HbF-Inducing Cytokines and BCL11A shRNA Have Combined Effects Upon Globin Gene Reprogramming In Adult Human Erythroblasts

Abstract Abstract 2076 Persistent expression of HbF ameliorates the clinical symptoms of β-thalassemia and sickle cell disease; therefore, reactivation of the γ-globin gene in adults is of substantial interest for the clinical management of β-hemoglobinopathies. The recently identified γ-globin repressor, BCL11A, has been shown to regulate HbF expression level in adult erythroblasts. Cytokine signal transduction also increases HbF expression in cultured erythroblasts due in part to reducing the chromatin occupancy of BCL11A within the β-globin locus. Cultured CD34+ cells from at least six healthy research subjects were utilized to investigate the potential for combined effects of genetic manipulation of BCL11A with signal transduction upon globin gene regulation. The cells were transduced with BCL11A shRNA lentiviral vectors under culture conditions with and without addition of HbF-inducing cytokines. Under all culture conditions, nuclear BCL11A protein levels were reduced to nearly undetectable levels by the shRNA lentiviral vectors. The combination effects of BCL11A knockdown and HbF-inducing cytokines were quantitated by QRT-PCR. The absolute copy numbers of the eight globin genes (β-like genes; ε-, γ-, δ-, β-globins and α-like genes; ζ-, μ-, α- and θ-globins) at the proerythroblast stage of maturation were determined using standard curve methods. As previously reported, cytokine-mediated induction of HbF significantly increased the expression of γ-, ε-, and ζ-globin mRNAs while β- and δ-globins were down-regulated (p-value <0.05). BCL11A knockdown similarly increased in γ-globin mRNA, decreased β- and δ-globin mRNAs, as well as an unexpected increase in ε- and ζ-globin mRNAs. A significant reduction in α-globin mRNA was additionally detected in association with BCL11A knockdown. Although the transcript levels of several individual globin genes were affected, the total α- to non-α-globin ratio remained stable. The combination of BCL11A knockdown with cytokine signal transduction resulted in further increased γ-globin mRNA to levels of 78.1 ± 3.5% of the total transcripts from the β-globin cluster. Strongly synergistic effects of BCL11A knockdown and cytokine signal transduction were also detected in the embryonic genes. As a result, ε-globin mRNA reached levels of 8.7 ± 4.7% of the total transcripts from the β-globin cluster compared with 0.0003% in baseline cultures, 0.07 ± 0.03% in HbF-inducing cytokines alone, and 0.04 ± 0.03% in BCL11A knockdown alone. Hemoglobin expression was also examined in matured cells from the same donors using high pressure liquid chromatography (HPLC). The additive effects of BCL11A knockdown and HbF-inducing cytokines were shown by HPLC: %HbF = 2.0 ± 0.7 at baseline, 28.2 ± 14.1 in HbF-inducing cytokines alone, 47.6 ± 5.7 in BCL11A knockdown alone, and 74.9 ± 2.5 in BCL11A knockdown combined with HbF-inducing cytokines. Cation exchange HPLC further revealed reduced HbA along with an unidentified peak downstream of HbA2 encompassing 1–5% of the total hemoglobin that is being investigated for embryonic globin content. These data demonstrate that synergistic and robust effects upon globin gene reprogramming in adult human erythroblasts may be achieved by combined mechanisms of signal transduction and genetic manipulation of BCL11A. Disclosures: No relevant conflicts of interest to declare.

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During Terminal Differentiation, HbF-Inducing Cytokines Cause Decreased Expression and Reduced Globin Locus Occupancy of BCL11A in Human Erythroblasts.

Blood ◽

10.1182/blood.v114.22.457.457 ◽

2009 ◽

Vol 114 (22) ◽

pp. 457-457

Author(s):

Orapan Sripichai ◽

Christine M. Kiefer ◽

Y. Terry Lee ◽

Emily Riehm Meier ◽

Colleen Byrnes ◽

...

