The Thalassemias

Orkin, Stuart H.; Nathan, David G.

doi:10.1056/nejm197609232951306

Cited by 28 publications

(9 citation statements)

References 19 publications

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“…These include (i) the analysis of the coordination of a and f3 globin gene expression in erythroleukemic cells (4) and mouse erythropoietic spleen cells (26); (ii) the search for possible globin expression in non-erythroid tissues (27); and (iii) the measurement of the relative synthetic rates of individual globin RNAs in human erythropoietic cells, from both normal individuals and those with thalassemia syndromes (28).…”

Section: Resultsmentioning

confidence: 99%

Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation.

Orkin¹,

Swerdlow²

1977

Proc. Natl. Acad. Sci. U.S.A.

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Murine erythroleukemic cells accumulate cytoplasmic globin mRNA during differentiation induced in tissue culture by dimethyl sulfoxide. Cellular accumulation of globin RNA may reflect transcriptional activation of the globin genes and/or posttranscriptional stabilization of globin RNA during differentiation. To evaluate possible transcriptional controls directly, globin RNA synthesis by isolated erythroleukemic cell nuclei was studied.Conditions were established for optimal nuclear RNA synthesis in vitro in the presence of a mercurinucleotide (Hg-CTP Isolation of Nuclei. Washed cell pellets were suspended in 0.3 M sucrose (Schwarz/Mann ultrapure) containing 2 mM Mg(OAc)2, 3 mM CaCI2, 10 mM Tris-HCl at pH 8.0, and 2 mM 2-mercaptoethanol. After addition of Triton X-100 to 0.25%, the cell suspension was stirred on a vortex mixer for 30 sec. The crude nuclei were gently sedimented at 1000 X g for 5 min at 40, and then resuspended in the same buffer minus Triton X-100. An equal volume of 2 M sucrose containing 5 mM Mg(OAc)2, 10 mM Tris at pH 8.0, and 2 mM mercaptoethanol was immediately added, and the mixture was homogenized with five strokes of a tight-fitting Teflon-glass homogenizer. Purified nuclei were sedimented through a cushion of 2 M sucrose containing 2 mM Mg(OAc)2, 3 mM CaCl2, and 10 mM Tris at pH 8.0 by centrifugation at 25,000 rpm in a Beckman SW-40 rotor for 45 min at 40. The nuclear pellet was resuspended in 25% (vol/vol) glycerol/5 mM Mg(OAc)2/50 mM Tris at pH 8.0/2 mM mercaptoethanol at 1 to 2 X 108 nuclei per ml, and frozen in liquid nitrogen.RNA Synthesis by Isolated Nuclei. Conditions for RNA synthesis were modified slightly from those described for other systems (8, 9). Each reaction mixture contained 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 8.0, 5 mM Mg(OAc)2, 1 mM MnCl2, 150 mM KCI, 12.5% glycerol, 4 mM phosphoenolpyruvate, pyruvate kinase (3 units/ml), 0.185 mM of three ribonucleoside triphosphates, 0.037 mM of the fourth ribonucleoside triphosphate labeled with 3H, and approximately 5 X 107 rapidly thawed nuclei per ml. Mercurinucleotide, either Hg-CTP or Hg-UTP (P.L. Biochemicals), replaced the corresponding unmodified nucleotide at the same concentration in the reaction mixture when RNA containing mercury (Hg-RNA) was prepared. Mercaptans, as noted below, were present at 10 mM final concentration in the reaction mixture. The reaction mixture was incubated at 230 with intermittent, gentle shaking and aliquots were withdrawn at appropriate times, spotted on Whatman 3MM paper, and processed as described by Ernest et al. (9) to monitor RNA synthesis. No adjustment was made for background cpm at time zero.Preparative Isolation of RNA after Nuclear Synthesis. After incubation, nuclei were diluted 10-fold with 20 mM Tris at pH 7.5/50 mM NaCl/1 mM EDTA/1% sodium dodecyl sulfate and extracted vigorously in a vortex mixer with an equal volume of 600 phenol previously equilibrated with Tris buffer. The aqueous phase was reextracted three times with chloroform/is...

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Section: Resultsmentioning

confidence: 99%

Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation.

Orkin¹,

Swerdlow²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…[1][2][3] Caused by more than 200 mutations affecting the human ␤-globin gene, they are most prevalent in the Mediterranean region, the Middle East, the Indian subcontinent, and South East Asia, representing a serious health problem in certain areas where gene frequencies reach 3% to 10% of the population. The severity of ␤-thalassemia is directly linked to the degree of imbalance in the production of ␣-and ␤-globin chains.…”

Section: Introductionmentioning

confidence: 99%

“…The severity of ␤-thalassemia is directly linked to the degree of imbalance in the production of ␣-and ␤-globin chains. [1][2][3] The excess ␣-globin chains that are not incorporated into adult hemoglobin (␣ 2 :␤ 2 A ) precipitate in red blood cell (RBC) precursors, impairing erythroid maturation and causing mechanical damage, oxidative membrane destruction, and eventually apoptosis. However, the ␤-thalassemic phenotype is heterogeneous, depending on the genotype as well as the degree of ␥-globin chain expression.…”

Section: Introductionmentioning

confidence: 99%

Successful treatment of murine β-thalassemia intermedia by transfer of the human β-globin gene

