Globin Messenger RNA in Hemoglobin H Disease

Functional messenger RNA (mRNA) forhuman globin synthesis was isolated fromreticulocytes of each of two patients withhemoglobin H disease. The RNA wastested for its capacity to direct globinsynthesis in a messenger RNA-dependentcell-free system derived from Krebs Type IImouse ascites tumor cells. In each case,hemoglobin H disease mRNA directed thesynthesis of a great excess of -globinchains relative to -globin chains of hemoglobin A. The / synthetic ratios obtained in the cell-free system at saturatingconcentrations of mRNA were >22 and>15, respectively, for the two hemoglobinH disease mRNA preparations, whereasthe / synthetic ratios obtained by incubation of intact reticulocytes from thesesame patients were 2.6 and 2.8, respectively. The / synthetic ratio obtained inthe cell-free system did not vary whenlower concentrations of hemoglobin Hdisease mRNA were used. A marked decrease in the amount of functional -globin-chain mRNA relative to -chainmRNA is therefore associated with the decreased -chain synthesis observed inhemoglobin H disease. This decrease in-chain-specific mRNA activity is greaterthan expected from the / synthetic ratioof intact reticulocytes in hemoglobin Hdisease.

Submitted on May 1, 1973 Revised on June 15, 1973 Accepted on June 16, 1973     

Synthesis of Hemoglobin Abraham Lincoln (beta 32 leu rarr pro)

Globin chain synthesis was studied in vitrowith reticulocytes from a patient heterozygous for Hb Abraham Lincoln, an unstable beta chain variant. Synthesis of-chains by the reticulocytes exceededtotal -chain synthesis, and a substantialfraction (about 16%) of radioactivityincorporated into globin was recoveredfrom the cells as uncombined subunits.In a time-course study, the ratio of : -chain specific activity was found to increase progressively in a nearly linearmanner, suggesting that a fraction ofnewly synthesized -chains had undergone rapid destruction. The specific activity of the abnormal -chain was nearlythree times that of ^A. The rate of synthesis of the -chain of Hb AbrahamLincoln appeared to be approximately halfthat of the -chain of Hb A.

Submitted on September 13, 1973 Revised on November 1, 1973 Accepted on November 6, 1973     

The Interaction of Hemoglobin E With beta Thalassemia: A Study of Hemoglobin Synthesis in a Family of Mixed Burmese and Iranian Origin

A 5-yr-old girl with hemoglobin E- thalassemia was discovered in a family of mixedorigin. The father is Iranian (-thalassemiatrait) and the mother is Burmese (hemoglobin-E trait). Hemoglobin synthesis wasstudied in vitro in the blood of theproposita and family members. In the subjects with hemoglobin E trait the ratio ofthe quantity of hemoglobin A to hemoglobin E was 3:1. However. the ^A/^Esynthesis ratio in reticulocytes was in therange of 1.5-2.18, and the specific activityof ^E was 31%-49% greater than ^A, suggesting instability of hemoglobin E withpreferential destruction of abnormal hemoglobin. The blood of the proposita exhibitedonly hemoglobin F and hemoglobin E andreticulocytes and bone marrow showed no^A synthesis. This Iranian -thalassemiagene is therefore of the ° type. The ^E/synthesis ratio (approximately 0.74) inblood of the proposita was similar to the^A/ ratio in mildly affected relatives withthalassemia trait. These results suggestthat the severity of the hemoglobin E-thalassemia syndrome is attributable toboth instability and defective synthesis ofhemoglobin E in association with absent^A synthesis due to a ° thalassemia gene.

Submitted on March 19, 1973 Revised on May 4, 1973 Accepted on May 8, 1973     

Bone Marrow and Peripheral Blood Globin Synthesis in an American Black Family With Beta Thalassemia

Synthesis of globin chains in bonemarrow and peripheral blood samplesfrom a black family with mild betathalassemia was compared with similar studies in white people. Blood andbone marrow were incubated with ¹⁴C-leucine, globin chains were isolated,and / and / ratios were calculated.The results of studies of globin synthesis in homozygotes of differentraces were similar, despite the differences in severity of clinical disease. Inthe heterozygotes, there was a significant defect in beta synthesis in theperipheral blood of white subjects,while in two of three black patients the/ ratio was in the normal range. Although there was no evidence of segregation of an alpha thalassemia genein this black family to explain the unusual / ratios, the presence of sucha gene in the heterozygotes could notbe excluded.