Keyword(s):

Signal Transduction ◽

Protein Expression ◽

Culture Condition ◽

Conflicts Of Interest ◽

Association Studies ◽

Globin Gene ◽

Terminal Differentiation ◽

Fetal Hemoglobin ◽

Beta Globin ◽

Adult Human

Abstract Abstract 457 Genetic association studies and gene regulation studies demonstrate that the transcription factor BCL11A is a regulator of fetal hemoglobin (HbF) expression in humans. Cytokine signal transduction also regulates fetal hemoglobin expression in cultured adult human erythroblasts. To further explore the potential for BCL11A in the cytokine-mediated induction of HbF during adult erythropoiesis, transcript and protein expression levels of BCL11A were measured during erythroblast differentiation. BCL11A expression was detected at all stages of erythroid differentiation with the highest level expression in proerythroblasts during the first week in culture under both low-HbF (%HbF ≤3) and high-HbF (%HbF ≥30) culture conditions. Despite a reduction in BCL11A mRNA expression, Western analyses failed to demonstrate reduced levels of BCL11A nuclear protein expression at the proerythroblast stage of differentiation. However, BCL11A protein expression in the high-HbF producing cells was reduced relative to the low-HbF cells during the later period of culture as the cells underwent terminal differentiation. During this later culture period, hemoglobinization occurred, and cells grown in the high-HbF condition revealed a pancellular distribution of HbF compared with a heterocellular distribution in the low-HbF culture condition. Chromatin immunoprecipitation further demonstrated that the addition of HbF-inducing cytokines caused a nearly complete loss of BCL11A chromatin occupancy within the beta-globin locus under the high-HbF culture condition. Specifically, the loss of chromatin occupancy was detected in a region approximately 3 kb downstream of the (A)gamma-globin gene. Further examination of this genomic region demonstrated several BCL11A binding domains located on a cluster of non-coding, intronless RNAs previously named “BGL3” that possess an expression pattern in vivo that is largely restricted to the fetal-liver. In addition to increased and pancellular expression of fetal hemoglobin in the high-HbF erythroblasts, the loss of BCL11A chromatin occupancy in that region of the beta-globin locus was associated with increased expression of BGL3 mRNA (GenBank: AY034471) measured by RT-PCR. These findings demonstrate that defined combinations of cytokines regulate the expression level and chromatin occupancy of BCL11A in adult human erythroblasts as they undergo terminal differentiation. In addition to inheritance and ontogeny, the data also support a role for BCL11A in the regulation of HbF by cytokine signal transduction. Disclosures: No relevant conflicts of interest to declare.

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Diagnostic Challenges in a Low a2 β-Thalassemia Case

Blood ◽

10.1182/blood.v124.21.4898.4898 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4898-4898

Author(s):

Niren Patel ◽

Natalia Dixon ◽

Lina Zhuang ◽

Shanequa Bryant ◽

Ferdane Kutlar ◽

...

Keyword(s):

