M; Pawlik, Kevin M; Townes, Tim M

    2003-12-15

    Although sickle cell anemia was the first hereditary disease to be understood at the molecular level, there is still no adequate long-term treatment. Allogeneic bone marrow transplantation is the only available cure, but this procedure is limited to a minority of patients with an available, histocompatible donor. Autologous transplantation of bone marrow stem cells that are transduced with a stably expressed, antisickling globin gene would benefit a majority of patients with sickle cell disease. Therefore, the development of a gene therapy protocol that corrects the disease in an animal model and is directly translatable to human patients is critical. A method is described in which unmobilized, highly purified bone marrow stem cells are transduced with a minimum amount of self-inactivating (SIN) lentiviral vector containing a potent antisickling beta-globin gene. These cells, which were transduced in the absence of cytokine stimulation, fully reconstitute irradiated recipients and correct the hemolytic anemia and organ pathology that characterize the disease in humans. The mean increase of hemoglobin concentration was 46 g/L (4.6 g/dL) and the average lentiviral copy number was 2.2; therefore, a 21-g/L /vector copy increase (2.1-g/dL) was achieved. This transduction protocol may be directly translatable to patients with sickle cell disease who cannot tolerate current bone marrow mobilization procedures and may not safely be exposed to large viral loads.

  13. Beta-globin gene cluster haplotypes in sickle cell patients from southwest Iran.

    PubMed

    Rahimi, Z; Karimi, M; Haghshenass, M; Merat, A

    2003-11-01

    Sickle cell anemia in Iran is accompanied by a high level of HbF and mild clinical presentation. Here we report haplotypes of the beta gene cluster found in 81 randomly selected sickle cell patients, including 47 sickle cell anemia (SS), 17 sickle cell trait (AS), and 17 sickle/thalassemia (S/thal) from southwest Iran. We found all five common typical haplotypes as well as five atypical haplotypes in our patients. Except for four patients with homozygous Benin haplotype, none of the other African typical haplotypes were found in a homozygous state. Arab-Indian was found to be the most prevalent haplotype in the study population. This haplotype accounted for 51.1% as the homozygous form in SS patients, where 69.1% of chromosomes in these patients had the Arab-Indian haplotype. Bantu A2 was the second most prevalent haplotype among all patients. The mean %HbF in SS patients was 27.83 and in the homozygous Arab-Indian haplotype it was still higher (30.40%), while in AS patients the %HbF was only 1.20. The high %Ggamma chain (71.81) in the Arab-Indian homozygous haplotype was concomitant with the presence of an Xmn I site in both chromosomes. The presence of the Arab-Indian haplotype as the predominant haplotype might be suggestive of a gene flow to/from Saudi Arabia or India. More haplotype investigations of a normal population can clarify the high incidence of Bantu A2 haplotype in our population.

  14. Morbidity, beta S haplotype and alpha-globin gene patterns among sickle cell anemia patients in Kuwait.

    PubMed

    Adekile, A D; Haider, M Z

    1996-01-01

    Admission records of children with sickle cell anemia (SS), in the two main teaching hospitals in Kuwait, were reviewed for a 1-year period. The haplotypes of 92 beta s chromosomes (from 39 SS, 11 AS, 2 S beta-thalassemia [S beta-thal] and 1 SD individuals) were determined using an allele-specific oligonucleotide (ASO) hybridization technique, while the alpha-globin gene status of 27 SS and 33 AS individuals, i.e. 120 chromosomes, was determined with a combination of polymerase chain reaction and AS techniques. A vasooclusive crisis was the most common (60.0%) cause of hospitalization, followed by infections (20%). Hospital admissions were most common during the hottest month of the year (July). Few complications of the disease were seen among patients on follow-up; however, splenomegaly was present in 24.0%, hepatomegaly in 15.2%, gallstones in 15.2% and aseptic necrosis of the femoral head in 6.1%. Haplotype 31 (Saudi Arabia/India) is the most frequent in this community, being present in 80.4% of the chromosomes tested; Benin haplotype 19 was found in 12.0% and Bantu haplotype 20 in 6.5%. Hb F in the haplotype 31 homozygotes and heterozygotes ranged from 11.4 to 35.1% (mean 22.5 +/- 5.2%). The frequency of alpha-thal determinants in the study was 40.0%, the commonest being the -alpha 3.7-kb deletion (27.5%), the alpha 2 polyadenylation signal (AATAAA-> AATAAG) mutation (10.2%) and the IVS-I 5' end GAGGT-GAGG->GAGG pentanucleotide (5 nt) deletion (3.3%). SS patients with coexistent alpha-thal trait did not have severe recurrent infections and none had gallstones. The high frequencies of the Saudi Arabia/India beta s haplotype and alpha-thalassemia trait contribute to the mild nature of SS disease among Kuwaiti Arabs comparable to that in eastern Saudi Arabia.

  15. A Phylogenetic Analysis of the Globins in Fungi

    PubMed Central

    Hoogewijs, David; Dewilde, Sylvia; Vierstraete, Andy; Moens, Luc; Vinogradov, Serge N.

    2012-01-01

    Background All globins belong to one of three families: the F (flavohemoglobin) and S (sensor) families that exhibit the canonical 3/3 α-helical fold, and the T (truncated 3/3 fold) globins characterized by a shortened 2/2 α-helical fold. All eukaryote 3/3 hemoglobins are related to the bacterial single domain F globins. It is known that Fungi contain flavohemoglobins and single domain S globins. Our aims are to provide a census of fungal globins and to examine their relationships to bacterial globins. Results Examination of 165 genomes revealed that globins are present in >90% of Ascomycota and ∼60% of Basidiomycota genomes. The S globins occur in Blastocladiomycota and Chytridiomycota in addition to the phyla that have FHbs. Unexpectedly, group 1 T globins were found in one Blastocladiomycota and one Chytridiomycota genome. Phylogenetic analyses were carried out on the fungal globins, alone and aligned with representative bacterial globins. The Saccharomycetes and Sordariomycetes with two FHbs form two widely divergent clusters separated by the remaining fungal sequences. One of the Saccharomycete groups represents a new subfamily of FHbs, comprising a previously unknown N-terminal and a FHb missing the C-terminal moiety of its reductase domain. The two Saccharomycete groups also form two clusters in the presence of bacterial FHbs; the surrounding bacterial sequences are dominated by Proteobacteria and Bacilli (Firmicutes). The remaining fungal FHbs cluster with Proteobacteria and Actinobacteria. The Sgbs cluster separately from their bacterial counterparts, except for the intercalation of two Planctomycetes and a Proteobacterium between the Fungi incertae sedis and the Blastocladiomycota and Chytridiomycota. Conclusion Our results are compatible with a model of globin evolution put forward earlier, which proposed that eukaryote F, S and T globins originated via horizontal gene transfer of their bacterial counterparts to the eukaryote ancestor, resulting from

  16. The Prevalence and Molecular Spectrum of α- and β-Globin Gene Mutations in 14,332 Families of Guangdong Province, China

    PubMed Central

    Xu, Longchang; Wu, Li; Zhang, Liang; Ma, Yuanzhu; Chen, Tingting; Gao, Shuang; Liang, Juqing; Guo, Hao; Qin, Danqing; Wang, Jicheng; Yuan, Tenglong; Wang, Yixia; Huang, Wei-wei; He, Wen-Fei; Zhang, Yanxia; Liu, Chang; Xia, Sujian; Chen, Qingshan; Zhao, Qingguo; Zhang, Xiaozhuang

    2014-01-01

    Objective To reveal the familial prevalence and molecular variation of α- and β-globin gene mutations in Guangdong Province. Methods A total of 40,808 blood samples from 14,332 families were obtained and analyzed for both hematological and molecular parameters. Results A high prevalence of α- and β-globin gene mutations was found. Overall, 17.70% of pregnant women, 15.94% of their husbands, 16.03% of neonates, and 16.83% of couples (pregnant women and their husbands) were heterozygous carriers of α- or β-thalassemia. The regions with the highest prevalence were the mountainous and western regions, followed by the Pearl River Delta; the region with the lowest prevalence was Chaoshan. The total familial carrier rate (both spouses were α- or β-thalassemia carriers) was 1.87%, and the individual carrier rates of α- and β-thalassemia were 1.68% and 0.20%, respectively. The total rate of moderate-to-severe fetal thalassemia was 12.78% among couples in which both parents were carriers. Conclusions There was a high prevalence of α- and β-thalassemia in Guangdong Province. This study will contribute to the development of thalassemia prevention and control strategies in Guangdong Province. PMID:24587075

  17. A randomized Phase I/II Trial of HQK-1001, an oral fetal globin gene inducer, in β–thalassaemia intermedia and HbE/β–thalassaemia

    PubMed Central

    Fucharoen, Suthat; Inati, Adlette; Siritanaratku, Noppadol; Thein, Swee Lay; Wargin, William C.; Koussa, Suzanne; Taher, Ali; Chaneim, Nattawara; Boosalis, Michael; Berenson, Ronald; Perrine, Susan P.

    2014-01-01

    β–thalassemia intermedia syndromes (BTI) cause hemolytic anemia, ineffective erythropoiesis, and widespread complications. Higher fetal globin expression within genotypes reduces globin imbalance and ameliorates anemia. Sodium 2,2 dimethylbutyrate (HQK-1001), an orally bioavailable short-chain fatty acid derivative, induces γ-globin expression experimentally and is well-tolerated in normal subjects. Accordingly, a randomized, blinded, placebo-controlled, Phase I/II trial was performed in 21 adult BTI patients (14 with HbE/β0 thalassemia and 7 with β+/β0 thalassemia intermedia, to determine effective doses for fetal globin induction, safety, and tolerability. HQK-1001 or placebo were administered once daily for 8 weeks at four dose levels (10, 20, 30, or 40 mg/kg/day), and subjects were monitored for laboratory and clinical events. Pharmacokinetic profiles demonstrated a t1/2 of 10–12 hours. Adverse events with HQK-1001 treatment were not significantly different from placebo treatment. Median HbF increased with the 20 mg/kg treatment doses above baseline levels by 6.6% and 0.44 g/dL (p <0.01) in 8/9 subjects; total hemoglobin (Hgb) increased by a mean of 1.1 gm/dL in 4/9 subjects. These findings identify a safe oral therapeutic which induces fetal globin in BTI. Further investigation of HQK-1001 with longer dosing to definitively evaluate its hematologic potential appears warranted. PMID:23530969

  18. β-Globin locus control region HS2 and HS3 interact structurally and functionally

    PubMed Central

    Jackson, David A.; McDowell, Jennifer C.; Dean, Ann

    2003-01-01

    The overall structure of the DNase I hypersensitive sites (HSs) that comprise the β-globin locus control region (LCR) is highly conserved among mammals, implying that the HSs have conserved functions. However, it is not well understood how the LCR HSs, either individually or collectively, activate transcription. We analyzed the interactions of HS2, HS3 and HS4 with the human ε- and β-globin genes in chromatinized episomes in fetal/embryonic K562 cells. Only HS2 activates transcription of the ε-globin gene, while all three HSs activate the β-globin gene. HS3 stimulates the β-globin gene constitutively, but HS2 and HS4 transactivation requires expression of the transcription factor EKLF, which is not present in K562 cells but is required for β-globin expression in vivo. To begin addressing how the individual HSs may interact with one another in a complex, we linked the β-globin gene to both the HS2 and HS3. HS2 and HS3 together resulted in synergistic stimulation of β-globin transcription. Unexpectedly, mutated, inactive forms of HS2 impeded the activation of the β-globin gene by HS3. Thus, there appear to be distinct interactions among the HSs and between the HSs and the globin genes. These preferential, non-exclusive interactions may underlie an important structural and functional cooperativity among the regulatory sequences of the β-globin locus in vivo. PMID:12582237

  19. A novel (A)γδβ(0)-thalassemia caused by DNA deletion-inversion-insertion of the β-globin gene cluster and five olfactory receptor genes: Genetic interactions, hematological phenotypes and molecular characterization.

    PubMed

    Singha, Kritsada; Fucharoen, Goonnapa; Hama, Abdulloh; Fucharoen, Supan

    2015-07-01

    To report the phenotypes and genetic basis of a novel (A)γδβ(0)-thalassemia found in Thai individuals with several forms of thalassemia. An initial study was done in an adult Thai woman who had hypochromic microcytic red cells with unusually 100% Hb F. Extended study was carried out on her parents and another 17 unrelated individuals with elevated Hb F. Hb analysis was performed by capillary electrophoresis and DNA analysis was done using PCR. A novel diagnostic method based on multiplex PCR assays was developed. DNA analysis of the proband revealed the homozygosity for a novel deletion of 118.3 kb, removing the entire (A)γ, ψβ, δ-, β-globin and five olfactory receptor (OR) genes with an insertion of a 179 bp inverted DNA sequence located behind the OR52A5 gene located downstream and an insertion of 7 orphan nucleotides. Her parents were both carriers of this mutation. Further screening in suspected cases in our series unexpectedly led to identification of an additional 17 cases with this mutation in different genotypes including plain heterozygote, homozygote, compound heterozygote with Hb E, and double heterozygote with several forms of α-thalassemia. Hematological features associated with these genetic interactions are presented. Haplotype analysis indicated a single origin of this novel deletion-inversion-insertion (A)γδβ(0)-thalassemia in the Thai population. Differentiation of this mutation and other high Hb F determinants documented previously could be done by using a developed multiplex PCR assay. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  20. Genetic origin of Hb D-Los Angeles [beta121(GH4)Glu-->Gln, GAA-->CAA] according to the beta-globin gene cluster haplotypes.

    PubMed

    Atalay, Erol O; Atalay, Ayfer; Ustel, Emre; Yildiz, Sanem; Oztürk, Onur; Köseler, Aylin; Bahadir, Anzel

    2007-01-01

    Hb D-Los Angeles (also known as D-Punjab, D-North Carolina, D-Portugal, D-Chicago and Oak Ridge) is an abnormal hemoglobin (Hb) with an amino acid substitution of glutamine for glutamic acid at codon 121 of the beta-globin gene. The origin and spread of Hb D-Los Angeles is not known. This is due to lack of information and remains to be elucidated. According to published reports, the Hb D-Los Angeles mutation is mostly linked with Mediterranean haplotype I [+ - - - - + +]. Besides the Mediterranean haplotype, a novel haplotype was also reported from Thailand [- - + + - - + + +]. Here we report a new haplotype from Turkey [- + -- + + +] that has not been described before. These results suggest that the Hb D-Los Angeles mutation has at least three different genetic origins.

  1. Molecular analysis of the beta-globin gene cluster in the Niokholo Mandenka population reveals a recent origin of the beta(S) Senegal mutation.

    PubMed

    Currat, Mathias; Trabuchet, Guy; Rees, David; Perrin, Pascale; Harding, Rosalind M; Clegg, John B; Langaney, André; Excoffier, Laurent

    2002-01-01

    A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.

  2. β-Globin chain abnormalities with coexisting α-thalassemia mutations

    PubMed Central

    Canataroglu, Abdullah; Unsal, Cagatay; Yildiz, Sule Menziletoglu; Turhan, Ferda Tekin; Bozdogan, Sevcan Tug; Dincer, Suleyman; Erkman, Hakan

    2012-01-01

    Introduction The frequency of hemoglobinopathies is still high in Adana, the biggest city of the Cukurova Region that is located in the southern part of Turkey. Our aim was to identify the concomitant mutations in α- and β-globin genes which lead to complex hemoglobinopathies and to establish an appropriate plan of action for each subject, particularly when prenatal diagnosis is necessary. Material and methods We studied the association between the β-globin gene and α-thalassemia genotypes. The reverse hybridization technique was employed to perform molecular analysis, and the results were confirmed by amplification refractory mutation system (ARMS) or restriction fragment length polymorphism (RFLP) technique. Results We evaluated 36 adult subjects (28 female and 8 male; age range: 18-52 years) with concomitant mutations in their α- and β-globin genes. The –α3.7/αα deletion was the commonest defect in the α-chain as expected, followed by α3.7/–α3.7 deletion. Twenty-five of 36 cases were sickle cell trait with coexisting α-thalassemia, while seven Hb S/S patients had concurrent mutations in their α-genes. The coexistence of αPolyA-2α/αα with Hb A/D and with Hb S/D, which is very uncommon, was also detected. There was a subject with compound heterozygosity for β-globin chain (–α3.7/αα with IVSI.110/S), and also a case who had –α3.7/αα deletion with IVSI.110/A. Conclusions Although limited, our data suggest that it would be valuable to study coexisting α-globin mutations in subjects with sickle cell disease or β-thalassemia trait during the screening programs for premarital couples, especially in populations with a high frequency of hemoglobinopathies. PMID:23056075

  3. Electrophoretic separation of a class of nucleosomes enriched in HMG 14 and 17 and actively transcribed globin genes.

    PubMed Central

    Albanese, I; Weintraub, H

    1980-01-01

    Monomer nucleosomes from chick erythrocytes can be fractionated according to their electrophoretic mobility in (comparatively) high salt acrylamide gels. We show that the fractionation is based predominantly on differences in charge. The monomer heterogeneity persists even when the nucleosomes are trimmed down to 145 bp with Exo III or when H1 and H5 are removed. The slowest migrating monomers are associated with HMG 14 and 17; however, we do not believe that these proteins are entirely responsible for the altered mobility since the nucleosome heterogeneity persists even after removal of HMG 14 and 17. The DNA associated with the HMG 14 and 17 containing nucleosomes is shown to be enriched in actively transcribed globin sequences. Images PMID:6448987

  4. Hb Bronte or alpha93(FG5)Val-->Gly: a new unstable variant of the alpha2-globin gene, associated with a mild alpha(+)-thalassemia phenotype.

    PubMed

    Lacerra, Giuseppina; Testa, Rosario; De Angioletti, Maria; Schilirò, Gino; Carestia, Clementina

    2003-08-01

    We report a new unstable variant identified in three carriers of a family from East Sicily; it was named Hb Bronte after the place from which the family originated. DNA sequencing from nucleotides -181 to +894 (alpha1) and to +884 (alpha2) revealed a GTG-->GGG substitution at codon 93 of the alpha2-globin gene. The MCV and MCH values were at the lower end of the normal range in the carriers. On cation exchange high performance liquid chromatography (HPLC), the Hb A2 level was apparently increased to around 6%, and a small abnormal peak (0.3-0.4%) was detected after Hb A2. Two abnormal bands were detected by cellulose acetate electrophoresis: a major band (about 3-4%) migrated between Hb A and Hb F; a minor band (<1%) migrated between Hb A2 and carbonic anhydrase. Normal values of Hb A2 were detected by DEAE microchromatography. On reversed phase HPLC the variant chain was not detected, and most likely it was eluted with the alpha chain peak. The isopropanol stability test was very slightly positive in the carriers. Hemolytic symptoms were absent with the exception of indirect bilirubin, which was at high borderline in 2/3 carriers. In biosynthesis in vitro, the specific activity of the alpha chains was much higher than that of the beta-globin chains, and the alpha/beta biosynthetic ratio in the mother and proband was of the beta-thalassemia (thal) type (2.24 and 2.54, respectively). Time course experiments showed that the increase of the 3H-specific activity of the peak containing normal and variant alpha chains was not linear and was much higher than that of beta chains; moreover, the alpha/beta biosynthetic ratio varied during the 2 hours incubation.

  5. Prevalence of β(S)-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil.

    PubMed

    Shimauti, Eliana LitsukoTomimatsu; Silva, Danilo Grunig Humberto; de Souza, Eniuce Menezes; de Almeida, Eduardo Alves; Leal, Francismar Prestes; Bonini-Domingos, Claudia Regina

    2015-01-01

    The aim of this study was to determine the frequency of beta S-globin gene (β(S) globin) haplotypes and alpha thalassemia with 3.7 kb deletion (-α(3.7kb) thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of β(S) globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The β(S)globin haplotypes and -α(3.7kb) thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the -α(3.7kb) mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity.

  6. Molecular spectrum of α- and β-globin gene mutations detected in the population of Guangxi Zhuang Autonomous Region, People's Republic of China.

    PubMed

    Zheng, Chen-Guang; Liu, Ming; Du, Juan; Chen, Ke; Yang, Yige; Yang, Ze

    2011-01-01

    We studied 6,023 individuals diagnosed with anemia on the basis of hematological examinations. The study showed that the frequency of α-thalassemia (α-thal) carriers was 26.9% and β-thal carriers comprised 19.9% of the population of Guangxi Zhuang Autonomous Region, People's Republic of China (PCR). The diagnosed α-thal anomalies were related to six gene mutations and 16 genotypes, whereas the β-thal were related to 10 gene mutations and 65 genotypes. The four most common mutations [codons 41/42 (-TTCT), codon 17 (A>T), -28 (A>G) and IVS-II-654 (C>T)] accounted for 86.38% of the β-globin gene mutations. Risk analysis of mutation alleles in thalassemia cases identified four mutations (-α(3.7), -α(4.2), αα(Westmead) and αα(CS)) that were associated with α-thal intermedia, with an odds ratio (OR) of 62.41-32.68. Four high-risk mutations, namely, codon 26 (G>A), -28, codons 41/42 and codon 17, were associated with β-thal major (β-TM), with an OR of 3.93-2.20. The present study provides important genetic information on thalassemia in this population.

  7. Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

    PubMed

    Trabuchet, G; Elion, J; Baudot, G; Pagnier, J; Bouhass, R; Nigon, V M; Labie, D; Krishnamoorthy, R

    1991-06-01

    Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.

  8. Molecular variations linked to the grouping of beta- and alpha-globin genes in neonatal patients with sickle cell disease in the State of Pernambuco, Brazil.

    PubMed

    Bezerra, Marcos André C; Santos, Magnun N N; Araújo, Aderson S; Gomes, Yara M; Abath, Frederico G C; Bandeira, Flavia M G C

    2007-01-01

    Various factors have been described as phenotypic modulators of sickle cell disease, such as levels of fetal hemoglobin (Hb F), presence of alpha-thalassemia (thal), and haplotypes of the beta-globin genes. In order to characterize and determine the frequency of the betaS and betaC mutations and the prevalence of -alpha3.7-thal, 74 patients with sickle cell disease detected during neonatal screening in the State of Pernambuco, Brazil, were studied. The haplotypes of the beta gene and -alpha3.7-thal were determined using polymerase chain reaction (PCR), and specific restriction endonucleases were used to establish the polymorphic sites of the haplotypes. The results showed the high frequency of the Central African Republic (CAR) or Bantu haplotype in the State of Pernambuco, Brazil. The low frequency of the Benin haplotype recorded in this study, in comparison with other states in northeast Brazil, suggests the diversity of origins of Afro-Brazilians in this region.

  9. Detection of β-globin Gene Mutations Among β-thalassaemia Carriers and Patients in Malaysia: Application of Multiplex Amplification Refractory Mutation System–Polymerase Chain Reaction

    PubMed Central

    Hassan, Syahzuwan; Ahmad, Rahimah; Zakaria, Zubaidah; Zulkafli, Zefarina; Abdullah, Wan Zaidah

    2013-01-01

    Background: β-thalassaemia is one of the most common single-gene disorders worldwide. Each ethnic population has its own common mutations, accounting for the majority of cases, with a small number of mutations for the rarer alleles. Due to the heterogeneity of β-thalassaemia and the multi-ethnicity of Malaysians, molecular diagnostics may be expensive and time consuming. Methods: A simple polymerase chain reaction (PCR) approach involving a multiplex amplification refractory mutation system (MARMS) and one amplification refractory mutation system (ARMS), consisting of 20 β-globin gene mutations, were designed and employed to investigate β-thalassaemia patients and carriers. Results: Out of 169 carriers tested with the MARMS, Cd 41/42 (–TTCT), Cd 26 (A–G) HbE, IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 78.1%. Among the Malays, Cd 26 (A–G) HbE, Cd 41/42 (–TTCT), IVS 1–1 (G–T), and IVS 1–5 (G–C) were the most common mutations, accounting for 81.4%, whereas Cd 41/42 (–TTCT) and IVS 2–654 (C–T) were most common among the Chinese (79.1%). Conclusion: We propose the use of this cheap, easy to interpret, and simple system for the molecular diagnostics of β-thalassaemia among Malaysians at the Institute for Medical Research (IMR). PMID:23613656

  10. Erythroid-specific Expression of β-globin by Sleeping Beauty Transposon for Sickle Cell Disease

    PubMed Central

    Zhu, Jianhui; Kren, Betsy T.; Park, Chang Won; Bilgim, Rasim; Wong, Phillip Y-P.; Steer, Clifford J.

    2013-01-01

    Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild type β- or γ-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel non-viral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild type β-globin expression cassette into the human genome for sustained expression of β-globin. We initially constructed a β-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer: chicken β-actin promoter (CAGGS) and full length β-globin cDNA, as well as truncated forms lacking either the 3′ or 5′ untranslated regions (UTRs), to optimize efficient expression of β-globin. β-globin with its 5′ UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-β-globin plasmids using a minimal β-globin gene driven by the hybrid promoters; IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human αLCR, ankyrin-1 promoter); IHβp (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human αLCR, β-globin promoter;) or HS3βp (HS3 core element from human βLCR, β-globin promoter) to establish erythroid-specific expression of β-globin. Stable genomic insertion of the minimal gene and expression of the β-globin transgene for > 5 months at a level comparable to the endogenous γ-globin gene were achieved using a SB-Tn β-globin cis construct. Interestingly, erythroid-specific expression of β-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable

  11. The application of the human beta-globin gene locus control region and murine erythroleukemia cell system to the expression and pharmacological characterization of human endothelin receptor subtypes.