Submitted on September 2, 1971 Revised on November 5, 1971 Accepted on December 15, 1971     

Globin Synthesis in Iron-deficiency Anemia

The to globin chain ratio has beendetermined in the peripheral red cells of11 patients with iron deficiency anemia.The mean ratio was found to be 0.74 ±0.07, which is significantly lower than theratio of 0.97 ± 0.07 obtained in normals.When the stroma-free hemolysates werepurified prior to globin preparation the to ratio did not change. On the otherhand, globin extracted from whole iron-deficient cells, including the stroma, had ahigher to ratio, 0.88 ± 0.04, but stillsignificantly lower than normal. These results suggest that in iron deficiency thereis a decrease in -chain synthesis relativeto -chain and that there are membrane-bound globin chains, but no excessive increase in the free -chain pool. Similarfindings have been reported previously inother states of heme deficiency like sideroblastic anemia and lead poisoning.

Submitted on January 9, 1974 Accepted on March 19, 1974     

Globin Chain Synthesis in the Marrow and Reticulocytes of Beta Thalassemia, Hemoglobin H Disease, and Beta Delta Thalassemia

, , and globin chain synthesis inbone marrow and peripheral bloodreticulocytes were studied in two patients with thalassemia major, two withthalassemia intermedia, one with thalassemia minor, one with Hb H disease,and one with homozygous -thalassemia. Nine nonthalassemic patientsserved as controls. In thalassemiamajor, a marked imbalance of - to -chain synthesis was found in the bonemarrow as well as in reticulocytes. Theimbalance, however, was slightly moreevident in the latter. In the patientswith thalassemia intermedia and minorthe - to -globin chain ratios in thereticulocytes were of the same orderof magnitude, despite the marked clinical differences between thalassemiaintermedia and minor. A balanced synthesis was found in the bone marrowof the patient with thalassemia minor.The bone marrow globin synthesis inthalassemia intermedia was not studied. Contrary to that in Hb H diseaseand -thalassemia, the imbalancewas more apparent in the bone marrow. In the latter, no evidence for imbalance was detected in the reticulocytes. These results point out the needfor further studies on globin chain synthesis in the bone marrow and reticulocytes of patients With the variousthalassemia syndromes and the effectof the free globin chain pool on thoseresults.

Submitted on March 9, 1971 Revised on November 29, 1971 Accepted on February 12, 1972     

A Fetal Hemoglobin Variant of Unusual Genetic Interest

A fetal hemoglobin variant abnormal in the peptide chains has beenfound in an umbilical cord blood sample obtained from an infant who hadinherited Hb-G (presumably identical with G_Phila.) from his father. This provides evidence that chain synthesis in fetal and adult life is under a singlegenetic control and that chain mutations, unlike chain mutations, may bemanifested in intrauterine life.

     

Hemoglobin D Iran agr2A beta222-GlurarrGln in Association With Thalassemia

A 25-yr-old Indian (Asiatic) woman investigated for a life-long anemia was foundto have a hitherto undescribed structuralhemoglobin variant ₂^A₂^22-glugln, whichwas found independently and designatedHb D Iran by Rahbar in members of afamily from Iran. In the present case, Hb DIran was found in association with high A₂thalassemia. The replacement of glutamicacid by glutamine at 22 (helical residueB4) was demonstrated by thin-layer chromatography after automated Edman sequencing. This is the fourth substitution tobe described at 22; the previous substitutions were (1) G Coushatta (ala); (2) ESaskatoon (lys); (3) G Taipei (gly). Helicalresidue B4 is an external residue, does notparticipate in - or protein-heme contactsand hemoglobin D Iran resembles Hb A invisible spectra, O₂ equilibria in dilute solutions and heat stability. Since the propositus and her mother who was also heterozygous for thalassemia had similardegrees of anemia, no interaction betweenthis variant and thalassemia was evident.The presence of this Hb variant in Iraniansand in Western Indians may reflect themigrations of populations in these areascenturies ago.

Submitted on November 15, 1972 Revised on February 5, 1973 Accepted on February 6, 1973     

Unbalanced Globin Chain Synthesis in Congenital Dyserythropoietic Anemia

Hematologic evaluation of a 5-yr-old girlwith lifelong anemia demonstrated thecharacteristic findings of congenital dyserythropoietic anemia (CDA) type II.Globin chain synthesis was studied in vitroby measuring the incorporation of L-leucine-¹⁴C into globin by peripheral bloodand bone marrow erythroid cells. In cellsfrom the child and from both of her parentsan abnormal balance between the synthesis of the and non- globin componentsof hemoglobin was observed, the chainsbeing synthesized in excess. Neitherparent demonstrated microcytosis, hypochromia, or other findings suggestive of-thalassemia trait.