Los Angeles ◽

Gene Mutation ◽

Conflicts Of Interest ◽

Direct Sequencing ◽

Globin Gene ◽

Fe Deficiency ◽

Globin Genes ◽

Globin Mrna ◽

Thalassemia Trait ◽

Thalassemia Mutation

Abstract Inheritance of two β-thal alleles results in transfusion dependent anemia, growth retardation, and skeletal abnormalities (β-thal major), whereas heterozygosity (β-thal minor or β-thal trait) is clinically asymptomatic. β-thal trait leads to hypochromia, microcytosis, and in some cases, mild anemia. The characteristic finding in β-thal trait, however, is an elevated level of Hb A2 (>3.5%). Many factors are known to influence Hb A2 levels: α-thalassemia and Fe deficiency are two well-established factors. However, co-inheritance of α-thal with β-thal trait or concurrent Fe deficiency does not decrease Hb A2 to normal levels (1.8-3.5%). The differential diagnosis of “normal Hb A2 β-thal trait” includes δβ thalassemias resulting in the deletion of both δ and β globin genes, rare, mild, “silent” β-thal alleles (such as the -101 C→T mutation in the β-globin promoter), and rarely coinheritance of δ-thal alleles. We present a novel case of low Hb A2 β-thalassemia trait, wherein the value of Hb A2 is affected by the presence of δ gene mutation. The proband was never diagnosed to have a β-thalassemia trait until recently when her child was observed to have Hb FXA (variant hemoglobin + β-thalassemia compound heterozygosity). A 6-week-old Caucasian baby was referred to the pediatric hematology/oncology section of North Carolina Baptist Hospital for routine evaluation of Hb FXA pattern observed on newborn screening by electrophoresis. The parents were counseled by the physician to learn that the father is a known case of Hb D-Los Angeles trait but the mother was never diagnosed to have β-thalassemia. Initial CBC of the child revealed: Hb 11.7g/dL, Hct 34.9%, MCV 88.3fl, MCH 29.5pg, and MCHC 33.4g/dl. An EDTA blood sample on the child, mother, and father was sent to the T.H.J. Huisman Hemoglobinopathy Laboratory at Georgia Regents University in Augusta, Georgia for definitive identification of the Hb variant. Hemoglobin electrophoresis was performed and different hemoglobin fractions were calculated using cation exchange high performance liquid chromatography (HPLC) on a Synchropak CM-300 column Eprogen, Darien, IL, USA]. Genetic testing was performed to characterize the specific β-globin gene mutation. Amplification of the β-globin gene by polymerase chain reaction (PCR) and direct sequencing were used as a fast and reliable method for identification of the β-globin gene mutations. The HEP and DNA results are shown below. TableHemoglobin TypeInfantFatherMotherHb A%:1.456.696.3Hb A2%:1.33.72.6Hb F%:76.50.41.1Hb X%: (D-Los Angeles)20.839.3-β-globin gene sequencingIVS I-5, G→T β121(GH4) GAA→CAAβ121(GH4) GAA→CAAIVS I-5, G→T The Hemoglobin phenotype and genotype of the child is consistent with Hb D-Los Angeles - β+thalassemia. A heterozygous β+ thalassemia mutation was found [IVS I-5, G→T] along with a heterozygous Hb D-Los Angeles mutation [β121(GH4)Glu→Gln, GAA→CAA] on the infant’s sample. The father’s phenotype and genotype is consistent with Hb A – D Los Angeles trait; however, the mother’s genotype analysis is not consistent with the phenotype analysis. Her HEP shows a normal hemoglobin pattern but a heterozygous β+ thalassemia mutation [IVS I-5, G→T] was observed in her DNA. IVS I-5, G→T mutation is known to produce a high Hb A2 phenotype, but in our case, it was found to be within normal range. We further investigated to look for the presence of α-thalassemia that turned out to be negative (αα/αα). We further amplified and sequenced the δ globin gene and found a heterozygous Hb A2-Yialousa mutation [δ27(B9)Ala → Ser; HBD: c.82G→T] leading to δ+ thalassemia. This mutation activates a cryptic splice site at the boundary of Exon-1 and intron 1, thus leading to a decrease in correctly spliced δ-globin mRNA and hence, δ-thal phenotype. Hb A2-Yialousa explains the presence of a normal level of Hb A2 in the mother’s sample. The phenotype of increased Hb A2 typical of β-thalassemia carriers can be reduced to normal or borderline values because of the coinheritance of a δ-globin gene mutation, which may lead to misinterpretation of diagnostic results. Thus, Hb A2 values alone cannot be relied upon for the diagnosis of β-thalassemia cases. Disclosures No relevant conflicts of interest to declare.

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In situ hybridization reveals co-expression of embryonic and adult alpha globin genes in the earliest murine erythrocyte progenitors

Development ◽

10.1242/dev.116.4.1041 ◽

1992 ◽

Vol 116 (4) ◽

pp. 1041-1049 ◽

Cited By ~ 2

Author(s):

A. Leder ◽

A. Kuo ◽

M.M. Shen ◽

P. Leder

Keyword(s):