    PubMed

    Davies, A; Whiting, E; Bath, C; Tang, E; Brennand, J

    1995-06-01

    The cDNAs encoding both A and B subtypes of the human endothelin receptor have been inserted into mammalian cell expression vectors that utilize the human globin gene, locus control region. These constructs have been introduced into murine erythroleukemia cells and inducible high level expression of the receptors has been achieved (approximately 1.5-pM/mg membrane protein and approximately 13,500 binding sites/cell for both receptor subtypes). Cell lines expressing these receptors were obtained on a rapid time scale (3-4 weeks), facilitated by the need for the analysis of only small numbers of cell clones/receptor (approximately 6). Competitive binding assays with endothelin-1 gave IC50s of 130 +/- 30 pM for endothelin-A receptor and 160 +/- 30 pM for endothelin-B receptor. Similar studies with the different isoforms of endothelin, sarafatoxin-S6b and -S6c, BQ123 and BQ3020, all gave the expected selectivity profiles. The IC50s for all compounds were in close agreement with those reported for native receptors. Thus, this expression system, which has several advantages over other described expression systems, is capable of rapidly providing large quantities of receptor for detailed pharmacological analyses or drug screening. In addition, the expressed receptors display the expected pharmacological profiles in the absence of any complicating, competing interactions from other subtypes or binding sites.

  12. Prevalence of βS-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil

    PubMed Central

    Shimauti, Eliana LitsukoTomimatsu; Silva, Danilo Grunig Humberto; de Souza, Eniuce Menezes; de Almeida, Eduardo Alves; Leal, Francismar Prestes; Bonini-Domingos, Claudia Regina

    2015-01-01

    The aim of this study was to determine the frequency of beta S-globin gene (βS globin) haplotypes and alpha thalassemia with 3.7 kb deletion (−α3.7kb thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of βS globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The βSglobin haplotypes and −α3.7kb thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the −α3.7kb mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity. PMID:26500435

  13. Combination of a triple alpha-globin gene with beta-thalassemia in a gypsy family: importance of the genetic testing in the diagnosis and search for a donor for bone marrow transplantation for one of their children.

    PubMed

    Yus Cebrian, Flor; Recasens Flores, María del Valle; Izquierdo Álvarez, Silvia; Parra Salinas, Ingrid; Rodriguez-Vigil Iturrate, Carmen

    2016-04-14

    The simultaneous presence of a heterozygous β-thalassemia with α-gene triplication may cause anything from a thalassemia trait to thalassemia intermedia of mild to moderate severity. An 8-month-old ethnic Gypsy male infant with failure to thrive from birth, mild jaundice and splenomegaly. Clinical signs were compatible with severe microcytic anemia requiring bi-monthly blood transfusions. The β-thalassemia gene analysis found homozygous mutation IVS-I-110 (G>A) (c.93-21G>A) in intron 1 of the hemoglobin beta globin gene and a non-pathogenic sequence variant (single nucleotide polimorfism (SNP) Rs1609812). In addition, the patient had α gene triplication (ααα(anti 3.7)/αα) caused by double heterozygosity for a 3.7 kb fragment that contained only the hemoglobin alpha globin gene-2 gene. This finding led to screening and follow up in first-degree relatives, twin brothers and a sister and parents to provide them with appropriate genetic counseling. Nowadays, new horizons could open a new therapeutic management until definitive cure of these diseases through gene therapy or mutation-specific genome editing. Genetic testing can provide an early diagnosis and facilitates the search for a suitable donor for transplantation.

  14. The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells

    PubMed Central

    Stavrou, Eleana F.; Lazaris, Vassileios M.; Giannakopoulos, Aristeidis; Papapetrou, Eirini; Spyridonidis, Alexandros; Zoumbos, Nikolas C.; Gkountis, Antonis; Athanassiadou, Aglaia

    2017-01-01

    Specific human chromosomal elements enhance the performance of episomal gene-transfer vectors. S/MAR-based episomal vector pEPI-eGFP transfects CD34+ haematopoietic cells, but only transiently. To address this issue we reinforced (1) transgene transcription by replacing the CMV promoter driving eGFP with the EF1/HTLV or SFFV promoters to produce vectors pEPI-EF1/HTLV and pEPI-SFFV, respectively; and (2) plasmid replication by inserting the replication-Initiation Region (IR) from the β-globin locus into vector pEPI-SFFV to produce vector pEP-IR. All vectors supported stable transfections in K562 cells. Transfections of CD34+ cells from peripheral blood of healthy donors reached 30% efficiency. Upon evaluation of CD34+/eGFP+ cells in colony-forming cell (CFC) assays, vector pEP-IR showed superior performance after 14 days, by fluorescent microscopy: 100% eGFP+-colonies against 0% for pEPI-eGFP, 56.9% for pEPI-SFFV and 49.8% for pEPI-EF1/HTLV; 50% more plasmid copies per cell and 3-fold eGFP expression compared to the latter two constructs, by quantitative (q)PCR and RT-qPCR, respectively. Importantly, the establishment rate in CFC assays was 15% for pEP-IR against 5.5% for pEPI-SFFV and 5% for pEPI-EF1/HTLV. Vector pEP-IR shows extremely low delivery rate but supports eGFP expression in thalassaemic mouse haematopoietic progenitor cells. The IR is a novel human control element for improved episomal gene transfer into progenitor cells. PMID:28106085

  15. Erythroid Krüppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5'HS3 of the beta-globin locus control region.

    PubMed

    Tewari, R; Gillemans, N; Wijgerde, M; Nuez, B; von Lindern, M; Grosveld, F; Philipsen, S

    1998-04-15

    Disruption of the gene for transcription factor EKLF (erythroid Krüppel-like factor) results in fatal anaemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid-specific genes, including the embryonic epsilon- and fetal gamma-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the beta-gene promoter, even though it is already present in embryonic red cells. Here, we show that a beta-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF-/- embryos. Thus, embryonic beta-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5'HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5'HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from gamma- to beta-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in beta-globin gene activation.

  16. Globin-coupled sensors, protoglobins, and the last universal common ancestor.

    PubMed

    Freitas, Tracey Allen K; Saito, Jennifer A; Hou, Shaobin; Alam, Maqsudul

    2005-01-01

    The strategy for detecting oxygen, carbon monoxide, nitric oxide, and sulfides is predominantly through heme-based sensors utilizing either a globin domain or a PAS domain. Whereas PAS domains bind various cofactors, globins bind only heme. Globin-coupled sensors (GCSs) were first described as regulators of the aerotactic responses in Bacillus subtilis and Halobacterium salinarum. GCSs were also identified in diverse microorganisms that appear to have roles in regulating gene expression. Functional and evolutionary analyses of the GCSs, their protoglobin ancestor, and their relationship to the last universal common ancestor (LUCA) are discussed in the context of globin-based signal transduction.

  17. Replication initiation patterns in the beta-globin loci of totipotent and differentiated murine cells: evidence for multiple initiation regions.

    PubMed

    Aladjem, Mirit I; Rodewald, Luo Wei; Lin, Chii Mai; Bowman, Sarah; Cimbora, Daniel M; Brody, Linnea L; Epner, Elliot M; Groudine, Mark; Wahl, Geoffrey M

    2002-01-01

    The replication initiation pattern of the murine beta-globin locus was analyzed in totipotent embryonic stem cells and in differentiated cell lines. Initiation events in the murine beta-globin locus were detected in a region extending from the embryonic Ey gene to the adult betaminor gene, unlike the restricted initiation observed in the human locus. Totipotent and differentiated cells exhibited similar initiation patterns. Deletion of the region between the adult globin genes did not prevent initiation in the remainder of the locus, suggesting that the potential to initiate DNA replication was not contained exclusively within the primary sequence of the deleted region. In addition, a deletion encompassing the six identified 5' hypersensitive sites in the mouse locus control region had no effect on initiation from within the locus. As this deletion also did not affect the chromatin structure of the locus, we propose that the sequences determining both chromatin structure and replication initiation lie outside the hypersensitive sites removed by the deletion.

  18. alpha-globin gene deletion and point mutation analysis among in Iranian patients with microcytic hypochromic anemia.

    PubMed

    Garshasbi, Masoud; Oberkanins, Christian; Law, Hai Yang; Neishabury, Maryam; Kariminejad, Roxana; Najmabadi, Hossein

    2003-10-01

    We tested 67 Iranian individuals, presenting with low mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels, normal hemoglobin electrophoresis and iron status, for the presence of twelve common alpha-thalassemia gene deletions and point mutations. Five different mutations (-alpha(3.7), -alpha(4.2), --MED, -(alpha)20.5, Hb Constant Spring) were identified in a total of 43 cases

  19. Why are some genetic diseases common? Distinguishing selection from other processes by molecular analysis of globin gene variants.

    PubMed

    Flint, J; Harding, R M; Clegg, J B; Boyce, A J

    1993-03-01

    Various processes (selection, mutation, migration and genetic drift) are known to determine the frequency of genetic disease in human populations, but so far it has proved almost impossible to decide to what extent each is responsible for the presence of a particular genetic disease. The techniques of gene and haplotype analysis offer new hope in addressing this issue, and we review relevant studies of three haemoglobinopathies: sickle cell anaemia, and alpha and beta thalassaemia. We show how for each disease it is possible to recognize a pattern of regionally specific mutations, found in association with one or a few haplotypes, that is best explained as the result of selection; other patterns are due to population migration and genetic drift. However, we caution that such conclusions can be drawn in special circumstances only. In the case of the haemoglobinopathies it is possible because a selective agent (malaria) was already suspected, and the investigations could be carried out in relatively genetically homogenous populations whose migratory histories are known. Moreover, some data reviewed here suggest that gene conversion and the haplotype composition of a population may affect the frequency of a mutation, making interpretation of gene frequencies difficult on the basis of standard population genetics theory. Hence attempts to use the same approaches with other genetic diseases are likely to be frustrated by a lack of suitably untrammelled populations and by difficulties accounting for poorly understood genetic processes. We conclude that although this combination of molecular and population genetics is successful when applied to the study of haemoglobinopathies, it may not be so easy to apply it to the study of other genetic diseases.

  20. Masking of a β-thalassemia determinant by a novel δ-globin gene defect [Hb A2-Saurashtra or δ100(G2)Pro→Ser; HBD: c.301C>T] in Cis.

    PubMed

    Colaco, Stacy; Trivedi, Amee; Colah, Roshan B; Ghosh, K; Nadkarni, Anita H

    2014-01-01

    Abstract The molecular basis of β-thalassemia (β-thal) syndromes have been well documented, while the spectrum of mutations causing δ-thalassemia (δ-thal) has not been well characterized. δ-Thalassemia has no clinical symptoms but its coinheritance with heterozygous β-thal may cause misdiagnosis, especially in countries with a high prevalence of β-thal where prevention programs have been implemented. The coinheritance of β- and δ-globin mutations in India is not common. This association may interfere with correct diagnosis and genetic counseling of β-thal in screening programs. Here we report two families showing borderline Hb A2 levels belonging to the Koli Community, indigenous to the Saurashtra Province of Gujarat, India. They were referred to us for thalassemia molecular screening as they had children clinically presenting before 2 years of age and requiring regular blood transfusions. Interestingly, both families carried a novel δ-globin gene mutation at codon 100 (C > T) linked to a polyadenylation (polyA) site [AATAAA > A(-AATAA)] 5 bp deletional β-thal mutation, never before reported in the Indian population. This report highlights the importance of considering δ-globin gene analysis during β-thal screening to avoid false-negative results in the detection of at-risk couples. It also highlights how incomplete diagnosis of a borderline or normal Hb A2 level may lead to the probable birth of a β-thal major (β-TM) child. This has important implications in prenatal diagnosis.

  1. Hb St. Jozef, A Val-->Leu N-terminal mutation leading to retention of the methionine, and partial acetylation found in the globin gene in Cis with a -alpha3.7 thalassemia deletion.

    PubMed

    Harteveld, Cornelis L; Versteegh, Florens G A; van Leer, Eduard H G; Starreveld, Jaap S; Kok, Peter J M J; van Rooijen-Nijdam, Irene; van Delft, Peter; Zanella-Cleon, Isabelle; Becchi, Michel; Wajcman, Henri; Giordano, Piero C

    2007-01-01

    We report a new hemoglobin (Hb) variant found in a 6-year-old girl of Moroccan origin, living in the Dutch city of Gouda. The child was referred because of microcytic and hypochromic parameters. A normal zinc protoporphyirin (ZPP) value excluded iron deficiency and gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the common -alpha(3.7) thalassemia deletion, partially justifying the hematological picture. The Hb pattern on alkaline electrophoresis and capillary electrophoresis was normal, while a fraction of 9% preceding the Hb A peak, remained visible on different high performance liquid chromatography (HPLC) devices. This fraction, located in front of the Hb A peak, is usually considered as a Hb A derivate that becomes more expressed in older samples. However, the sample was freshly collected and the peak unusually evident. Therefore, direct sequencing of the alpha-globin genes was performed revealing a GTG-->CTG transversion at codon 1 of the alpha1-globin gene or of the hybrid gene. This point mutation induces a single amino acid substitution from valine to leucine. Electrospray-mass spectrometry (ES-MS) analysis revealed, in addition to this substitution, that the N-terminal methionine was retained and that about 20% of the variant was acetylated. As expected for an association with a -alpha(3.7)-thalassemia (thal) deletion, the non acetylated and acetylated abnormal alpha chain amounted to 32% of the total alpha chains. Family studies revealed that the mutated codon was located in cis of the deletion.

  2. DNAase I hypersensitive site 3' to the beta-globin gene cluster containing two TAA insertions and a G-->A polymorphism is predominantly associated with the beta+-thalassemia IVS-I-6 (T-->C) mutation.

    PubMed

    Martins, Juliana T N; Bordin, Silvana; de Albuquerque, Dulcinéia M; Saad, Sara T O; Costa, Fernando F

    2005-01-01

    Analysis of DNA polymorphic sites is an important tool for the detection of gene flow in human evolutionary studies and to study the genetic background for gene mutations. The beta-globin locus contains several single-base restriction fragment length polymorphism (RFLP) sites throughout chromosome 11. In addition to these polymorphic sequence repeats, others are being studied in order to expand our knowledge concerning the role between haplotype-genotype and phenotype associations. Far downstream of the expressed beta-globin genes, there is a hypersensitive site (HS) whose function remains obscure. We sequenced this region in 27 thalassemia patients and found a new pattern in the micro-satellite-like AT-rich region of this site: a new TAA insertion in addition to the one previously described in sickle cell patients with a concomitant polymorphism (G-->A). This new variation was found to be linked to the IVS-I-6 (T-->C) mutation. This polymorphism may be useful for studies concerning genotype and phenotype associations.

  3. Structural analysis of the 5 prime flanking region of the. beta. -globin gene in African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa

    SciTech Connect

    Chebloune, Y.; Pagnier, J.; Trabuchet, G.; Faure, C.; Verdier, G.; Labie, D.; Nigon, V. )

    1988-06-01

    Haplotype analysis of the {beta}-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT){sub n} and (AT){sub x}T{sub y}, which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT){sub n} and (AT){sub x}T{sub y} repeats. The authors found three additional structures for (AT){sub x}T{sub y} correlating with the geographic origin of the patients. Ten other nucleotide positions, 5{prime} and 3{prime} to the (AT){sub x}T{sub y} copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5{prime} flanking region of the {beta}-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

  4. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  5. Growth factor independence 1b (gfi1b) is important for the maturation of erythroid cells and the regulation of embryonic globin expression.

    PubMed

    Vassen, Lothar; Beauchemin, Hugues; Lemsaddek, Wafaa; Krongold, Joseph; Trudel, Marie; Möröy, Tarik

    2014-01-01

    Growth factor independence 1b (GFI1B) is a DNA binding repressor of transcription with vital functions in hematopoiesis. Gfi1b-null embryos die at midgestation very likely due to defects in erythro- and megakaryopoiesis. To analyze the full functionality of Gfi1b, we used conditionally deficient mice that harbor floxed Gfi1b alleles and inducible (Mx-Cre, Cre-ERT) or erythroid specific (EpoR-Cre) Cre expressing transgenes. In contrast to the germline knockout, EpoR-Cre mediated erythroid specific ablation of Gfi1b allows full gestation, but causes perinatal lethality with very few mice surviving to adulthood. Both the embryonic deletion of Gfi1b by EpoR-Cre and the deletion in adult mice by Mx-Cre or Cre-ERT leads to reduced numbers of erythroid precursors, perturbed and delayed erythroid maturation, anemia and extramedullary erythropoiesis. Global expression analyses showed that the Hba-x, Hbb-bh1 and Hbb-y embryonic globin genes were upregulated in Gfi1b deficient TER119+ fetal liver cells over the gestation period from day 12.5-17.5 p.c. and an increased level of Hbb-bh1 and Hbb-y embryonic globin gene expression was even maintained in adult Gfi1b deficient mice. While the expression of Bcl11a, a regulator of embryonic globin expression was not affected by Gfi1b deficiency, the expression of Gata1 was reduced and the expression of Sox6, also involved in globin switch, was almost entirely lost when Gfi1b was absent. These findings establish Gfi1b as a regulator of embryonic globin expression and embryonic and adult erythroid maturation.

  6. Differential expression of murine adult hemoglobins in early ontogeny

    SciTech Connect

    Wawrzyniak, C.J.; Lewis, S.E.; Popp, R.A.

    1985-01-01

    A hemoglobin mutation is described that permits study of the expression of the two adult ..beta..-globin genes throughout fetal and postnatal development. Mice with a mutation at the Hbb/sup s/, ..beta..-globin locus, were used to study the relative levels of ..beta..-s2major and ..beta..-sminor globins specified by the mutant Hbb/sup s2/ haplotype during development. At 11.5 days of gestation ..beta..-sminor comprised over 80% and ..beta..-s2major under 20% of the adult beta-globin. The relative level of ..beta..-sminor decreased through fetal development; at birth ..beta..-sminor represented 33.7% of the ..beta..-globin. The adult values of 71.0% ..beta..-s2major and 29.0% ..beta..-sminor globin are expressed in mice six days after birth. Because the two ..beta..-globin genes are expressed in mice of the Hbb/sup 2s/ haplotype, both the ..beta..-smajor and ..beta..-sminor genes must be expressed in mice of the Hbb/sup s/ haplotype. Expression of the ..beta..-sminor gene is elevated to 35.6% in Hbb/sup s2/ mice that have been bled repeatedly. Thus, the 5' ..beta..-s2major and 3' ..beta..-sminor genes of the Hbb/sup s2/ haplotype and, presumably the 5' ..beta..-smajor and 3' ..beta..-sminor genes of the Hbb/sup s/ haplotype, are regulated independently and are homologous to the 5' ..beta..-dmajor and 3' ..beta..-dminor genes of the Hbb/sup d/ haplotype. Mice of the Hbb/sup s2/ haplotype are better than mice of the Hbb/sup d/ haplotytpe for studying the mechanisms of hemoglobin switching because the Hbb/sup s2/ each of the three embryonic and two adult hemoglobins can be separated by electrophoresis. 17 refs., 3 figs.

  7. Original Research: Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: -175 Black HPFH and -195 Brazilian HPFH.

    PubMed

    Braghini, Carolina A; Costa, Flavia C; Fedosyuk, Halyna; Neades, Renee Y; Novikova, Lesya V; Parker, Matthew P; Winefield, Robert D; Peterson, Kenneth R

    2016-04-01

    Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the (A)γ-globin -175 T > C or -195 C > G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult -175 and -195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In -175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the (A)γ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of (A)γ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to

  8. Original Research: Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: -175 Black HPFH and -195 Brazilian HPFH

    PubMed Central

    Braghini, Carolina A; Costa, Flavia C; Fedosyuk, Halyna; Neades, Renee Y; Novikova, Lesya V; Parker, Matthew P; Winefield, Robert D

    2016-01-01

    Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the Aγ-globin -175 T > C or -195 C > G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult -175 and -195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In -175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the Aγ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of Aγ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to study

  9. The globins of Campylobacter jejuni.

    PubMed

    Tinajero-Trejo, Mariana; Shepherd, Mark

    2013-01-01

    Campylobacter jejuni is a zoonotic Gram-negative bacterial pathogen that is exposed to reactive nitrogen species, such as nitric oxide, from a variety of sources. To combat the toxic effects of this nitrosative stress, C. jejuni upregulates a small regulon under the control of the transcriptional activator NssR, which positively regulates the expression of a single-domain globin protein (Cgb) and a truncated globin protein (Ctb). Cgb has previously been shown to detoxify nitric oxide, but the role of Ctb remains contentious. As C. jejuni is amenable to genetic manipulation, and its globin proteins are easily expressed and purified, a combination of mutagenesis, complementation, transcriptomics, spectroscopic characterisation and structural analyses has been used to probe the regulation, function and structure of Cgb and Ctb. This ability to study Cgb and Ctb with such a multi-pronged approach is a valuable asset, especially since only a small fraction of known globin proteins have been functionally characterised.

  10. A novel 26 bp deletion [HBB: c.20_45del26bp] in exon 1 of the β-globin gene causing β-thalassemia major.

    PubMed

    Edison, Eunice S; Venkatesan, Rajkumar S; Govindanattar, Sankari Devi; George, Biju; Shaji, Ramachandran V

    2012-01-01

    Molecular characterization of β-thalassemia (β-thal) is essential in prevention and in understanding the biology of the disease. Deletion mutations are relatively uncommon in β-thal. In this report, we describe a novel 26 bp deletion from codon 6 to codon 14 in the β-globin in a consanguineous family from Tamil Nadu, India. This novel mutation causes a shift in the normal reading frame of the β-globin coding sequence, and consequently, a premature chain termination of translation due to the creation of a stop codon at the position of codon 21. The identification of this novel deletional mutation adds to the repertoire of β-thal mutations in India.

  11. Frequency and origin of haplotypes associated with the beta-globin gene cluster in individuals with trait and sickle cell anemia in the Atlantic and Pacific coastal regions of Colombia

    PubMed Central

    Fong, Cristian; Lizarralde-Iragorri, María Alejandra; Rojas-Gallardo, Diana; Barreto, Guillermo

    2013-01-01

    Sickle cell anemia is a genetic disease with high prevalence in people of African descent. There are five typical haplotypes associated with this disease and the haplotypes associated with the beta-globin gene cluster have been used to establish the origin of African-descendant people in America. In this work, we determined the frequency and the origin of haplotypes associated with hemoglobin S in a sample of individuals with sickle cell anemia (HbSS) and sickle cell hemoglobin trait (HbAS) in coastal regions of Colombia. Blood samples from 71 HbAS and 79 HbSS individuals were obtained. Haplotypes were determined based on the presence of variable restriction sites within the β-globin gene cluster. On the Pacific coast of Colombia the most frequent haplotype was Benin, while on the Atlantic coast Bantu was marginally higher than Benin. Eight atypical haplotypes were observed on both coasts, being more diverse in the Atlantic than in the Pacific region. These results suggest a differential settlement of the coasts, dependent on where slaves were brought from, either from the Gulf of Guinea or from Angola, where the haplotype distributions are similar. Atypical haplotypes probably originated from point mutations that lost or gained a restriction site and/or by recombination events. PMID:24385850

  12. Frequency and origin of haplotypes associated with the beta-globin gene cluster in individuals with trait and sickle cell anemia in the Atlantic and Pacific coastal regions of Colombia.

    PubMed

    Fong, Cristian; Lizarralde-Iragorri, María Alejandra; Rojas-Gallardo, Diana; Barreto, Guillermo

    2013-12-01

    Sickle cell anemia is a genetic disease with high prevalence in people of African descent. There are five typical haplotypes associated with this disease and the haplotypes associated with the beta-globin gene cluster have been used to establish the origin of African-descendant people in America. In this work, we determined the frequency and the origin of haplotypes associated with hemoglobin S in a sample of individuals with sickle cell anemia (HbSS) and sickle cell hemoglobin trait (HbAS) in coastal regions of Colombia. Blood samples from 71 HbAS and 79 HbSS individuals were obtained. Haplotypes were determined based on the presence of variable restriction sites within the β-globin gene cluster. On the Pacific coast of Colombia the most frequent haplotype was Benin, while on the Atlantic coast Bantu was marginally higher than Benin. Eight atypical haplotypes were observed on both coasts, being more diverse in the Atlantic than in the Pacific region. These results suggest a differential settlement of the coasts, dependent on where slaves were brought from, either from the Gulf of Guinea or from Angola, where the haplotype distributions are similar. Atypical haplotypes probably originated from point mutations that lost or gained a restriction site and/or by recombination events.

  13. Erythroid-specific expression of beta-globin by the sleeping beauty transposon for Sickle cell disease.

    PubMed

    Zhu, Jianhui; Kren, Betsy T; Park, Chang Won; Bilgim, Rasim; Wong, Phillip Y-P; Steer, Clifford J

    2007-06-12

    Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient

  14. Globin-based redox signaling

    PubMed Central

    De Henau, Sasha; Braeckman, Bart P.

    2016-01-01

    ABSTRACT In recent years, moderate levels of reactive oxygen species (ROS) have become recognized as signaling cues that participate at all levels of cellular organization. Globins, with their redox-active heme iron and ubiquitous presence, seem ideally suited to participate in ROS metabolism. Here we comment on our recent findings that show the participation of a globin, GLB-12, in a redox signaling pathway in Caenorhabditis elegans. We found that GLB-12 produces superoxide, a type of ROS, after which this is converted to what appears to be a hydrogen peroxide gradient over the plasma membrane by the activity of intracellular and extracellular superoxide dismutases. In the first part, we discuss in more detail the different regulatory mechanisms that increase the effectiveness of this redox signal. In the second part, we comment on how specific structural and biochemical properties allow this globin to perform redox reactions. Interestingly, these properties are also observed in 2 other C. elegans globins that appear to be involved in redox biology. We therefore hypothesize that globins involved in redox signaling display similar structural and biochemical characteristics and propose that a subgroup of globins can be added to the group of proteins that play a vital role in redox signaling. PMID:27695650

  15. Globin-based redox signaling.

    PubMed

    De Henau, Sasha; Braeckman, Bart P

    2016-01-01

    In recent years, moderate levels of reactive oxygen species (ROS) have become recognized as signaling cues that participate at all levels of cellular organization. Globins, with their redox-active heme iron and ubiquitous presence, seem ideally suited to participate in ROS metabolism. Here we comment on our recent findings that show the participation of a globin, GLB-12, in a redox signaling pathway in Caenorhabditis elegans. We found that GLB-12 produces superoxide, a type of ROS, after which this is converted to what appears to be a hydrogen peroxide gradient over the plasma membrane by the activity of intracellular and extracellular superoxide dismutases. In the first part, we discuss in more detail the different regulatory mechanisms that increase the effectiveness of this redox signal. In the second part, we comment on how specific structural and biochemical properties allow this globin to perform redox reactions. Interestingly, these properties are also observed in 2 other C. elegans globins that appear to be involved in redox biology. We therefore hypothesize that globins involved in redox signaling display similar structural and biochemical characteristics and propose that a subgroup of globins can be added to the group of proteins that play a vital role in redox signaling.