Submitted on December 13, 1972 Revised on June 8, 1973 Accepted on June 11, 1973     

Hemichromes in Single Inclusion Bodies in Red Cells of Beta Thalassemia

Inclusion bodies in the red cells of patients with thalassemia syndromes mayresult from precipitation of thosehemoglobin subunits that are producedin relative excess. In hemoglobin H disease, a form of -thalassemia, the inclusions are precipitated -subunits,while in the -thalassemias they are -subunits. It has been shown that theinclusions in hemoglobin H diseasehave the spectral characteristics ofhemichrome. In the present study, microspectrophotometric examination ofsingle inclusion bodies in ghosts prepared from the red cells of two patientswith -thalassemia major revealed theabsorption spectrum of hemichrome.These results suggest that hemichrome formation is an importantpathogenetic event in the production ofred cell inclusions and consequenthemolysis in the thalassemias.

Submitted on August 2, 1971 Revised on December 27, 1971 Accepted on December 29, 1971     

Cloning and Characterization of the Human Interleukin-3 (IL-3)/IL-5/ Granulocyte-Macrophage Colony-Stimulating Factor Receptor beta c Gene: Regulation by Ets Family Members

High-affinity receptors for interleukin-3 (IL-3), IL-5, andgranulocyte-macrophage colony-stimulating factor (GM-CSF) are composedof two distinct subunits, a ligand-specific chain and a common  chain (c). Whereas the mouse has two homologous subunits (cand IL-3), in humans, only a single  chain is identified. Wedescribe here the isolation and characterization of the gene encodingthe human IL-3/IL-5/GM-CSF receptor  subunit. The gene spans about25 kb and is divided into 14 exons, a structure very similar to that ofthe murine c/IL-3 genes. Surprisingly, we also found the remnantsof a second c chain gene directly downstream of c. We identifieda functional promoter that is active in the myeloid cell lines U937 andHL-60, but not in HeLa cells. The proximal promoter region, locatedfrom 103 to +33 bp, contains two GGAA consensus binding sites formembers of the Ets family. Single mutation of those sites reducespromoter activity by 70% to 90%. The 5 element specificallybinds PU.1, whereas the 3 element binds a yet-unidentifiedprotein. These findings, together with the observation thatcotransfection of PU.1 and other Ets family members enhances cpromoter activity in fibroblasts, reinforce the notion that GGAAelements play an important role in myeloid-specific gene regulation.     

Delivery of Co57B12 to Erythrocytes from agr and beta Globulin of Normal, B12-Deficient, and Chronic Myeloid Leukemia Serum

In a study of Co⁵⁷B₁₂ uptake by reticulocyte-rich erythrocytes, transfer fromchronic myeloid leukemia (CML) serum was less efficient than from normalserum. Decreased transfer of Co⁵⁷B₁₂ was not due to excessive transfer ofendogenous B₁₂ (which is normally elevated in CML) but was associatedwith an -globulin B₁₂-binder (isolated from "baby" DEAE columns) unableto normally deliver B₁₂ to erythrocytes. Delayed plasma clearance of intravenously injected B₁₂ in CML may be due to this phenomenon.

B₁₂-deficient serum delivered slightly more added Co⁵⁷B₁₂ to erythrocytesthan did normal serum, possibly due to less interference by endogenous B₁₂.- and -globulin B₁₂ binders from B₁₂-deficient serum transferred boundCo⁵⁷B₁₂ as efficiently as did normal serum - and -binders; the -binders delivered more than the -binders. CML -binder delivered as well as did - binder from pernicious anemia and normal serum. These findings suggest thatthe -binder of CML serum is physiologically and therefore chemically differentfrom the -binder in normal and pernicious anemia serum.

Reticulocyte-rich, B₁₂-deficient erythrocytes from pernicious anemia patientstook up Co⁵⁷B₁₂ less well than did B₁₂-sufficient reticulocyte-rich erythrocytes.This may partially explain delayed plasma clearance of B₁₂ in B₁₂ deficiency.

Rat liver homogenate uptake of Co⁵⁷B₁₂ was much greater when B₁₂ wastransferred from normal serum than from normal gastric juice or saline. Preliminary investigations showed that both - and -binders from normal serumtransferred B₁₂ to liver homogenate but CML -binder transferred poorly.