In Situ Hybridization ◽

Embryonic Development ◽

Fetal Liver ◽

Globin Gene ◽

Yolk Sac ◽

Globin Genes ◽

Globin Mrna ◽

Mature Adult ◽

Alpha Globin

Murine erythropoiesis begins with the formation of primitive red blood cells in the blood islands of the embryonic yolk sac on day 7.5 of gestation. By analogy to human erythropoiesis, it has been thought that there is a gradual switch from the exclusive expression of the embryonic alpha-like globin (zeta) to the mature adult form (alpha) in these early mouse cells. We have used in situ hybridization to assess expression of these two globin genes during embryonic development. In contrast to what might have been expected, we find that there is simultaneous expression of both zeta and alpha genes from the very onset of erythropoiesis in the yolk sac. At no time could we detect expression of embryonic zeta globin mRNA without concomitant expression of adult alpha globin mRNA. Indeed, adult alpha transcripts exceed those of embryonic zeta in the earliest red cell precursors. Moreover, the pattern of hybridization reveals co-expression of both genes within the same cells. Even in the fetal liver, which supersedes the yolk sac as the major site of murine fetal erythropoiesis, there is a brief co-expression of zeta and alpha genes followed by the exclusive expression of the adult alpha genes. These data indicate an important difference in hematopoietic ontogeny between mouse and that of human, where zeta expression precedes that of alpha. In addition to resolving the embryonic expression of these globin genes, our results suggest that the embryonic alpha-like globin gene zeta may be physiologically redundant, even during the earliest stages of embryonic development.

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Characterization, expression, and evolution of the mouse embryonic zeta-globin gene.

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.1025 ◽

1985 ◽

Vol 5 (5) ◽

pp. 1025-1033 ◽

Cited By ~ 23

Author(s):

A Leder ◽

L Weir ◽

P Leder

Keyword(s):

Amino Acid ◽

Polypeptide Chain ◽

Globin Gene ◽

Complete Sequence ◽

Globin Genes ◽

Globin Mrna ◽

A Cell ◽

Erythroleukemic Cell ◽

Zeta Globin Gene ◽

Cloned Gene

We have determined the complete sequence of the embryonic alpha-like, zeta (zeta)-globin gene of the BALB/c mouse. The structure of this gene establishes the amino acid sequence of the mouse embryonic zeta-globin polypeptide chain and allows us to identify sequences within the gene that may be important for its expression. One of these is a 300-base segment that is tightly conserved between mice and humans and is located at the 5' end of the zeta-globin gene. By introducing the cloned gene into permanently transfected mouse erythroleukemic cell lines and comparing its transcript with that of zeta-globin mRNA derived from embryonic yolk sac erythrocytes, we are able to show that the cloned gene is transcriptionally active and that its transcript is correctly initiated and processed. Interestingly, the zeta-globin gene is also active when permanently transfected into an immunoglobulin-producing B-cell, a cell that presumably has tissue-specific requirements for gene expression. Further, a comparison of the amino acid coding sequence of the mouse zeta-globin gene to that of zeta-like globin genes of other species supports a revised evolutionary lineage in which goats and humans are closely related, whereas mice are further removed.

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Spontaneous delta- to beta-globin switching in K562 human leukemia cells

Blood ◽

10.1182/blood.v79.3.820.820 ◽

1992 ◽

Vol 79 (3) ◽

pp. 820-825 ◽

Cited By ~ 7

Author(s):

B Mookerjee ◽

MO Arcasoy ◽

GF Atweh

Keyword(s):

Cell Line ◽

Messenger Rna ◽

Culture Conditions ◽

Human Leukemia ◽

Globin Genes ◽

Beta Globin ◽

Globin Mrna ◽

Hemoglobin Switching ◽

Erythroleukemia Cell ◽

Globin Promoter

Abstract Previous analysis of the hemoglobin phenotype of the K562 human erythroleukemia cell line showed regulated expression of the epsilon-, zeta-, gamma-, alpha-, and delta-globin genes. Expression of the beta- globin genes has not been previously detected in this cell line. In this report, we describe the isolation of a variant of the K562 cell line that actively expresses beta-globin messenger RNA (mRNA) and polypeptide and shows greatly reduced expression of the delta-globin genes. This phenotype developed spontaneously in culture while two other K562 isolates grown under the same culture conditions have not undergone the same delta- to beta-globin switch. Analysis of this unique K562 variant shows that a construct containing a beta-globin promoter is quite active upon transient transfection into these cells. This finding suggests that the activation of the endogenous beta-globin genes results from changes in the trans-acting environment of these cells. The regulation of the beta-globin genes in this variant is characterized by a paradoxical decrease in the level of beta-globin mRNA after exposure to hemin. Other globin genes of this variant are appropriately regulated and show increased expression after hemin induction. Further study of this variant may shed light on mechanisms of gene regulation that are involved in hemoglobin switching.