  16. HEMOGLOBIN POLYMORPHISMS IN DEER MICE (PEROMYSCUS MANICULATUS): PHYSIOLOGY OF BETA-GLOBIN VARIANTS AND ALPHA-GLOBIN RECOMBINANTS.

    PubMed

    Chappell, Mark A; Hayes, Jack P; Snyder, Lee R G

    1988-07-01

    Wild populations of deer mice (Peromyscus maniculatus) contain hemoglobin polymorphisms at both alpha-globin (Hba, Hbc) and beta-globin (Hbd) loci. Population gene frequencies of beta-globin variants (d(0) and d(1) haplotypes) are not correlated with altitude, whereas a(1) c(1) alpha-globin haplotypes are fixed in low-altitude populations, and a(0) c(0) haplotypes reach near fixation at high altitudes. We examined the effects of alpha- and beta-globin variants on blood oxygen affinity and on aerobic performance, measured as maximum oxygen consumption (V˙O2max). Exercise and cold exposure were used to elicit V˙O2max. Experiments were performed at low (340 m) and high (3,800 m) altitude to include the range of oxygen partial pressures encountered by wild deer mice. Beta-globin variants had little effect on blood oxygen affinity or V˙O2max. Oxygen-dissociation curves from a(0) c(0) and a(1) c(1) homozygotes and heterozygotes had similar shapes, but the P50 of a(0) c(0) homozygotes was significantly lower than that of other genotypes. Mice carrying a(1) c(1) /a(1) c(1) genotypes had the highest V˙O2max at low altitude, but mice with a(0) c(0) /a(0) c(0) genotypes had the highest V˙O2max at high altitude. Mice carrying rare recombinant alpha-globin haplotypes (a(0) c(1) ) had lower V˙O2max than nonrecombinant genotypes as a whole but in most cases were not significantly different from nonrecombinant heterozygotes (a(0) c(0) /a(1) c(1) ). We conclude that genetic adaptation to different altitudes was important in the evolution of deer mouse alpha-globin polymorphisms and in the maintenance of linkage disequilibrium in the alpha-globin loci but was not a significant factor in the evolution of beta-globin polymorphisms. © 1988 The Society for the Study of Evolution.

  17. Evolution and Expression of Tissue Globins in Ray-Finned Fishes

    PubMed Central

    Gallagher, Michael D.

    2017-01-01

    The globin gene family encodes oxygen-binding hemeproteins conserved across the major branches of multicellular life. The origins and evolutionary histories of complete globin repertoires have been established for many vertebrates, but there remain major knowledge gaps for ray-finned fish. Therefore, we used phylogenetic, comparative genomic and gene expression analyses to discover and characterize canonical “non-blood” globin family members (i.e., myoglobin, cytoglobin, neuroglobin, globin-X, and globin-Y) across multiple ray-finned fish lineages, revealing novel gene duplicates (paralogs) conserved from whole genome duplication (WGD) and small-scale duplication events. Our key findings were that: (1) globin-X paralogs in teleosts have been retained from the teleost-specific WGD, (2) functional paralogs of cytoglobin, neuroglobin, and globin-X, but not myoglobin, have been conserved from the salmonid-specific WGD, (3) triplicate lineage-specific myoglobin paralogs are conserved in arowanas (Osteoglossiformes), which arose by tandem duplication and diverged under positive selection, (4) globin-Y is retained in multiple early branching fish lineages that diverged before teleosts, and (5) marked variation in tissue-specific expression of globin gene repertoires exists across ray-finned fish evolution, including several previously uncharacterized sites of expression. In this respect, our data provide an interesting link between myoglobin expression and the evolution of air breathing in teleosts. Together, our findings demonstrate great-unrecognized diversity in the repertoire and expression of nonblood globins that has arisen during ray-finned fish evolution. PMID:28173090

  18. Sequence variations in the 5' flanking and IVS-II regions of the G gamma- and A gamma-globin genes of beta S chromosomes with five different haplotypes.

    PubMed

    Lanclos, K D; Oner, C; Dimovski, A J; Gu, Y C; Huisman, T H

    1991-06-01

    We have amplified and sequenced the 5' flanking and the second intervening sequence (IVS-II) regions of both the G gamma- and A gamma-globin genes of the beta S chromosomes from sickle cell anemia (SS) patients with homozygosities for five different haplotypes. The sequencing data, compared with previously published sequences for the normal chromosomes A and B, show many similarities to chromosome B for haplotypes 19, 20, and 17, while haplotypes 3 and 31 are remarkably similar to chromosome A and also similar to each other. Several unique mutations were found in the 5' flanking regions (G gamma and A gamma) of haplotypes 19 and 20 and in the IVS-II segments of the same genes of haplotypes 19, 20, and 17; the IVS-II of haplotypes 3 and 31 were identical to those of chromosome A. Dot-blot analyses of amplified DNA from additional SS patients with specific probes have confirmed that these mutations are unique for each haplotype. The two general patterns that have been observed among the five haplotypes have most probably arisen by gene conversion events between the A and B type chromosomes in the African population. These patterns correlate with high and low fetal hemoglobin expression, and it is speculated that these and other yet unknown gene conversions may contribute to the variations in hemoglobin F and G gamma levels observed among SS patients. In vitro expression experiments involving the approximately 1.3-kb 5' flanking regions of the G gamma- and A gamma-globin genes of the beta S chromosomes with the five different haplotypes failed to detect differences between the levels of expression, suggesting that the sequence variations observed between these segments of DNA are not the primary cause of the differences in hemoglobin F levels among the SS patients.

  19. The gene for familial Mediterranean fever in both Armenians and non-Ashkenzai Jews is linked to the [alpha]-globin complex on 16p: Evidence for locus homogeneity

    SciTech Connect

    Shohat, M.; Shohat, T.; Magal, N.; Danon, Y. ); Xiangdong Bu; Fischel-Ghodsian, N.; Schwabe, A.D.; Rotter, J.I. ); Nakamura, Yusuke ); Schlezinger, M. )

    1992-12-01

    Familial Mediterranean fever (FMF) is a recurrent inflammatory disorder characterized by short episodes of fever, peritonitis, pleuritis, and arthritis. While FMF has been shown to be inherited in an autosomal recessive fashion in both non-Ashkenazi Jews and Armenian families, clinical differences have raised the possibility of genetic heterogeneity. As its pathogenesis is unknown, mapping of the gene for FMF may provide the first objective method for early and accurate diagnosis of this disease. After excluding 45% of the entire human genome, the authors studied 14 Armenian and 9 non-Ashkenazi Jewish families with FMF and tested linkage with the [alpha]-globin locus on chromosome 16. Analysis of the PvuII length polymorphism of the 3[prime] HVR (hypervariable region) probe showed significant linkage with the FMF gene (maximum lod score [lod[sub max

  20. Two new δ-globin gene variants: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro (HBD: c.44T>C)] and Hb A(2)-Marseille [δ22(B4) Ala→Lys (HBD: c.67G>A;68C>A)].

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Desbrée, Aurélie; Couprie, Nicole; Francina, Alain

    2013-01-01

    We report two new variants of the δ-globin gene: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro] and Hb A(2)-Marseille [δ22(B4)Ala→Lys]. The first variant has a low rate of expression, the second results from a double nucleotide mutation on the same codon.

  1. trans-Activation of a globin promoter in nonerythroid cells.

    PubMed Central

    Evans, T; Felsenfeld, G

    1991-01-01

    We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1 bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple and sensitive system with which to study gene activation by a tissue-specific factor. Images PMID:1990287

  2. Genome scan identifies a locus affecting gamma-globin expression in human beta-cluster YAC transgenic mice

    SciTech Connect

    Lin, S.D.; Cooper, P.; Fung, J.; Weier, H.U.G.; Rubin, E.M.

    2000-03-01

    Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression of human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.

  3. The β-globin locus control region in combination with the EF1α short promoter allows enhanced lentiviral vector-mediated erythroid gene expression with conserved multilineage activity.

    PubMed

    Montiel-Equihua, Claudia A; Zhang, Lin; Knight, Sean; Saadeh, Heba; Scholz, Simone; Carmo, Marlene; Alonso-Ferrero, Maria E; Blundell, Michael P; Monkeviciute, Aiste; Schulz, Reiner; Collins, Mary; Takeuchi, Yasuhiro; Schmidt, Manfred; Fairbanks, Lynette; Antoniou, Michael; Thrasher, Adrian J; Gaspar, H Bobby

    2012-07-01

    Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the β-globin gene (β-LCR). We show that the β-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3-7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the β-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the β-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

  4. Preliminary identification of hemoglobin q-iran in an Iranian family from central province of Iran by globin chain analysis on HPLC.

    PubMed

    Khatami, Shohreh; Najmabadi, Hossein; Rouhi, Soghra; Mirzazadeh, Roghieh; Bayat, Parastoo; Sadeghi, Sedigheh

    2013-12-01

    Many abnormal α-chain hemoglobins (Hbs) are caused by single nucleotide mutations in α1- or α2-goblin genes. One of these Hbs is Hb Q-Iran which is resulted from a point mutation at codon 75 of the α1-globin gene (Asp→His). The identification of Hb Q-Iran was observed in two members of a family from the Central Province of Iran. In this study, Globin chain analysis on high performance liquid chromatography (HPLC) and DNA sequencing were applied. An unusual Hb variant, like HbS on alkaline pH electrophoresis was identified from samples of a father and his son from Arak city in the Central Province of Iran. The variant was further characterized by globin chain analysis and DNA sequencing methods. Globin chain analysis revealed an unknown globin chain peak after α-globin chain peak with a different retention time from βs-globin chain, as the control in both samples. Genetic analysis led to the identification of an unknown Hb variant, Hb Q-Iran. Globin chain analysis showed the presence of an unknown globin chain, and likewise DNA sequencing revealed HbQ-Iran. In other words, Globin chain analysis procedure could preliminarily detect an unknown globin chain.

  5. A precise termination site in the mouse beta major-globin transcription unit.

    PubMed Central

    Salditt-Georgieff, M; Darnell, J E

    1983-01-01

    Nascent labeled RNA from induced, globin-producing mouse erythroleukemia cells was hybridized to cloned regions of the beta major-globin gene. Transcription ceases about 1,000 bases downstream from the poly(A) site as indicated by protection from nuclease digestion of a discrete-sized RNA fragment that it shorter than the protecting cloned DNA fragment. This defines an apparently unique termination site for a protein-coding gene that is transcribed by RNA polymerase II. Images PMID:6192441

  6. Co-inheritance of novel ATRX gene mutation and globin (α & β) gene mutations in transfusion dependent beta-thalassemia patients.

    PubMed

    Al-Nafie, Awatif N; Borgio, J Francis; AbdulAzeez, Sayed; Al-Suliman, Ahmed M; Qaw, Fuad S; Naserullah, Zaki A; Al-Jarrash, Sana; Al-Madan, Mohammed S; Al-Ali, Rudaynah A; AlKhalifah, Mohammed A; Al-Muhanna, Fahad; Steinberg, Martin H; Al-Ali, Amein K

    2015-06-01

    α-Thalassemia X-linked mental retardation syndrome is a rare inherited intellectual disability disorder due to mutations in the ATRX gene. In our previous study of the prevalence of β-thalassemia mutations in the Eastern Province of Saudi Arabia, we confirmed the widespread coinheritance of α-thalassemia mutation. Some of these subjects have a family history of mental retardation, the cause of which is unknown. Therefore, we investigated the presence or absence of mutations in the ATRX gene in these patients. Three exons of the ATRX gene and their flanking regions were directly sequenced. Only four female transfusion dependent β-thalassemia patients were found to be carriers of a novel mutation in the ATRX gene. Two of the ATRX gene mutations, c.623delA and c.848T>C were present in patients homozygous for IVS I-5(G→C) and homozygous for Cd39(C → T) β-thalassemia mutation, respectively. While the other two that were located in the intronic region (flanking regions), were present in patients homozygous for Cd39(C → T) β-thalassemia mutation. The two subjects with the mutations in the coding region had family members with mental retardation, which suggests that the novel frame shift mutation and the missense mutation at coding region of ATRX gene are involved in ATRX syndrome. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. The higher structure of chromatin in the LCR of the beta-globin locus changes during development.

    PubMed

    Fang, Xiangdong; Yin, Wenxuan; Xiang, Ping; Han, Hemei; Stamatoyannopoulos, George; Li, Qiliang

    2009-11-27

    The beta-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant beta-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.

  8. Characterization of histone H3K27 modifications in the {beta}-globin locus

    SciTech Connect

    Kim, Yea Woon; Kim, AeRi

    2011-02-11

    Research highlights: {yields} The {beta}-globin locus control region is hyperacetylated and monomethylated at histone H3K27. {yields} Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. {yields} Association of PRC2 subunits is comparable with H3K27me3 pattern. {yields} Modifications of histone H3K27 are established in an enhancer-dependent manner. -- Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human {beta}-globin locus using the ChIP assay. The LCR of the human {beta}-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the {beta}-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer.

  9. Differential Loss and Retention of Cytoglobin, Myoglobin, and Globin-E during the Radiation of Vertebrates

    PubMed Central

    Hoffmann, Federico G.; Opazo, Juan C.; Storz, Jay F.

    2011-01-01

    If rates of postduplication gene retention are positively correlated with levels of functional constraint, then gene duplicates that have been retained in a restricted number of taxonomic lineages would be expected to exhibit relatively low levels of sequence conservation. Paradoxical patterns are presented by gene duplicates that have been retained in a small number of taxa but which are nonetheless subject to strong purifying selection relative to paralogous members of the same multigene family. This pattern suggests that such genes may have been co-opted for novel, lineage-specific functions. One possible example involves the enigmatic globin-E gene (GbE), which appears to be exclusively restricted to birds. Available data indicate that this gene is expressed exclusively in the avian eye, but its physiological function remains a mystery. In contrast to the highly restricted phyletic distribution of GbE, the overwhelming majority of jawed vertebrates (gnathostomes) possess copies of the related cytoglobin (Cygb) and myoglobin (Mb) genes. The purpose of the present study was 1) to assess the phyletic distribution of the Cygb, Mb, and GbE genes among vertebrates, 2) to elucidate the duplicative origins and evolutionary histories of these three genes, and 3) to evaluate the relative levels of functional constraint of these genes based on comparative sequence analysis. To accomplish these objectives, we conducted a combined phylogenetic and comparative genomic analysis involving taxa that represent each of the major lineages of gnathostome vertebrates. Results of synteny comparisons and phylogenetic topology tests revealed that GbE is clearly not the product of a recent, bird-specific duplication event. Instead, GbE originated via duplication of a proto-Mb gene in the stem lineage of gnathostomes. Unlike the Mb gene, which has been retained in all major gnathostome lineages other than amphibians, the GbE gene has been retained only in the lineage leading to modern

  10. Erythroid-Specific Expression of β-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

    PubMed Central

    Sjeklocha, Lucas M.; Park, Chang-Won; Wong, Phillip Y-P; Roney, Mark J.; Belcher, John D.; Kaufman, Dan S.; Vercellotti, Gregory M.; Hebbel, Robert P.; Steer, Clifford J.

    2011-01-01

    Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease. PMID:22216176

  11. Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

    PubMed

    Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen

    2011-01-01

    Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

  12. Integrated protein quality-control pathways regulate free α-globin in murine β-thalassemia

    PubMed Central

    Khandros, Eugene; Thom, Christopher S.; D'Souza, Janine

    2012-01-01

    Cells remove unstable polypeptides through protein quality-control (PQC) pathways such as ubiquitin-mediated proteolysis and autophagy. In the present study, we investigated how these pathways are used in β-thalassemia, a common hemoglobinopathy in which β-globin gene mutations cause the accumulation and precipitation of cytotoxic α-globin subunits. In β-thalassemic erythrocyte precursors, free α-globin was polyubiquitinated and degraded by the proteasome. These cells exhibited enhanced proteasome activity, and transcriptional profiling revealed coordinated induction of most proteasome subunits that was mediated by the stress-response transcription factor Nrf1. In isolated thalassemic cells, short-term proteasome inhibition blocked the degradation of free α-globin. In contrast, prolonged in vivo treatment of β-thalassemic mice with the proteasome inhibitor bortezomib did not enhance the accumulation of free α-globin. Rather, systemic proteasome inhibition activated compensatory proteotoxic stress-response mechanisms, including autophagy, which cooperated with ubiquitin-mediated proteolysis to degrade free α-globin in erythroid cells. Our findings show that multiple interregulated PQC responses degrade excess α-globin. Therefore, β-thalassemia fits into the broader framework of protein-aggregation disorders that use PQC pathways as cell-protective mechanisms. PMID:22427201

  13. [γ-Globin Inductive Therapy of β-thalassemia and Its Relationship with MicroRNA].

    PubMed

    Li, Yao-Yao; Gu, Jian; Yu, Duo-Nan

    2016-04-01

    β-thalassemia is a chronic hemolytic anemia characterized by the reduction or absence of synthesis of β-globin chains because of the β-globin gene mutations. β-thalassemia belongs to the inherited hemoglobin disease, and occurs in some provinces of China, such as in Guangdong, Guangxi, Fujian, its prevalence is about 2%. The treatment of this disease include transfusion, iron chelating agent, hematopoietic stem cell transplantation, splenectomy, induced expression of Fetal Hemoglobin (HbF) and gene therapies. However, the mortality rate of this disease is still higher, thus some new treatments are urgently needed. In recent years, the study was mainly concentrated in 2 aspects: the normal β-globin gene transfer and endogenous γ-globin re-activation. Some studies showed that the expression of miRNAs was dysregulated in β-thalassemia. Some miRNAs could regulate γ-globin at posttranscriptional level, thus, the clarification of relationship between miRNAs and β-thalassemia is expected to provide experimental bases to β-thalassemia therapy. In this review, the induced therapy of γ-globin for β-thalassemia and its relationship with the miRNA are summarized.

  14. Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression

    PubMed Central

    Lee, Tze Yan; Muniandy, Logeswaran; Teh, Lai Kuan; Abdullah, Maha; George, Elizabeth; Sathar, Jameela; Lai, Mei I

    2016-01-01

    Objective: The diverse clinical phenotype of hemoglobin E (HbE)/β-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes. Materials and Methods: A total of 47 HbE/β-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data. Results: The BACH1 expression among the β-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, β-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy. Conclusion: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/β-thalassemia. PMID:26377036

  15. RNA Trans-Splicing Targeting Endogenous β-Globin Pre-Messenger RNA in Human Erythroid Cells.

    PubMed

    Uchida, Naoya; Washington, Kareem N; Mozer, Brian; Platner, Charlotte; Ballantine, Josiah; Skala, Luke P; Raines, Lydia; Shvygin, Anna; Hsieh, Matthew M; Mitchell, Lloyd G; Tisdale, John F

    2017-02-14

    Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34(+) cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.

  16. A Membrane-Bound Vertebrate Globin

    PubMed Central

    Blank, Miriam; Wollberg, Jessica; Gerlach, Frank; Reimann, Katja; Roesner, Anja; Hankeln, Thomas; Fago, Angela; Weber, Roy E.; Burmester, Thorsten

    2011-01-01

    The family of vertebrate globins includes hemoglobin, myoglobin, and other O2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O2 binding with a variable affinity (P50∼1.3–12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein. PMID:21949889

  17. The globins of cyanobacteria and algae.

    PubMed

    Johnson, Eric A; Lecomte, Juliette T J

    2013-01-01

    Approximately, 20 years ago, a haemoglobin gene was identified within the genome of the cyanobacterium Nostoc commune. Haemoglobins have now been confirmed in multiple species of photosynthetic microbes beyond N. commune, and the diversity of these proteins has recently come under increased scrutiny. This chapter summarizes the state of knowledge concerning the phylogeny, physiology and chemistry of globins in cyanobacteria and green algae. Sequence information is by far the best developed and the most rapidly expanding aspect of the field. Structural and ligand-binding properties have been described for just a few proteins. Physiological data are available for even fewer. Although activities such as nitric oxide dioxygenation and oxygen scavenging are strong candidates for cellular function, dedicated studies will be required to complete the story on this intriguing and ancient group of proteins.

  18. A synthetic model of human beta-thalassemia erythropoiesis using CD34+ cells from healthy adult donors.

    PubMed

    Lee, Y Terry; Kim, Ki Soon; Byrnes, Colleen; de Vasconcellos, Jaira F; Noh, Seung-Jae; Rabel, Antoinette; Meier, Emily R; Miller, Jeffery L

    2013-01-01

    Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14-21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.

  19. A Synthetic Model of Human Beta-Thalassemia Erythropoiesis Using CD34+ Cells from Healthy Adult Donors

    PubMed Central

    Byrnes, Colleen; de Vasconcellos, Jaira F.; Noh, Seung-Jae; Rabel, Antoinette; Meier, Emily R.; Miller, Jeffery L.

    2013-01-01

    Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14–21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia. PMID:23861885

  20. Genetic origin of Behçet's disease population in Denizli, Turkey; population genetics data analysis; historical demography and geographical perspectives based on β-globin gene cluster haplotype variation.

    PubMed

    Ozturk, O; Arikan, S; Bahadir, A; Atalay, A; Atalay, E O

    2017-01-01

    In our study, we aimed to investigate the possible genetic drift, relationships, expansion and historical origin based on haplotype frequencies of the β-globin gene cluster of normal and Behçet's disease (BD) population in Denizli, Turkey. We examined blood DNA samples obtained from our DNA bank. The association of population genetic parameters such as haplotypes, diversity, differentiation, Hardy-Weinberg equilibrium and demographic analysis for two populations was performed by Arlequin ver. 3.5. Our results show that both populations have high similarity in genetic parameters in terms of development and expansion based on haplotype diversity through the history. We found that historical levels of gene flow were significantly higher between the two populations. According to historical population, growth parameter of τ values for normal and BD populations dated approximately 42 000 to 38 000 ybp, respectively. In conclusion, historically, two populations show similar genetic parameters and unimodal growth distribution. Our results are consistent with the view that the BD may have occurred in area, independent from Silk Road.

  1. Differentiation of the mRNA transcripts originating from the alpha 1- and alpha 2-globin loci in normals and alpha-thalassemics.

    PubMed

    Liebhaber, S A; Kan, Y W

    1981-08-01

    The alpha-globin polypeptide is encoded by two adjacent genes, alpha 1 and alpha 2. In the normal diploid state (alpha alpha/alpha alpha) all four alpha-globin genes are expressed. Loss or dysfunction of one or more of these genes leads to deficient alpha-globin production and results in alpha-thalassemia. We present a technique to differentially assess the steady-state levels of the alpha 1- and alpha-2-globin messenger RNA (mRNA) transcripts and thus delineate the relative level of expression of the two alpha-globin loci in a variety of alpha-thalassemia states. Only alpha 1 mRNA was produced in the alpha-thalassemia-2 haplotype (-alpha) (one of the two alpha-globin genes deleted from chromosome 16). This confirms previous gene mapping data which demonstrate deletion of the alpha 2 gene. The triple alpha-globin gene haplotype (alpha alpha alpha) is the reciprocal of the alpha-thalassemia-2 haplotype and thus contains an extra alpha 2-globin gene. RNA from this haplotype contained a greater than normal level of alpha 2-relative to alpha 1-globin mRNA. This data implies that the extra alpha 2 gene in the triple alpha-globin haplotype is functional. We detected a relative instability of the alpha 2-globin mRNA encoding the alpha-globin structural mutant Constant Spring. This instability may contribute to the low level of expression of the alpha-Constant Spring protein. In a Chinese patient with nondeletion hemoglobin-H disease (- -/alpha alpha T) (both alpha-globin genes are present but not fully functional) a normal ratio was maintained between the levels of alpha 1- and alpha 2-globin mRNA, implying that mRNA production from both alpha-globin genes is suppressed in a balanced manner. These observations extended previous findings concerning the structural rearrangements in the deletion types of alpha-thalassemia and the pathophysiology of two nondeletion variants.

  2. VNTR internal structure mapping at the {alpha}-globin 3{prime}HVR locus reveals a hierachy of related lineages in oceania

    SciTech Connect

    Martinson, J.J.; Clegg, J.B.; Boyce, A.J.

    1994-09-01

    Analysis of the {alpha}-globin gene complex in Oceania has revealed many different rearrangements which remove one of the adult globin genes. Frequencies of these deletion chromosomes are elevated by malarial resistance conferred by the resulting {alpha}-thalassaemia. One particular deletion chromosome, designated -{alpha}{sup 3.7}III, is found at high levels in Melanesia and Polynesia: RFLP haplotype analysis shows that this deletion is always found on chromosomes bearing the IIIa haplotype and is likely to be the product of one single rearrangement event. A subset of the -{alpha}{sup 3.7}III chromosomes carries a more recent mutation which generates the haemoglobin variant HbJ{sup Tongariki}. We have characterized the allelic variation at the 3{prime}HVR VNTR locus located 6 kb from the globin genes in each of these groups of chromosomes. We have determined the internal structure of these alleles by RFLP mapping of PCR-amplified DNA: within each group, the allelic diversity results from the insertion and/or deletion of small {open_quotes}motifs{close_quotes} of up to 6 adjacent repeats. Mapping of 3{prime}HVR alleles associated with other haplotypes reveals that these are composed of repeat arrays that are substantially different to those derived from IIIa chromosomes, indicating that interchromosomal recombination between heterologous haplotypes does not account for any of the diversity seen to date. We have recently shown that allelic size variation at the two VNTR loci flanking the {alpha}-globin complex is very closely linked to the haplotypes known to be present at this locus. Here we show that, within a haplotype, VNTR alleles are very closely related to each other on the basis of internal structure and demonstrate that intrachromosomal mutation processes involving small numbers of tandem repeats are the main cause of variation at this locus.