Submitted on September 9, 1966 Accepted on November 24, 1966     

Recombination Breakpoints in the Human beta -Globin Gene Cluster

The human -globin gene complex spans a region of 70 kb andcontains numerous sequence variants. These variant sites form a 5cluster (5 -haplotype) and a 3 cluster (3 -haplotype) withstrong linkage disequilibrium among the sites within each cluster, butnot between the two clusters. The 9-kb region between the 5 and 3clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a`hotspot' of recombination. We describe three families with evidenceof meiotic recombination within this `hotspot' of the -globin genecluster and in which the cross-over breakpoints have been defined atthe sequence level. In one family, the recombination has occurred inthe maternal chromosome within a region of 361 bp between positions911 and 550 5 to the -globin gene. In the other two families,the recombination has occurred in the paternal chromosome within aregion of approximately 1,100 bp between positions 542 and +568relative to the -globin gene cap site. Both regions occur within the2-kb region of replication initiation (IR) in the -globin genedomain with no overlap. The IR region contains a consensus sequence fora protein (Pur), which binds preferentially tosingle-stranded DNA, a role implicated in recombination events.     

Vitamin B12-binding Protein of Leukocytes as a Possible Major Source of the Third Vitamin B12-binding Protein of Serum

Much evidence suggests that granulocytes are a major source of serumvitamin B₁₂-binding protein (BBP). Thelatter has three components: -1-globulin BBP (Transcobalamin I, TC I),-globulin BBP (TC II), and a recentlydescribed third BBP. Granulocytic BBPhas appeared to be identical to TC Iexcept in electrophoretic mobility. Inthe present study, the dominant BBPof leukocyte extracts from subjectswith and without myeloproliferativedisease behaved like the third serumBBP. With a few exceptions, morethan half the leukocytic binder elutedwith the " globulins" on rapid DEAE-cellulose chromatography. At pH 8.6,electrophoretic mobility of the leukocytic BBP was always ₂ or . At ph4.5, normal and chronic myelogenousleukemia leukocytic BBP, unlike TC Iand TC II, showed little electrophoretic migration. These findings suggestthat leukocytic BBP is probably heterogenous and that its major componentresembles the third serum BBP morethan it does TC I. The third serumBBP, levels of which are elevated insome states of leukocytic proliferation,may derive directly from maturegranulocytes. TC I may arise by addition of sialic acid to the third (granulocytic) BBP under certain circumstances or be released from othercells, such as less mature granulocytes. Much of the confusion in theliterature regarding source and significance of serum BBP may relate toseparating it into only two fractions( globulin and " globulin," or "TC I"and TC II) instead of into three fractions.

Submitted on March 2, 1972 Accepted on April 26, 1972     

Hemoglobin Rush [beta101 (G3) Glutamine]: A New Unstable Hemoglobin Causing Mild Hemolytic Anemia

A mild hemolytic anemia in a 43-yr-old blackwoman was attributed to the presence of an abnormal hemoglobin (Hb Rush) which migratedcathodically to Hb A at pH 8.0. Its structuralabnormality was found to be in the -chain,101 (G3) glu gln. Another electrophoreticband at pH 8.0 proved to be a hybrid tetramer(^A₂^ARush). Hb Rush is heat unstable. Alikely explanation of the instability is thepresence of an uncovered positive charge in thecentral cavity where normally glutamic acid inposition 101 neutralizes arginine in position104 contributing to the net neutrality in thisregion. This neutrality is disturbed by the substitution of glutamic acid by glutamine in HbRush.

Submitted on May 10, 1973 Revised on June 25, 1973 Accepted on July 16, 1973     

The Characterization of Hemoglobin Shimonoseki

Hemoglobin Shimonoseki, discovered in western Japan in 1960, has beenfurther characterized as a mutant with abnormal -polypeptide chains on thebasis of:

(1) The presence of a minor hemoglobin component migrating cathodallyat pH 8.6 to Hb A₂, presumably ₂^Sh₂^A₂.

(2) Hybridization studies.

(3) Fingerprinting of isolated -polypeptide chains.

Hemoglobin Sh is characterized by the substitution of arginine for glutamine at residue 54 and can therefore be designated as ₂^{54 Arg}₂^A.

Submitted on December 17, 1963 Accepted on March 7, 1964     

Variable protection of beta 3-integrin--deficient mice from thrombosis initiated by different mechanisms