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Cytokine-mediated increases in fetal hemoglobin are associated with globin gene histone modification and transcription factor reprogramming

Blood ◽

10.1182/blood-2009-05-219386 ◽

2009 ◽

Vol 114 (11) ◽

pp. 2299-2306 ◽

Cited By ~ 38

Author(s):

Orapan Sripichai ◽

Christine M. Kiefer ◽

Natarajan V. Bhanu ◽

Toshihiko Tanno ◽

Seung-Jae Noh ◽

...

Keyword(s):

Signal Transduction ◽

Transcription Factor ◽

Transcription Factors ◽

Protein Expression ◽

Histone Modification ◽

Globin Gene ◽

Expression Patterns ◽

Fetal Hemoglobin ◽

Transcriptome Profiling ◽

Globin Genes

Abstract Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

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Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6634 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6634-6640 ◽

Cited By ~ 30

Author(s):

Denise E. Sabatino ◽

Amanda P. Cline ◽

Patrick G. Gallagher ◽

Lisa J. Garrett ◽

George Stamatoyannopoulos ◽

...

Keyword(s):

Transgenic Mice ◽

Fetal Liver ◽

Globin Gene ◽

Gene Promoter ◽

Yolk Sac ◽

Globin Genes ◽

Erythroid Cells ◽

Globin Mrna ◽

Globin Promoter ◽

In Erythroid Cells

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.

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Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽

10.1182/blood.v108.11.555.555 ◽

2006 ◽

Vol 108 (11) ◽

pp. 555-555 ◽

Cited By ~ 2

Author(s):

Hassana Fathallah ◽

Ali Taher ◽

Ali Bazarbachi ◽

George F. Atweh

Keyword(s):

Gene Expression ◽

Sickle Cell Disease ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Mrna Levels ◽

Globin Genes ◽

Thalassemia Intermedia ◽

Cell Disease ◽

Globin Mrna ◽

Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

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Globin Genes Induction by Nitric Oxide of Stromal Cell Origin.

Blood ◽

10.1182/blood.v110.11.4102.4102 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4102-4102

Author(s):

Vladan P. Cokic ◽

Bojana B. Beleslin-Cokic ◽

Constance Tom Noguchi ◽

Alan N. Schechter

Keyword(s):

Progenitor Cells ◽

Globin Gene ◽

Erythroid Cell ◽

Mrna Levels ◽

Globin Genes ◽

Globin Mrna ◽

Erythroid Progenitor ◽

Gamma Globin ◽

Erythroid Progenitor Cells ◽

Globin Gene Expression

Abstract We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). Here we report that co-culture of human bone marrow endothelial cells with erythroid progenitor cells induced gamma-globin mRNA expression (1.8 fold), and was further elevated (2.4 fold) in the presence of hydroxyurea (40 μM). Based on these results, NOS-dependent stimulation of NO levels by bradykinin and lipopolysaccharide has been observed in endothelial (up to 0.3 μM of NO) and macrophage cells (up to 6 μM of NO), respectively. Bradykinin slightly increased gamma-globin mRNA levels in erythroid progenitor cells, but failed to increase gamma-globin mRNA levels in endothelial/erythroid cell co-cultures indicating that stimulation of endothelial cell production of NO alone is not sufficient to induce gamma-globin expression. In contrast, lipopolysaccharide and interferon-gamma mutually increased gamma-globin gene expression (2 fold) in macrophage/erythroid cell co-cultures. In addition, hydroxyurea (5–100 μM) induced NOS-dependent production of NO in human (up to 0.7 μM) and mouse macrophages (up to 1.2 μM). Co-culture studies of macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to 3 fold) in the presence of hydroxyurea (20–100 μM). These results demonstrate a mechanism by which hydroxyurea may induce globin genes and affect changes in the phenotype of hematopoietic cells via the common paracrine effect of bone marrow stromal cells.

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Diagnostic Complications of Thalassemic Hb Showa-Yakushiji (β110[G12] Leu→Pro),Observed in An African American Child with Co-Inherited Hb B2 (δ16[A13]Gly→Arg) and α-Thal-2 (-α3.7 deletion): Effects of Triple Globin Gene Abnormality On Phenotype.