  3. Globin domain interactions control heme pocket conformation and oligomerization of globin coupled sensors.

    PubMed

    Rivera, Shannon; Burns, Justin L; Vansuch, Gregory E; Chica, Bryant; Weinert, Emily E

    2016-11-01

    Globin coupled sensors (GCS) are O2-sensing proteins used by bacteria to monitor the surrounding gaseous environment. To investigate the biphasic O2 dissociation kinetics observed for full-length GCS proteins, isolated globin domains from Pectobacterium carotovorum ssp. carotovorum (PccGlobin), and Bordetella pertussis (BpeGlobin), have been characterized. PccGlobin is found to be dimeric, while BpeGlobin is monomeric, indicating key differences in the globin domain dimer interface. Through characterization of wild type globin domains and globin variants with mutations at the dimer interface and within the distal pocket, dimerization of the globin domain is demonstrated to correlate with biphasic dissociation kinetics. Furthermore, a distal pocket tyrosine is identified as the primary hydrogen bond donor, while a secondary hydrogen bond donor within the distal heme pocket is involved in conformation(s) that lead to the second O2 dissociation rate. These findings highlight the role of the globin dimer interface in controlling properties of both the heme pocket and full-length GCS proteins.

  4. Hemoglobin genetics: recent contributions of GWAS and gene editing.

    PubMed

    Smith, Elenoe C; Orkin, Stuart H

    2016-10-01

    The β-hemoglobinopathies are inherited disorders resulting from altered coding potential or expression of the adult β-globin gene. Impaired expression of β-globin reduces adult hemoglobin (α2β2) production, the hallmark of β-thalassemia. A single-base mutation at codon 6 leads to formation of HbS (α2β(S)2) and sickle cell disease. While the basis of these diseases is known, therapy remains largely supportive. Bone marrow transplantation is the only curative therapy. Patients with elevated levels of fetal hemoglobin (HbF, α2γ2) as adults exhibit reduced symptoms and enhanced survival. The β-globin gene locus is a paradigm of cell- and developmental stage-specific regulation. Although the principal erythroid cell transcription factors are known, mechanisms responsible for silencing of the γ-globin gene were obscure until application of genome-wide association studies (GWAS). Here, we review findings in the field. GWAS identified BCL11A as a candidate negative regulator of γ-globin expression. Subsequent studies have established BCL11A as a quantitative repressor. GWAS-related single-nucleotide polymorphisms lie within an essential erythroid enhancer of the BCL11A gene. Disruption of a discrete region within the enhancer reduces BCL11A expression and induces HbF expression, providing the basis for gene therapy using gene editing tools. A recently identified, second silencing factor, leukemia/lymphoma-related factor/Pokemon, shares features with BCL11A, including interaction with the nucleosome remodeling deacetylase repressive complex. These findings suggest involvement of a common pathway for HbF silencing. In addition, we discuss other factors that may be involved in γ-globin gene silencing and their potential manipulation for therapeutic benefit in treating the β-hemoglobinopathies.

  5. Brain globins in physiology and pathology

    PubMed Central

    Xie, Luo-kun; Yang, Shao-hua

    2016-01-01

    Globins are globular proteins for either transport or storage of oxygen which are critical for cellular metabolism. Four globins have been identified in rodent and human brains. Among them, neuroglobin, cytoglobin and hemoglobin chains are constitutively expressed in normal brain, while myoglobin is only expressed in some neurological disorders. Studies on the molecular structure, expression and functional features of these brain globins indicated that they may play crucial roles in maintenance of neural cell survival and activity, including neurons and astrocytes. Their regulation in neurological disorders may help thoroughly understand initiation and progression of ischemia, Alzheimer's disease and glioma, etc. Elucidation of the brain globin functions might remarkably improve medical strategies that sustain neurological homeostasis and treat neurological diseases. Here the expression pattern and functions of brain globins and their involvement in neurological disorders are reviewed. PMID:27867483

  6. Globin-like proteins in Caenorhabditis elegans: in vivo localization, ligand binding and structural properties

    PubMed Central

    2010-01-01

    Background The genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression. Results We expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 → Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three α-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding. Conclusion The presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes. PMID:20361867

  7. Globin-like proteins in Caenorhabditis elegans: in vivo localization, ligand binding and structural properties.

    PubMed

    Geuens, Eva; Hoogewijs, David; Nardini, Marco; Vinck, Evi; Pesce, Alessandra; Kiger, Laurent; Fago, Angela; Tilleman, Lesley; De Henau, Sasha; Marden, Michael C; Weber, Roy E; Van Doorslaer, Sabine; Vanfleteren, Jacques; Moens, Luc; Bolognesi, Martino; Dewilde, Sylvia

    2010-04-02

    The genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression. We expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 --> Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three alpha-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding. The presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes.

  8. Fine structure genetic analysis of a beta-globin promoter.

    PubMed

    Myers, R M; Tilly, K; Maniatis, T

    1986-05-02

    A novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse beta-major globin gene. The effects of these promoter substitutions on transcription were determined by transfecting the cloned mutant genes into HeLa cells on plasmids containing an SV40 transcription enhancer, and measuring the levels of correctly initiated beta-globin transcripts after 2 days. Mutations in three regions of the promoter resulted in a significant decrease in the level of transcription: (i) the CACCC box, located between -87 and -95, (ii) the CCAAT box, located between -72 and -77, and (iii) the TATA box, located between -26 and -30 relative to the start site of transcription. In contrast, two different mutations in nucleotides immediately upstream from the CCAAT box resulted in a 3- to 3.5-fold increase in transcription. With two minor exceptions, single base substitutions in all other regions of the promoter had no effect on transcription. These results precisely delineate the cis-acting sequences required for accurate and efficient initiation of beta-globin transcription, and they establish a general approach for the fine structure genetic analysis of eukaryotic regulatory sequences.

  9. [Beta globin haplotypes in hemoglobin S carriers in Colombia].

    PubMed

    Durán, Claudia Liliana; Morales, Olga Lucía; Echeverri, Sandra Johanna; Isaza, Mario

    2012-01-01

    The hemoglobin S (HbS) mutation is accompanied by other mutations in the region of chromosome 11 known as "beta globin cluster". The pattern of combination of these polymorphisms giving rise to the haplotypes that co-inherit the HbS mutation, are called haplotypes bs, and are of great epidemiological and clinical significance. The frequencies of major haplotypes associated with S beta-globin gene was determined in Colombian patients heterozygous for hemoglobin S. As part of the national neonatal screening program at Clínica Colsanitas, located in major cities of Colombia, nearly 1,200 children from different areas of the country were examined for hemoglobinopathies. The sickle cell trait was identified as the most common. S beta-globin gene haplotypes were determined by PCR and restriction enzymes in 33 children with AS hemoglobin electrophoretic patterns (carrier state). In addition, electrophoretic patterns of hemoglobin, fetal hemoglobin levels and hematologic parameters of each individual were identified. The most frequent haplotypes in Colombia were the Bantú haplotype (36.4 %), followed by Senegal (30.3 %), Benin (21.2 %) and Cameroon (12.1 %) haplotypes. Hemoglobin electrophoresis confirmed the AS phenotype in all patients, and fetal hemoglobin levels below 1%. Other hematological parameters were normal in all cases. The HbS haplotypes found more frequently in the sample were of African origin, and their distribution varied according to the place of origin of the individual. The most frequent corresponded to the Bantu haplotype.

  10. Effect of β‑globin MAR characteristic elements on transgene expression.

    PubMed

    Li, Qin; Dong, Weihua; Wang, Tianyun; Liu, Zhonghe; Wang, Fang; Wang, Xiaoyin; Zhao, Chunpeng; Zhang, Junhe; Wang, Li

    2013-06-01

    The aim of the present study was to investigate the effect of the characteristic elements of matrix attachment region (MAR) on transgene expression. Human β‑globin MAR was obtained by PCR amplification. A splicing MAR fragment containing all the characteristic elements of β‑globin MAR was artificially synthesized and then cloned into the eukaryotic expression vector. Following digestion and sequence identification, we transfected Chinese hamster ovary (CHO) cells with the two vectors, and then screened for the transformation of stable cells. The transgene expression level was analyzed by ELISA, and the copy numbers of the CAT gene were analyzed by real‑time fluorescent quantitative PCR. β‑globin MAR enhanced CAT reporter gene expression by 2.1489‑fold, whereas the β‑globin MAR characteristic elements did not enhance this expression. The real‑time fluorescent quantitative PCR analysis demonstrated that the relative copy numbers of the CAT gene of the β‑globin MAR expression vector were 1.2‑fold higher compared with those of the non‑MAR expression vector. MAR was able to improve the transgene expression level to a certain extent. The MAR characteristic elements did not improve the transgene expression alone. The transgenic expression levels were not linear with the transgene copy number; however, the enhancement of transgenic expression was relative to the increase in the gene copy number.

  11. Globin chain synthesis ratios in sideroblastic anaemia.

    PubMed

    Peters, R E; May, A; Jacobs, A

    1983-02-01

    Globin synthesis ratios were measured on reticulocytes from nine patients with primary acquired sideroblastic anaemia (SA), four patients with hereditary or congenital SA, two patients with secondary acquired SA and three patients with iron deficiency (ID). Ten of the samples from patients with SA and all the samples from patients with ID had normal ratios. Samples from three patients had significantly abnormal ratios, one from a patient with SA and acquired Hb H disease (alpha/beta 0 X 26), one from a patient with secondary acquired SA (alpha/beta 0 X 88), and one from a patient who went on to develop acute myeloblastic leukaemia (alpha/beta 1 X 36). Globin synthesis was stimulated by 100 microM haem similarly in normal, SA and ID reticulocytes. Any limitation of globin synthesis in SA and ID is therefore not easily reversible by adding haem. Inhibition of haem synthesis in nonsideroblastic reticulocytes using 4 mM isonicotinic acid hydrazide for 1 h incubation affected neither total globin synthesis nor the alpha/beta ratio. These results contradict the view that decreased haem synthesis decreases globin chain synthesis and decreases the alpha/beta globin chain synthesis ratios in human reticulocytes. Previously reported findings that haem could reverse globin chain synthesis inhibition in SA were good evidence for a primary deficiency of haem synthesis in the erythroblasts of these patients. Our inability to substantiate these findings emphasizes the need for a re-evaluation of the aetiology of sideroblastic anaemia.

  12. Non-methylated CpG-rich islands at the human alpha-globin locus: implications for evolution of the alpha-globin pseudogene.

    PubMed Central

    Bird, A P; Taggart, M H; Nicholls, R D; Higgs, D R

    1987-01-01

    We have analysed CpG frequency and CpG methylation across part of the human alpha-globin locus. Clusters of CpG at the alpha 1 and alpha 2 genes resemble the 'HpaII tiny fragment (HTF) islands' that are characteristic of mammalian 'housekeeping' genes: CpG frequency is not suppressed; testable CpGs are not methylated in DNA from erythroid or nonerythroid tissues, although flanking CpGs are methylated; CpG clusters are approximately 1.5 kb long and extend both upstream and downstream of the alpha-globin transcription start site. These features are not found at genes of the beta-globin locus. The alpha-globin pseudogene (psi alpha 1) is highly homologous to the alpha 2 and alpha 1 genes, but it lacks an HTF island. Sequence comparison shows that a high proportion of CpGs in the alpha 2 gene are substituted by TpG or CpA in the pseudogene. This strongly suggests that an ancestral HTF island at the pseudogene became methylated in the germline, and was lost due to the mutability of 5-methylcytosine. Images Fig. 2. Fig. 3. Fig. 5. PMID:3595568

  13. Recombinant AAV2-mediated β-globin expression in human fetal hematopoietic cells from the aborted fetuses with β-thalassemia major.

    PubMed

    Tian, Jing; Wang, Feng; Xue, Jin-Feng; Zhao, Fei; Song, Liu-Jiang; Tan, Meng-Qun

    2011-06-01

    Genetic correction of autologous hematopoietic stem cells has been proposed as an attractive treatment method for β-thalassemia. Our previous study has shown that recombinant adeno-associated virus 2 (rAAV2) efficiently transduces human fetal liver hematopoietic cells, and mediates the expression of the human β-globin gene in vivo. In this study, we investigated whether rAAV2 could also mediate the expression of normal β-globin gene in human hematopoietic cells from β-thalassemia patients. Human hematopoietic cells were isolated from aborted β-thalassemia major fetuses, transduced with rAAV2-β-globin, and then transplanted into nude mice. We found that rAAV2-β-globin transduced human fetal hematopoietic cells, as determined by allele-specific PCR analysis. Furthermore, β-globin transgene expression was detected in human hematopoietic cells up to 70 days post-transplantation in the recipient mice. High-pressure liquid chromatography analysis showed that human β-globin expression levels increased significantly compared with control, as indicated by a 1.2-2.8-fold increase in the ratio of β/α-globin chain. These novel data demonstrate that rAAV2 can transduce and mediate the normal β-globin gene expression in fetal hematopoietic cells from β-thalassemia patients. Our findings further support the potential use of rAAV-based gene therapy in the treatment of human β-thalassemia.

  14. β-globin-expressing definitive erythroid progenitor cells generated from embryonic and induced pluripotent stem cell-derived sacs

    PubMed Central

    Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J.; Winkler, Thomas; Tisdale, John

    2016-01-01

    Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ε-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ~120 erythroid cells per single ES cell. Both primitive (ε-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of GPA or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. PMID:26866725

  15. Characterization of Hydroxymethylation Patterns in the Promoter of β-globin Clusters in Murine Fetal Livers.

    PubMed

    Zhou, Shasha; Li, Liantao; Yan, Zhonghai; Li, Wenxiu; Shen, Yihang

    2015-02-27

    DNA methylation of 5-methylcytosine (5mC) is a key epigenetic regulator in mammals; the dynamic balance between methylation and demethylation affects the transcriptional activity of β-globin. However, the dynamic cytosine methylation of β-globin in vivo during the different stages of embryogenesis and in developing liver has not been fully established. 5-Hydroxymethylcytosine (5hmC) is a newly discovered epigenetic modification that is presumably generated by oxidation of 5mC by the ten-eleven translocation (TET) family and it has not been fully identified in β-globin clusters. Here, we determined the 5hmC modifications in the promoter of murine β-globin from fetal livers during normal embryonic development with the methods of bisulfite (BS) and oxidative bisulfite (oxBS)-based pyrosequencing techniques, with the combination of methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR). The results characterized the 5hmC modification at the CpG sites of -426, -388, and -151 of ɛ(y) promoters and -50 and -487 CpG of β(h1) from transcriptional start sites from E15.5 and E17.5 livers, while 5hmC modification was not observed in the adult β-globin promoters. These observations were validated by the induction of TET transcription after being treated with a potent demethylating agent 5-azacytidine, and TET-mediated hydroxymethylation of ɛ(y) and β(h1) from E13.5 livers was also confirmed in our study. These results suggested the 5hmC modification in promoters of ɛ(y) and β(h1) and indicated that the 5hmC modification is essential for the β-globin switching before the embryonic globin reactivation.

  16. Activator-mediated Recruitment of the MLL2 Methyltransferase Complex to the β-globin Locus

    PubMed Central

    Demers, Celina; Chaturvedi, Chandra-Prakash; Ranish, Jeffrey A.; Juban, Gaetan; Lai, Patrick; Morle, Francois; Aebersold, Ruedi; Dilworth, F. Jeffrey; Groudine, Mark; Brand, Marjorie

    2007-01-01

    Summary MLL-containing complexes methylate histone H3 at lysine 4 (H3K4) and have been implicated in the regulation of transcription. However, it is unclear how MLL complexes are targeted to specific gene loci. Here, we show that the MLL2 complex associates with the hematopoietic activator NF-E2 in erythroid cells and is important for H3K4 trimethylation and maximal levels of transcription at the β-globin locus. Furthermore, recruitment of the MLL2 complex to the β-globin locus is dependent upon NF-E2 and coincides spatio-temporally with NF-E2 binding during erythroid differentiation. Thus a DNA-bound activator is important initially for guiding MLL2 to a particular genomic location. Interestingly, while the MLL2-associated subunit Ash2L is restricted to the β-globin locus control region 38 kb upstream of the βmaj-globin gene, the MLL2 protein spreads across the β-globin locus, suggesting a previously undefined mechanism by which an activator influences transcription and H3K4 trimethylation at a distance. PMID:17707229

  17. Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds*

    PubMed Central

    Grispo, Michael T.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.

    2012-01-01

    The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. PMID:22962007

  18. Gene duplication and the evolution of hemoglobin isoform differentiation in birds.

    PubMed

    Grispo, Michael T; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E; Storz, Jay F

    2012-11-02

    The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the α(A)-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the α(D)-globin gene). The α(D)-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O(2) affinity in the presence of allosteric effectors such as organic phosphates and Cl(-) ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O(2) affinity stems primarily from changes in the O(2) association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the α(D)-globin gene that is shared with the embryonic α-like globin gene.

  19. Common 5' beta-globin RFLP haplotypes harbour a surprising level of ancestral sequence mosaicism.

    PubMed

    Webster, Matthew T; Clegg, John B; Harding, Rosalind M

    2003-07-01

    Blocks of linkage disequilibrium (LD) in the human genome represent segments of ancestral chromosomes. To investigate the relationship between LD and genealogy, we analysed diversity associated with restriction fragment length polymorphism (RFLP) haplotypes of the 5' beta-globin gene complex. Genealogical analyses were based on sequence alleles that spanned a 12.2-kb interval, covering 3.1 kb around the psibeta gene and 6.2 kb of the delta-globin gene and its 5' flanking sequence known as the R/T region. Diversity was sampled from a Kenyan Luo population where recent malarial selection has contributed to substantial LD. A single common sequence allele spanning the 12.2-kb interval exclusively identified the ancestral chromosome bearing the "Bantu" beta(s) (sickle-cell) RFLP haplotype. Other common 5' RFLP haplotypes comprised interspersed segments from multiple ancestral chromosomes. Nucleotide diversity was similar between psibeta and R/T-delta-globin but was non-uniformly distributed within the R/T-delta-globin region. High diversity associated with the 5' R/T identified two ancestral lineages that probably date back more than 2 million years. Within this genealogy, variation has been introduced into the 3' R/T by gene conversion from other ancestral chromosomes. Diversity in delta-globin was found to lead through parts of the main genealogy but to coalesce in a more recent ancestor. The well-known recombination hotspot is clearly restricted to the region 3' of delta-globin. Our analyses show that, whereas one common haplotype in a block of high LD represents a long segment from a single ancestral chromosome, others are mosaics of short segments from multiple ancestors related in genealogies of unsuspected complexity.

  20. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells.

    PubMed

    Aimola, Idowu A; Inuwa, Hajiya M; Nok, Andrew J; Mamman, Aisha I; Bieker, James J

    2016-04-05

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer.

  1. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

    PubMed Central

    Aimola, Idowu A.; Inuwa, Hajiya M.; Nok, Andrew J.; Mamman, Aisha I.; Bieker, James J.

    2017-01-01

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24 h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ9 desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ9-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer PMID:26879870

  2. Bone marrow and peripheral blood globin chain synthesis in sickle cell beta zero thalassaemia.

    PubMed Central

    Costa, F F; Zago, M A

    1986-01-01

    A similar imbalance of globin chain synthesis, with low non-alpha/alpha ratios, was shown in peripheral blood and in bone marrow of compound heterozygotes for both the Hb S and beta zero thalassaemia genes (S/beta zero thalassaemia). Previous purification of whole cell globin obtained from the bone marrow did not change the non-alpha/alpha ratio. The mean non-alpha/alpha ratios were 0.57 +/- 0.13 (means +/- SD) for the peripheral blood of 12 patients, 0.52 +/- 0.10 for five patients using bone marrow globin purified on Sephadex G100, and 0.55 +/- 0.16 for the unfiltered bone marrow globin of five patients. The data show that patients with S/beta zero thalassaemia have a similar beta chain deficiency in reticulocytes and in bone marrow cells, provided whole cell globin is used which avoids the removal of the free alpha chains. The non-alpha/alpha ratios in the peripheral blood of an S/beta zero thalassaemia patient and a beta thalassaemia heterozygote from the same family were compared in seven families and no significant difference was found. PMID:3723554

  3. Beta-globin gene haplotypes among cameroonians and review of the global distribution: is there a case for a single sickle mutation origin in Africa?

    PubMed

    Bitoungui, Valentina J Ngo; Pule, Gift D; Hanchard, Neil; Ngogang, Jeanne; Wonkam, Ambroise

    2015-03-01

    Studies of hemoglobin S haplotypes in African subpopulations have potential implications for patient care and our understanding of genetic factors that have shaped the prevalence of sickle cell disease (SCD). We evaluated HBB gene cluster haplotypes in SCD patients from Cameroon, and reviewed the literature for a global distribution. We reviewed medical records to obtain pertinent socio-demographic and clinical features for 610 Cameroonian SCD patients, including hemoglobin electrophoresis and full blood counts. RFLP-PCR was used to determine the HBB gene haplotype on 1082 chromosomes. A systematic review of the current literature was undertaken to catalogue HBB haplotype frequencies in SCD populations around the world. Benin (74%; n = 799) and Cameroon (19%; n = 207) were the most prevalent haplotypes observed among Cameroonian patients. There was no significant association between HBB haplotypes and clinical life events, anthropometric measures, hematological parameters, or fetal hemoglobin (HbF) levels. The literature review of the global haplotype distributions was consistent with known historical migrations of the people of Africa. Previously reported data from Sudan showed a distinctly unusual pattern; all four classical haplotypes were reported, with an exceptionally high proportion of the Senegal, Cameroon, and atypical haplotypes. We did not observe any significant associations between HBB haplotype and SCD disease course in this cohort. Taken together, the data from Cameroon and from the wider literature suggest that a careful reassessment of African HBB haplotypes may shed further light on the evolutionary dynamics of the sickle allele, which could suggest a single origin of the sickle mutation.

  4. The population genetics of the alpha-2 globin locus of orangutans (Pongo pygmaeus).

    PubMed

    Steiper, Michael E; Wolfe, Nathan D; Karesh, William B; Kilbourn, Annelisa M; Bosi, Edwin J; Ruvolo, Maryellen

    2005-03-01

    In this study, the molecular population genetics of the orangutan's alpha-2 globin (HBA2) gene were investigated in order to test for the action of natural selection. Haplotypes from 28 orangutan chromosomes were collected from a 1.46-kilobase region of the alpha-2 globin locus. While many aspects of the data were consistent with neutrality, the observed heterogeneous distribution of polymorphisms was inconsistent with neutral expectations. Furthermore, a single amino acid variant, found in both the Bornean and the Sumatran orangutan subspecies, was associated with different alternative synonymous variants in each subspecies, suggesting that the allele may have spread separately through the two subspecies after two distinct origination events. This variant is not in Hardy-Weinberg equilibrium (HWE). These observations are consistent with neutral models that incorporate population structure and models that invoke selection. The orangutan Plasmodium parasite is a plausible selective agent that may underlie the variation at alpha-2 globin in orangutans.

  5. beta (+)-Thalassaemia in the Po river delta region (northern Italy): genotype and beta globin synthesis.

    PubMed Central

    Del Senno, L; Pirastu, M; Barbieri, R; Bernardi, F; Buzzoni, D; Marchetti, G; Perrotta, C; Vullo, C; Kan, Y W; Conconi, F

    1985-01-01

    Six beta(+)-thalassaemic patients from the Po river delta region have been studied. Using synthetic oligonucleotides as specific hybridisation probes, the beta(+) IVS I mutation (G----A at position 108) was demonstrated. This lesion and the enzyme polymorphism pattern in the subjects examined are the same as have been described for other Mediterranean beta(+)-thalassaemias. Antenatal diagnosis through DNA analysis of beta(+)-thalassaemia is therefore possible. The production of beta globin in a beta(+), homozygote and in a beta (+), beta(0) 39 (nonsense mutation at codon 39) double heterozygote is approximately 20% and 10% respectively of total non-alpha globin synthesis. Despite some overlapping of the results, similar beta globin synthesis levels have been obtained in 43 beta(+)-thalassaemia patients. This suggests that in the Po river delta region the most common thalassaemic genes are beta(0) 39 and beta(+) IVS I. Images PMID:2580095

  6. Genetic relationship of a Mexican Afromestizo population through the analysis of the 3' haplotype of the beta globin gene in betaA chromosomes.

    PubMed

    Magaña, María Teresa; Perea, F Javier; González, Juan Ramón; Ibarra, Bertha

    2007-01-01

    We analyzed 112 beta(A) chromosomes from the Costa Chica region, with the aim of determining the 3' haplotype (3'Hp) in Afromestizo individuals and its relationship with the reported populations. Thirty polymorphic sites were identified by sequencing and two by restriction fragment length polymorphisms. Genetic variability, genetic distances and neutrality tests were performed with the computer program Arlequin 3.0. Three groups were constructed, which we named 3 kb-Hp, 330 bp-Hp and 2.67 kb-Hp with 32, 15 and 17 polymorphic sites respectively. In 3 kb-Hp, 34 different 3' haplotypes (14 of them new) were found; the three most common were 7B1 (17.8%), 7A1 (17.0%) and 1C1 (14.3%). 330 bp-Hp revealed 18 different allelic sequences; the most frequent were the 1 (AT(9)T(5), 22.3%), 2 (AT(8)T(5), 20.5%) and 3 (AT(7)T(7), 20.5%). 2.67 kb-Hp displayed 14 distinct haplotypes, with B1 (30.3%), A1 (28.6%) and C1 (21.4%) having the highest frequencies. The gene diversity of the Costa Chica population was only significantly different to Gambia. In the genetic distances, the p values were not significant for Vanuatu, Sumatra and Central African Republic. The neutrality tests showed that the patterns of diversity in the Costa Chica population deviate significantly from the expectations of the standard neutral model. This is the first work performed in Mexico in which the extended 3'Hp was analyzed in beta(A) chromosomes. The study showed clearly the presence of African and Asian genes in the Costa Chica population.

