scholarly journals Efficacy of ruxolitinib as inducer of fetal hemoglobin in primary erythroid cultures from sickle cell and beta-thalassemia patients

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Alice Pecoraro ◽  
Antonio Troia ◽  
Aurelio Maggio ◽  
Rosalba Di Marzo
Keyword(s):  
Mrna Expression ◽  
Sickle Cell ◽  
Cultured Cells ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Gamma Globin

High levels of HbF may ameliorate the clinical course of β-thalassaemia and SCD. Hydroxyurea (HU) is the only HbF inducer approved for the treatment of patients. However not all patients respond to the treatment, for this reason it is noteworthy to identify new HbF inducers. Ruxolitinib is a JAK inhibitor that decreases the phosphorilation of STAT proteins. In particular STAT3 is a repressor of gamma-globin gene. The decrease of STAT3 phosphorilation could derepress gamma-globin gene and reactivate its trascription. In this study we evaluated the efficacy of ruxolitinib as inducer of HbF production. The analyses were performed in cultured erythroid progenitors from 16 beta-thalassemia intermedia (TI) and 4 sickle cell disease (SCD) patients. The use of quantitative RT-PCR technique allowed us to determine the increase of gamma-globin mRNA expression in human erythroid cultured cells treated with ruxolitinib. The results of our study demonstrated an increase in vitro of gamma-globin mRNA expression in almost all patients. These data suggest that ruxolitinib could be a good candidate to be used in vivo for the treatment of hemoglobinopathies.

scholarly journals Study on the Efficacy of a JAK Inhibitor Pharmacological Agent As Inducer of Fetal Hemoglobin Production in Cultured Erythroid Precursors from Sickle Cell and Beta-Thalassemia Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2048-2048
Author(s):  
Alice Pecoraro ◽  
Antonio Troia ◽  
Angela Vitrano ◽  
Rosario Di Maggio ◽  
Massimiliano Sacco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Long Term Treatment ◽  
Globin Gene Expression

Abstract Phenotypic improvement of hemoglobinopathies such as sickle cell disease and beta-thalassemia (beta-thal) has been shown in patients with high levels of fetal hemoglobin (HbF). In sickle cell disease (SCD) the beneficial effects of HbF are due to the inhibition of HbS polymerization and to the dilution of HbS determining the reduction of sickling and vascular occlusion. Moreover, in beta-thal, high levels of gamma-chains combined with the redundant alpha-chains, lead to a reduction of dyserythropoiesis and of the requirement for blood transfusions. The only drug approved for the treatment of adult patients with SCD and that has been entered in clinical practice of patients affected by beta-thal is hydroxyurea (HU); however there is a great variability in the responses of patients to HU, in fact some patients are good responder, while others exhibit little or no change in HbF levels after HU treatment; moreover a decrease in the efficacy during long term treatment was observed. Other pharmacological compounds, including 5-azacytidine and thalidomide have been shown to increase HbF production. Due to concerns about the safety of this agents, their use was limited to severe cases for whom conventional therapy was unfeasible. For this reason the search of new inducers of HbF production is important. Ruxolitinib is a JAK inhibitor and decreases the phosphorilation of STAT (Signal transducers and activators of transcription) family proteins, in particular STAT5 and STAT3. Phosphorylation of STAT5 is essential for basal erythropoiesis and for its acceleration during stress erythropoiesis. STAT3 plays an essential role in regulating gene expression of several genes involved in cell growth and apoptosis, in particular it was demonstrated to inhibit gamma-globin gene expression. The decrease of STAT3 phoshorilation could decrease the inhibition of gamma-globin gene expression; for this reason we considered ruxolitinib a candidate as inducer of HbF production. In our laboratory an ex vivo system was developed predictive of the in vivo response to hydroxyurea treatment by using liquid erythroid cultures, an in vitro culture system that recapitulates the process of human erythropoiesis. To evaluate the efficacy of ruxolitinib in increasing gamma-globin gene expression we carried out a study in vitro using liquid erythroid cultures. In this study we developed and exposed to ruxolitinib liquid erythroid precursors from 4 SCD and 17 beta-thal intermedia (beta-TI) patients. The use of quantitative Real-Time-polymerase chain reaction allowed us to determine the increase in gamma-globin mRNA expression in human erythroid cells treated with ruxolitinib compared to untreated cells. The results are summarized in Table 1 and showed that ruxolitinib at 200nM is able to determine a significant increase of gamma-globin gene expression (3.4±0.1)compared to HU (2.0± 0.2). In conclusion our study suggests that ruxolitinib could be considered an inducer of HbF and could be used in vivo for the treatment of hemoglobinopathies, particularly in patients who do not respond to HU therapy or who show a decreased response after long-term treatment. Table 1. Fold increase of Gamma-globin gene expression in presence of Ruxolitinib in erythroid cultured cells. Patient Sex Genotype gamma-globin mRNA fold increasein the presence of ruxolitinib #1 M b039/aaa +1 #2 F b039/aaa +1.65 #3 F b039/b039 +1.9 #4 F b039/IVS1,110 +1.5 #5 M IVS1,1/aaa +2.5 #6 M IVS1,110/IVS1,1 +9.2 #7 M b039/bs +6 #8 F bs/b039 +1.6 #9 F b039/IVS1,6 +1.7 #10 M IVS1,6/frcd6 +3 #11 M IVS1,6/bs +2.5 #12 M IVS1,6/frcd6 +8 #13 F IVS1,6/b039 +9 #14 M IVS1,1/b039 +2.2 #15 M db/IVS1,110 +8 #16 F db/IVS1,110 +1.8 #17 F IVS2,1/aaa +3.9 #18 M b039/-101 +1.4 #19 M IVS1,6/b039 +1 #20 M bs/IVS1,110 +1.4 #21 M IVS1,6/IVS1,6 +1.9 Disclosures No relevant conflicts of interest to declare.