May

Rivella

Chadburn

et al. 2002

Blood

156

103

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The ␤-thalassemias are caused by more than 200 mutations that reduce or abolish ␤-globin production. The severity of the resulting anemia can lead to lifelong transfusion dependency. A genetic treatment based on globin gene transfer would require that transgene expression be erythroid specific, elevated, and sustained over time. We report here that long-term synthesis of chimeric hemoglobin (mu␣ 2 :hu␤ 2 A ) could be achieved in mice with ␤-thalassemia intermedia following engraftment with bone marrow cells transduced with a lentiviral vector encoding the human ␤-globin gene. In the absence of any posttransduction selection, the treated chimeras exhibit durably increased hemoglobin levels without diminution over 40 weeks. Ineffective erythropoiesis and extramedullary hematopoiesis (EMH) regress, as reflected by normalization of spleen size, architecture, hematopoietic colony formation, and disappearance of liver EMH. These findings establish that a sustained increase of 3 to 4 g/dL hemoglobin is sufficient to correct ineffective erythropoiesis. Hepatic iron accumulation is markedly decreased in 1-year-old chimeras, indicating persistent protection from secondary organ damage. These results demonstrate for the first time that viral-mediated globin gene transfer in hematopoietic stem cells effectively treats a severe hemoglobin disorder. (Blood. 2002;99:1902-1908 © 2002 by The American Society of Hematology IntroductionThe ␤-thalassemias are inherited anemias caused by mutations that reduce or abolish production of the ␤-globin chain of hemoglobin. [1][2][3] Caused by more than 200 mutations affecting the human ␤-globin gene, they are most prevalent in the Mediterranean region, the Middle East, the Indian subcontinent, and South East Asia, representing a serious health problem in certain areas where gene frequencies reach 3% to 10% of the population. The severity of ␤-thalassemia is directly linked to the degree of imbalance in the production of ␣-and ␤-globin chains. 1-3 The excess ␣-globin chains that are not incorporated into adult hemoglobin (␣ 2 :␤ 2 A ) precipitate in red blood cell (RBC) precursors, impairing erythroid maturation and causing mechanical damage, oxidative membrane destruction, and eventually apoptosis. However, the ␤-thalassemic phenotype is heterogeneous, depending on the genotype as well as the degree of ␥-globin chain expression. 4 In the most severe form found in homozygotes and compound heterozygotes, ␤-thalassemia major, massive medullary cell proliferation and expansion lead to severe skeletal deformities, while extramedullary hematopoiesis (EMH) 1 develops in spleen and liver. Erythropoiesis is nonetheless ineffective and the profound anemia requires regular lifelong blood transfusions; without treatment, the condition is lethal within the first years of life. 5 However, transfusion therapy leads to iron overload, which is itself lethal if untreated. 6 At present, the only means to definitively cure the disease is through hematopoietic stem cell (HSC) replacement. 7,8 But allogeneic b...

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“…The common form of ,B-thalassemia that is associated with an increased Hb A2 level can be classified into two types according to the presence or absence of ,B-globin chain synthesis in the homozygous state (1)(2)(3)(4). In homozygous 3+-thalassemia, a decreased amount of fl-globin mRNA consistent with the degree of reduction in f-globin chain synthesis has been demonstrated by several groups of investigators (5)(6)(7)(8).…”

mentioning

confidence: 95%

Authentic beta-globin mRNA sequences in homozygous betaO-thalassemia.

Temple¹,

Chang²,

Kan³

1977

Proc. Natl. Acad. Sci. U.S.A.

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In a patient with homozygous PO-thalassemia in whom studies of reticulocyte hemoglobin synthesis showed no fl-globin chain synthesis in vivo and in vitro, molecular hybridization studies revealed RNA sequences complementary to -globin cDNA. The fact that these sequences were authentic P-globin mRNA was shown by fingerprint analysis of T1 ribonuclease-digested mRNA and by sequencing of oligonucleotides unique to P-globin mRNA. The Blood used as a nonthalassemic control was obtained from a patient with sickle cell anemia. Blood from a fetus that died from homozygous a-thalassemia was used as a source of ymRNA because it contained predominantly y-and a small amount of ,B-globin chain sequences but no a-globin sequences.Preparation of RNA and DNA. Peripheral blood was washed three times with isotonic saline and the erythrocytes were lysed with the Orskov reaction according to the method of Boyer et al. (16). With this technique, only erythrocytes were lysed and 98% of the leukocytes remained intact. The latter were saved for the DNA preparation as described (17). Ribonucleoproteins from the erythrocytes lysate were precipifated at pH 5, and the RNA was extracted with phenol, chloroform, and isoamyl alcohol (12). Approximately 15 A260 units of total RNA were obtained per 50 ml of blood. (One unit is the amount of material that when dissolved in 1 ml of solvent gives an absorbance of 1 with a path length of 1 cm.) The RNA was twice passed through oligo(dT)-cellulose columns (18) and the poly(A)-containing RNA eluted with low-salt buffer was separated by centrifugation on a 15-30% (wt/vol) sucrose density gradient. Fractions between the 8S and 12S positions were pooled.To prepare mRNA rich in fl-globin sequences, we separated the sickle cell mRNA by electrophoresis on 5% polyacrylamide gels in 98% formamide (19). The slowly migrating band in the 10S region containing more ,B than a sequences was isolated and the RNA was eluted from the gel, extracted with phenol and purified on oligo(dT)-cellulose (20).Hybridization Analysis. Radioactive DNAs complementary to a-and f,-globin mRNA were synthesized according to the methods of Verma et al. (21), as described (17). a-and fl-globin sequences in the mRNA were measured by hybridization with a-and f-globin cDNAs that were labeled with [a-32P]dCTP (specific activity 120 Ci-mmolh') and [3H]dCTP (specific activity 23 Ci-mmolh'), respectively. The ,B-cDNA was a tran-

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The Thalassemias

Cited by 28 publications

References 19 publications

Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation.

Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation.

Successful treatment of murine β-thalassemia intermedia by transfer of the human β-globin gene

Authentic beta-globin mRNA sequences in homozygous betaO-thalassemia.

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