  7. Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous b1/b2 globin gene deletion) mice

    PubMed Central

    Gelderman, Monique P.; Baek, Jin Hyen; Yalamanoglu, Ayla; Puglia, Michele; Vallelian, Florence; Burla, Bo; Vostal, Jaroslav; Schaer, Dominik J.; Buehler, Paul W.

    2015-01-01

    Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbbth3/+ mice. Mice with the Hbbth3/+ phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbbth3/+ mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice. PMID:25616571

  8. Beta-Globin Gene Haplotypes Among Cameroonians and Review of the Global Distribution: Is There a Case for a Single Sickle Mutation Origin in Africa?

    PubMed Central

    Bitoungui, Valentina J. Ngo; Pule, Gift D.; Hanchard, Neil; Ngogang, Jeanne

    2015-01-01

    Abstract Studies of hemoglobin S haplotypes in African subpopulations have potential implications for patient care and our understanding of genetic factors that have shaped the prevalence of sickle cell disease (SCD). We evaluated HBB gene cluster haplotypes in SCD patients from Cameroon, and reviewed the literature for a global distribution. We reviewed medical records to obtain pertinent socio-demographic and clinical features for 610 Cameroonian SCD patients, including hemoglobin electrophoresis and full blood counts. RFLP-PCR was used to determine the HBB gene haplotype on 1082 chromosomes. A systematic review of the current literature was undertaken to catalogue HBB haplotype frequencies in SCD populations around the world. Benin (74%; n=799) and Cameroon (19%; n=207) were the most prevalent haplotypes observed among Cameroonian patients. There was no significant association between HBB haplotypes and clinical life events, anthropometric measures, hematological parameters, or fetal hemoglobin (HbF) levels. The literature review of the global haplotype distributions was consistent with known historical migrations of the people of Africa. Previously reported data from Sudan showed a distinctly unusual pattern; all four classical haplotypes were reported, with an exceptionally high proportion of the Senegal, Cameroon, and atypical haplotypes. We did not observe any significant associations between HBB haplotype and SCD disease course in this cohort. Taken together, the data from Cameroon and from the wider literature suggest that a careful reassessment of African HBB haplotypes may shed further light on the evolutionary dynamics of the sickle allele, which could suggest a single origin of the sickle mutation. PMID:25748438

  9. Recent trends in the gene therapy of β-thalassemia

    PubMed Central

    Finotti, Alessia; Breda, Laura; Lederer, Carsten W; Bianchi, Nicoletta; Zuccato, Cristina; Kleanthous, Marina; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases. PMID:25737641

  10. Recent trends in the gene therapy of β-thalassemia.

    PubMed

    Finotti, Alessia; Breda, Laura; Lederer, Carsten W; Bianchi, Nicoletta; Zuccato, Cristina; Kleanthous, Marina; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases.

  11. Characterization of Clinical and Laboratory Profiles of the Deletional α2-Globin Gene Polyadenylation Signal Sequence (AATAAA > AATA- -) in an Indian Population.

    PubMed

    Deshpande, Prashant; Kamalanathan, Neelagandan; Sampath, Eswari; George, Biju; Shaji, Ramachandran V; Edison, Eunice S

    2015-01-01

    α-Thalassemia (α-thal) is characterized by large deletions involving the variable regions of α2 and/or α1 genes. Nondeletional mutations and polyadenylation (polyA) signal sequence motif mutations are less common. In this retrospective study, we describe a fragment length analysis-based polymerase chain reaction (PCR) assay for screening the T(Indian) (AATAAA > AATA- -; HBA2: c.*93_*94delAA) polyA signal deletion along with its clinical and laboratory presentation in 21 patients. Most of the patients were diagnosed in early adulthood with a clinical presentation ranging from asymptomatic in the heterozygous state to severe Hb H disease with a prominent hemolytic component in the homozygous state. On genetic analysis, 14 patients were found to be homozygotes, five were compound heterozygotes and two were heterozygotes. Thus, the T(Indian) polyA signal deletion is common in the Indian population and should be screened for in patients with nondeletional α-thal mutations.

  12. A New Intergenic α-Globin Deletion (α-αΔ125) Found in a Kabyle Population.

    PubMed

    Singh, Amrathlal Rabbind; Lacan, Philippe; Cadet, Estelle; Bignet, Patricia; Dumesnil, Cécile; Vannier, Jean-Pierre; Joly, Philippe; Rochette, Jacques

    2016-01-01

    We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a β-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact.

  13. The relative cellular levels of CP2a and CP2b potentiates erythroid cell-specific expression of the {alpha}-globin gene by regulating the nuclear localization of CP2c

    SciTech Connect

    Chae, Ji Hyung; Kang, Ho Chul; Kim, Chul Geun

    2009-03-20

    CP2b activates {alpha}-globin expression in an erythroid cell-specific manner, through interaction with CP2c and PIAS1. Although CP2a is identical to CP2b except for lacking an exon encoding additional 36 amino acids and has the intrinsic DNA binding and transactivation properties, it does not exert any role in {alpha}-globin expression. Investigation of subcellular localization of exogenous CP2 proteins revealed that CP2a and CP2b were exclusively localized in the cytosol and nucleus, respectively. The CP2b-specific exon was in charge of the nuclear localization of CP2b. Interestingly, subcellular localization of CP2c was either in the nucleus or cytosol depending on the relative level of CP2a and CP2b although CP2c intrinsically localized in the cytosol in the absence of CP2a/CP2b. Finally, dramatic increment of hemoglobin expression was correlated with nuclear translocation of CP2c during MEL cell differentiation. Our data suggest that CP2b potentiate erythroid cell-specific {alpha}-globin expression by recruiting CP2c into the nucleus.

  14. The role of transcriptional activator GATA-1 at human β-globin HS2

    PubMed Central

    Cho, Youngran; Song, Sang-Hyun; Lee, Jong Joo; Choi, Narae; Kim, Chul Geun; Dean, Ann; Kim, AeRi

    2008-01-01

    GATA-1 is an erythroid activator that binds β-globin gene promoters and DNase I hypersensitive sites (HSs) of the β-globin locus control region (LCR). We investigated the direct role of GATA-1 interaction at the LCR HS2 enhancer by mutating its binding sites within minichromosomes in erythroid cells. Loss of GATA-1 in HS2 did not compromise interaction of NF-E2, a second activator that binds to HS2, nor was DNase I hypersensitivity at HS2 or the promoter of a linked ε-globin gene altered. Reduction of NF-E2 using RNAi confirmed the overall importance of this activator in establishing LCR HSs. However, recruitment of the histone acetyltransferase CBP and RNA pol II to HS2 was diminished by GATA-1 loss. Transcription of ε-globin was severely compromised with loss of RNA pol II from the transcription start site and reduction of H3 acetylation and H3K4 di- and tri-methylation in coding sequences. In contrast, widespread detection of H3K4 mono-methylation was unaffected by loss of GATA-1 in HS2. These results support the idea that GATA-1 interaction in HS2 has a prominent and direct role in co-activator and pol II recruitment conferring active histone tail modifications and transcription activation to a target gene but that it does not, by itself, play a major role in establishing DNase I hypersensitivity. PMID:18586828

  15. Globin synthesis in fractionated Normoblasts of beta-thalassemia heterozygotes.

    PubMed Central

    Wood, W G; Stamatoyannopoulos, G

    1975-01-01

    Globin chain synthesis was examined in erythroid cells of increasing maturity, fractionated from the whole bone marrow of beta-thalassemia heterozygotes by a density gradient centrifugation procedure. In experiments using total cell "globin," a gradient of alpha/beta chain ratios was observed, increasing with erythroid cell maturation from unity in the basophilic cells up to 2.0 in reticulocytes. Gel filtration of the lysates from these marrow fractions revealed the presence of free alpha chains even in the most immature cells, the amount of which increased with erythroid cell age; the total alpha/beta ratio derived from gel filtration experiments showed a gradient similar to that observed in the total globin experiments. However, the alpha/beta ratio of the hemoglobin fraction obtained by gel filtration remained constant throughout maturation at an average of 0.65. This latter finding is incompatible with balanced synthesis at any stage of red cell development and excludes the possibility that total beta chain production is higher in the early cells than in the later cells or that alpha chain production in the early cells is reduced to the level of beta chain synthesis. Furthermore, in a Hb S/beta-thalassemia marrow examined, the beta A/beta S ratio remained constant throughout maturation while the alpha/non-alpha ratio showed an increase like that observed in the simple beta-thalassemia heterozygotes. This argues strongly against increased synthesis from either the thalassemic or nonthalassemic beta chain gene being responsible for the balanced synthesis in the immature cells. These findings lead us to suggest that, in beta-thalassemia heterozygotes, a large alpha chain pool is present throughout erythroid cell maturation and that the observed increase in alpha/beta ratios is a function of the ability of those cells to degrade the excess alpha chains. Images PMID:1167870

  16. Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

    PubMed

    Li, Hu; Xie, Wensheng; Gore, Elizabeth R; Montoute, Monica N; Bee, Weilin Tiger; Zappacosta, Francesca; Zeng, Xin; Wu, Zining; Kallal, Lorena; Ames, Robert S; Pope, Andrew J; Benowitz, Andrew; Erickson-Miller, Connie L

    2013-12-01

    Sickle cell anemia (SCA) is a genetic disorder of the β-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.

  17. SIRT1 deacetylates SATB1 to facilitate MAR HS2-MAR ε interaction and promote ε-globin expression.

    PubMed

    Xue, Zheng; Lv, Xiang; Song, Wei; Wang, Xing; Zhao, Guang-Nian; Wang, Wen-Tian; Xiong, Jian; Mao, Bei-Bei; Yu, Wei; Yang, Ben; Wu, Jie; Zhou, Li-Quan; Hao, De-Long; Dong, Wen-Ji; Liu, De-Pei; Liang, Chih-Chuan

    2012-06-01

    The higher order chromatin structure has recently been revealed as a critical new layer of gene transcriptional control. Changes in higher order chromatin structures were shown to correlate with the availability of transcriptional factors and/or MAR (matrix attachment region) binding proteins, which tether genomic DNA to the nuclear matrix. How posttranslational modification to these protein organizers may affect higher order chromatin structure still pending experimental investigation. The type III histone deacetylase silent mating type information regulator 2, S. cerevisiae, homolog 1 (SIRT1) participates in many physiological processes through targeting both histone and transcriptional factors. We show that MAR binding protein SATB1, which mediates chromatin looping in cytokine, MHC-I and β-globin gene loci, as a new type of SIRT1 substrate. SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MAR(HS2) and MAR(ε). We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation. Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

  18. Nutritive value of globin-amino acid and complementary globin-cereal mixtures.

    PubMed

    Landmann, W A; Dill, C W; Young, C R

    1980-11-01

    Protein efficiency ratios (PER) were determined using male weanling rats fed diets containing bovine globin alone and with wheat and corn gluten. Simultaneous equations and graphical methods were devised for selecting combinations of globin and cereal proteins to provide optimal and suboptimal profiles of limiting amino acids. Supplementation of the globin with amino acids established isoleucine and methionine as limiting amino acids. Addition of globin, whose amino acid pattern is complementary to that of the cereal proteins, markedly improved the PER values of the proteins. However, growth rates of rats fed various combinations of proteins were not identical, even though PER values differed only slightly. The PER of combined proteins were not predictable from amino acid composition or from correlations between PER and chemical score based on National Research Council (NRC) requirements for essential amino acids. The inability of the optimal mixtures to meet expected nutritional performance clearly indicated that other factors affecting availability of amino acids are implicated. Nevertheless, mutual improvement of two incomplete proteins such as globin and wheat or corn gluten was demonstrated. Addition of globin to widely used diets consisting mainly of corn or wheat can be nutritionally beneficial.

  19. Combining gene therapy and fetal hemoglobin induction for treatment of β-thalassemia.

    PubMed

    Breda, Laura; Rivella, Stefano; Zuccato, Cristina; Gambari, Roberto

    2013-06-01

    β-thalassemias are caused by nearly 300 mutations of the β-globin gene, leading to a low or absent production of adult hemoglobin (HbA). Two major therapeutic approaches have recently been proposed: gene therapy and induction of fetal hemoglobin (HbF) with the objective of achieving clinically relevant levels of Hbs. The objective of this article is to describe the development of therapeutic strategies based on a combination of gene therapy and induction of HbFs. An increase of β-globin gene expression in β-thalassemia cells can be achieved by gene therapy, although de novo production of clinically relevant levels of adult Hb may be difficult to obtain. On the other hand, an increased production of HbF is beneficial in β-thalassemia. The combination of gene therapy and HbF induction appears to be a pertinent strategy to achieve clinically relevant results.

  20. Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer

    PubMed Central

    Wilber, Andrew; Hargrove, Phillip W.; Kim, Yoon-Sang; Riberdy, Janice M.; Sankaran, Vijay G.; Papanikolaou, Eleni; Georgomanoli, Maria; Anagnou, Nicholas P.; Orkin, Stuart H.; Nienhuis, Arthur W.

    2011-01-01

    β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α2β2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34+ cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-thalassemic CD34+ cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency. PMID:21156846

  1. Replication of alpha and beta globin DNA sequences occurs during early S phase in murine erythroleukemia cells.

    PubMed Central

    Epner, E; Rifkind, R A; Marks, P A

    1981-01-01

    Murine erythroleukemia cells (MELC) can be induced to express the characteristics of erythroid differentiation by a variety of agents. Previous studies indicate that an action of inducer, occurring during early S phase, may be critical to the expression of differentiated characteristics such as initiation of accumulation of newly synthesized alpha and beta globin mRNAs. In this investigation, the time of replication of globin genes in MELC was studied. DNA was isolated from synchronous populations of cells obtained by centrifugal elutriation. Newly replicated DNA sequences were prepared from synchronized cells cultured for 1 1/2 hr with 5-bromodeoxyuridine; bromodeoxyuridine-containing DNA was isolated by CsCl gradient centrifugation. By employing cloned probes for hybridization to newly synthesized DNA, it was found that alpha and beta globin gene sequences are replicated early in S phase, while ribosomal RNA gene sequences are replicated to about the same extent in early, middle, and late S phases. PMID:6942415

  2. High-density SNP genotyping to define beta-globin locus haplotypes.

    PubMed

    Liu, Li; Muralidhar, Shalini; Singh, Manisha; Sylvan, Caprice; Kalra, Inderdeep S; Quinn, Charles T; Onyekwere, Onyinye C; Pace, Betty S

    2009-01-01

    Five major beta-globin locus haplotypes have been established in individuals with sickle cell disease (SCD) from the Benin, Bantu, Senegal, Cameroon, and Arab-Indian populations. Historically, beta-haplotypes were established using restriction fragment length polymorphism (RFLP) analysis across the beta-locus, which consists of five functional beta-like globin genes located on chromosome 11. Previous attempts to correlate these haplotypes as robust predictors of clinical phenotypes observed in SCD have not been successful. We speculate that the coverage and distribution of the RFLP sites located proximal to or within the globin genes are not sufficiently dense to accurately reflect the complexity of this region. To test our hypothesis, we performed RFLP analysis and high-density single nucleotide polymorphism (SNP) genotyping across the beta-locus using DNA samples from healthy African Americans with either normal hemoglobin A (HbAA) or individuals with homozygous SS (HbSS) disease. Using the genotyping data from 88 SNPs and Haploview analysis, we generated a greater number of haplotypes than that observed with RFLP analysis alone. Furthermore, a unique pattern of long-range linkage disequilibrium between the locus control region and the beta-like globin genes was observed in the HbSS group. Interestingly, we observed multiple SNPs within the HindIII restriction site located in the Ggamma-globin intervening sequence II which produced the same RFLP pattern. These findings illustrated the inability of RFLP analysis to decipher the complexity of sequence variations that impacts genomic structure in this region. Our data suggest that high-density SNP mapping may be required to accurately define beta-haplotypes that correlate with the different clinical phenotypes observed in SCD.

  3. High-resolution analysis of cis-acting regulatory networks at the α-globin locus.

    PubMed

    Hughes, Jim R; Lower, Karen M; Dunham, Ian; Taylor, Stephen; De Gobbi, Marco; Sloane-Stanley, Jacqueline A; McGowan, Simon; Ragoussis, Jiannis; Vernimmen, Douglas; Gibbons, Richard J; Higgs, Douglas R

    2013-01-01

    We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster. In addition, we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster.

  4. Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

    PubMed Central

    Boosalis, Michael S.; Sangerman, Jose I.; White, Gary L.; Wolf, Roman F.; Shen, Ling; Dai, Yan; White, Emily; Makala, Levi H.; Li, Biaoru; Pace, Betty S.; Nouraie, Mehdi; Faller, Douglas V.; Perrine, Susan P.

    2015-01-01

    High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies. PMID:26713848

  5. Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice.

    PubMed

    Boosalis, Michael S; Sangerman, Jose I; White, Gary L; Wolf, Roman F; Shen, Ling; Dai, Yan; White, Emily; Makala, Levi H; Li, Biaoru; Pace, Betty S; Nouraie, Mehdi; Faller, Douglas V; Perrine, Susan P

    2015-01-01

    High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

  6. Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

    PubMed Central

    2014-01-01

    Background Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in β-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV). Results Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/μL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR. Conclusions HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results. PMID:24456650

  7. Oxygen supply from the bird's eye perspective: globin E is a respiratory protein in the chicken retina.

    PubMed

    Blank, Miriam; Kiger, Laurent; Thielebein, Anke; Gerlach, Frank; Hankeln, Thomas; Marden, Michael C; Burmester, Thorsten

    2011-07-29

    The visual process in the vertebrate eye requires high amounts of metabolic energy and thus oxygen. Oxygen supply of the avian retina is a challenging task because birds have large eyes, thick retinae, and high metabolic rates but neither deep retinal nor superficial capillaries. Respiratory proteins such as myoglobin may enhance oxygen supply to certain tissues, and thus the mammalian retina harbors high amounts of neuroglobin. Globin E (GbE) was recently identified as an eye-specific globin of chicken (Gallus gallus). Orthologous GbE genes were found in zebra finch and turkey genomes but appear to be absent in non-avian vertebrate classes. Analyses of globin phylogeny and gene synteny showed an ancient origin of GbE but did not help to assign it to any specific globin type. We show that the photoreceptor cells of the chicken retina have a high level of GbE protein, which accumulates to ∼10 μM in the total eye. Quantitative real-time RT-PCR revealed an ∼50,000-fold higher level of GbE mRNA in the eye than in the brain. Spectroscopic analysis and ligand binding kinetics of recombinant chicken GbE reveal a penta-coordinated globin with an oxygen affinity of P(50) = 5.8 torrs at 25 °C and 15 torrs at 41 °C. Together these data suggest that GbE helps to sustain oxygen supply to the avian retina.

  8. Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells

    PubMed Central

    Papapetrou, Eirini P; Lee, Gabsang; Malani, Nirav; Setty, Manu; Riviere, Isabelle; Tirunagari, Laxmi M S; Kadota, Kyuichi; Roth, Shoshannah L; Giardina, Patricia; Viale, Agnes; Leslie, Christina; Bushman, Frederic D; Studer, Lorenz; Sadelain, Michel

    2012-01-01

    Realizing the therapeutic potential of human induced pluripotent stem (iPS) cells will require robust, precise and safe strategies for genetic modification, as cell therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify human iPS cells at ‘safe harbor’ sites in the genome, which fulfill five criteria based on their position relative to contiguous coding genes, microRNAs and ultraconserved regions. We demonstrate that ~10% of integrations of a lentivirally encoded β-globin transgene in β-thalassemia-patient iPS cell clones meet our safe harbor criteria and permit high-level β-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. This approach, combining bioinformatics and functional analyses, should be broadly applicable to introducing therapeutic or suicide genes into patient-specific iPS cells for use in cell therapy. PMID:21151124

  9. Characterization of the genomic structure, chromosomal location, promoter, and development expression of the alpha-globin transcription factor CP2.

    PubMed

    Swendeman, S L; Spielholz, C; Jenkins, N A; Gilbert, D J; Copeland, N G; Sheffery, M

    1994-04-15

    We recently cloned murine and human cDNAs that encode CP2, a cellular transcription factor that interacts with the alpha-globin promoter as well as with additional cellular and viral promoter elements. We have now characterized the genomic structure, chromosome location, promoter, and expression pattern of the factor. Genes for the murine and human mRNAs contained 16 and 15 exons, respectively. Both genes spanned approximately 30 kilobases of chromosomal DNA, and among coding exons, all exon/intron boundaries were conserved. The human gene for CP2 was found to reside on chromosome 12 while the murine gene mapped to the distal end of chromosome 15, near Gdc-1, Wnt-1, and Rarg, a region syntenic with human chromosome 12. The murine and human promoters initiated mRNAs at multiple start sites in a conserved region that spanned more than 450 nucleotides. Lastly, a study of the pattern of CP2 gene expression showed that the factor was expressed in all adult and fetal murine tissues examined from at least day 9.5 of development.

  10. A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference.

    PubMed

    Samakoglu, Selda; Lisowski, Leszek; Budak-Alpdogan, Tulin; Usachenko, Yelena; Acuto, Santina; Di Marzo, Rosalba; Maggio, Aurelio; Zhu, Ping; Tisdale, John F; Rivière, Isabelle; Sadelain, Michel

    2006-01-01

    The application of RNA interference (RNAi) to stem cell-based therapies will require highly specific and lineage-restricted gene silencing. Here we show the feasibility and therapeutic potential of coregulating transgene expression and RNAi in hematopoietic stem cells. We encoded promoterless small-hairpin RNA (shRNA) within the intron of a recombinant gamma-globin gene. Expression of both gamma-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage-specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34(+) cells from patients with sickle cell anemia led to erythroid-specific expression of the gamma-globin transgene and concomitant reduction of endogenous beta(S) transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny.

  11. Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization.

    PubMed

    Larsen, Rasmus Dines; Schønau, Andreas; Thisted, Marianne; Petersen, Kenneth Heesche; Lohse, Jesper; Christensen, Britta; Philip, John; Pluzek, Karl-Johan

    2003-01-01

    Fetal nucleated red blood cells (NRBC) that enter the peripheral blood of the mother are suitable for non-invasive prenatal diagnosis. The application of peptide nucleic acid (PNA) probes for tyramide amplified flow fluorescence in situ hybridization (FISH) detection of gamma-globin mRNA in fixed fetal NRBC is investigated. Hemin-induced K562 cells or nucleated blood cells (NBC) from male cord blood were mixed with NBC from non-pregnant women and analysed using both slide and flow FISH protocols. Post-chorionic villus sampling (CVS) blood samples from pregnant females carrying male fetuses were flow-sorted (2 x 10(6) NBC/sample). Y chromosome-specific PNA FISH was used to confirm that the identified gamma-globin mRNA stained cells were of fetal origin. Flow FISH isolated gamma-globin mRNA positive NBCs showing characteristic cytoplasmic staining were all Y positive. The amplification system generated a population of false positive cells that were, however, easy to distinguish from the NRBCs in the microscope. The gamma-globin mRNA specific PNA probes can be used for detection and isolation of fetal NRBCs from maternal blood. The method has additional potential for the study of gamma-globin mRNA levels or the frequency of adult NRBC (F cells) in patients with hemoglobinopathies. Copyright 2003 John Wiley & Sons, Ltd.

  12. YY1 and GATA-1 interaction modulate the chicken 3'-side alpha-globin enhancer activity.

    PubMed

    Rincón-Arano, Héctor; Valadez-Graham, Viviana; Guerrero, Georgina; Escamilla-Del-Arenal, Martín; Recillas-Targa, Félix

    2005-06-24

    Studying the chicken alpha-globin domain as a model system of gene regulation, we have previously identified contiguous silencer-enhancer elements located on the 3'-side of the domain. To better characterize the enhancer we performed a systematic functional analysis to define its expression influence range and the ubiquitous and stage-specific transcriptional regulators interacting with this control element. In contrast to previous reports, we found that, in addition to a core element that includes three GATA-1 binding sites, the enhancer incorporates a 120 base-pair DNA fragment where EKLF, NF-E2 and a fourth GATA-1 factor could interact. Functional experiments demonstrate that the enhancer activity over the adult alpha(D) promoter is differentially regulated. We found that the transcriptional factor Ying Yang 1 (YY1) binds to the 120 base-pair DNA fragment and its effect over the enhancer activity is GATA-1-dependent. In addition, we characterize a novel physical interaction between GATA-1 and YY1 that influences the enhancer function. Experiments using a histone deacetylation inhibitor indicate that, in pre-erythroblasts, the enhancer down-regulation could be influenced by a closed chromatin conformation. Our observations show that the originally defined enhancer possesses a more complex composition than previously assumed. We propose that its activity is modulated through differential nuclear factor interactions and chromatin modifications at distinct erythroid stages.

  13. Transcriptional regulation of fetal to adult hemoglobin switching: new therapeutic opportunities

    PubMed Central

    Wilber, Andrew; Nienhuis, Arthur W.

    2011-01-01

    In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing erythroblasts during ontogeny. For the past 40 years, this process has been the subject of intensive study because of its value to enlighten the biology of developmental gene regulation and because fetal hemoglobin can significantly ameliorate the clinical manifestations of both sickle cell disease and β-thalassemia. Understanding the normal process of loss of fetal globin expression and activation of adult globin expression could potentially lead to new therapeutic approaches for these hemoglobin disorders. Herein, we briefly review the history of the study of hemoglobin switching and then focus on recent discoveries in the field that now make new therapeutic approaches seem feasible in the future. Erythroid-specific knockdown of fetal gene repressors or enforced expression of fetal gene activators may provide clinically applicable approaches for genetic treatment of hemoglobin disorders that would benefit from increased fetal hemoglobin levels. PMID:21321359

  14. O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter.

    PubMed

    Zhang, Zhen; Costa, Flávia C; Tan, Ee Phie; Bushue, Nathan; DiTacchio, Luciano; Costello, Catherine E; McComb, Mark E; Whelan, Stephen A; Peterson, Kenneth R; Slawson, Chad

    2016-07-22

    One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells. When WT β-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2β at the (A)γ-globin promoter is increased. In addition, OGT and Mi2β recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2β is modified with O-GlcNAc, and both OGT and OGA interact with Mi2β, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2β repressor complex at the -566 GATA motif within the promoter.