Blood ◽

10.1182/blood.v114.22.2558.2558 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2558-2558

Author(s):

Chinwe Obiaga ◽

Niren Patel ◽

Hernan Sabio ◽

Natalia Dixon ◽

Steffen E. Meiler ◽

...

Keyword(s):

African American ◽

Conflicts Of Interest ◽

Globin Gene ◽

Molecular Characteristics ◽

African American Child ◽

Globin Genes ◽

Heterozygous State ◽

Globin Chain ◽

American Child ◽

Globin Chains

Abstract Abstract 2558 Poster Board II-535 Hemoglobinopathies can usually be classified under two major categories. Qualitative abnormalities resulting from missense mutations in the globin genes, leading to the production of mostly asymptomatic Hb variants, and quantitative defects, which result in the synthesis of structurally normal globin chains in reduced quantities (thalassemias). However there are known globin chain variants that cause alterations of the globin structure as well as a decrease in synthesis, leading to a thalassemic phenotype. The occurrence of multiple abnormalities of α, β and δ globin chains can lead to an unusual and complex phenotype. We report here the inheritance of triple globin gene abnormalities in an African American child with a genotype that is heterozygous for three abnormalities: α-thal-2 (-α3.7 deletion), thalassemic Hb Showa-Yakushiji (β110[G12] Leu→Pro), and a δ-chain variant Hb B2 (δ16[A13]Gly→ Arg) . Although Hb Showa Yakushiji presents with a severe hemolytic anemia and a thalassemia-like phenotype in the heterozygous state; when co-inherited with Hb B2 and α-thal-2, a milder phenotype was observed. We report the diagnostic approach, molecular characteristics and genotype/phenotype correlations of this complex hemoglobinopathy syndrome. A 2 year old African American boy presented with anemia which was not responsive to iron therapy. CBC revealed: Hb 9.9 g/dL, Hct 31.3 %, MCV 62.5 fl, MCH 19.8 pg, MCHC 31.7 g/dl. The reticulocyte count was 1.1%. The iron profile showed a TIBC of 368; Iron 119; Transferrin 257, Ferritin 30; and % Iron saturation 32. The peripheral blood smear revealed a microcytic anemia suggestive of a thalassemic phenotype. The patient's hemolysate was analyzed by isoelectric focusing (IEF) showed Hb's A, F, A2, and a minor peak Hb X which was significantly slower than Hb A2 . Quantitative values by high performance liquid chromatography (HPLC) were: Hb F : 5.0%, Hb A: 91.0%, Hb A2: 2. 0% and Hb X (B2): 2.0%. Reverse Phase HPLC was also performed and no additional abnormality was detected. Sequencing of the β-globin genes revealed a heterozygous T→C mutation at the codon 110 consistent with Hb Showa-Yakushiji (β110[G12] Leu →Pro) which was not detectable with IEF and HPLC. Sequencing of the δ-globin genes showed a heterozygous G→C mutation at codon 16, Hb B2 (δ16[A13] Gly →Arg) which was also not detectible by IEF or HPLC unless over applied. A 590 bp long fragment of the β-globin gene (Accession # EU605697/APR-2008) and a 780 bp long fragment of the δ-globin gene (Accession # EU605698/APR-2008) sequences have been submitted to NCBI/GenBank. Detection of alpha thalasemia (α−3.7) deletion by PCR analysis, revealed one alpha gene deletion (−3.7α/αα). The leucine to proline substitution at residue 110 of β-globin chain, disrupts the G helix and the α1β1 contact of the hemoglobin molecule. As a result, an extremely unstable Hb variant will be produced, which leads to a thalassemic phenotype because of the reduced stability/viability of the mutant beta chain. Previously reported cases of Hb Showa-Yakushiji showed a more severe clinical picture in the heterozygous state than that observed in our patient. This is the first time Hb Showa-Yakushiji is identified in an African American child who presented with a moderate anemia and a thalassemia-like phenotype. The milder phenotype observed in our case may be due to the co-inheritance of α-thal-2 (α−3.7) deletion. The decreased production of α- globin chains may ameliorate the effect of the chain imbalances thus leading to milder clinical and hematologic manifestations. Disclosures: No relevant conflicts of interest to declare.

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