scholarly journals AUF-1 and YB-1 are critical determinants of β-globin mRNA expression in erythroid cells

Blood ◽  
2012 ◽  
Vol 119 (4) ◽  
pp. 1045-1053 ◽  
Author(s):  
Sebastiaan van Zalen ◽  
Grace R. Jeschke ◽  
Elizabeth O. Hexner ◽  
J. Eric Russell
Keyword(s):  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Affinity Enrichment

Abstract The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

scholarly journals The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1470-1474 ◽  
Author(s):  
GF Atweh ◽  
XX Zhu ◽  
HE Brickner ◽  
CH Dowling ◽  
HH Jr Kazazian ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Mutation ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
New Type

A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

scholarly journals Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 454-459 ◽  
Author(s):  
SP Perrine ◽  
BA Miller ◽  
DV Faller ◽  
RA Cohen ◽  
EP Vichinsky ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Sodium Butyrate ◽  
Clinical Symptoms ◽  
Therapeutic Potential ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Gamma Globin

Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.

Next Generation Clinical-Stage HbF Inducers Exhibit Multiple Molecular Actions In Vitro and In Vivo and Offer Personalized Therapeutic Regimens

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 560-560
Author(s):  
Betty S. Pace ◽  
Jose I Sangerman ◽  
Michael S Boosalis ◽  
Roman Wolf ◽  
Yan Dai ◽  
...  
Keyword(s):  
Globin Gene ◽  
Clinical Stage ◽  
Hdac Inhibitors ◽  
Gene Promoter ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
F Cells ◽  
High Level