  15. Electron transfer function versus oxygen delivery: a comparative study for several hexacoordinated globins across the animal kingdom.

    PubMed

    Kiger, Laurent; Tilleman, Lesley; Geuens, Eva; Hoogewijs, David; Lechauve, Christophe; Moens, Luc; Dewilde, Sylvia; Marden, Michael C

    2011-01-01

    Caenorhabditis elegans globin GLB-26 (expressed from gene T22C1.2) has been studied in comparison with human neuroglobin (Ngb) and cytoglobin (Cygb) for its electron transfer properties. GLB-26 exhibits no reversible binding for O(2) and a relatively low CO affinity compared to myoglobin-like globins. These differences arise from its mechanism of gaseous ligand binding since the heme iron of GLB-26 is strongly hexacoordinated in the absence of external ligands; the replacement of this internal ligand, probably the E7 distal histidine, is required before binding of CO or O(2) as for Ngb and Cygb. Interestingly the ferrous bis-histidyl GLB-26 and Ngb, another strongly hexacoordinated globin, can transfer an electron to cytochrome c (Cyt-c) at a high bimolecular rate, comparable to those of inter-protein electron transfer in mitochondria. In addition, GLB-26 displays an unexpectedly rapid oxidation of the ferrous His-Fe-His complex without O(2) actually binding to the iron atom, since the heme is oxidized by O(2) faster than the time for distal histidine dissociation. These efficient mechanisms for electron transfer could indicate a family of hexacoordinated globin which are functionally different from that of pentacoordinated globins.

  16. Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia.

    PubMed

    Sen Santara, Sumit; Roy, Jayasree; Mukherjee, Supratim; Bose, Moumita; Saha, Rina; Adak, Subrata

    2013-10-15

    Globin and adenylate cyclase play individually numerous crucial roles in eukaryotic organisms. Comparison of the amino acid sequences of globins and adenylate cyclase from prokaryotic to eukaryotic organisms suggests that they share an early common ancestor, even though these proteins execute different functions in these two kingdoms. The latest studies of biological signaling molecules in both prokaryotic and eukaryotic organisms have discovered a new class of heme-containing proteins that act as sensors. The protein of the globin family is still unknown in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, globin-coupled heme containing adenylate cyclase is undescribed in the literature. Here we report a globin-coupled heme containing adenylate cyclase (HemAC-Lm) in the unicellular eukaryotic organism Leishmania. The protein exhibits spectral properties similar to neuroglobin and cytoglobin. Localization studies and activity measurements demonstrate that the protein is present in cytosol and oxygen directly stimulates adenylate cyclase activity in vivo and in vitro. Gene knockdown and overexpression studies suggest that O2-dependent cAMP signaling via protein kinase A plays a fundamental role in cell survival through suppression of oxidative stress under hypoxia. In addition, the enzyme-dependent cAMP generation shows a stimulatory as well as inhibitory role in cell proliferation of Leishmania promastigotes during normoxia. Our work begins to clarify how O2-dependent cAMP generation by adenylate cyclase is likely to function in cellular adaptability under various O2 tensions.

  17. Diversity of [beta]-globin mutations in Israeli ethnic groups reflects recent historic events

    SciTech Connect

    Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. )

    1994-05-01

    The authors characterized nearly 500 [beta]-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. They found 28 different mutations in the [beta]-globin gene, including three mutations ([beta][sup S], [beta][sup C], and [beta][sup O-Arab]) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates - Druze and Samaritans - had a single mutation each. Fifteen of the [beta]-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele-nonsense codon 37-appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of [beta]-globin mutations can be largely explained by migration events that occurred in the past millennium. 26 refs., 2 figs., 3 tabs.

  18. Diversity of β-Globin Mutations in Israeli Ethnic Groups Reflects Recent Historic Events

    PubMed Central

    Filon, Dvora; Oron, Varda; Krichevski, Svetlana; Shaag, Avraham; Shaag, Yechezkel; Warren, Tina C.; Goldfarb, Ada; Shneor, Yona; Koren, Ariel; Aker, Mehmet; Abramov, Ayala; Rachmilewitz, Eliezer A.; Rund, Deborah; Kazazian, Haig H.; Oppenheim, Ariella

    1994-01-01

    We characterized nearly 500 β-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. We found 28 different mutations in the β-globin gene, including three mutations (βS, βC, and βO-Arab) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates—Druze and Samaritans—had a single mutation each. Fifteen of the β-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele—nonsense codon 37—appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of β-globin mutations can be largely explained by migration events that occurred in the past millennium. PMID:8178823

  19. Spectrum of Common α-Globin Deletion Mutations in the Southern Region of Vietnam.

    PubMed

    Bui Thi Kim, Ly; Phu Chi, Dung; Hoang Thanh, Chi

    2016-06-01

    The common deletion mutations of α-globin genes in the Vietnamese population is not well known. Here we report the presence of five deletional mutations of Southeast Asia in the southern region of Vietnam. The - -(SEA) (NG_000006.1: g.26264_45564del19301) mutation is the most common type of deletion (87.35%), followed by the -α(3.7) (rightward) (NG_000006.1: g.34164_37967del3804) deletion (9.64%), -α(4.2) (leftward) (AF221717) deletion (2.41%) and - -(THAI) (NG_000006.1: g.10664_44164del33501) (0.6%) mutation in this region. The - -(FIL) (NG_000006.1: g.11684_43534del31581) mutation was not detected in this study. This result provided a view of the distribution of common α-globin gene mutations in Vietnam and could serve as a baseline for further investigations into these genetic defects.

  20. HisE11 and HisF8 provide bis-histidyl heme hexa-coordination in the globin domain of Geobacter sulfurreducens globin-coupled sensor.

    PubMed

    Pesce, Alessandra; Thijs, Liesbet; Nardini, Marco; Desmet, Filip; Sisinni, Lorenza; Gourlay, Louise; Bolli, Alessandro; Coletta, Massimiliano; Van Doorslaer, Sabine; Wan, Xuehua; Alam, Maqsudul; Ascenzi, Paolo; Moens, Luc; Bolognesi, Martino; Dewilde, Sylvia

    2009-02-13

    Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS(162)) is reported. A combination of X-ray crystallography (crystal structure at 1.5 A resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS(162) as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS(162) is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS(162) is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS(162) is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.

  1. HMGA2 Moderately Increases Fetal Hemoglobin Expression in Human Adult Erythroblasts

    PubMed Central

    de Vasconcellos, Jaira F.; Lee, Y. Terry; Byrnes, Colleen; Tumburu, Laxminath; Rabel, Antoinette; Miller, Jeffery L.

    2016-01-01

    Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with beta-hemoglobin disorders. Previous studies showed that let-7 microRNAs (miRNAs) are highly regulated in erythroid cells during the fetal-to-adult developmental transition, and that targeting let-7 mediated the up-regulation of HbF to greater than 30% of the total globin levels in human adult cultured erythroblasts. HMGA2 is a member of the high-mobility group A family of proteins and a validated target of the let-7 family of miRNAs. Here we investigate whether expression of HMGA2 directly regulates fetal hemoglobin in adult erythroblasts. Let-7 resistant HMGA2 expression was studied after lentiviral transduction of CD34(+) cells. The transgene was regulated by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2-OE). HMGA2-OE caused significant increases in gamma-globin mRNA expression and HbF to around 16% of the total hemoglobin levels compared to matched control transductions. Interestingly, no significant changes in KLF1, SOX6, GATA1, ZBTB7A and BCL11A mRNA levels were observed. Overall, our data suggest that expression of HMGA2, a downstream target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in adult human erythroblasts. PMID:27861570

  2. Widespread occurrence of N-terminal acylation in animal globins and possible origin of respiratory globins from a membrane-bound ancestor.

    PubMed

    Blank, Miriam; Burmester, Thorsten

    2012-11-01

    Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or

  3. α-Globin as a molecular target in the treatment of β-thalassemia.

    PubMed

    Mettananda, Sachith; Gibbons, Richard J; Higgs, Douglas R

    2015-06-11

    The thalassemias, together with sickle cell anemia and its variants, are the world's most common form of inherited anemia, and in economically undeveloped countries, they still account for tens of thousands of premature deaths every year. In developed countries, treatment of thalassemia is also still far from ideal, requiring lifelong transfusion or allogeneic bone marrow transplantation. Clinical and molecular genetic studies over the course of the last 50 years have demonstrated how coinheritance of modifier genes, which alter the balance of α-like and β-like globin gene expression, may transform severe, transfusion-dependent thalassemia into relatively mild forms of anemia. Most attention has been paid to pathways that increase γ-globin expression, and hence the production of fetal hemoglobin. Here we review the evidence that reduction of α-globin expression may provide an equally plausible approach to ameliorating clinically severe forms of β-thalassemia, and in particular, the very common subgroup of patients with hemoglobin E β-thalassemia that makes up approximately half of all patients born each year with severe β-thalassemia. © 2015 by The American Society of Hematology.

  4. α-Globin as a molecular target in the treatment of β-thalassemia

    PubMed Central

    Mettananda, Sachith; Gibbons, Richard J.

    2015-01-01

    The thalassemias, together with sickle cell anemia and its variants, are the world’s most common form of inherited anemia, and in economically undeveloped countries, they still account for tens of thousands of premature deaths every year. In developed countries, treatment of thalassemia is also still far from ideal, requiring lifelong transfusion or allogeneic bone marrow transplantation. Clinical and molecular genetic studies over the course of the last 50 years have demonstrated how coinheritance of modifier genes, which alter the balance of α-like and β-like globin gene expression, may transform severe, transfusion-dependent thalassemia into relatively mild forms of anemia. Most attention has been paid to pathways that increase γ-globin expression, and hence the production of fetal hemoglobin. Here we review the evidence that reduction of α-globin expression may provide an equally plausible approach to ameliorating clinically severe forms of β-thalassemia, and in particular, the very common subgroup of patients with hemoglobin E β-thalassemia that makes up approximately half of all patients born each year with severe β-thalassemia. PMID:25869286

  5. Why the DNA self-depurination mechanism operates in HB-β but not in β-globin paralogs HB-δ, HB-ɛ1, HB-γ1 and HB-γ2.

    PubMed

    Amosova, Olga; Alvarez-Dominguez, Juan R; Fresco, Jacques R

    2015-08-01

    The human β-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5'GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze depurination of the loop 5'G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of β-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and β-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-depurination sites, which differ by only one or two base pairs in the stem region from that of the β-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-depurination site in β-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA depurination associated with variability of mutation incidence among human β-globin paralogs.

  6. REST regulation of gene networks in adult neural stem cells.

    PubMed

    Mukherjee, Shradha; Brulet, Rebecca; Zhang, Ling; Hsieh, Jenny

    2016-11-07

    Adult hippocampal neural stem cells generate newborn neurons throughout life due to their ability to self-renew and exist as quiescent neural progenitors (QNPs) before differentiating into transit-amplifying progenitors (TAPs) and newborn neurons. The mechanisms that control adult neural stem cell self-renewal are still largely unknown. Conditional knockout of REST (repressor element 1-silencing transcription factor) results in precocious activation of QNPs and reduced neurogenesis over time. To gain insight into the molecular mechanisms by which REST regulates adult neural stem cells, we perform chromatin immunoprecipitation sequencing and RNA-sequencing to identify direct REST target genes. We find REST regulates both QNPs and TAPs, and importantly, ribosome biogenesis, cell cycle and neuronal genes in the process. Furthermore, overexpression of individual REST target ribosome biogenesis or cell cycle genes is sufficient to induce activation of QNPs. Our data define novel REST targets to maintain the quiescent neural stem cell state.

  7. REST regulation of gene networks in adult neural stem cells

    PubMed Central

    Mukherjee, Shradha; Brulet, Rebecca; Zhang, Ling; Hsieh, Jenny

    2016-01-01

    Adult hippocampal neural stem cells generate newborn neurons throughout life due to their ability to self-renew and exist as quiescent neural progenitors (QNPs) before differentiating into transit-amplifying progenitors (TAPs) and newborn neurons. The mechanisms that control adult neural stem cell self-renewal are still largely unknown. Conditional knockout of REST (repressor element 1-silencing transcription factor) results in precocious activation of QNPs and reduced neurogenesis over time. To gain insight into the molecular mechanisms by which REST regulates adult neural stem cells, we perform chromatin immunoprecipitation sequencing and RNA-sequencing to identify direct REST target genes. We find REST regulates both QNPs and TAPs, and importantly, ribosome biogenesis, cell cycle and neuronal genes in the process. Furthermore, overexpression of individual REST target ribosome biogenesis or cell cycle genes is sufficient to induce activation of QNPs. Our data define novel REST targets to maintain the quiescent neural stem cell state. PMID:27819263

  8. Evolution of Hemoglobin and Its Genes

    PubMed Central

    Hardison, Ross C.

    2012-01-01

    Insights into the evolution of hemoglobins and their genes are an abundant source of ideas regarding hemoglobin function and regulation of globin gene expression. This article presents the multiple genes and gene families encoding human globins, summarizes major events in the evolution of the hemoglobin gene clusters, and discusses how these studies provide insights into regulation of globin genes. Although the genes in and around the α-like globin gene complex are relatively stable, the β-like globin gene clusters are more dynamic, showing evidence of transposition to a new locus and frequent lineage-specific expansions and deletions. The cis-regulatory modules controlling levels and timing of gene expression are a mix of conserved and lineage-specific DNA, perhaps reflecting evolutionary constraint on core regulatory functions shared broadly in mammals and adaptive fine-tuning in different orders of mammals. PMID:23209182

  9. Oxygen association-dissociation and stability analysis on mouse hemoglobins with mutant alpha- and beta-globins.

    PubMed

    D'Surney, S J; Popp, R A

    1992-10-01

    Oxygen association-dissociation and hemoglobin stability analysis were performed on mouse hemoglobins with amino acid substitutions in an alpha-globin (alpha 89, His to Leu) and a beta-globin (beta 59, Lys to Ile). The variant alpha-globin, designated chain 5m in the Hbag2 haplotype, had an high oxygen affinity and was stable. The variant beta-globin, (beta s2) of the Hbbs2 haplotype, also had an elevated oxygen affinity and in addition was moderately unstable in 19% isopropanol. Hemoglobins from the expected nine (Hbag2/Hbag2;Hbbs/Hbbs x Hbaa/Hbaa;Hbbs2/Hbbs2) F2 genotypes can be grouped into five classes of P50 values characterized by strict additivity and dependency on mutant globin gene dosage; physiologically, both globin variants gave indistinguishable effects on oxygen affinity. The hemoglobin of normal mice (Hbaa/Hbaa;Hbbs/Hbbs) had a P50 = 40 mm Hg and the hemoglobin of Hbag2/Hbag2;Hbbs2/Hbbs2 F2 mice had a P50 = 25 mm Hg (human P50 = 26 mm Hg). Peripheral blood from Hbag2/Hbag2;Hbbs/Hbbs, Hbaa/Hbaa;Hbbs2/Hbbs2 and Hbag2/Hbag2;Hbbs2/Hbbs2 mice exhibited normal hematological values except for a slightly higher hematocrit for Hbag2/Hbag2;Hbbs/Hbbs and Hbag2/Hbag2;Hbbs2/Hbbs2 mice, slightly elevated red cell counts for mice of the three mutant genotypes, and significantly lower values for the mean corpuscular volume and mean corpuscular hemoglobin for Hbag2/Hbag2;Hbbs2/Hbbs2 mice.

  10. Determination of Ligand Pathways in Globins

    PubMed Central

    Salter, Mallory D.; Blouin, George C.; Soman, Jayashree; Singleton, Eileen W.; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S.

    2012-01-01

    Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  11. Globins Scavenge Sulfur Trioxide Anion Radical.

    PubMed

    Gardner, Paul R; Gardner, Daniel P; Gardner, Alexander P

    2015-11-06

    Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600, and ≥ 7,500 × 10(6) m(-1) s(-1), respectively, and correlate with redox potentials. Measured rate constants for O2, GSH, ascorbate, and NAD(P)H are also large at ∼100, 10, 130, and 30 × 10(6) m(-1) s(-1), respectively, but nevertheless allow for favorable competition by globins and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O2-dependent growth impairment with nonfermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP(+)-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical, and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested.

  12. Globins Scavenge Sulfur Trioxide Anion Radical*

    PubMed Central

    Gardner, Paul R.; Gardner, Daniel P.; Gardner, Alexander P.

    2015-01-01

    Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600, and ≥ 7,500 × 106 m−1 s−1, respectively, and correlate with redox potentials. Measured rate constants for O2, GSH, ascorbate, and NAD(P)H are also large at ∼100, 10, 130, and 30 × 106 m−1 s−1, respectively, but nevertheless allow for favorable competition by globins and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O2-dependent growth impairment with nonfermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP+-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical, and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested. PMID:26381408

  13. Induction of fetal hemoglobin through enhanced translation efficiency of γ-globin mRNA.

    PubMed

    Hahn, Cynthia K; Lowrey, Christopher H

    2014-10-23

    Fetal hemoglobin (HbF) induction can ameliorate the clinical severity of sickle cell disease and β-thalassemia. We previously reported that activation of the eukaryotic initiation factor 2α (eIF2α) stress pathway increased HbF through a posttranscriptional mechanism. In this study, we explored the underlying means by which salubrinal, an activator of eIF2α signaling, enhances HbF production in primary human erythroid cells. Initial experiments eliminated changes in globin messenger RNA (mRNA) stability or cellular location and reduction of adult hemoglobin as possible salubrinal mechanisms. We then determined that salubrinal selectively increased the number of actively translating ribosomes on γ-globin mRNA. This enhanced translation efficiency occurred in the recovery phase of the stress response as phosphorylation of eIF2α and global protein synthesis returned toward baseline. These findings highlight γ-globin mRNA translation as a novel mechanism for regulating HbF production and as a pharmacologic target for induction of HbF. © 2014 by The American Society of Hematology.

  14. A five prime splice-region G yields C mutation in exon 1 of the human. beta. -globin gene inhibits pre-mRNA splicing: A mechanism for. beta. sup + -thalassemia

    SciTech Connect

    Vidaud, M.; Vidaud, D.; Amselem, S.; Rosa, J.; Goossens, M. ); Gattoni, R.; Stevenin, J. ); Chibani, J. )

    1989-02-01

    The authors have characterized a Mediterranean {beta}-thalassemia allele containing a sequence change at codon 30 that alters both {beta}-globin pre-mRNA splicing and the structure of the homoglobin product. Presumably, this G {yields} C transversion at position {minus}1 of intron 1 reduces severely the utilization of the normal 5{prime} splice site since the level of the Arg {yields} Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position {minus}1 of the CAG/GTAAGT consensus sequence had been isolated previously. They investigated the role of this nucleotide in the constitution of an active 5{prime} splice site by studying the splicing of the pre-mRNA in cell-free extracts. They demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Their results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5.

  15. Hemoglobin switching in sheep: Only the γ gene is in the active conformation in fetal liver but all the β and γ genes are in the active conformation in bone marrow

    PubMed Central

    Young, Neal S.; Benz, Edward J.; Kantor, Judith A.; Kretschmer, Peter; Nienhuis, Arthur W.

    1978-01-01

    Differential expression of the closely linked γ, βA (or βB), and βC globin genes in sheep results in the production of fetal hemoglobin (Hb F, α2γ2) during gestation and the adult hemoglobins (Hb A, α2β2A, and Hb B, α2β2B) after birth. Erythropoietic stress in certain animals leads to production of Hb C (α2β2C). The molecular mechanism of differential expression of these genes in nuclei of fetal and adult erythroid cells has been investigated by analysis of their susceptibility to digestion by DNase I (genes that are in the conformation associated with active transcription are sensitive to this nuclease). The concentration of globin gene sequences in DNA from control and DNase I-digested nuclei was determined by annealing to synthetic DNAs and analogous cDNA probes derived from recombinant plasmids containing one of the sheep globin genes. In nuclei from sheep fetal liver erythroid cells, the γ genes but not the β genes were digested by DNase I; the γ locus was open but the βA or βC loci was closed, consistent with synthesis of only Hb F by these cells. DNase I digestion of nuclei from bone marrow of anemic sheep making only Hb C or Hb B resulted in equivalent digestion of the β and γ gene sequences, although γ mRNA was not detected in these cells. Digestion by DNase I did not decrease the globin gene sequence concentration in residual DNA of spleen nuclei. As a further control, DNA from digested bone marrow and spleen nuclei were shown to anneal equally well to a cDNA prepared from liver polysomal mRNA. Differential expression of the γ and β globin genes in sheep fetal erythroid cell appears to be based on differences in chromatin structure. The γ globin gene remains in the active conformation in adult erythroid cells; failure of γ mRNA to accumulate in these cells probably reflects transcriptional or post-transcriptional regulation. PMID:282609

  16. The Mammalian Adult Neurogenesis Gene Ontology (MANGO) Provides a Structural Framework for Published Information on Genes Regulating Adult Hippocampal Neurogenesis

    PubMed Central

    Overall, Rupert W.; Paszkowski-Rogacz, Maciej; Kempermann, Gerd

    2012-01-01

    Background Adult hippocampal neurogenesis is not a single phenotype, but consists of a number of sub-processes, each of which is under complex genetic control. Interpretation of gene expression studies using existing resources often does not lead to results that address the interrelatedness of these processes. Formal structure, such as provided by ontologies, is essential in any field for comprehensive interpretation of existing knowledge but, until now, such a structure has been lacking for adult neurogenesis. Methodology/Principal Findings We have created a resource with three components 1. A structured ontology describing the key stages in the development of adult hippocampal neural stem cells into functional granule cell neurons. 2. A comprehensive survey of the literature to annotate the results of all published reports on gene function in adult hippocampal neurogenesis (257 manuscripts covering 228 genes) to the appropriate terms in our ontology. 3. An easy-to-use searchable interface to the resulting database made freely available online. The manuscript presents an overview of the database highlighting global trends such as the current bias towards research on early proliferative stages, and an example gene set enrichment analysis. A limitation of the resource is the current scope of the literature which, however, is growing by around 100 publications per year. With the ontology and database in place, new findings can be rapidly annotated and regular updates of the database will be made publicly available. Conclusions/Significance The resource we present allows relevant interpretation of gene expression screens in terms of defined stages of postnatal neuronal development. Annotation of genes by hand from the adult neurogenesis literature ensures the data are directly applicable to the system under study. We believe this approach could also serve as an example to other fields in a ‘bottom-up’ community effort complementing the already successful

  17. The mammalian adult neurogenesis gene ontology (MANGO) provides a structural framework for published information on genes regulating adult hippocampal neurogenesis.

    PubMed

    Overall, Rupert W; Paszkowski-Rogacz, Maciej; Kempermann, Gerd

    2012-01-01

    Adult hippocampal neurogenesis is not a single phenotype, but consists of a number of sub-processes, each of which is under complex genetic control. Interpretation of gene expression studies using existing resources often does not lead to results that address the interrelatedness of these processes. Formal structure, such as provided by ontologies, is essential in any field for comprehensive interpretation of existing knowledge but, until now, such a structure has been lacking for adult neurogenesis. We have created a resource with three components 1. A structured ontology describing the key stages in the development of adult hippocampal neural stem cells into functional granule cell neurons. 2. A comprehensive survey of the literature to annotate the results of all published reports on gene function in adult hippocampal neurogenesis (257 manuscripts covering 228 genes) to the appropriate terms in our ontology. 3. An easy-to-use searchable interface to the resulting database made freely available online. The manuscript presents an overview of the database highlighting global trends such as the current bias towards research on early proliferative stages, and an example gene set enrichment analysis. A limitation of the resource is the current scope of the literature which, however, is growing by around 100 publications per year. With the ontology and database in place, new findings can be rapidly annotated and regular updates of the database will be made publicly available. The resource we present allows relevant interpretation of gene expression screens in terms of defined stages of postnatal neuronal development. Annotation of genes by hand from the adult neurogenesis literature ensures the data are directly applicable to the system under study. We believe this approach could also serve as an example to other fields in a 'bottom-up' community effort complementing the already successful 'top-down' approach of the Gene Ontology.

  18. Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

    PubMed Central

    Corti, Paola; Xue, Jianmin; Tejero, Jesús; Wajih, Nadeem; Sun, Ming; Stolz, Donna B.; Tsang, Michael; Kim-Shapiro, Daniel B.; Gladwin, Mark T.

    2016-01-01

    The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, six-coordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins. PMID:27407144

  19. Neuroglobins, Pivotal Proteins Associated with Emerging Neural Systems and Precursors of Metazoan Globin Diversity

    PubMed Central

    Lechauve, Christophe; Jager, Muriel; Laguerre, Laurent; Kiger, Laurent; Correc, Gaëlle; Leroux, Cédric; Vinogradov, Serge; Czjzek, Mirjam; Marden, Michael C.; Bailly, Xavier

    2013-01-01

    Neuroglobins, previously thought to be restricted to vertebrate neurons, were detected in the brain of a photosymbiotic acoel, Symsagittifera roscoffensis, and in neurosensory cells of the jellyfish Clytia hemisphaerica. For the neuroglobin of S. roscoffensis, a member of a lineage that originated either at the base of the bilateria or of the deuterostome clade, we report the ligand binding properties, crystal structure at 2.3 Å, and brain immunocytochemical pattern. We also describe in situ hybridizations of two neuroglobins specifically expressed in differentiating nematocytes (neurosensory cells) and in statocytes (ciliated mechanosensory cells) of C. hemisphaerica, a member of the early branching animal phylum cnidaria. In silico searches using these neuroglobins as queries revealed the presence of previously unidentified neuroglobin-like sequences in most metazoan lineages. Because neural systems are almost ubiquitous in metazoa, the constitutive expression of neuroglobin-like proteins strongly supports the notion of an intimate association of neuroglobins with the evolution of animal neural systems and hints at the preservation of a vitally important function. Neuroglobins were probably recruited in the first protoneurons in early metazoans from globin precursors. Neuroglobins were identified in choanoflagellates, sponges, and placozoans and were conserved during nervous system evolution. Because the origin of neuroglobins predates the other metazoan globins, it is likely that neuroglobin gene duplication followed by co-option and subfunctionalization led to the emergence of globin families in protostomes and deuterostomes (i.e. convergent evolution). PMID:23288852

  20. Characterization of the molecularly cloned murine alpha-globin transcription factor CP2.

    PubMed

    Lim, L C; Fang, L; Swendeman, S L; Sheffery, M

    1993-08-25

    We recently cloned human and murine cDNAs that encode CP2, a transcription factor that interacts with the murine alpha-globin promoter. In this report, we exploited our ability to express CP2 in bacteria and eukaryotic cells to further investigate factor activities in vitro and in vivo. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assays. The results suggest that CP2 binds a hyphenated recognition sequence motif that spans one DNA helix turn. In addition, the enriched bacterial protein activated transcription of alpha-globin promoter templates approximately 3- to 4-fold in vitro. We then tested the effect of elevating CP2 levels 2.5- to 5.5-fold in vivo using both transient and stable transformation assays. When a reporter construct comprised of the intact murine alpha-globin promoter driving the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into these overexpressing cells, we observed a 3- to 6-fold increase in CAT activity when compared to cells expressing normal levels of CP2. These results define the CP2 factor binding site in more detail and help characterize the activities of the factor in vivo.