Platelet integrin IIb3 (GPIIb/IIIa) plays a central role inthe initiation of arterial thrombosis, but its contribution todisseminated microvascular thrombosis is less well defined. Therefore,wild-type mice (3^+/+), 3-integrin-deficient mice(3^/), and wild-type mice treated with a hamstermonoclonal antibody (1B5) that blocks murine IIb3 function weretested in models of large-vessel and microvascular thrombosis. In thelarge-vessel model, ferric chloride was used to injure the carotidartery, and the time to thrombosis was measured. In 3^+/+mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the 3^/ mice tested(P < .001). Fab and F(ab')₂ fragments of1B5 increased the median time to occlusion. To initiate systemicintravascular thrombosis, prothrombotic agents were administeredintravenously, and platelet thrombus formation was monitored by thedecrease in circulating platelet count. Three minutes after theinjection of adenosine diphosphate (ADP), collagen + epinephrine,or tissue factor, the platelet counts in 3^+/+ micedecreased by 289, 424, and 429 × 10³/µL, respectively.3^/ mice and wild-type mice pretreated with 1B5 Fab(1 mg/kg, IP) were nearly completely protected from the effects of ADP.In contrast, 3^/ mice were only partially protectedfrom the effects of collagen + epinephrine and minimally protectedfrom the effects of tissue factor. In all cases, less fibrin becamedeposited in the lungs of 3^/ mice than in wild-typemice. These results suggest that though IIb3 plays adominant role in large-vessel thrombosis, it plays a variable role insystemic intravascular thrombosis.     

Hemoglobin Andrew-Minneapolis agrA2beta144 Lys rarr Asn2: A New High-Oxygen-Affinity Mutant Human Hemoglobin

The functional properties and primarystructure of a new -chain mutant ofhuman hemoglobin are described. Themutant was transmitted as an autosomaldominant characteristic. Affected members of the kindred exhibited markederythrocytosis due to the high oxygenaffinity of the resultant hemoglobin. Theabnormality is associated with a substitution of an asparaginyl residue for lysinein the 144 position of the -chain,^A₂^144LysAsn₂, presumably due to anAAA/G to AAA/U transversion. The mutant hemoglobin displayed a profound increase in oxygen affinity, with a P₅₀ of thefresh whole blood of 14 mm Hg. The isolated mutant hemoglobin exhibited nearnormal heme—heme interaction, a half-normal Bohr effect, and normal reactivitywith 2,3-diphosphoglycerate.

Submitted on January 17, 1974 Accepted on April 15, 1974     

A New Abnormal Hemoglobin O Padova, agr 30 (B11) Glu rarr Lys, and a Dyserythropoietic Anemia With Erythroblastic Multinuclearity Coexisting in the Same Patient

A patient with a not previously describedabnormal hemoglobin (^{30Glu Lys}) anddyserythropoietic anemia with erythroblastic multinuclearity is reported. Theheat stability and the functional propertiesof the new abnormal hemoglobin, namedhemoglobin O Padova, are normal, although the replacement lies in the ₁₁ interchain contact. The hemolytic condition,which was much improved by splenectomy, therefore appears to be linked tothe hereditary erythroblastic multinuclearity similar to hereditary erythroblasticmultinuclearity with positive acidifiedserum test (HEMPAS). In addition to theleading features observed in publishedcases of this entity, our case exhibitedsome immunologic peculiarities.

Submitted on November 8, 1973 Accepted on June 11, 1974     

Coinduction of Embryonic and Adult-Type Globin mRNAs by Sodium Butyrate and Trichostatin A in Two Murine Interleukin-3-Dependent Bone Marrow-Derived Cell Lines

Using an RNase protection assay, globin mRNA species expressed inclones derived from Ba/F3 and B6SUtA cells transfected with theerythropoietin receptor (EpoR) and selected with erythropoietin (Epo)were compared with globin mRNA species induced in corresponding parental cells by sodium butyrate (SB) and trichostatin A (TSA). ^Major/^minor- and -1/-2-globinmRNAs were the major species, with trace amounts of -globin mRNA,formed in Epo-stimulated EpoR⁺ Ba/F3 clones, whereas SBand TSA allowed expression of all species of globin mRNAs, ie, ,h1, ^major/^minor, , and -1/-2,in parental Ba/F3 cells. In contrast, - and -1/-2-globinmRNAs were the major species present in Epo-stimulated EpoR⁺ B6SUtA clones, whereas SB and TSA activated -,h1-, ^S/^T-, and -1/-2-globingenes in parental B6SUtA cells; -globin mRNA was not detected in SB-and TSA-treated B6SUtA cells. Because TSA is a specific inhibitor ofhistone deacetylase, the mimicry of action exhibited by SB and TSAsuggests that the effects of SB are mediated through its ability toinhibit histone deacetylase and that histone deacetylase is an integralpart of the repression of globin genes in theseinterleukin-3-dependent cells. Efficient coinduction of embryonic andadult types of globin mRNA in bone marrow cell lines derived from adultmice indicates that adult hematopoietic precursors possess an embryonicnature. These cell lines are useful models to study the mechanism(s) ofdevelopmental globin gene switching.