Abstract High-level expression of fetal (Υ) globin reduces clinical complications in sickle cell disease and this is achieved with hydroxyurea (HU) in young children. However, non-cytotoxic high-potency therapeutics, particularly which can be utilized in combination with HU, are needed for most adolescent and adult patients who have continued serious clinical events. We have identified additional pharmaceutical candidates which induce HbF without cytotoxicity, using a Υ-globin gene promoter linked to GFP for robotic high-throughput screening, and screening five diverse chemical libraries. From a library of US and EU drugs which are approved for treatment of other medical conditions, a small panel of approved therapeutics were found to induce Υ globin expression, and have benign safety profiles, are orally active, and are suitable for long-term use. Three orally active candidates were evaluated in anemic baboons, and two, DLT and PB-04, induced Υ-globin mRNA by 15- to 33-fold over baseline levels. In 3/3 beta-globin locus YAC transgenic mice, one candidate (PB-04; 20 mg/kg) given by intra-peritoneal (IP) injections (for experimental feasibility) 3 times/ week for 5 wks significantly increased F-cells from 0.1 to 9%, 0.4 to 18%, and 0.13 to 12% respectively; and mean fluorescence intensity (MFI) increased by 10- to 33-fold. Responses were observed within one week. In hydroxyurea treated mice (100 mg/kg; IP, 5 days/ wk) F-cells increased from 0.3 to 2.3% on average (p<0.05), and MFI increased by 6- to 7-fold, while water vehicle did not increase F-cells significantly. PB-04 has been used clinically for decades as an excipient solely to prolong the half-life of another pharmaceutical, and is suitable for repurposing. In ChIP assays in K562 cells, PB-04 treatment demonstrated dual actions of displacing HDAC3 by 20-fold and LSD-1 by 3-fold from the Υ globin gene promoter. To investigate potential effects of genetic modifiers of HbF on responses to different HbF inducer classes, erythroid progenitors from 40 sickle and beta thalassemic subjects were sub-genotyped for 3 major quantitative trait loci (QTL) (Bcl-11A, HMIP, Xmn-I) and cultured +/- 7 HbF inducers which are in clinical trials or approved. Most HbF inducers, including decitabine and butyrate used as positive controls, are active in 50-70% of progenitors, with differential Υ-globin mRNA responses observed. Only 10% of progenitors did not respond to any inducing agent. Most progenitors with the Xmn-1 variant responded with higher Υ globin transcription to all inducers. Sodium dimethylbutyrate (HQK-1001) and decitabine, produced 6-fold overall mean induction. PB-04 produced 9-fold mean induction above untreated control levels from the same subject. HDAC inhibitors (Butyrate, MS-275) which suppress Bcl-11A expression, demonstrate higher activity in progenitors from subjects without an underlying SNP in Bcl-11A. Another HDAC inhibitor, SB939, produced responses in 80% of progenitors. 25-30% of subjects’ progenitors exhibit high induction, 12-to 40-fold above untreated controls, to dimethylbutyrate, PB-04, decitabine, and an HDAC1/2 inhibitor 14F, suggesting a “high responder genotype” of which only half had a recognized favorable QTL. Taken together, these in vitro and in vivo studies identify a mini-pipeline of clinical-stage HbF-inducing therapeutics, with both epigenetic and targeted molecular actions, which can be investigated clinically to develop tailored therapeutics and therapeutic combinations for high-level induction of HbF in subgenotyped hemoglobinopathy patients. Seven new and established inducers have been evaluated in erythroid progenitors cultured from sub-genotyped hemoglobinopathy patients, 3 new drugs (PB-04, DLT, RSV) and two HDAC inhibitors (MS-275 and SB939), induced 3- to 40-fold higher Υ-globin mRNA above untreated control levels. Disclosures: Faller: Phoenicia BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Perrine:Phoenicia BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

scholarly journals Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 454-459 ◽  
Author(s):  
SP Perrine ◽  
BA Miller ◽  
DV Faller ◽  
RA Cohen ◽  
EP Vichinsky ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Sodium Butyrate ◽  
Clinical Symptoms ◽  
Therapeutic Potential ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Gamma Globin

Abstract Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.

scholarly journals The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1470-1474 ◽  
Author(s):  
GF Atweh ◽  
XX Zhu ◽  
HE Brickner ◽  
CH Dowling ◽  
HH Jr Kazazian ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Mutation ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
New Type

Abstract A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

Targeting BCL11A and KLF1 For The Treatment Of Sickle Cell Disease and β-Thalassemia In Vitro using Antisense Oligonucleotides

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1022-1022 ◽  
Author(s):  
Raechel Peralta ◽  
Audrey Low ◽  
Aneeza Kim ◽  
Sue Murray ◽  
Shuling Guo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mrna Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Antisense Oligonucleotides ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Mrna ◽  
Dose Dependent

Abstract Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein with greater oxygen binding affinity in the fetus during the last months of embryonic development and the first few months of life after birth. HbF inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD as well as β-thalassemia. In β-thalassemia, there is reduced or absent synthesis of the β-globin gene, causing ineffective erythropoiesis. B-cell lymphoma/leukemia 11A (BCL11A) is a transcription factor in the zinc-finger protein family and is expressed in B cells and erythroid cells. BCL11A represses fetal hemoglobin expression by binding to the GGCCCGG motif in the β-globin promoter region. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of BCL11A and KLF1, we can promote production of HbF through the upregulation of γ-globin expression. To demonstrate upregulation of γ-globin mRNA expression in vitro, we used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse BCL11A and KLF1 (Tim Townes, Univ. of Alabama at Birmingham). Antisense oligonucleotides (ASOs) targeting mouse BCL11A or mouse KLF1 were added to the cells in a dose-dependent manner. Seven days later, with free uptake of the ASOs into the cells, we observed dose-dependent reduction of mouse BCL11A mRNA (IC50 = 0.7 μM) and mouse KLF1 mRNA (IC50 = 3 μM). Consequently, we observed a 300 +/- 8% upregulation of human γ-globin mRNA expression after achieving ∼90% reduction in BCL11A mRNA expression after ASO treatment compared to the untreated control cells. Similarly, KLF1 ASO treatment caused a 500 +/- 58% up regulation of human γ-globin mRNA expression after achieving ∼80% mRNA reduction in KLF expression. These data indicate that targeting BCL11A and/or KLF1 with ASO treatment can cause an increase in γ-globin expression that is necessary for the upregulation of fetal hemoglobin and may be used for the treatment of sickle-cell anemia and β-thalassemia. Disclosures: Peralta: Isis Pharmaceuticals, Inc.: Employment. Low: Isis Pharmaceuticals, Inc.: Employment. Kim: Isis Pharmaceuticals, Inc.: Employment. Murray: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Freier: Isis Pharmaceuticals, Inc.: Employment. Townes: University of Alabama at Birmingham: Employment. Hung: Isis Pharmaceuticals, Inc.: Employment.