  1. Genetics Home Reference: methemoglobinemia, beta-globin type

    MedlinePlus

    ... blood cells. Specifically, it alters a molecule called hemoglobin within these cells. Hemoglobin within red blood cells attaches (binds) to oxygen ... in tissues throughout the body. Instead of normal hemoglobin, people with methemoglobinemia, beta-globin type have an ...

  2. Target-based drug discovery for [Formula: see text]-globin disorders: drug target prediction using quantitative modeling with hybrid functional Petri nets.

    PubMed

    Mehraei, Mani; Bashirov, Rza; Tüzmen, Şükrü

    2016-10-01

    Recent molecular studies provide important clues into treatment of [Formula: see text]-thalassemia, sickle-cell anaemia and other [Formula: see text]-globin disorders revealing that increased production of fetal hemoglobin, that is normally suppressed in adulthood, can ameliorate the severity of these diseases. In this paper, we present a novel approach for drug prediction for [Formula: see text]-globin disorders. Our approach is centered upon quantitative modeling of interactions in human fetal-to-adult hemoglobin switch network using hybrid functional Petri nets. In accordance with the reverse pharmacology approach, we pose a hypothesis regarding modulation of specific protein targets that induce [Formula: see text]-globin and consequently fetal hemoglobin. Comparison of simulation results for the proposed strategy with the ones obtained for already existing drugs shows that our strategy is the optimal as it leads to highest level of [Formula: see text]-globin induction and thereby has potential beneficial therapeutic effects on [Formula: see text]-globin disorders. Simulation results enable verification of model coherence demonstrating that it is consistent with qPCR data available for known strategies and/or drugs.

  3. Regulation and function of adult neurogenesis: from genes to cognition.

    PubMed

    Aimone, James B; Li, Yan; Lee, Star W; Clemenson, Gregory D; Deng, Wei; Gage, Fred H

    2014-10-01

    Adult neurogenesis in the hippocampus is a notable process due not only to its uniqueness and potential impact on cognition but also to its localized vertical integration of different scales of neuroscience, ranging from molecular and cellular biology to behavior. This review summarizes the recent research regarding the process of adult neurogenesis from these different perspectives, with particular emphasis on the differentiation and development of new neurons, the regulation of the process by extrinsic and intrinsic factors, and their ultimate function in the hippocampus circuit. Arising from a local neural stem cell population, new neurons progress through several stages of maturation, ultimately integrating into the adult dentate gyrus network. The increased appreciation of the full neurogenesis process, from genes and cells to behavior and cognition, makes neurogenesis both a unique case study for how scales in neuroscience can link together and suggests neurogenesis as a potential target for therapeutic intervention for a number of disorders. Copyright © 2014 the American Physiological Society.

  4. Regulation and Function of Adult Neurogenesis. From Genes to Cognition

    SciTech Connect

    Aimone, J. B.; Li, Y.; Lee, S. W.; Clemenson, G. D.; Deng, W.; Gage, F. H.

    2014-10-01

    Adult neurogenesis in the hippocampus is a notable process due not only to its uniqueness and potential impact on cognition but also to its localized vertical integration of different scales of neuroscience, ranging from molecular and cellular biology to behavior. Our review summarizes the recent research regarding the process of adult neurogenesis from these different perspectives, with particular emphasis on the differentiation and development of new neurons, the regulation of the process by extrinsic and intrinsic factors, and their ultimate function in the hippocampus circuit. Arising from a local neural stem cell population, new neurons progress through several stages of maturation, ultimately integrating into the adult dentate gyrus network. Furthermore, the increased appreciation of the full neurogenesis process, from genes and cells to behavior and cognition, makes neurogenesis both a unique case study for how scales in neuroscience can link together and suggests neurogenesis as a potential target for therapeutic intervention for a number of disorders.

  5. Regulation and Function of Adult Neurogenesis. From Genes to Cognition

    DOE PAGES

    Aimone, J. B.; Li, Y.; Lee, S. W.; ...

    2014-10-01

    Adult neurogenesis in the hippocampus is a notable process due not only to its uniqueness and potential impact on cognition but also to its localized vertical integration of different scales of neuroscience, ranging from molecular and cellular biology to behavior. Our review summarizes the recent research regarding the process of adult neurogenesis from these different perspectives, with particular emphasis on the differentiation and development of new neurons, the regulation of the process by extrinsic and intrinsic factors, and their ultimate function in the hippocampus circuit. Arising from a local neural stem cell population, new neurons progress through several stages ofmore » maturation, ultimately integrating into the adult dentate gyrus network. Furthermore, the increased appreciation of the full neurogenesis process, from genes and cells to behavior and cognition, makes neurogenesis both a unique case study for how scales in neuroscience can link together and suggests neurogenesis as a potential target for therapeutic intervention for a number of disorders.« less

  6. Regulation and Function of Adult Neurogenesis: From Genes to Cognition

    PubMed Central

    Aimone, James B.; Li, Yan; Lee, Star W.; Clemenson, Gregory D.; Deng, Wei; Gage, Fred H.

    2014-01-01

    Adult neurogenesis in the hippocampus is a notable process due not only to its uniqueness and potential impact on cognition but also to its localized vertical integration of different scales of neuroscience, ranging from molecular and cellular biology to behavior. This review summarizes the recent research regarding the process of adult neurogenesis from these different perspectives, with particular emphasis on the differentiation and development of new neurons, the regulation of the process by extrinsic and intrinsic factors, and their ultimate function in the hippocampus circuit. Arising from a local neural stem cell population, new neurons progress through several stages of maturation, ultimately integrating into the adult dentate gyrus network. The increased appreciation of the full neurogenesis process, from genes and cells to behavior and cognition, makes neurogenesis both a unique case study for how scales in neuroscience can link together and suggests neurogenesis as a potential target for therapeutic intervention for a number of disorders. PMID:25287858

  7. Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.

    PubMed

    Cavazzana-Calvo, Marina; Payen, Emmanuel; Negre, Olivier; Wang, Gary; Hehir, Kathleen; Fusil, Floriane; Down, Julian; Denaro, Maria; Brady, Troy; Westerman, Karen; Cavallesco, Resy; Gillet-Legrand, Beatrix; Caccavelli, Laure; Sgarra, Riccardo; Maouche-Chrétien, Leila; Bernaudin, Françoise; Girot, Robert; Dorazio, Ronald; Mulder, Geert-Jan; Polack, Axel; Bank, Arthur; Soulier, Jean; Larghero, Jérôme; Kabbara, Nabil; Dalle, Bruno; Gourmel, Bernard; Socie, Gérard; Chrétien, Stany; Cartier, Nathalie; Aubourg, Patrick; Fischer, Alain; Cornetta, Kenneth; Galacteros, Frédéric; Beuzard, Yves; Gluckman, Eliane; Bushman, Frederick; Hacein-Bey-Abina, Salima; Leboulch, Philippe

    2010-09-16

    The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound β(E)/β(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The β(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated β(E)-globin with partial instability. When this is compounded with a non-functional β(0) allele, a profound decrease in β-globin synthesis results, and approximately half of β(E)/β(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe β(E)/β(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

  8. Fever of unclear origin and cytopenia because of acute splenic sequestration in a young immunocompetent carrier of beta-globin mutation for Hb Valletta.

    PubMed

    Parrinello, Gaspare; Torres, Daniele; Paterna, Salvatore; Di Pasquale, Pietro; Licata, Giuseppe

    2008-12-01

    Fever of unclear origin is a clinical challenge in medical practice. Infectious diseases, neoplasms, and collagen vascular illnesses are its main causes in adults and children. Acute splenic sequestration crises, a known potentially fatal complication of sickle cell disease and sickle beta-thalassemia, are uncommon in beta-heterozygosis. We describe a case of prolonged recurrent episodes of fever with spontaneous resolution, commencing at age 10 in a 15-year-old boy with a history of hypochromic microcytic anemia attributed to a thalassemic trait. He was admitted twice to our university hospital for continuous-remittent fever with a pruritic, macular evanescent Still's skin rash, severe splenomegaly, leucopenia, thrombocytopenia, and sudden aggravation of anemia. Infectious, rheumatologic, autoimmune, and hematologic illnesses were excluded. A genetic-based study revealed heterozygosis of the beta-globin gene for a A>C (Thr>Pro) substitution at position 87 called Hemoglobin Valletta (alpha 2 beta 2 87 PRO) with a C>G transition in homozygosis in beta-globin intronic polymorphism intervening sequence 2 at nucleotide 745. After a follow-up period of 1 year without treatment, the young patient remains apyretic and in good general clinical health with persistent microcythemia and hepatosplenomegaly. Acute splenic sequestration crisis and related cytopenia may be an unusual complication of fever of unclear origin in a beta-thalassemic carrier of a Hemoglobin Valletta mutation and polymorphism in homozygosis of intervening sequence 2 at nucleotide 745. This hemoglobinopathy may predispose to a clinical phenotype of minor or intermediate thalassemia and, during a febrile illness, to hemoglobin instability and splenic sequestration.

  9. Therapeutic hemoglobin levels after gene transfer in β-thalassemia mice and in hematopoietic cells of β-thalassemia and sickle cells disease patients.

    PubMed

    Breda, Laura; Casu, Carla; Gardenghi, Sara; Bianchi, Nicoletta; Cartegni, Luca; Narla, Mohandas; Yazdanbakhsh, Karina; Musso, Marco; Manwani, Deepa; Little, Jane; Gardner, Lawrence B; Kleinert, Dorothy A; Prus, Eugenia; Fibach, Eitan; Grady, Robert W; Giardina, Patricia J; Gambari, Roberto; Rivella, Stefano

    2012-01-01

    Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34(+) cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials

  10. Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

    PubMed Central

    Breda, Laura; Casu, Carla; Gardenghi, Sara; Bianchi, Nicoletta; Cartegni, Luca; Narla, Mohandas; Yazdanbakhsh, Karina; Musso, Marco; Manwani, Deepa; Little, Jane; Gardner, Lawrence B.; Kleinert, Dorothy A.; Prus, Eugenia; Fibach, Eitan; Grady, Robert W.; Giardina, Patricia J.; Gambari, Roberto; Rivella, Stefano

    2012-01-01

    Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials

  11. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

    Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

    2003-01-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

  12. Insulation of the chicken beta-globin chromosomal domain from a chromatin-condensing protein, MENT.

    PubMed

    Istomina, Natalia E; Shushanov, Sain S; Springhetti, Evelyn M; Karpov, Vadim L; Krasheninnikov, Igor A; Stevens, Kimberly; Zaret, Kenneth S; Singh, Prim B; Grigoryev, Sergei A

    2003-09-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken beta-globin domain. We observed two sharp transitions of MENT concentration coinciding with the beta-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.

  13. Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia.

    PubMed

    Origa, Raffaella; Paglietti, Maria E; Sollaino, Maria C; Desogus, Maria F; Barella, Susanna; Loi, Daniela; Galanello, Renzo

    2014-01-01

    α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for β-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family. The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with β-thalassemia, is important for a correct correlation of genotype-phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present.

  14. Nucleotide sequence from the coding region of rabbit β-globin messenger RNA

    PubMed Central

    Proudfoot, N.J.

    1976-01-01

    A sequence of 89 nucleotides from rabbit β-globin mRNA has been determined and is shown to code for residues 107 to 137 of the β-globin protein. In addition, a sequence heterogeneity has been identified within this 89 nucleotide long sequence which corresponds to a known polymorphic variant of rabbit β-globin. Images PMID:61580

  15. Characterization of the embryonic globin chains of the marsupial Tammar wallaby, Macropus eugenii.

    PubMed

    Holland, R A; Gooley, A A

    1997-09-15

    The embryonic hemoglobins of the marsupial Tammar wallaby (Macropus eugenii) are known to aggregate, which was shown by the finding that the Hill coefficient, h, was greater than 4.0 in the upper part of the oxygen equilibrium curve. Here, we have undertaken a detailed primary structure analysis of the Tammar wallaby pouch young hemoglobin complement, which we hoped might provide clues into the residues that cause aggregation and a high embryonic h. The Tammar wallaby embryonic hemoglobin complement is principally four major hemoglobins each with a different isoelectric point. Two early expressed hemoglobins contain the same embryonic beta-like chain, epsilon (epsilon), but two separate alpha-like chains, termed zeta and zeta prime (zeta and zeta') both of which are N-terminally blocked. The later two expressed hemoglobins contain the same adult alpha-chain, but different beta-like chains. The latest expressed hemoglobin contains the same beta-like chain, epsilon, as the two early expressed forms, but the third expressed hemoglobin contains a unique beta-like chain which we have termed omega (omega). A protein database similarity search using the first 54 N-terminal amino acids of the omega-chain showed a range of sequence identities of 57-72% to all known mammalian beta-like chains, including the other marsupial epsilon-chains. The closest identity, reflected by both the highest percentage identity and Smith-Waterman score, was with the embryonic beta-chains of the aves. While the primary structures of the hemoglobins reported here do not explain the low hemoglobin-oxygen affinity in embryonic marsupial blood, the finding of the similarity with the bird globin-like sequence with one of the marsupial chains has implications on mammalian globin evolution. How many other marsupials and placental mammals are harboring a bird-like globin in their embryos?

  16. DNaseI hypersensitive sites 1, 2 and 3 of the human beta-globin dominant control region direct position-independent expression.

    PubMed Central

    Fraser, P; Hurst, J; Collis, P; Grosveld, F

    1990-01-01

    The human beta-globin dominant control region (DCR) which flanks the multigene beta-globin locus directs high level, site of integration independent, copy number dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukemia (MEL) cells. We have assayed each of the individual DNaseI hypersensitive regions present in the full 15kb DCR for position independence and copy number dependence of a linked beta-globin gene in transgenic mice. The results show that at least three of the individual DNaseI hypersensitive site regions (sites 1, 2 and 3), though expressing at lower levels than the full DCR, are capable of position independent, copy number dependent expression. Site 2 alone directs the highest level of expression of the single site constructs, producing nearly 70% of the level of the full DCR. Sites 1 and 3 each provide 30% of the full activity. Deletion of either site 2 or 3 from the complete set significantly reduces the level of expression, but does not effect position independence or copy number dependence. This demonstrates that sites 2 and 3 are required for full expression and suggests that all the sites are required for the full expression of even a single gene from this multigene locus. Images PMID:2362805

  17. GATA-1 Modulates the Chromatin Structure and Activity of the Chicken α-Globin 3′ Enhancer▿ †

    PubMed Central

    Escamilla-Del-Arenal, Martín; Recillas-Targa, Félix

    2008-01-01

    Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken α-globin silencer-enhancer elements located 3′ of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken α-globin enhancer plays a central role in its capacity to differentially regulate α-globin gene expression during erythroid differentiation and development. PMID:17984219

  18. Virally mediated gene manipulation in the adult CNS

    PubMed Central

    Edry, Efrat; Lamprecht, Raphael; Wagner, Shlomo; Rosenblum, Kobi

    2011-01-01

    Understanding how the CNS functions poses one of the greatest challenges in modern life science and medicine. Studying the brain is especially challenging because of its complexity, the heterogeneity of its cellular composition, and the substantial changes it undergoes throughout its life-span. The complexity of adult brain neural networks results also from the diversity of properties and functions of neuronal cells, governed, inter alia, by temporally and spatially differential expression of proteins in mammalian brain cell populations. Hence, research into the biology of CNS activity and its implications to human and animal behavior must use novel scientific tools. One source of such tools is the field of molecular genetics—recently utilized more and more frequently in neuroscience research. Transgenic approaches in general, and gene targeting in rodents have become fundamental tools for elucidating gene function in the CNS. Although spectacular progress has been achieved over recent decades by using these approaches, it is important to note that they face a number of restrictions. One of the main challenges is presented by the temporal and spatial regulation of introduced genetic manipulations. Viral vectors provide an alternative approach to temporally regulated, localized delivery of genetic modifications into neurons. In this review we describe available technologies for gene transfer into the adult mammalian CNS that use both viral and non-viral tools. We discuss viral vectors frequently used in neuroscience, with emphasis on lentiviral vector (LV) systems. We consider adverse effects of LVs, and the use of LVs for temporally and spatially controllable manipulations. Especially, we highlight the significance of viral vector-mediated genetic manipulations in studying learning and memory processes, and how they may be effectively used to separate out the various phases of learning: acquisition, consolidation, retrieval, and maintenance. PMID:22207836

  19. Definition of the ovalbumin gene promoter by transfer of an ovalglobin fusion gene into cultured cells.

    PubMed Central

    Knoll, B J; Zarucki-Schulz, T; Dean, D C; O'Malley, B W

    1983-01-01

    In order to study the initiation of transcription from the ovalbumin gene promoter, we constructed a hybrid gene (ovalglobin) in which 753 bps of ovalbumin gene 5'-flanking sequence were joined to the chicken adult beta-globin gene. When transfected into HeLa S3 cells, ovalglobin gene transcription initiated at the ovalbumin gene cap site, as measured by S1 nuclease and primer extension analysis. Deletion of 5'-flanking sequences to position -95 had little effect on transcription; deletion to -77 reduced transcription to about 20% of the wild type level and deletion to -48 reduced the level to about 2%. A deletion to -24, removing the sequence TATATAT, abolished transcription entirely. Hormonal regulation of the ovalglobin gene was observed when primary oviduct cells were used as recipients for DNA transfection. Under these conditions, addition of progesterone increased the level of ovalglobin transcripts to more than 10 times the uninduced level. Images PMID:6314256

  20. Erythropoiesis in the Absence of Adult Hemoglobin

    PubMed Central

    Liu, Shanrun; McConnell, Sean C.

    2013-01-01

    During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucleated erythrocytes. Although Hb is one of the most extensively studied proteins, the role of Hb in erythroid lineage commitment, differentiation, and maturation remains unclear. In this study, we generate mouse embryos and embryonic stem (ES) cells with all of the adult α and β globin genes deleted (Hb Null). While Hb Null embryos die in midgestation, adult globin genes are not required for primitive or definitive erythroid lineage commitment. In vitro differentiation of Hb Null ES cells generates viable definitive proerythroblasts that undergo apoptosis upon terminal differentiation. Surprisingly, all stages of Hb Null-derived definitive erythroblasts develop normally in vivo in chimeric mice, and Hb Null erythroid cells undergo enucleation to form reticulocytes. Free heme toxicity is not observed in Hb Null-derived erythroblasts. Transplantation of Hb Null-derived bone marrow cells provides short-term radioprotection of lethally irradiated recipients, whose progressive anemia results in an erythroid hyperplasia composed entirely of Hb Null-derived erythroblasts. This novel experimental model system enables the role played by Hb in erythroid cell enucleation, cytoskeleton maturation, and heme and iron regulation to be studied. PMID:23530053

  1. The beta-globin domain in immature chicken erythrocytes: enhanced solubility is coincident with histone hyperacetylation.

    PubMed

    Nelson, D A; Ferris, R C; Zhang, D E; Ferenz, C R

    1986-02-25

    A 60 minute exposure of chicken immature erythrocytes to n-butyrate shifts actively acetylated and deacetylated histones to hypermodified forms. Micrococcal nuclease digestion of nuclei from n-butyrate treated cells and subsequent fractionation of the chromatin releases 40-45% of the adult beta-globin (beta A) nucleohistone into a soluble fraction. This is an eleven fold enrichment over the soluble chromatin from untreated cells (Ferenz and Nelson (1985) Nucleic Acids Res. 13, 1977-1995). The enhanced beta A chromatin solubility and induced histone hyperacetylation are coincident. Removal of n-butyrate from the cell incubation medium allows rapid histone deacetylation and a striking reduction in beta A chromatin solubility. Chromatin from cells incubated in the absence of n-butyrate, or in medium containing 10 mM NaCl or 2% dimethylsulfoxide, does not exhibit histone hyperacetylation, or the acquired solubility of beta A chromatin. We show that the H4 histone co-isolated with the beta A DNA is in a hyperacetylated state and present evidence that the n-butyrate incubation increases the solubility of both coding and noncoding chromatin regions in the beta-globin domain.

  2. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene.

    PubMed

    Nuez, B; Michalovich, D; Bygrave, A; Ploemacher, R; Grosveld, F

    1995-05-25

    Erythroid Krüppel-like factor (EKLF) was originally isolated from erythroid cell RNA by differential screening and shown to be erythroid-specific, although a low level of EKLF was found in mast cell lines. EKLF contains three zinc-fingers homologous to those found in the Krüppel family of transcription factors. Because it binds the sequence CCACACCCT, EKLF may affect erythroid development as a result of its ability to bind to the CAC box in the promoter of the beta-globin gene. Mutation of this element leads to reduced beta-globin expression and it appears to mediate the effect of the globin locus control region on the promoter. Here we inactivate the EKLF gene through insertion of a lacZ reporter gene by homologous recombination in embryonic stem (ES) cells. Heterozygous EKLF+/- mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin. We conclude that the transcription factor EKLF is essential for the final steps of definitive erythropoiesis in fetal liver.

  3. Accumulation of gamma-globin mRNA and induction of irreversible erythroid differentiation after treatment of CML cell line K562 with new doxorubicin derivatives.

    PubMed

    Szulawska, Agata; Arkusinska, Justyna; Czyz, Malgorzata

    2007-01-15

    Human chronic myelogenous leukemia (CML) cell line K562 can be chemically induced to differentiate and express embryonic and fetal globin genes. In this study, the effects of doxorubicin (DOX), an inducer of K562 cell erythroid differentiation, with those of epidoxorubicin (EDOX) as well as newly synthesized derivatives of both drugs (DOXM, DOXH, and EDOXM) on cell growth and differentiation were compared. Our results revealed that DOX, EDOX and their derivatives caused irreversible differentiation of K562 cells into more mature hemoglobin-containing cells. This phenomenon was linked to time-dependent inhibition of cell proliferation. Considering the impact of the structure of newly synthesized anthracyclines on their cellular activity, our data clearly indicated that among tested anthracyclines DOXM, a morpholine derivative of DOX exerted the highest antiproliferative and differentiating activity. An increase of gamma-globin mRNA level caused both by high transcription rate and by mRNA stabilization, as well as an enhancement of expression but not activity of erythroid transcription factor GATA-1 were observed. Therefore, a high level of hemoglobin-containing cells in the presence of DOXM resulted from transcriptional and post-transcriptional events on gamma-globin gene regulation. The same morpholine modification introduced to EDOX did not cause, however, similar effects on cellular level. Characterization of new powerful inducers of erythroid differentiation may contribute to the development of novel compounds for pharmacological approach by differentiation therapy to leukemia or to beta-globin disorder, beta-thalassemia.

  4. Distribution of beta-globin haplotypes among the tribes of southern Gujarat, India.

    PubMed

    Aggarwal, Aastha; Khurana, Priyanka; Mitra, Siuli; Raicha, Bhavesh; Saraswathy, K N; Italia, Yazdi M; Kshatriya, Gautam K

    2013-06-01

    The present study was carried out in Indo-European speaking tribal population groups of southern Gujarat (India) to elucidate the allelic and haplotypic content of β-globin system in individuals with HbAA genotypes. 6 neutral restriction sites of the β-globin system were analysed and various statistical parameters were estimated to draw meaningful interpretations. All the 6 sites were found to be polymorphic and most were in Hardy-Weinberg Equilibrium in the studied group. Haplotypes were constructed using two different combinations of the 6 restriction sites analysed. Analysis of the 5 sites revealed a set of three predominant haplotypes, '+----', '-++-+' and '-+-++'; and haplotypes '+--', '++-' and '+++' were found to be the most frequent when the 3 sites were used to construct the haplotypes. Haplotypic heterozygosity levels (>83%) observed in the present study group were comparable to those observed in African and Afro-American populations and greater than other world populations. All the ancestral haplotypes, +-----, -++-+, -+-++ and ----+ were found in the study group. The distribution pattern of various haplotypes was consistent with the global pattern. The paucity of comparable data from other Indian populations restricted one from making interpretations about the study group's relationships with other Indian populations but the results were indicative of older population histories or experience of gene flow by the study group and their affinities with populations of southern India.

  5. Inactivation of HDAC1 or HDAC2 induces gamma globin expression without altering cell cycle or proliferation.

    PubMed

    Esrick, Erica B; McConkey, Marie; Lin, Katherine; Frisbee, Alyse; Ebert, Benjamin L

    2015-07-01

    Other than hydroxyurea, no pharmacologic agents are clinically available for fetal hemoglobin (HbF) induction in sickle cell disease (SCD). An optimal candidate would induce HbF without causing cell cycle inhibition and would act independently of hydroxyurea in order to yield additional HbF induction when combined. We explored whether inhibition of histone deacetylase (HDAC) 1 or HDAC2 could achieve these goals. In human erythroid progenitor cells, shRNA knockdown of the HDAC1 or HDAC2 genes induced gamma globin, without altering cellular proliferation in vitro, and without altering cell cycle phase. Treatment with hydroxyurea in combination with HDAC2 knockdown yielded a further increase in gamma globin expression. Additionally, when CD34+ cells were treated with both hydroxyurea and MS-275 (an inhibitor of HDAC 1, 2, and 3), an additive induction of relative gamma globin expression was achieved. Our findings support further clinical investigation of HDAC inhibitors in combination with hydroxyurea in SCD patients. © 2015 Wiley Periodicals, Inc.