scholarly journals Sickle Cell Anemia and Babesia Infection

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1435
Author(s):  
Divya Beri ◽  
Manpreet Singh ◽  
Marilis Rodriguez ◽  
Karina Yazdanbakhsh ◽  
Cheryl Ann Lobo
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Current Knowledge ◽  
Globin Gene ◽  
Environmental Niche ◽  
Parasite Resistance ◽  
The Usa ◽  
Human Infections

Babesia is an intraerythrocytic, obligate Apicomplexan parasite that has, in the last century, been implicated in human infections via zoonosis and is now widespread, especially in parts of the USA and Europe. It is naturally transmitted by the bite of a tick, but transfused blood from infected donors has also proven to be a major source of transmission. When infected, most humans are clinically asymptomatic, but the parasite can prove to be lethal when it infects immunocompromised individuals. Hemolysis and anemia are two common symptoms that accompany many infectious diseases, and this is particularly true of parasitic diseases that target red cells. Clinically, this becomes an acute problem for subjects who are prone to hemolysis and depend on frequent transfusions, like patients with sickle cell anemia or thalassemia. Little is known about Babesia’s pathogenesis in these hemoglobinopathies, and most parallels are drawn from its evolutionarily related Plasmodium parasite which shares the same environmental niche, the RBCs, in the human host. In vitro as well as in vivo Babesia-infected mouse sickle cell disease (SCD) models support the inhibition of intra-erythrocytic parasite proliferation, but mechanisms driving the protection of such hemoglobinopathies against infection are not fully studied. This review provides an overview of our current knowledge of Babesia infection and hemoglobinopathies, focusing on possible mechanisms behind this parasite resistance and the clinical repercussions faced by Babesia-infected human hosts harboring mutations in their globin gene.

scholarly journals The inactive beta globin gene on a gamma delta beta thalassemia chromosome has a normal structure and functions normally in vitro

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 766-770
Author(s):  
PT Curtin ◽  
YW Kan
Keyword(s):  
Globin Gene ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Ribonuclease Protection Assay ◽  
Beta Globin ◽  
Gamma Delta ◽  
Beta Globin Gene ◽  
Thalassemia Minor

We have previously described an English family with gamma delta beta- thalassemia in which a large deletion stops 25 kilobases (kb) upstream from the beta-globin gene locus, and yet the beta-globin gene is inactive in vivo. Affected family members had a beta-thalassemia minor phenotype with a normal hemoglobin A2 level. Gene mapping showed that these subjects were heterozygous for a chromosome bearing a large deletion that began in the G gamma-globin gene, extended through the epsilon-globin gene, and continued upstream for at least 75 kb. The A gamma-, delta-, and beta-globin gene loci on this chromosome were intact. To examine the possibility that an additional defect was present in the beta-globin gene, we cloned, sequenced, and examined the expression of the beta-globin gene from the affected chromosome. No mutation was found in the beta-globin gene sequence from 990 base-pairs 5′ to the cap site to 350 basepairs 3′ to the polyadenylation signal. The gene was subcloned into an expression vector and introduced into HeLa cells. Analysis of RNA derived from these cells, using a ribonuclease protection assay, revealed qualitatively and quantitatively normal transcription. Thus a structurally and functionally normal beta-globin gene is inactive in the presence of a large deletion more than 25 kb upstream. The loss of beta-globin gene function may be due to disturbance of chromatin conformation caused by the deletion or may be the result of loss of upstream sequences that are necessary for beta-globin gene expression in vivo.

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