  6. A Digital Gene Expression-Based Bovine Gene Atlas Evaluating 92 Adult, Juvenile and Fetal Cattle Tissues

    USDA-ARS?s Scientific Manuscript database

    A comprehensive transcriptome survey, or “Gene Atlas,” provides information essential for a complete understanding of the genomic biology of an organism. Using a digital gene expression approach, we developed a Gene Atlas of RNA abundance in 92 adult, juvenile and fetal cattle tissues. The samples...

  7. Bone status of adult female butyrylcholinesterase gene-deficient mice.

    PubMed

    Haupt, Malte; Kauschke, Vivien; Sender, Jonas; Kampschulte, Marian; Kovtun, Anna; Dürselen, Lutz; Heiss, Christian; Lips, Katrin Susanne

    2015-11-01

    Butyrylcholinesterase (BChE) degrades acetylcholine in addition to acetylcholinesterase (AChE) which is involved in embryonic development of limbs. Since BChE is expressed by osteoblast-like cells we asked whether it is functional in adult bone remodeling. We addressed this issue by analyzing BChE gene-deficient mice (BChE-KO). Bones were extracted from 16-week old female BChE-KO and corresponding wild type mice (WT). Femoral bones were used for biomechanical testing and μCT evaluation of cancellous and cortical bone. Also vertebrae Th12 and L1 were investigated with μCT while L3 was used for tartrate-resistant acidic phosphatase (TRAP) histomorphometry and Th10 for gene expression analysis by means of real-time RT-PCR. BChE-KO did not reveal significant differences in biomechanical bone strength and bone mineral density determined by μCT. Microarchitecture of cancellous and cortical bone showed an increase in μCT parameters like trabecular thickness, trabecular separation, and relative cortical bone area of femoral BChE-KO bone compared to WT. In vertebrae no changes of microstructure and mRNA expression were detected. However, osteoclast histomorphometry with TRAP stained sections demonstrated a significant increase in relative osteoclast number. In conclusion, in adult murine bone the role of BChE is limited to bone specific changes in microarchitecture and to an increase in relative number of bone resorbing osteoclasts whereas the main collagen resorbing enzyme Cathepsin-K (CtsK) was stably expressed. Besides, AChE might be able to compensate the lack of BChE. Thus, further analyses using bone tissue specific AChE BChE cre-lox double knockout mice would be helpful.

  8. Genetic dissection of the α-globin super-enhancer in vivo

    DOE PAGES

    Hay, Deborah; Hughes, Jim R.; Babbs, Christian; ...

    2016-07-04

    Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. Furthermore, these super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation,more » without clear evidence of synergistic or higher-order effects. This study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.« less

  9. Genetic dissection of the α-globin super-enhancer in vivo

    SciTech Connect

    Hay, Deborah; Hughes, Jim R.; Babbs, Christian; Davies, James O. J.; Graham, Bryony J.; Hanssen, Lars L. P.; Kassouf, Mira T.; Oudelaar, A. Marieke; Sharpe, Jacqueline A.; Suciu, Maria C.; Telenius, Jelena; Williams, Ruth; Rode, Christina; Li, Pik-Shan; Pennacchio, Len A.; Sloane-Stanley, Jacqueline A.; Ayyub, Helena; Butler, Sue; Sauka-Spengler, Tatjana; Gibbons, Richard J.; Smith, Andrew J. H.; Wood, William G.; Higgs, Douglas R.

    2016-07-04

    Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. Furthermore, these super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. This study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

  10. Genetic dissection of the α-globin super-enhancer in vivo

    PubMed Central

    Hay, Deborah; Hughes, Jim R.; Rode, Christina; Li, Pik-Shan; Pennacchio, Len A.; Sloane-Stanley, Jacqueline A.; Ayyub, Helena; Butler, Sue; Sauka-Spengler, Tatjana; Gibbons, Richard J.; Smith, Andrew J.H.; Wood, William G.; Higgs, Douglas R.

    2016-01-01

    Many genes determining cell identity are regulated by clusters of mediator-bound enhancer elements collectively referred to as super-enhancers. These have been proposed to manifest higher-order properties important in development and disease. Here, we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer singly and in informative combinations, we demonstrate that each constituent enhancer appears to act independently and in an additive fashion with respect to hematologic phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation. PMID:27376235

  11. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase.

  12. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed Central

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase. PMID:284320

  13. Gene expression during development of fetal and adult Leydig cells.

    PubMed

    Dong, Lei; Jelinsky, Scott A; Finger, Joshua N; Johnston, Daniel S; Kopf, Gregory S; Sottas, Chantal M; Hardy, Matthew P; Ge, Ren-Shan

    2007-12-01

    In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.

  14. Acute chest syndrome is associated with single nucleotide polymorphism-defined beta globin cluster haplotype in children with sickle cell anaemia.

    PubMed

    Bean, Christopher J; Boulet, Sheree L; Yang, Genyan; Payne, Amanda B; Ghaji, Nafisa; Pyle, Meredith E; Hooper, W Craig; Bhatnagar, Pallav; Keefer, Jeffrey; Barron-Casella, Emily A; Casella, James F; Debaun, Michael R

    2013-10-01

    Genetic diversity at the human β-globin locus has been implicated as a modifier of sickle cell anaemia (SCA) severity. However, haplotypes defined by restriction fragment length polymorphism sites across the β-globin locus have not been consistently associated with clinical phenotypes. To define the genetic structure at the β-globin locus more thoroughly, we performed high-density single nucleotide polymorphism (SNP) mapping in 820 children who were homozygous for the sickle cell mutation (HbSS). Genotyping results revealed very high linkage disequilibrium across a large region spanning the locus control region and the HBB (β-globin gene) cluster. We identified three predominant haplotypes accounting for 96% of the β(S) -carrying chromosomes in this population that could be distinguished using a minimal set of common SNPs. Consistent with previous studies, fetal haemoglobin level was significantly associated with β(S) -haplotypes. After controlling for covariates, an association was detected between haplotype and rate of hospitalization for acute chest syndrome (ACS) (incidence rate ratio 0·51, 95% confidence interval 0·29-0·89) but not incidence rate of vaso-occlusive pain or presence of silent cerebral infarct (SCI). Our results suggest that these SNP-defined β(S) -haplotypes may be associated with ACS, but not pain or SCI in a study population of children with SCA.

  15. Splicing and 3' end formation in the definition of nonsense-mediated decay-competent human beta-globin mRNPs.

    PubMed

    Neu-Yilik, G; Gehring, N H; Thermann, R; Frede, U; Hentze, M W; Kulozik, A E

    2001-02-01

    Premature translation termination codons are common causes of genetic disorders. mRNAs with such mutations are degraded by a surveillance mechanism termed nonsense-mediated decay (NMD), which represents a phylogenetically widely conserved post-transcriptional mechanism for the quality control of gene expression. How NMD-competent mRNPs are formed and specified remains a central question. Here, we have used human beta-globin mRNA as a model system to address the role of splicing and polyadenylation for human NMD. We show that (i) splicing is an indispensable component of the human beta-globin NMD pathway, which cannot be compensated for by exonic beta-globin 'failsafe' sequences; (ii) the spatial requirements of human beta-globin NMD, as signified by the maximal distance of the nonsense mutation to the final exon-exon junction, are less constrained than in yeast; and (iii) non-polyadenylated mRNAs with a histone 3' end are NMD competent. Thus, the formation of NMD-competent mRNP particles critically depends on splicing but does not require the presence of a poly(A) tail.

  16. Plasmodium falciparum: effects of proteinase inhibitors on globin hydrolysis by cultured malaria parasites.

    PubMed

    Rosenthal, P J

    1995-03-01

    The effects of peptide proteinase inhibitors on globin hydrolysis by cultured malaria parasites were studied. All of the four cysteine proteinase inhibitors evaluated blocked globin hydrolysis, as documented by the development of a morphological abnormality in which parasite food vacuoles filled with undegraded globin and by SDS-PAGE showing that the cysteine proteinase inhibitor-treated parasites accumulated large quantities of globin. The aspartic proteinase inhibitor pepstatin did not block globin hydrolysis by cultured parasites. None of seven antimalarial drugs tested elicited the food vacuole abnormality caused by cysteine proteinase inhibitors, indicating that this morphological alteration was not simply a sign of nonspecific parasite toxicity. Our results indicate that a trophozoite cysteine proteinase is required for initial cleavages of globin by intact malaria parasites.

  17. Neuroglobin and cytoglobin: two new members of globin family

    PubMed Central

    Tosqui, Priscilla; Colombo, Marcio Francisco

    2011-01-01

    The globin family has long been defined by myoglobin and hemoglobin, proteins with the functions of oxygen storage and transportation, respectively. Recently, two new members of this family were discovered: neuroglobin present in neurons and retinal cells and cytoglobin found in various types of tissue. The increased expression of these proteins in hypoxic conditions first suggested a role in oxygen supply. However structural and functional differences, such as the hexacoordinated heme, a high autoxidation rate and different concentrations between different cellular types, have dismissed this hypothesis. The protective role of these globins has already been established. In vitro and in vivo studies have demonstrated increased survival of neurons under stress in the presence of neuroglobin and increased resistance to neurodegenerative diseases. However the mechanism remains unknown. Functions, including detoxification of nitric oxide, free radical scavenging and as an antioxidant and signaling of apoptosis, have also been suggested for neuroglobin and an antifibrotic function for cytoglobin. PMID:23049323

  18. The Evolution of the Globins: We Thought We Understood It

    NASA Astrophysics Data System (ADS)

    Lesk, Arthur M.

    Protein crystallography achieved its first results in the late 1950s with the structure determinations of sperm whale myoglobin and human haemoglobin. These gave us our first glimpse of the structural changes that take place during protein evolution. Many other structures of proteins in the globin family have continued to reveal interesting and important details of the coordinated divergence during evolution of amino acid sequences and protein structures and functions.

  19. Finding an average core structure: Application to the globins

    SciTech Connect

    Altman, R.B.; Gerstein, M.

    1994-12-31

    We present a procedure for automatically identifying from a set of aligned protein structures a subset of atoms with only a small amount of structural variation, i.e., a core. We apply this procedure to the globin family of proteins. Based purely on the results of the procedure, we show that the globin fold can be divided into two parts. The part with greater structural variation consists of the residues near the heme (the F helix and parts of the G and H helices), and the part with lesser structural variation (the core) forms a structural framework similar to that of the repressor protein (A, B, and E helices and remainder of the G and H helices). Such a division is consistent with many other structural and biochemical findings. In addition, we find further partitions within the core that may have biological significance. Finally, using the structural core of the globin family as a reference point, we have compared structural variation to sequence variation and shown that a core definition based on sequence conservation does not necessarily agree with one based on structural similarity.

  20. Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products.

    PubMed Central

    Lim, S K; Sigmund, C D; Gross, K W; Maquat, L E

    1992-01-01

    Human beta zero-thalassemic beta-globin genes harboring either a frameshift or a nonsense mutation that results in the premature termination of beta-globin mRNA translation have been previously introduced into the germ line of mice (S.-K. Lim, J.J. Mullins, C.-M. Chen, K. Gross, and L.E. Maquat, EMBO J. 8:2613-2619, 1989). Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and II. In this communication, we show by using genetics and S1 nuclease transcript mapping that the premature termination of beta-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human beta-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs. Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of beta-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of

  1. Investigation of genes important in neurodevelopment disorders in adult human brain.

    PubMed

    Maussion, Gilles; Diallo, Alpha B; Gigek, Carolina O; Chen, Elizabeth S; Crapper, Liam; Théroux, Jean-Francois; Chen, Gary G; Vasuta, Cristina; Ernst, Carl

    2015-10-01

    Several neurodevelopmental disorders (NDDs) are caused by mutations in genes expressed in fetal brain, but little is known about these same genes in adult human brain. Here, we test the hypothesis that genes associated with NDDs continue to have a role in adult human brain to explore the idea that NDD symptoms may be partially a result of their adult function rather than just their neurodevelopmental function. To demonstrate adult brain function, we performed expression analyses and ChIPseq in human neural stem cell(NSC) lines at different developmental stages and adult human brain, targeting two genes associated with NDDs, SATB2 and EHMT1, and the WNT signaling gene TCF7L2, which has not been associated with NDDs. Analysis of DNA interaction sites in neural stem cells reveals high (40-50 %) overlap between proliferating and differentiating cells for each gene in temporal space. Studies in adult brain demonstrate that consensus sites are similar to NSCs but occur at different genomic locations. We also performed expression analyses using BrainSpan data for NDD-associated genes SATB2, EHMT1, FMR1, MECP2, MBD5, CTNND2, RAI1, CHD8, GRIN2A, GRIN2B, TCF4, SCN2A, and DYRK1A and find high expression of these genes in adult brain, at least comparable to developing human brain, confirming that genes associated with NDDs likely have a role in adult tissue. Adult function of genes associated with NDDs might be important in clinical disease presentation and may be suitable targets for therapeutic intervention.

  2. Interaction of hemoglobin Grey Lynn (Vientiane) with a non-deletional α(+)-thalassemia in an adult Thai proband.

    PubMed

    Singha, Kritsada; Fucharoen, Goonnapa; Fucharoen, Supan

    2014-01-01

    Hemoglobin (Hb) Grey Lynn is a Hb variant caused by a substitution of Phe for Leu at position 91 of α1-globin chain, originally described in individual of unknown ethnic background. This article addresses the interaction of Hb Grey Lynn with a non-deletional α(+)-thalassemia found in Thailand, a hitherto un-described condition. The proband was adult Thai woman referred for investigation of mild anemia with Hb 90 g/L. Hb analyses using low pressure liquid chromatography raised a suspicion of abnormal Hb presence, which was failed to demonstrate by cellulose acetate electrophoresis and capillary electrophoresis. DNA sequencing identified a CTT (Leu) to TTT (Phe) mutation at codon 91 corresponding to the Hb Grey Lynn (Vientiane) [α91(FG3)Leu>Phe (α1) on α1-globin gene and a C deletion between codons 36 and 37 on α2-globin gene causing α(+)-thalassemia. As compared to those observed in a compound heterozygote for Hb Grey Lynn / α(0)-thalassemia reported previously, higher MCV (81.7 fL) and MCH (26.3 pg) values with a lower level of Hb Grey Lynn (19.7%) were observed in the proband. The normochromic normocytic anemia observed could be due to the interaction of Hb Grey Lynn with α(+)-thalassemia. The two mutations could be identified using PCR-RFLP and allele-specific PCR assays developed.

  3. Stepwise colonization of the Andes by ruddy ducks and the evolution of novel β-globin variants.

    PubMed

    Muñoz-Fuentes, V; Cortázar-Chinarro, M; Lozano-Jaramillo, M; McCracken, K G

    2013-03-01

    Andean uplift played a key role in Neotropical bird diversification, yet past dispersal and genetic adaptation to high-altitude environments remain little understood. Here we use multilocus population genetics to study population history and historical demographic processes in the ruddy duck (Oxyura jamaicensis), a stiff-tailed diving duck comprising three subspecies distributed from Canada to Tierra del Fuego and inhabiting wetlands from sea level to 4500 m in the Andes. We sequenced the mitochondrial DNA, four autosomal introns and three haemoglobin genes (α(A), α(D), β(A)) and used isolation-with-migration (IM) models to study gene flow between North America and South America, and between the tropical and southern Andes. Our analyses indicated that ruddy ducks dispersed first from North America to the tropical Andes, then from the tropical Andes to the southern Andes. While no nonsynonymous substitutions were found in either α globin gene, three amino acid substitutions were observed in the β(A) globin. Based on phylogenetic reconstruction and power analysis, the first β(A) substitution, found in all Andean individuals, was acquired when ruddy ducks dispersed from low altitude in North America to high altitude in the tropical Andes, whereas the two additional substitutions occurred more recently, when ruddy ducks dispersed from high altitude in the tropical Andes to low altitude in the southern Andes. This stepwise colonization pattern accompanied by polarized β(A) globin amino acid replacements suggest that ruddy ducks first acclimatized or adapted to the Andean highlands and then again to the lowlands. In addition, ruddy ducks colonized the Andean highlands via a less common route as compared to other waterbird species that colonized the Andes northwards from the southern cone of South America. © 2013 Blackwell Publishing Ltd.

  4. Hox genes in the adult skeleton: Novel functions beyond embryonic development.

    PubMed

    Rux, Danielle R; Wellik, Deneen M

    2016-12-27

    Hox genes encode evolutionarily conserved transcription factors that control skeletal patterning in the developing embryo. They are expressed in regionally restricted domains and function to regulate the morphology of specific vertebral and long bone elements. Recent work has provided evidence that Hox genes continue to be regionally expressed in adult tissues. Fibroblasts cultured from adult tissues show broadly maintained Hox gene expression patterns. In the adult skeleton, Hox genes are expressed in progenitor-enriched populations of mesenchymal stem/stromal cells (MSCs), and genetic loss-of-function analyses have provided evidence that Hox genes function during the fracture healing process. This review will highlight our current understanding of Hox expression in the adult animal and its function in skeletal regeneration. Developmental Dynamics, 2017. © 2017 Wiley Periodicals, Inc.

  5. Understanding the contrasting spatial haplotype patterns of malaria-protective β-globin polymorphisms

    PubMed Central

    Hockham, Carinna; Piel, Frédéric B.; Gupta, Sunetra; Penman, Bridget S.

    2015-01-01

    The malaria-protective β-globin polymorphisms, sickle-cell (βS) and β0-thalassaemia, are canonical examples of human adaptation to infectious disease. Occurring on distinct genetic backgrounds, they vary markedly in their patterns of linked genetic variation at the population level, suggesting different evolutionary histories. βS is associated with five classical restriction fragment length polymorphism haplotypes that exhibit remarkable specificity in their geographical distributions; by contrast, β0-thalassaemia mutations are found on haplotypes whose distributions overlap considerably. Here, we explore why these two polymorphisms display contrasting spatial haplotypic distributions, despite having malaria as a common selective pressure. We present a meta-population genetic model, incorporating individual-based processes, which tracks the evolution of β-globin polymorphisms on different haplotypic backgrounds. Our simulations reveal that, depending on the rate of mutation, a large population size and/or high population growth rate are required for both the βS- and the β0-thalassaemia-like patterns. However, whilst the βS-like pattern is more likely when population subdivision is high, migration low and long-distance migration absent, the opposite is true for β0-thalassaemia. Including gene conversion has little effect on the overall probability of each pattern; however, when inter-haplotype fitness variation exists, gene conversion is more likely to have contributed to the diversity of haplotypes actually present in the population. Our findings highlight how the contrasting spatial haplotype patterns exhibited by βS and β0-thalassaemia may provide important indications as to the evolution of these adaptive alleles and the demographic history of the populations in which they have evolved. PMID:26394108

  6. β-Globin Matrix Attachment Region Improves Stable Genomic Expression of the Sleeping Beauty Transposon

    PubMed Central

    Daly, Meghan C.; Steer, Clifford J.; Kren, Betsy T.

    2014-01-01

    The liver is an attractive target for gene therapy due to its extensive capability for protein production and the numerous diseases resulting from a loss of gene function it normally provides. The Sleeping Beauty Transposon (SB-Tn)1 system is a non-viral vector capable of delivering and mediating therapeutic transgene(s) insertion into the host genome for long-term expression. A current challenge for this system is the low efficiency of integration of the transgene. In this study we use a human hepatoma cell line (HuH-7) and primary human blood outgrowth endothelial cells (BOECs) to test vectors containing DNA elements to enhance transposition without integrating themselves. We employed the human β-globin matrix attachment region (MAR) and the Simian virus 40 (SV40) nuclear translocation signal to increase the percent of HuH-7 cells persistently expressing a GFP::Zeo reporter construct by ~50% for each element; while combining both did not show an additive effect. Interestingly, both elements together displayed an additive effect on the number of insertion sites, and in BOECs the SV40 alone appeared to have an inhibitory effect on transposition. In long-term cultures the loss of plasmid DNA, transposase expression and mapping of insertion sites demonstrated bona fide transposition without episomal expression. These results show that addition of the β-globin MAR and potentially other elements to the backbone of SB-Tn system can enhance transposition and expression of therapeutic transgenes. These findings may have a significant influence on the use of SB transgene delivery to liver for the treatment of a wide variety of disorders. PMID:21520245

  7. Understanding the contrasting spatial haplotype patterns of malaria-protective β-globin polymorphisms.

    PubMed

    Hockham, Carinna; Piel, Frédéric B; Gupta, Sunetra; Penman, Bridget S

    2015-12-01

    The malaria-protective β-globin polymorphisms, sickle-cell (β(S)) and β(0)-thalassaemia, are canonical examples of human adaptation to infectious disease. Occurring on distinct genetic backgrounds, they vary markedly in their patterns of linked genetic variation at the population level, suggesting different evolutionary histories. β(S) is associated with five classical restriction fragment length polymorphism haplotypes that exhibit remarkable specificity in their geographical distributions; by contrast, β(0)-thalassaemia mutations are found on haplotypes whose distributions overlap considerably. Here, we explore why these two polymorphisms display contrasting spatial haplotypic distributions, despite having malaria as a common selective pressure. We present a meta-population genetic model, incorporating individual-based processes, which tracks the evolution of β-globin polymorphisms on different haplotypic backgrounds. Our simulations reveal that, depending on the rate of mutation, a large population size and/or high population growth rate are required for both the β(S)- and the β(0)-thalassaemia-like patterns. However, whilst the β(S)-like pattern is more likely when population subdivision is high, migration low and long-distance migration absent, the opposite is true for β(0)-thalassaemia. Including gene conversion has little effect on the overall probability of each pattern; however, when inter-haplotype fitness variation exists, gene conversion is more likely to have contributed to the diversity of haplotypes actually present in the population. Our findings highlight how the contrasting spatial haplotype patterns exhibited by β(S) and β(0)-thalassaemia may provide important indications as to the evolution of these adaptive alleles and the demographic history of the populations in which they have evolved.

  8. The relative importance of the X-linked FCP locus and beta-globin haplotypes in determining haemoglobin F levels: a study of SS patients homozygous for beta S haplotypes.

    PubMed

    Chang, Y P; Maier-Redelsperger, M; Smith, K D; Contu, L; Ducroco, R; de Montalembert, M; Belloy, M; Elion, J; Dover, G J; Girot, R

    1997-03-01

    Five factors have been hypothesized to influence the 20-fold variation in fetal haemoglobin (Hb F) levels in sickle cell anaemia (SS): age sex, alpha-globin gene number, beta-globin haplotype, and the X-linked F-cell production locus (FCP) that regulates the production of Hb F containing erythrocytes (F cells). We analysed the association of these factors with Hb F levels in 112 SS patients living in France who are homozygous for the three common African beta-globin haplotypes (Benin, Bantu or Central African Republic and Senegal). We found that: (1) FCP accounts for about 40% of the overall variation in Hb F levels, (2) when the FCP influence is removed, beta-globin haplotype is associated with 14% of the remaining Hb F variation, and (3) the other factors have little influence. Comparison with our previous study of SS individuals in Jamaica leads to the following conclusions: (1) the X-linked FCP locus is a major determinant of Hb F levels in SS disease, (2) factors linked to the beta-globin haplotype have only a small effect on the variation in Hb F levels, in either the homozygous or heterozygous state, and (3)approximately half of the variation in Hb F levels still remains to be explained.

  9. Different Gene Expression Signatures in Children and Adults with Celiac Disease.

    PubMed

    Pascual, V; Medrano, L M; López-Palacios, N; Bodas, A; Dema, B; Fernández-Arquero, M; González-Pérez, B; Salazar, I; Núñez, C

    2016-01-01

    Celiac disease (CD) is developed after gluten ingestion in genetically susceptible individuals. It can appear at any time in life, but some differences are commonly observed between individuals with onset early in life or in adulthood. We aimed to investigate the molecular basis underlying those differences. We collected 19 duodenal biopsies of children and adults with CD and compared the expression of 38 selected genes between each other and with the observed in 13 non-CD controls matched by age. A Bayesian methodology was used to analyze the differences of gene expression between groups. We found seven genes with a similarly altered expression in children and adults with CD when compared to controls (C2orf74, CCR6, FASLG, JAK2, IL23A, TAGAP and UBE2L3). Differences were observed in 13 genes: six genes being altered only in adults (IL1RL1, CD28, STAT3, TMEM187, VAMP3 and ZFP36L1) and two only in children (TNFSF18 and ICOSLG); and four genes showing a significantly higher alteration in adults (CCR4, IL6, IL18RAP and PLEK) and one in children (C1orf106). This is the first extensive study comparing gene expression in children and adults with CD. Differences in the expression level of several genes were found between groups, being notorious the higher alteration observed in adults. Further research is needed to evaluate the possible genetic influence underlying these changes and the specific functional consequences of the reported differences.

  10. Molecular Study of Deletional and Nondeletional Mutations on the α-Globin Locus in the Azeri Population of Northwestern Iran.

    PubMed

    Derakhshan, Sima M; Khaniani, Mahmoud S; Afkhami, Fateme; PourFeizi, Abbasali H

    2016-09-01

    The aim of this study was to determine the molecular spectrum and frequency of deletional and nondeletional α-thalassemia (α-thal) mutations and the genotype-phenotype correlation in common mutations in the Azeri population of Northwestern Iran. A total of 1256 potential carriers with microcytic and hypochromic anemia and normal Hb A2 levels (<3.5%) and without iron deficiency anemia plus three fetuses were identified. Multiplex gap-polymerase chain reaction (gap-PCR) and sequencing for α-thal mutations were carried out. In 606 individuals, the α-globin gene was normal, but in 650 persons (51.6%) and three fetuses, 10 different mutations were detected. The most frequent deletional genotypes were as follo